Ionizable lipids comprising n,n-substituted hydroxylamine esters

Abstract

The present disclosure provides ionizable lipids that are particularly beneficial in formulations of lipid nanoparticles providing advantageous improvements in the delivery of nucleic acids to hepatic stellate cells with enhanced efficiency. The ionizable lipids are N,N-substituted hydroxylamine ester compound. The disclosure also provides lipid nanoparticles, pharmaceutical compositions, and methods including the provided ionizable lipids.

Description

PATENT Attorney Docket No.: 104290-1528239-G00710PC Client Reference No.: G-K007PC1_LK IONIZABLE LIPIDS COMPRISING N,N-SUBSTITUTED HYDROXYLAMINE ESTERS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 707,655 filed October 15, 2024, the full disclosure of which is incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] Lipid nanoparticles (LNPs) have emerged as a critical technology in the field of drug delivery, particularly for the encapsulation and systemic delivery of nucleic acids such as small interfering RNA (siRNA) and messenger RNA (mRNA). Their importance lies in their ability to protect these therapeutic agents from degradation, enhance cellular uptake, and control the release of the encapsulated agent.

[0003] As one example, the strategy of targeting LNPs to hepatic stellate cells (HSCs) for the delivery of nucleic acids is a promising advancement in the field of liver disease therapeutics. HSCs are a type of perisinusoidal cell in the liver that play a crucial role in the progression of liver diseases. In a healthy liver, HSCs are in a quiescent state. However, during liver injury, these cells become activated and transform into myofibroblasts, which are the principal collagen-producing cells in the liver. This transformation and subsequent collagen production contribute significantly to the development of liver fibrosis and hepatocellular carcinoma.

[0004] By directing LNPs to HSCs, nucleic acids such as siRNA or mRNA can be delivered directly to these cells. These nucleic acids can modulate the gene expression within HSCs, potentially inhibiting the process of fibrogenesis and slowing or even reversing the progression of liver fibrosis and hepatocellular carcinoma. This targeted approach has the potential to provide a more precise treatment, potentially reducing off-target effects and improving the therapeutic index.

[0005] While the targeted delivery of nucleic acids to HSCs using LNPs thus presents a promising therapeutic strategy for liver diseases, several limitations currently hinder its full potential. One of the primary challenges lies in achieving high delivery efficiency. Although1 80125197V.1systemically administered LNPs can access organs with fenestrated capillaries, such as the liver and spleen, ensuring that these LNPs efficiently target HSCs within these organs remains a complex task. This is further complicated by the heterogeneity of HSCs and the dynamic changes these cells undergo during activation. Furthermore, the field lacks sufficient research on HSC-specific targeted delivery, further indicating that the therapeutic cargo may not reach the HSCs in adequate quantities to exert a substantial effect. Lastly, LNP formulations must offer high tolerability, such as high biocompatibility and low toxicity, as otherwise potential adverse effects or immune responses associated with their use can arise. The present disclosure addresses these needs and provides associated and other advantages. BRIEF SUMMARY

[0006] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The 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 determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0007] In one aspect, the disclosure provides a compound having the formula:or the formula of a salt thereof. R1, R2, R3, and R4of formula (I) can each independently be C1-40alkyl, C2-40alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which can optionally be substituted with one or more R7groups. R5of formula (I) can be C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, each of which can optionally be substituted with one or more R7groups. Alternatively, R5can be combined with X1and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups. R6of formula (I) can be hydrogen or can be C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which can be substituted with one or more R7groups. Each R7can independently be C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, C1-6haloalkyl,2 80125197V.1hydroxy, halogen, oxo, -NH2, nitro, or cyano. Alternatively, any R7can be combined with another R7and the carbon to which they are attached to form cyclopropyl or cyclobutyl. X1of formula (I) can be C1-12alkylene, C3-12cycloalkylene, or 2- to 12-membered heteroalkylene, each of which can be substituted with one or more R7groups. Alternatively, X1can be combined with R5and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups. X2of formula (I) can be C1-12alkylene, 2- to 12-membered heteroalkylene, or 3- to 15-membered heterocyclylene. Subscripts m and n of formula (I) are each independently an integer from 1 to 12.

[0008] In another aspect, the disclosure provides a lipid nanoparticle. The lipid nanoparticle includes an ionizable lipid or a pharmaceutically acceptable salt thereof, where the ionizable lipid is a compound as disclosed herein. The lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof. The lipid nanoparticle further includes cholesterol or a derivative thereof. The lipid nanoparticle further includes a conjugated lipid.

[0009] In another aspect, the disclosure provides a composition, e.g., a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. The pharmaceutical composition further includes a lipid nanoparticle as disclosed herein or a compound as disclosed herein.

[0010] In another aspect, the disclosure provides a method of introducing a nucleic acid into a cell. The method includes contacting the cell with a lipid nanoparticle as disclosed herein, where the lipid nanoparticle includes the nucleic acid.

[0011] In another aspect, the disclosure provides an in vivo method of delivering a nucleic acid to a subject. The method includes administering to the subject a lipid nanoparticle as disclosed herein, or a pharmaceutical composition including a lipid nanoparticle as disclosed herein, where the lipid nanoparticle includes the nucleic acid.

[0012] In another aspect, the disclosure provides a method of preventing or treating a disease or disorder in a subject. The method includes administering to the subject an amount, e.g., a therapeutically effective amount, of a lipid nanoparticle as disclosed herein, a compound as disclosed herein, and / or a pharmaceutical composition as disclosed herein.3 80125197V.1DETAILED DESCRIPTION I. INTRODUCTION

[0013] The present disclosure generally relates to ionizable lipids and salts thereof. More specifically, the disclosure relates to ionizable lipids that are N,N-substituted hydroxylamine ester compounds, e.g.., lipids that contain two hydroxylamine ester groups each independently covalently bonded to two alkoxy, alkenoxy, or alkynoxy groups, and that further contain one, two, or more additional amine groups in their cores. When the ionizable lipids and salts disclosed herein are employed for example as components of lipid nanoparticles, the resulting lipid particles provide advantageous improvements in the targeting efficiency towards selected cell types. In particular, lipid nanoparticles including the provided ionizable lipids or salts thereof are beneficially effective in targeting hepatic stellate cells with enhanced efficiency.

[0014] The efficiency of lipid nanoparticles in delivering nucleic acids to certain cell types can be very important for many therapeutic applications, particularly when the nucleic acids delivered by these nanoparticles are used for silencing or reducing the expression of a gene in the targeted cell type. High-efficiency delivery can enhance the likelihood that a significant portion of the nucleic acids carried by the lipid nanoparticles reach their intended destination, thereby increasing the therapeutic potential of those nucleic acids. Conversely, inefficient delivery can lead to insufficient alteration of the gene expression to be silenced or reduced. Furthermore, the benefits of enhanced delivery efficiency can include not only improved effectiveness of the treatment, but also reductions to the amount of lipid nanoparticles and / or nucleic acids needed to produce the desired treatment effect, thereby minimizing potential toxicity and side effects.

[0015] While it has often proven challenging to efficiently deliver lipid nanoparticles to certain cell types, the materials and methods disclosed herein address these challenges with quaternary ammonium lipids and salts having particular structural features. By incorporating the provided ionizable lipids and salts into certain lipid nanoparticle formulations, the formulations are surprisingly efficient in targeting desired cell types, such as hepatic stellate cells. Furthermore, the lipid nanoparticles can be advantageously useful in therapies involving the down-regulation (e.g., the silencing or reducing) of gene expression in these targeted cells (e.g., hepatic stellate cells). Also, the compounds and formulations provided by the present disclosure exhibit these advantageous targeting efficiency properties while simultaneously4 80125197V.1beneficially demonstrating desirable tolerability, as indicated by a high biocompatibility and low toxicity. II. DEFINITIONS

[0016] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0017] The abbreviations used herein have their conventional meaning within the chemical and biological arts. Description of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds that are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, or physiological conditions. Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0018] Unless otherwise stated, the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds of the present invention may be labeled with isotopes, such as for example deuterium (2H), tritium (3H), iodine-125 (125I), carbon-13 (13C), or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0019] As used herein, the term “substitution,” when used in relation to a chemical substance, refers to replacement of a hydrogen atom with a non-hydrogen atom or covalently bonded group of atoms. The atom or group of atoms replacing the hydrogen atom is referred to as a “substituent.”

[0020] As used herein, the term “member,” when used in relation to a chemical substance, refers to a non-hydrogen atom of a covalently bonded group of atoms, e.g., a compound or substituent thereof.5 80125197V.1

[0021] When chemical structures are illustrated herein with a wavy line drawn across the middle or end of a bond of the structure, the wavy line signals that the illustration depicts a portion of a chemical compound (e.g., that the illustration depicts a substructure), and that further structure beyond the wavy line (e.g., further structure connected to the bond that is interrupted by the wavy line) is not shown. In this way, a wavy line placed in the middle of a bond indicates truncation of a larger structure. The structure that is not shown may be connected to the depicted structure at the location of the wavy line through a single bond, through a double bond, through a triple bond, or through multiple separate bonds connected to different atoms of the structure that is not shown.

[0022] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a phospholipid” optionally includes a combination of two or more phospholipids, and the like.

[0023] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0024] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0025] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0026] As used herein, the term “alkyl,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. A branched alkyl may include one or more branches having a geminal, vicinal, and / or isolated pattern. For example, an alkyl may include gem-methyl groups. Alkyl may include any number of carbons, such as C1-2, C1-3, C1-4, C1-5, C1-6, C1-7, C1-8, C1-9, C1-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, C1-6 alkyl includes, but is not limited to, methyl,6 80125197V.1ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl may also refer to alkyl groups having up to 40 carbons atoms or more, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. In some aspects, alkyl groups may be substituted. Unless otherwise specified, “substituted alkyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0027] As used herein, the term “alkylene,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene may be linked to the same atom or different atoms of the alkylene group. For instance, a straight chain alkylene may be the bivalent radical of -(CH2)n-, where n is 1, 2, 3, 4, 5 or 6. A branched alkylene may include one or more branches having a geminal, vicinal, and / or isolated pattern. For example, an alkylene may include gem-methyl groups. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. In some aspects, alkylene groups may be substituted. Unless otherwise specified, “substituted alkylene” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0028] As used herein, the term “alkenyl,” by itself or as part of another substituent, refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one carbon- carbon double bond. Alkenyl may include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Alkenyl groups may 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 (ethenyl), 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-hexatrienyl. In some aspects, alkenyl groups may be substituted. Unless otherwise specified, “substituted alkenyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0029] As used herein, the term “alkynyl,” by itself or as part of another substituent, refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple7 80125197V.1carbon-carbon bond. Alkynyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. In some aspects, alkynyl groups may be substituted. Unless otherwise specified, “substituted alkynyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0030] As used herein, the term “alkoxy,” by itself or as part of another substituent, refers to a substituted alkyl group, as defined above, having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O-. As for the unsubstituted portion of the alkyl group, alkoxy groups may have any suitable number of carbon atoms, such as C1-6. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.

[0031] As used herein, the term “heteroalkyl,” by itself or as part of another substituent, refers to an alkyl group of any suitable length and having any number of heteroatoms selected from N, O and S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyl may include ethers, thioethers and alkyl-amines. The heteroatom portion of the heteroalkyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms. In some aspects, heteroalkyl groups may be substituted. Unless otherwise specified, “substituted heteroalkyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0032] As used herein, the term “heteroalkylene,” by itself or as part of another substituent, refers to a heteroalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heteroalkylene may be linked to the same atom or different atoms of the heteroalkylene. In some aspects, heteroalkylene groups may be substituted. Unless otherwise specified, “substituted heteroalkylene” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.8 80125197V.1

[0033] As used herein, the term “heteroalkenyl,” by itself or as part of another substituent, refers to an alkenyl group of any suitable length and having any number of heteroatoms selected from N, O, and S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkenyl may include ethers, thioethers and alkyl-amines. The heteroatom portion of the heteroalkenyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms. In some aspects, heteroalkenyl groups may be substituted. Unless otherwise specified, “substituted heteroalkenyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0034] As used herein, the term “heteroalkynyl,” by itself or as part of another substituent, refers to an alkynyl group of any suitable length and having any number of heteroatoms selected from N, O, and S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkynyl may include ethers, thioethers and alkyl-amines. The heteroatom portion of the heteroalkynyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms. In some aspects, heteroalkynyl groups may be substituted. Unless otherwise specified, “substituted heteroalkynyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0035] As used herein, the term “cycloalkyl,” by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, spiro polycyclic, or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl may include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, bicyclo[1.1.1]pentane, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene, and adamantane. Cycloalkyl groups may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated 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 norbornadiene. When cycloalkyl is a saturated monocyclic C3-8cycloalkyl, exemplary groups9 80125197V.1include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In some aspects, cycloalkyl groups may be substituted. Unless otherwise specified, “substituted cycloalkyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0036] As used herein, the term “cycloalkylene,” by itself or as part of another substituent, refers to a cycloalkyl group having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent radical. The two moieties linked to the cycloalkylene may be linked to the same atom or different atoms of the cycloalkylene group. Examples of cycloalkylene rings include cyclopropylene, cyclobutylene, cyclopentylene and cyclohexylene, among others. Cycloalkylene groups may be linked 1,1, 1,2, 1,3, or 1,4. The cyclohexylene ring, for example, may adopt a number of conformations, including the boat and chair conformations. The chair conformation of cyclohexylene may have substituents in an axial or equatorial orientation. The divalent nature of the cycloalkylenes results in cis and trans formations where cis refers to both substituents being on the same side (top or bottom) of the cycloalkylene ring, and where trans refers to the substituents being on opposite sides of the cycloalkylene ring. For example, cis-1,2- and cis-1,4-cyclohexylene may have one substituent in the axial orientation and the other substituent in the equatorial orientation, while trans-1,2- and trans-1,4-cyclohexylene have both substituents in the axial or equatorial orientation. cis- 1,3-cyclohexylene have both substituents in the axial or equatorial orientation, and trans-1,3- cyclohexylene may have one substituent in the axial orientation and the other substituent in the equatorial orientation. In some aspects, cycloalkylene groups may be substituted. Unless otherwise specified, “substituted cycloalkylene” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0037] As used herein the term “heterocyclyl,” by itself or as part of another substituent, refers to a saturated heterocyclyl ring system having from 3 to 15 ring members, a partially unsaturated non-aromatic ring, or a partially unsaturated, non-aromatic fused, spiro, or bridged multiple-ring system in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms selected from N, O, and S. The heteroatoms may be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heterocyclyl groups may include any number of ring atoms, such as, C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10,10 80125197V.1C3-11, C3-12, or C3-15, where at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms may be replaced with heteroatoms in the heterocyclyl groups, 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. The heterocyclyl group may include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, dithiane and the like. One or more heterocyloalkyl rings of the heterocyclyl groups may also be fused to aromatic or non-aromatic rings to form members including, but not limited to, indoline. Heterocyclyl groups thus 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 dihydroquinolines, e.g., 3,4- dihydroquinoline, dihydroisoquinolines, e.g., 1,2-dihydroisoquinoline, tetrahydroquinolines, e.g., 1,2,3,4-tetrahydroquinoline, tetrahydroisoquinoline, dihydroimidazole, tetrahydroimidazole, etc., indoline, isoindoline, isoindolones (e.g., isoindolin-1-one), isatin, dihydrophthalazine, quinolinone, spiro[cyclopropane-1,1'-isoindolin]-3'-one, and the like. Heterocyclyl groups may have 3-15 members, or 3-12 members, or 3-10 members, or 3-7 members, or 5-6 members. In some aspects, heterocyclyl groups may be substituted. Unless otherwise specified, “substituted heterocyclyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, oxo, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0038] As used herein, the term “heterocyclylene,” by itself or as part of another substituent, refers to a heterocyclyl group, as defined above, linking at least two other groups. The two moieties linked to the heterocyclyl can be linked to the same atom or different atoms of the heterocyclyl. In some aspects, heterocyclylene groups may be substituted. Unless otherwise specified, “substituted heterocyclylene” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.

[0039] As used herein, the term “hydroxy” by itself or as part of another substituent refers to a group consisting of hydrogen bonded to oxygen: -OH.

[0040] As used herein, the terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.11 80125197V.1

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

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

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

[0044] As used herein, the terms “lipid particle,” “lipid nanoparticle,” and “LNP” refer to a particle comprising a phospholipid and an ionizable lipid. A lipid particle may comprise additional lipid components, such as a sterol and / or a conjugated lipid, and may further comprise a nucleic acid, where the nucleic acid may be encapsulated within the particle. Lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Application Publication Nos. 2004 / 0142025 and 2007 / 0042031, and International Patent Application Publication No. WO 2012 / 000104.

[0045] As used herein, the term “phospholipid” refers to a lipid species having a phosphate- containing hydrophilic “head group” and a hydrophobic moiety. The hydrophobic moiety can comprise one or more hydrophobic groups, most typically two hydrophobic groups. The hydrophobic groups are also referred to as hydrophobic “tails,” and can be derived from fatty acids and joined by an alcohol residue, e.g., glycerol. Exemplary structures of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0046] As used herein, the term “ionizable lipid” refers to a lipid species that carries a net positive charge at a selected pH, such as an acidic pH or physiological pH. In some cases, an ionizable lipid includes an ionizable primary, secondary, or tertiary amine (e.g., pH titratable) head group. In some instances, ionizable lipids promote encapsulation of a negatively charged nucleic acid (e.g., mRNA or siRNA) payload during particle formation. In some instances, ionizable lipids promote endosomal fusion and cytoplasmic release of a payload following cellular uptake of a lipid nanoparticle.

[0047] As used herein, the terms “permanently charged lipid,” “permanently charged cationic lipid,” and “permanently charged ionic lipid” generally refer to a lipid species that carries a net positive charge regardless of the pH of its biological environment.12 80125197V.1

[0048] As used herein, the term “conjugated lipid” refers to a polymer-conjugated lipid, e.g., a polymer-conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), polysarcosine-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Pat. No.5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the polymer to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.

[0049] As used herein, the term “salt” refers to acid or base salts of the compounds of the present disclosure. A “pharmaceutically acceptable salt” is one that is compatible with other ingredients of a formulation composition containing the compound, and that is not deleterious to a recipient thereof, i.e., a subject. It is thus understood that the pharmaceutically acceptable salts do not cause a significant adverse toxicological effect on the subject.

[0050] As used herein, the term “nucleic acid” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, that have similar binding properties as the reference nucleotide and are metabolized in a manner similar to reference nucleotides. Non-limiting examples of nucleotide analogs are described in, e.g., International Patent Application No. WO 2007 / 024708. Examples of nucleic acids including such nucleotide analogs, modified backbone residues, or linkages include, without limitation, those containing phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine (Batzer et al.,13 80125197V.1(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605; and Rossolini et al., (1994) Mol. Cell. Probes 8:91).

[0051] Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or noncoding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), small nucleolar RNA(snoRNA), ribozymes, deoxynucleotides (dNTPs), or dideoxynucleotides (ddNTPs). Polynucleotides can also include complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification. Polynucleotides can also include DNA molecules produced synthetically or by amplification, genomic DNA (gDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides, nucleotide analogs, and / or nucleosides suitable for reducing the immunogenicity of RNA, such as those described in International Patent Application No. WO 2007 / 024708. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Polynucleotide sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise.

[0052] Nucleic acids or polynucleotides can be double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive, for example, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.

[0053] Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications include base modifications such as 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of S-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.14 80125197V.1

[0054] Nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription such as described in, e.g., International Patent Application Publication No. WO 2007 / 024708, or from a combination of those processes.

[0055] As used herein in the context of lipid nanoparticles and nucleic acids, the term “fully encapsulated” indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also suggests that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0056] As used herein in the context of lipid nanoparticles and cells, the term “contacting” refers to either the direct or indirect in vitro or in vivo delivering of lipid nanoparticles to the surfaces of cells, e.g., by providing lipid nanoparticles at or proximate to a location of the cells to be contacted. The in vitro contacting may involve, for example, cells in a cell culture or tissue culture. The cell culture or tissue culture may include cells in a suspension and / or adherent cells. The contacted cells may be of same cell type or of two or more different cell types. Merely for illustration, different contacted cell types may include, for example, hepatocytes and hepatic stellate cells. In some example, different cell types are cultured together before and / or during the contacting with lipid nanoparticles. In other examples, different cell types are cultured separately. In further examples, one cell type of two or more cell types to be contacted is specifically cultured in the absence of any other cell types of the two or more cell types. In certain examples, hepatic stellate cells are cultured, either alone or in combination with other cell types from the liver, such as, e.g., liver cells, prior to and / or during the contacting. In some examples, the tissue culture is a liver tissue culture. The in vivo contacting of cells typically involves administering the lipid nanoparticles to a subject, where the cells are within the body of the subject. In some examples, the administering is proximate to the location of the contacted cells within the subject’s body. In other examples, the administering is distal to the location of the contacted cells, and the lipid nanoparticles migrate to the cell location, e.g., via the targeting capabilities of the particles.15 80125197V.1

[0057] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is 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 above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0058] As used herein, the terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like. One of skill in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in the present disclosure.

[0059] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, intrathecal, intracerebroventricular, intraparenchymal, subretinal, or intravitreal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0060] As used herein, the term “therapeutically effective amount” refers to an amount or dose of a compound, composition, or formulation that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., 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).16 80125197V.1

[0061] As used here, the terms “treat”, “treating” and “treatment” refers to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination or laboratory test. III. IONIZABLE LIPIDS

[0062] In one aspect, the present disclosure provides various ionizable lipids and salts thereof. In the context of the present disclosure, the ionizable lipids are N,N-substituted hydroxylamine ester compounds. The particular structures of these ionizable lipids provide surprising improvements to the properties of lipid nanoparticles formed from lipid compositions including the disclosed ionizable lipids or their salts. For example, lipid nanoparticles including the disclosed ionizable lipids are demonstrated herein as exhibiting high biocompatibility, good biodegradability, and low toxicity. Such lipid nanoparticles are also advantageously effective in efficiently targeting hepatic stellate cells.

[0063] The provided class of ionizable lipids is generally stable when exposed to a wide variety of chemical conditions. This general stability can be observed, for example, during synthesis of the ionizable lipids according to the procedures described in the Examples of this disclosure. During these syntheses, the ionizable lipid class demonstrates stability in environments including but not limited to aqueous solutions with a wide pH range, as well as upon exposure to a large variety of solvents including polar, protic, and nucleophilic solvents, e.g., methanol and ethanol. Hydroxylamine esters are not widely known to appear in nature, if at all, and consequently the inventors are not aware of enzymes reported to act on these substrates. However, in view of the observed chemical stability of the ionizable lipids, and without being bound to a particular theory, the inventors believe that this class of compounds may be degraded metabolically, rather than through chemical instability, when the compounds are in an in vivo environment.

[0064] The ionizable lipid generally has the formula:17 80125197V.1The R1, R2, R3, and R4groups of formula (I) can each independently be C1-40 alkyl, C2-40 alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. The R5group of formula (I) can be C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, each of which can optionally be substituted with one or more R7groups. Alternatively, R5can be combined with X1and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups. The R6group of formula (I) can be hydrogen or can be C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which can optionally be substituted with one or more R7groups. Each R7can independently be C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, C1-6 haloalkyl, hydroxy, halogen, oxo, -NH2, nitro, or cyano. Alternatively, one or more R7groups can each independently be combined with another R7and the carbon to which they are attached to form cyclopropyl or cyclobutyl. The X1group of formular (I) can be C1-12 alkylene, C3-12 cycloalkylene, or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups. Alternatively, X1can be combined with R5and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups. The X2group of formula (I) can be C1-12 alkylene, 2- to 12-membered heteroalkylene, or 3- to 15-membered heterocyclylene. Subscripts m and n can each independently be an integer from 1 to 12.

[0065] The R1, R2, R3, and R4groups of formula (I) can each be the same as or different from one another. In some examples, R1, R2, R3, and R4are identical. In other examples, two of R1, R2, R3, and R4are the same as one another, and these two groups are different from the other two of R1, R2, R3, and R4. In some embodiments, R1and R2are identical to one another and are different from each of R3and R4. For example, R1and R2can be identical to one another, while R3and R4are also identical to one another. Alternatively, R1and R2can be identical to one another while R3and R4are different from one another. In still other examples, R3and R4are identical to one another while R1and R2are different from one another. In some embodiments, R1and R3are identical to one another and are different from each of R2and R4. For example, R1and R3can be identical to one another, while R2and R4are also identical to one another. Alternatively, R1and R3can be identical to one another, while R2and R4are different from one18 80125197V.1another. In some embodiments, R1, R2, and R3are identical to one another and different from R4. In other embodiments, R1, R3,and R4are identical to one another and different from R2.

[0066] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkynyl, 2- to 40- membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, C2-40 alkynyl, 2- to 40- membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups.

[0067] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, C2-40 alkynyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, C2-40 alkynyl, or 2- to 40- membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40 alkenyl, C2-40 alkynyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R8groups. In some embodiments, one,19 80125197V.1two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, 2- to 40- membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkynyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40- membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, 2- to 40-membered heteroalkyl, or C2-40 alkynoxy, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkynyl, 2- to 40- membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkynyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40- membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkenyl, C2-40alkynyl, 2- to 40- membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally20 80125197V.1substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40- membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40- membered heteroalkynyl, each of which is optionally substituted with one or more R7groups.

[0068] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, or C2-40 alkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkenyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, C2-40alkynyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkynyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, C2-40 alkynyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some21 80125197V.1embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, C2- 40 alkynyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, C2-40 alkynyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkenyl, C2-40alkynyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40- membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups.

[0069] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl or C2-40 alkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, or C2-40 alkynyl, one, two, three, or all of R1, R2, R3, and R4are optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R8groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40 alkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkenyl or C2-40alkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two,22 80125197V.1three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkenyl or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkynyl or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkynyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkynyl or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkyl or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkyl or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups.

[0070] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C1-40alkyl, optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40 alkenyl, optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-40alkynyl, optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkyl, optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkenyl, optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently 2- to 40-membered heteroalkynyl, optionally substituted with one or more R7groups.23 80125197V.1

[0071] In any of the preceding embodiments in which one, two, three, or all of R1, R2, R3, and R4are C1-40 alkyl, it can be preferred that the alkyl is linear, i.e., unbranched. For example, R1, R2, R3, and R4can each independently be C2-40alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, 2- to 40-membered heteroalkynyl, or linear C1- 40 alkyl, each of which can be optionally substituted with one or more R7groups.

[0072] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-C2-40alkenyl optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl, (Z)-undec-5-enyl, (Z)-non-4-enyl, or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl, (Z)-undec-5-enyl, or (Z)-non-4-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl, (Z)-undec-5-enyl, or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl, (Z)-non-4-enyl, or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-undec-5-enyl, (Z)-non-4-enyl, or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl or (Z)-undec-5-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4-enyl or (Z)-non-4-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-dec-4- enyl or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-undec-5-enyl or (Z)-non-4-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently (Z)-undec-5-enyl or (Z)-undec-8-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are (Z)-dec-4-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are (Z)-undec-5-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are (Z)-non-4-enyl. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are (Z)-undec-8-enyl.

[0073] In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C2-35 alkyl optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C3-30 alkyl optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C4-25alkyl optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C5-20 alkyl optionally substituted with one or more R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are independently C6-15alkyl optionally substituted with one or more24 80125197V.1R7groups. In some embodiments, one, two, three, or all of R1, R2, R3, and R4are decyl. In some embodiments, R1is decyl. In some embodiments, R2is decyl. In some embodiments, R3is decyl. In some embodiments, R4is decyl.

[0074] The R5group of formula (I) can be, for example, C1-8 alkyl optionally substituted with one or more R7groups. In some embodiments, R5is C2-8 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-8 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C7-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C1-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-7alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-7alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C1-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-6 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-6 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C1-5 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-5alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-5 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-5 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C1-4alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-4alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-4 alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C1-3alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-3alkyl, optionally substituted with one or more R7groups. In some embodiments, R5is methyl. In some embodiments, R5is hydroxyethyl.

[0075] The R5group of formula (I) can be, for example, C2-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-8alkenyl, optionally substituted25 80125197V.1with one or more R7groups. In some embodiments, R5is C4-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-8alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C7-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-7 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-7 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-6alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-6 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-6 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-6alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-5 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-5 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-5alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-4alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-4 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-3alkenyl, optionally substituted with one or more R7groups.

[0076] The R5group of formula (I) can be, for example, C2-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-8alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C7-8alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-7 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C6-7 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-6alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-6alkynyl, optionally substituted26 80125197V.1with one or more R7groups. In some embodiments, R5is C4-6 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C5-6 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-5alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-5 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C4-5 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-4alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C3-4alkynyl, optionally substituted with one or more R7groups. In some embodiments, R5is C2-3 alkynyl, optionally substituted with one or more R7groups.

[0077] The R5group of formula (I) can be, for example, combined with X1and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclyl group optionally substituted with one or more R7groups. In some embodiments, R5is combined with X1and the nitrogen to which R5is attached to form a 4- to 11-membered heterocyclyl group optionally substituted with one or more R7groups.

[0078] In any of the preceding embodiments in which R5is combined with X1and the nitrogen to which R5is attached to form a heterocyclyl group, it can be preferred that the heterocyclyl is a 3- to 5-membered monocylclic heterocyclylene group, a 5- to 15-membered spiro or bridged polycyclic heterocyclylene group, or a 5- to 15-membered fused heterocyclylene diastereomer group, each of which is optionally substituted with one or more R7groups. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl, azetidindiyl, or hexahydro-2H-pyrrolo[3,4- b]pyridindiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl or azetidindiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl or hexahydro-2H-pyrrolo[3,4-b]pyridindiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form azetidindiyl or hexahydro-2H-pyrrolo[3,4- b]pyridindiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form azetidindiyl. In some embodiments, R5is combined with X and the nitrogen to which R5is attached to form hexahydro-2H-pyrrolo[3,4- b]pyridindiyl.27 80125197V.1

[0079] The R6group of formula (I) can be, for example, hydrogen. Alternatively, R6can be C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkenyl, C2-8alkynyl, or 5, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl, C2-8 alkynyl, 2- to 40- membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkenyl, or C2-8 alkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8 alkyl, C2-8 alkenyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkenyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8 alkyl, C2-8 alkynyl, or 2- to 40- membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl, C2-8alkynyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8 alkyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8alkenyl, C2-8 alkynyl, or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl, C2-8 alkynyl, or 2- to 40- membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8 alkyl or C2-8 alkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl or C2-8alkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C1-8alkyl or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R728 80125197V.1groups. In some embodiments, R6is C1-8 alkyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl or C2-8alkynyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkenyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8alkynyl or 2- to 40-membered heteroalkyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is C2-8 alkynyl or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is 2- to 40-membered heteroalkyl or 2- to 40- membered heteroalkenyl, each of which is optionally substituted with one or more R7groups. In some embodiments, R6is 2- to 40-membered heteroalkyl optionally substituted with one or more R7groups. In some embodiments, R6is 2- to 40-membered heteroalkenyl optionally substituted with one or more R7groups.

[0080] The R6group of formula (I) can be, for example, C1-8 alkyl optionally substituted with one or more R7groups. In some embodiments, R6is C2-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-8 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-8alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C7-8 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C1-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-7alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-7 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-7alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-7alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C1-6 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-6 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-6alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C1-5alkyl, optionally substituted with one or more R729 80125197V.1groups. In some embodiments, R6is C2-5 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-5 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-5alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C1-4 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-4 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-4alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C1-3alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-3 alkyl, optionally substituted with one or more R7groups. In some embodiments, R6is methyl. In some embodiments, R6is hydroxyethyl.

[0081] The R6group of formula (I) can be, for example, C2-8alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-8alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C7-8 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-7 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-7alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-6 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-6 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-6alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-6 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-5 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-5alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-5alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-4 alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-4alkenyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-3alkenyl, optionally substituted with one or more R7groups.

[0082] The R6group of formula (I) can be, for example, C2-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-8alkynyl, optionally substituted30 80125197V.1with one or more R7groups. In some embodiments, R6is C4-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-8alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C7-8 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-7 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-7 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C6-7alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-6alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-6 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-6 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C5-6alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-5 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-5 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C4-5alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-4alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C3-4 alkynyl, optionally substituted with one or more R7groups. In some embodiments, R6is C2-3alkynyl, optionally substituted with one or more R7groups.

[0083] The R5and R6groups of formula (I) can be the same as or different from one another. In some examples, R5and R6are identical. In other examples, R5and R6are not identical.

[0084] In some embodiments, the X1group of formula (I) is C1-12alkylene, C3-12cycloalkylene, or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups. In some embodiments, X1is C1-12 alkylene or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups. In some embodiments, X1is C1-12 alkylene or C3-12 cycloalkylene, each of which is optionally substituted with one or more R7groups. In some embodiments, X1is C3-12 cycloalkylene or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups. In some embodiments, X1is C1-12alkylene optionally substituted with one or more R7groups. In some embodiments, X1is C1-8 alkylene optionally substituted with one or more R7groups. In some embodiments, X1is C1-4 alkylene optionally substituted with one or more R7groups. In any of the preceding embodiments in which X1is alkylene, it can be preferred that31 80125197V.1the alkylene is methylene. In some embodiments, X1is C3-12 cycloalkylene optionally substituted with one or more R7groups. In some embodiments, X1is C4-10 cycloalkylene optionally substituted with one or more R7groups. In some embodiments, X1is C5-8cycloalkylene optionally substituted with one or more R7groups. In some embodiments, X1is cyclohexylene. In some embodiments, X1is 2- to 12-membered heteroalkylene optionally substituted with one or more R7groups.

[0085] In some embodiments, the X2group of formula (I) is C1-12alkylene or 2- to 12- membered heteroalkylene. In some embodiments, X2is C1-12 alkylene or 3- to 15-membered heterocyclylene. In some embodiments, X2is 2- to 12-membered heteroalkylene or 3- to 15- membered heterocyclylene. In some embodiments, X2is C1-12alkylene. In some embodiments, X2is 2- to 12-membered heteroalkylene. In some embodiments, X2is. In some embodiments, X2is. some. In some embodiments, X2is 2- to 12-membered heteroalkylene. In some embodiments, X2is 3- to 15- membered heterocyclylene.

[0086] The subscripts m and n of formula (I) can be the same as or different from one another. In some examples, m and n are identical. In other examples, m and n are not identical. In particular examples, n is one greater than m (i.e., n is equal to m + 1). In other certain examples, n is two greater than m (i.e., n is equal to m + 2). Subscripts m and n can each independently be an integer from 1 to 12, e.g., from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 12, from 2 to 11, from 2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3, from 3 to 12, from 3 to 11, from 3 to 10, from 3 to 9, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 12, from 4 to 11, from 4 to 10, from 4 to 9, from 4 to 8, from 4 to 7, from 4 to 6, from 4 to 5, from 5 to 12, from 5 to 11, from 5 to 10, from 5 to 9, from 5 to 8, from 5 to 7, from 5 to 6, from 6 to 12, from 6 to 11, from 6 to 10, from 6 to 9, from 6 to 8, from 6 to 7, from 7 to 12, from 7 to 11, from 7 to 10, from 7 to 9, from 7 to 8, from 8 to 12, from 8 to 11, from 8 to 10, from 8 to 9, from 9 to 12, from 9 to 11, from 9 to 10, from 10 to 12, from 10 to 11, or from 11 to 12. In some embodiments, m and n are each independently 2, 3, 4, 5, 6, or 7. In some embodiments, m and n are each 5. In some embodiments, m is 4 and n is 5. In some embodiments, m and n are each 7. In some embodiments, m and n are each 3. In32 80125197V.1some embodiments, m is 6 and n is 7. In some embodiments, m and n are each 6. In some embodiments, m is 3 and n is 5. In some embodiments, m is 5 and n is 7. In some embodiments, m and 4 are each 4. In some embodiments, m is 5 and n is 6.

[0087] In some embodiments, the ionizable lipid has the formula:(I)(a), or the formula of a salt thereof. The R1, R2, R3, R4, and X1groups and the m and n subscripts of formula (I)(a) are as defined for formula (I).

[0088] In some embodiments, the ionizable lipid has the formula:or the formula of a salt thereof. The R1, R2, R3, and R4groups and the m and n subscripts of formula (I)(b) are as defined for formula (I).

[0089] In some embodiments, the ionizable lipid has the formula:or the formula of a salt thereof. The R1, R2, R3, and R4groups and the m and n subscripts of formula (I)(c) are as defined for formula (I).

[0090] In some embodiments, the ionizable lipid has the formula:or the formula of a salt thereof. The R1, R2, R3, and R4groups and the m and n subscripts of formula (I)(d) are as defined for formula (I).

[0091] In some aspects, the present disclosure provides various salts of any of the ionizable lipids described herein. In some embodiments, the salts include or consist of pharmaceutically acceptable salts. The salts can include or consist of a bromide salt, a chloride salt, an iodide salt, or any combination thereof.33 80125197V.1

[0092] In some embodiments, the ionizable lipid is: ,34 80125197V.1,35 80125197V.136 80125197V.1, ,37 80125197V.1,38 80125197V.139 80125197V.1.

[0093] In some embodiments, the ionizable lipid of the present disclosure comprises a racemic mixture. In other embodiments, an ionizable lipid of the present disclosure comprises a mixture of one or more diastereomers. In certain embodiments, an ionizable lipid of the present disclosure comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. In certain other embodiments, an ionizable lipid of the present invention is enriched in one diastereomer, such that the lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. In certain additional embodiments, an ionizable lipid of the present invention is chirally pure (e.g., comprises a single optical isomer). In further embodiments, an ionizable lipid of the present invention is enriched in 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% isomeric excess. The present disclosure provides the synthesis of any of the ionizable lipids disclosed herein as a racemic mixture or in optically pure form.40 80125197V.1IV. LIPID NANOPARTICLES

[0094] In another aspect, the present disclosure provides various lipid nanoparticles that include any of the ionizable lipids or salts thereof described in Section III. The lipid nanoparticles generally further include a phospholipid or a pharmaceutically acceptable salt thereof, cholesterol or a derivative thereof, and a conjugated lipid. The particular selection, composition, and amounts (e.g., relative amounts) of these components provide surprising improvements in various properties of the lipid nanoparticles, e.g., properties advantageous for the delivery of nucleic acids. These improved properties include, for example, improved efficiency in targeting hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility.

[0095] In some examples of the provided lipid nanoparticles, the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between about 45 mol% and about 55 mol% of the total lipid of the lipid nanoparticle (e.g., between about 45 mol% and about 52.5 mol%, between about 45 mol% and about 50 mol%, between about 45 mol% and about 47.5 mol%, between about 47.5 mol% and about 55 mol%, between about 47.5 mol% and about 52.5 mol%, between about 47.5 mol% and about 50 mol%, between about 50 mol% and about 55 mol%, between about 50 mol% and about 52.5 mol%, or between about 52.5 mol% and about 55 mol%), the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 9 mol% and about 11 mol% of the total lipid of the lipid nanoparticle (e.g., between about 9 mol% and about 10.5 mol%, between about 9 mol% and about 10 mol%, between about 9 mol% and about 9.5 mol%, between about 9.5 mol% and about 11 mol%, between about 9.5 mol% and about 10.5 mol%, between about 9.5 mol% and about 10 mol%, between about 10 mol% and about 11 mol%, between about 10 mol% and about 10.5 mol%, or between about 10.5 mol% and about 11 mol%), the cholesterol or the derivative thereof comprises between about 36.5 mol% and about 40.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 36.5 mol% and about 39.5 mol%, between about 36.5 mol% and about 38.5 mol%, between about 36.5 mol% and about 37.5 mol%, between about 37.5 mol% and about 40.5 mol%, between about 37.5 mol% and about 39.5 mol%, between about 37.5 mol% and about 38.5 mol%, between about 38.5 mol% and about 40.5 mol%, between about 38.5 mol% and about 39.5 mol%, or between about 39.5 mol% and about 40.5 mol%), and the conjugated lipid comprising between about 0.85 mol% and about 2.6 mol% of the total lipid of the lipid nanoparticle (e.g., between about 0.85 mol% and about 2.16 mol%, between about 0.85 mol% and about 1.73 mol%, between about 0.85 mol% and about 1.29 mol%,41 80125197V.1between about 1.29 mol% and about 2.6 mol%, between about 1.29 mol% and about 2.16 mol%, between about 1.29 mol% and about 1.73 mol%, between about 1.73 mol% and about 2.6 mol%, between about 1.73 mol% and about 2.16 mol%, or between about 2.16 mol% and about 2.6 mol%).

[0096] The identity and relative amounts of the lipid nanoparticle components can also be selected to provide the lipid nanoparticle with a size that is beneficial for use in delivering a nucleic acid, e.g., to a hepatic stellate cell. The lipid nanoparticles can have a mean diameter that is, for example, between about 40 nm and about 150 nm, between about 50 nm and about 150 nm, between about 60 nm and about 130 nm, between about 70 nm and about 110 nm, between about 60 nm and about 100 nm, between about 50 and about 80 nm, between about 60 and about 80 nm, between about 60 and about 90 nm, or between about 70 and about 90 nm.

[0097] The specific configuration of the lipid nanoparticles described herein, and in particular the design of the ionizable lipid it contains, can also provide the nanoparticles with a pKa giving the nanoparticles targeting, stability, degradability, and toxicity properties advantageous for, e.g., the delivery of nucleic acids, in particular to hepatic stellate cells. For example, in some embodiments, the lipid nanoparticle has a pKa between about 5.6 and about 7.4, e.g., between about 5.6 and about 7.1, between about 5.6 and about 6.8, between about 5.6 and about 6.5, between about 5.6 and about 6.2, between about 5.6 and about 5.9, between about 5.9 and about 7.4, between about 5.9 and about 7.1, between about 5.9 and about 6.8, between about 5.9 and about 6.5, between about 5.9 and about 6.2, between about 6.2 and about 7.4, between about 6.2 and about 7.1, between about 6.2 and about 6.8, between about 6.2 and about 6.5, between about 6.5 and about 7.4, between about 6.5 and about 7.1, between about 6.5 and about 6.8 between about 6.8 and about 7.4, between about 6.8 and about 7.1, or between about 7.1 and about 7.4. In terms of upper limits, the nanoparticle pKa can be, for example, no greater than about 7.5, e.g., no greater than about 7.3, no greater than about 7.1, no greater than about 6.9, no greater than about 6.7, no greater than about 6.5, no greater than about 6.3, no greater than about 6.1, no greater than about 5.9, or no greater than about 5.7. In terms of lower limits, the nanoparticle pKa can be, for example, no less than about 5.5, e.g., no less than about 5.7, no less than about 5.9, no less than about 6.1, no less than about 6.3, no less than about 6.5, no less than about 6.7, no less than about 6.9, no less than about 7.1, or no less than about 7.3.42 80125197V.1

[0098] In some preferred examples, the lipid nanoparticle has a pKa between about 6.3 and about 6.6, e.g., between about 6.3 and about 6.55, between about 6.3 and about 6.5, between about 6.3 and about 6.45, between about 6.3 and about 6.4, between about 6.3 and about 6.35, between about 6.35 and about 6.6, between about 6.35 and about 6.55, between about 6.3 and about 6.5, between about 6.3 and about 6.45, between about 6.3 and about 6.4, between about 6.3 and about 6.35, between about 6.35 and about 6.6, between about 6.35 and about 6.55, between about 6.35 and about 6.5, between about 6.35 and about 6.45, between about 6.35 and about 6.4, between about 6.4 and about 6.6, between about 6.4 and about 6.55, between about 6.4 and about 6.5, between about 6.4 and about 6.45, between about 6.45 and about 6.6, between about 6.45 and about 6.55, between about 6.45 and about 6.5, between about 6.5 and about 6.6, between about 6.5 and about 6.55, or between about 6.55 and about 6.6. In terms of upper limits, the nanoparticle pKa can be, for example, no greater than about 6.6, e.g., no greater than about 6.55, no greater than about 6.5, no greater than about 6.45, no greater than about 6.4, or no greater than about 6.35. In terms of lower limits, the nanoparticle pKa can be, for example, no less than about 6.3, e.g., no less than about 6.35, no less than about 6.4, no less than about 6.45, no less than about 6.5, or no less than about 6.55.

[0099] In a preferred embodiment, the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at a temperature between about 34 °C and about 40 °C. In some embodiments, the lipid nanoparticle has a pKa, as determined using an assay performed at a temperature between about 34 °C and about 40 °C, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above. In particularly preferred embodiments, the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at a temperature of about 37 °C. In some embodiments, the lipid nanoparticle has a pKa, as determined using an assay performed at a temperature of about 37 °C, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above.

[0100] In a preferred embodiment, the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at more than 5 different pH levels. In some embodiments, the lipid nanoparticle has a pKa, as determined using an assay performed at more than 5 different pH levels, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above. In particularly preferred embodiments, the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at more than 15 different pH levels. In some embodiments, the lipid nanoparticle has a pKa, as determined using an assay43 80125197V.1performed at more than 15 different pH levels, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above.

[0101] In a preferred embodiment, the pKa of the provided lipid nanoparticle is determined using in situ fluorescence titration, and preferably TNS fluorescence titration. This assay typically yields a sigmoidal curve showing fluorescence, such as TNS fluorescence, wherein the pKa of the provided lipid nanoparticle is determined to be the value that corresponds to 0.5 normalized fluorescence, such as normalized TNS fluorescence, on a scale from 0 to 1.0.

[0102] For the purposes of determining the pKa of the lipid nanoparticle described herein, the lipid nanoparticle preferably includes an ionizable lipid (preferably comprising between about 30 mol% and about 70 mol% of the total lipid present in the lipid particle). In certain examples, the pKa of the lipid nanoparticle is determined when the lipid nanoparticle further includes a non-cationic lipid (preferably cholesterol or a cholesterol derivative comprising between about 20 mol% and about 60 mol% of the total lipid present in the lipid particle, and a phospholipid comprising between about 1 mol% and about 20 mol% of the total lipid present in the nanoparticle), and a conjugated lipid (preferably comprising between about 0.1 mol% and about 5 mol% of the total lipid present in the lipid particle). In particularly preferred embodiments, for determining the pKa of the lipid nanoparticle described herein, the lipid nanoparticle further includes a therapeutic agent, e.g., a nucleic acid.

[0103] The provided lipid nanoparticles can have in vivo clearance and degradation characteristics that are particularly beneficial when the nanoparticle is used to administer a nucleic acid to a subject, e.g., to provide a treatment to the subject and / or to silence genetic information of the subject. Clearance and degradation rates that are too high can prevent a lipid nanoparticle from protecting a cargo, such as a nucleic acid cargo, and from successfully delivering it to its targeted destination within the subject. Alternatively, clearance and degradation rates that are too low can be associated with increased toxicity or undesirable off- target effects. The provided lipid nanoparticles also can exhibit advantageous efficiency in delivering, e.g., nucleic acids, to a targeted site, e.g., hepatic stellate cells, in a subject when the lipid nanoparticle is administered to the subject. The provided lipid nanoparticles can further be configured, e.g., through the selection of their components, to be substantially non- toxic.44 80125197V.1A. Phospholipid

[0104] The lipid nanoparticles disclosed herein generally include at least one phospholipid or pharmaceutically acceptable salt thereof. The phospholipid can be selected or designed to provide the lipid nanoparticle with improved performance in the delivery of a nucleic acid, e.g., enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility. In some embodiments, the lipid nanoparticle includes one species of phospholipid. In other embodiments, the lipid nanoparticle includes two or more species of phospholipids, e.g., 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 species of phospholipids.

[0105] In some embodiments, the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylcholine. Phosphatidylcholine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn- glycero-3-phosphocholine, 1,3-dipalmitoyl-rac-glycero-2-phosphocholine, 1,2-dilauroyl-sn- glycero-3-phosphocholine (DLPC), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine, 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3- phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl- sn-glycero-3-phosphocholine, 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine, 1,2- diarachidoyl-sn-glycero-3-phosphocholine, 1,2-divaccenoyl -sn-glycero-3-phosphocholine, 1,2-di[(8Z)octadecenoyl]-sn-glycero-3-phosphocholine, 1,2-dimyristoleoyl-sn-glycero-3- phosphocholine, 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn- glycero-3-phosphocholine, 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine, 1,2- dipetroselenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dielaidoyl-sn-glycero-3-phosphocholine, 1,2-dilinoleoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-dieicosenoyl-sn-glycero- 3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1-pentadecanoyl-2- oleoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2- myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2- linoleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphocholine (PAPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1- stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-45 80125197V.1phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2- arachidonoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-myristoyl-sn-glycero-3- phosphocholine (OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2- stearoyl-sn-glycero-3-phosphocholine, 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3- phosphocholine, and combinations thereof.

[0106] In some embodiments, the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylethanolamine. Phosphatidylethanolamine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-diheptadecanoyl-sn- glycero-3-phosphoethanolamine, 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dipentadecanoyl-sn-glycero-3- phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-docosahexaenoyl-sn- glycero-3-phosphoethanolamine, and combinations thereof.

[0107] In some embodiments, the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylserine. Phosphatidylserine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3- phospho-L-serine (DSPS), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2- didecanoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dilauroyl-sn-glycero-3-phospho-L-serine (DLPS), 1,2-dilinoleoyl-sn-glycero-3- phospho-L-serine, 1,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine, 1-pentadecanoyl- 2-oleoyl-sn-glycero-3-phospho-L-serine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-46 80125197V.1serine (POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (SOPS), 1-palmitoyl-2- linoleoyl-sn-glycero-3-phospho-L-serine, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho- L-serine, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine, 1-stearoyl-2- linoleoyl-sn-glycero-3-phospho-L-serine, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L- serine, 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine, and combinations thereof.

[0108] In some embodiments, the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylglycerol. Phosphatidylglycerol lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycero-3-phospho-(1'- rac-glycerol) (DOPG), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2- didecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycero-3-phospho-(1'- rac-glycerol) (DLPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2- dipentadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3- phospho-(1'-rac-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dielaidoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycero-3- phospho-(1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2- diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 1,2-didocosahexaenoyl-sn-glycero- 3-[phospho-rac-(1-glycerol)], 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG), 1-palmitoyl-2- docosahexaenoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1-stearoyl-2-oleoyl-sn-glycero-3- phospho-(1'-rac-glycerol) (SOPG), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac- glycerol), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (SAPG), 1- stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-(1'-rac-glycerol), and combinations thereof.

[0109] In addition to the species of phospholipid, the amount of phospholipid included in the provided lipid nanoparticle has also been shown to provide the particle with its advantageous characteristics. The phospholipid present in the provided lipid nanoparticle can comprise, for example, between about 1 mol% and about 20 mol% of the total lipid present in the particle, e.g., between about 1 mol% and about 16.2 mol%, between about 1 mol% and about 12.4 mol%, between about 1 mol% and about 8.6 mol%, between about 1 mol% and about 4.8 mol%, between about 4.8 mol% and about 20 mol%, between about 4.8 mol% and about 16.2 mol%, between about 4.8 mol% and about 12.4 mol%, between about 4.8 mol% and about 8.647 80125197V.1mol% of the total lipid present in the particle, between about 8.6 mol% and about 20 mol%, between about 8.6 mol% and about 16.2 mol%, between about 8.6 mol% and about 12.4 mol%, between about 12.4 mol% and about 20 mol%, between about 12.4 mol% and about 16.2 mol%, or between about 16.2 mol% and about 20 mol% of the total lipid present in the lipid nanoparticle. B. Cholesterol

[0110] The lipid nanoparticles disclosed herein generally include a sterol, e.g., cholesterol, or one or more derivatives thereof. The sterol can be selected or designed to provide the lipid nanoparticle with improved performance in the delivery of a nucleic acid, e.g., enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility. In some embodiments, the lipid nanoparticle includes one species of sterol. In other embodiments, the lipid nanoparticle includes two or more species of sterols, e.g., 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 species of sterols. The lipid nanoparticle can include, for example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′- hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7-betahydroxycholesterol, 7-alphahydroxycholesterol, 4-betahydroxycholesterol, cholesterol PEG, beta sitosterol, or any combination thereof. In certain examples, the lipid nanoparticle includes cholesterol, but substantially no derivative of cholesterol.

[0111] In addition to the species of cholesterol, the amount of cholesterol included in the provided lipid nanoparticle has also been shown to provide the lipid nanoparticle with its advantageous characteristics. In some examples, the cholesterol comprises between about 20 mol% and about 60 mol% of the total lipid of the lipid nanoparticle, e.g., between about 20 mol% and about 52 mol%, between about 20 mol% and about 44 mol%, between about 20 mol% and about 36 mol%, between about 20 mol% and about 28 mol%, between about 28 mol% and about 60 mol%, between about 28 mol% and about 52 mol%, between about 28 mol% and about 44 mol%, between about 28 mol% and about 36 mol%, between about 36 mol% and about 60 mol%, between about 36 mol% and about 52 mol%, between about 36 mol% and about 44 mol%, between about 44 mol% and about 60 mol%, between about 44 mol% and about 52 mol%, or between about 52 mol% and about 60 mol% of the total lipid of the lipid nanoparticle.48 80125197V.1C. Conjugated Lipid

[0112] The lipid nanoparticles disclosed herein generally include at least one conjugated lipid. The conjugated lipid can be selected or designed to provide the lipid nanoparticle with improved performance in the delivery of a nucleic acid, e.g., enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility. In some embodiments, the lipid nanoparticle includes one species of conjugated lipid. In other embodiments, the lipid nanoparticle includes two or more species of conjugated lipids, e.g., 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 species of conjugated lipids. The one or more conjugated lipids can each independently be selected or configured to control the size of the particle during formation, and / or to prevent particle aggregation by sterically stabilizing the lipid nanoparticle. The conjugated lipid of the lipid nanoparticle can be situated at the surface of the particle, with the hydrophilic polymer of the conjugated lipid oriented outwardly and interfacing with the aqueous environment, and the lipid component of the conjugated lipid buried in the particle to anchor it in place.

[0113] Conjugated lipids suitable for use with the provided lipid nanoparticle include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), polyoxazoline (POZ)-lipids, polysarcosine (pSAR)- lipids and mixtures thereof. In certain embodiments, the lipid nanoparticle comprises either a PEG-lipid conjugate or an ATTA-lipid conjugate optionally together with a CPL.

[0114] In a preferred embodiment, the conjugated lipid includes or consists of a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., International Patent Application Publication No. WO 05 / 026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Application Publication Nos.2003 / 0077829 and 2005 / 0008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613, PEG conjugated to cholesterol or a derivative thereof, mPEG(2000)-N,N-ditetradecylacetamide (ALC-0159), mPEG(2000)-stearate, 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), R-3-[(ω- methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine (PEG-C- DOMG), any of the PEG lipids described in International Patent Application Publication No. WO 2022 / 133344, and mixtures thereof. The disclosures of these patent documents are49 80125197V.1incorporated herein by reference in their entirety for all purposes. The provided lipid nanoparticle can include one or more methyl capped PEG-lipids, one or more uncapped PEG- lipids, or a combination thereof.

[0115] PEG is a linear, water-soluble polymer of ethylene glycol repeating units. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. Such molecular weights are average molecular weights due to polydispersity. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG- NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs such as those described in U.S. Pat. Nos.6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.

[0116] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0117] In certain instances, the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester containing linker moiety. As used herein, the term “non-ester containing linker moiety” refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-). Suitable non-ester containing linker moieties50 80125197V.1include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulphide (-S-S-), ether (-O-), succinyl (- C(O)CH2CH2C(O)-), succinimidyl (-NHC(O)CH2CH2C(O)NH-), as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0118] In other embodiments, an ester containing linker moiety is used to couple the PEG to the lipid. Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0119] In addition to the foregoing, it will be readily apparent to those of skill in the art 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, poly (ethyl ethylene phosphate) (PEEP), and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0120] The conjugated lipid of the provided lipid nanoparticles can include or consist of, e.g., one or more of the following: a polyethylene glycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, or mixtures thereof. In one preferred embodiment, the nucleic acid- lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. The conjugated lipids may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof.

[0121] Additional PEG-lipid conjugates suitable for use in the provided lipid nanoparticle include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C- DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT / US08 / 88676, filed Dec. 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8′-(l,2-dimyristoyl-3-propanoxy)-carboxamido- 3′,6′-dioxaoctanyl]carbamoyl-w-methylpoly(ethylene glycol) (2 KPEG-DMG). The synthesis51 80125197V.1of 2 KPEG-DMG is described in U.S. Pat. No. 7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0122] In addition to the species of conjugated lipid, the amount of conjugated lipid included in the provided lipid nanoparticle has also been shown to provide the lipid nanoparticle with its advantageous characteristics. In some examples, the conjugated lipid comprises between about 0.1 mol% and about 5 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.1 mol% and about 4 mol%, between about 0.1 mol% and about 3 mol%, between about 0.1 mol% and about 2 mol%, between about 0.1 mol% and about 1 mol%, between about 1 mol% and about 5 mol%, between about 1 mol% and about 4 mol%, between about 1 mol% and about 3 mol%, between about 1 mol% and about 2 mol%, between about 2 mol% and about 5 mol%, between about 2 mol% and about 4 mol%, between about 2 mol% and about 3 mol%, between about 3 mol% and about 5 mol%, between about 3 mol% and about 4 mol%, or between about 4 mol% and about 5 mol% of the total lipid of the lipid nanoparticle.

[0123] By controlling the composition and concentration of the conjugated lipid in the provided lipid nanoparticle, one can control the rate at which the conjugated lipid exchanges out of the lipid nanoparticle and, in turn, the rate at which the lipid nanoparticle becomes fusogenic. For instance, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the conjugated lipid, the rate at which the lipid nanoparticle becomes fusogenic can be varied, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide. In addition, other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and / or control the rate at which the lipid nanoparticle becomes fusogenic. Other methods which can be used to control the rate at which the lipid nanoparticle becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure. D. Ionizable Lipid

[0124] The provided lipid nanoparticle generally includes at least one ionizable lipid or pharmaceutically acceptable salt thereof. The ionizable lipid or salt can any of those described in more detail in Section III, and thus be selected or designed to provide the lipid nanoparticle with a desired particle size, stability, and capacity for encapsulation of nucleic acids. For example, the ionizable lipid of the provided lipid nanoparticle can be an N,N-substituted hydroxylamine ester compounds, e.g., a hydroxylamine ester compound having the structure52 80125197V.1of formula (I). Lipid nanoparticles including the particular ionizable lipids described herein also exhibit, for example, enhanced targeting efficiency, optimized clearance rates, and decreased toxicity and immune stimulation. In some embodiments, the lipid nanoparticle includes one species of ionizable lipid. In other embodiments, the lipid nanoparticle includes two or more species of ionizable lipids, e.g., 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 species of ionizable lipids.

[0125] In addition to the species of ionizable lipid, the amount of ionizable lipid included in the provided lipid nanoparticle has also been shown to provide the lipid nanoparticle with its advantageous characteristics. In some examples, the ionizable lipid or the salt thereof comprises between about 30 mol% and about 70 mol% of the total lipid of the lipid nanoparticle, e.g., between about 30 mol% and about 62 mol%, between about 30 mol% and about 54 mol%, between about 30 mol% and about 46 mol%, between about 30 mol% and about 38 mol%, between about 38 mol% and about 70 mol%, between about 38 mol% and about 62 mol%, between about 38 mol% and about 54 mol%, between about 38 mol% and about 46 mol%, between about 46 mol% and about 70 mol%, between about 46 mol% and about 62 mol%, between about 46 mol% and about 54 mol%, between about 54 mol% and about 70 mol%, between about 54 mol% and about 62 mol%, or between about 62 mol% and about 70 mol% of the total lipid of the lipid nanoparticle. E. Nucleic Acid

[0126] In some embodiments, the provided lipid nanoparticles are associated with a nucleic acid. The composition of the lipid nanoparticle can be selected or configured such that nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Nucleic acid may be administered alone in the lipid nanoparticles described herein, or in combination (e.g., co-administered) with lipid particles comprising peptides, polypeptides, or small molecules such as conventional drugs. In some embodiments, the nucleic acid is at least 50% encapsulated within the lipid nanoparticle; in one embodiment, the nucleic acid is at least 75% encapsulated within the lipid nanoparticle; in one embodiment, the nucleic acid is at least 90% encapsulated within the lipid nanoparticle; and in one embodiment, the nucleic acid is fully encapsulated within the lipid nanoparticle.

[0127] In certain embodiments, the present disclosure provides a lipid nanoparticle formulation comprising a plurality or population of lipid nanoparticles. In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid nanoparticles such53 80125197V.1that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 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 fraction thereof or range therein) of the lipid nanoparticles in the plurality or population of lipid nanoparticles have the nucleic acid encapsulated therein.

[0128] The nucleic acid that is present in the lipid nanoparticles described herein can include or consist of any form of nucleic acid that is known. The nucleic acids used herein can 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, e.g., mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA).

[0129] Nucleic acids may be of various lengths, generally dependent upon the particular form of nucleic acid. For example, in particular embodiments, mRNA, plasmids, or genes may be from about 1,000 to about 100,000 nucleotides in length. In particular embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In various related embodiments, oligonucleotides, that can be single-stranded, double-stranded, and / or triple- stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length.

[0130] In some embodiments, the nucleic acid of a provided lipid nanoparticle comprises or consists of a modified or substituted polynucleotide or oligonucleotide. Modified or substituted polynucleotides and oligonucleotides can be preferred over native forms in some instances because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0131] In some embodiments, the nucleic acid is an RNA molecule comprising at least one modified nucleotide. In certain preferred embodiments, the RNA molecule comprises one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-54 80125197V.1stranded region. In certain instances, the RNA molecule (e.g., siRNA) has a double-stranded region that comprises from 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%) modified nucleotides in the double-stranded region. In preferred embodiments, less than about 25% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% to about 25% (e.g., from about 1% to 25%, 5% to 25%, 10% to 25%, 15% to 25%, 20% to 25%, or 10% to 20%) of the nucleotides in the double-stranded region comprise modified nucleotides.

[0132] 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′- deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In preferred embodiments, the RNA comprises 2′Ome nucleotides (e.g., 2′Ome purine and / or pyrimidine nucleotides) such as, for example, 2′Ome- guanosine nucleotides, 2′Ome-uridine nucleotides, 2′Ome-adenosine nucleotides, 2′Ome- cytosine nucleotides, and mixtures thereof. In certain instances, the RNA does not comprise 2′Ome-cytosine nucleotides. In other embodiments, the RNA comprises a hairpin loop structure.

[0133] The RNA may comprise modified nucleotides in one strand (i.e., sense or antisense) or both strands of a double-stranded region of the RNA molecule. Preferably, uridine and / or guanosine nucleotides are modified at selective positions in the double-stranded region of the RNA duplex. With regard to uridine nucleotide modifications, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and / or antisense strand can be a modified uridine nucleotide such as a 2′Ome-uridine nucleotide. In some embodiments, every uridine nucleotide in the sense and / or antisense strand is a 2′Ome-uridine nucleotide. With regard to guanosine nucleotide modifications, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and / or antisense strand can be a modified guanosine nucleotide such as a 2′Ome-guanosine nucleotide. In some embodiments, every guanosine nucleotide in the sense and / or antisense strand is a 2′Ome-guanosine nucleotide.

[0134] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5′-GU- 3′ motifs in an RNA sequence may be modified, e.g., 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′ motif can be in the sense strand, the antisense strand, or both strands of the RNA55 80125197V.1sequence. 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.

[0135] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of an oligonucleotide. Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.

[0136] In particular embodiments, an oligonucleotide (or a strand thereof) specifically hybridizes to or is complementary to a target polynucleotide sequence. The terms “specifically hybridizable” and “complementary” as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In some embodiments, an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted. Thus, the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.

[0137] In some embodiments, the nucleic acid of a provided lipid nanoparticle is configured or selected to downregulate, e.g., reduce or eliminate, expression of a gene in a cell, e.g., a cell targeted by the lipid nanoparticle. For example, the nucleic acid can be configured or selected to reduce or eliminate expression of a gene in a hepatic stellate cell. In some examples, the nucleic acid capable of downregulating gene expression includes or consists of small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense RNA (asRNA), Dicer-substrate small interfering RNA (DsiRNA), Piwi-interacting RNA (piRNA), an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.56 80125197V.11. siRNA

[0138] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of siRNA. siRNA is interfering RNA capable of silencing the expression of a target sequence in vitro and / or in vivo. The siRNA may be of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (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 in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5′ phosphate termini. Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double- stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0139] In some preferred embodiments, a modified siRNA molecule is less immunostimulatory than a 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 the introduction of minimal and selective 2′Ome modifications within the siRNA sequence such as, e.g., within the double-stranded region of the siRNA duplex. In certain instances, the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 3545%, 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. It will be readily apparent to those of skill in the art that the immunostimulatory properties of the modified siRNA molecule and the57 80125197V.1corresponding unmodified siRNA molecule can be determined by, for example, measuring INF-α and / or IL-6 levels from about two to about twelve hours after systemic administration in a mammal or transfection of a mammalian responder cell using an appropriate lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0140] In certain embodiments, a modified siRNA molecule has an IC50 (i.e., half-maximal inhibitory concentration) less than or equal to ten-fold that of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC50that is less than or equal to ten-times the IC50of the corresponding unmodified siRNA). In other embodiments, the modified siRNA has an IC50 less than or equal to three-fold that of the corresponding unmodified siRNA sequence. In yet other embodiments, the modified siRNA has an IC50less than or equal to two-fold that of the corresponding unmodified siRNA. It will be readily apparent to those of skill in the art that a dose-response curve can be generated and the IC50 values for the modified siRNA and the corresponding unmodified siRNA can be readily determined using methods known to those of skill in the art.

[0141] In yet another embodiment, a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 2080%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence.

[0142] In some embodiments, the siRNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and / or antisense strand of the double-stranded region. In other embodiments, the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and / or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not comprise phosphate backbone modifications. In further embodiments, the siRNA does not comprise 2′-deoxy nucleotides, e.g., in the sense and / or antisense strand of the double-stranded region. In yet further embodiments, the siRNA comprises one, two, three, four, or more 2′-deoxy nucleotides, e.g., in the sense and / or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not comprise 2′-deoxy nucleotides.

[0143] In certain 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 certain other instances, the nucleotides near the 3′-end (e.g., within one, two, three, or four nucleotides of the 3′-end) of the double-stranded region in the sense and / or antisense strand are not modified nucleotides.58 80125197V.1

[0144] The siRNA molecules described herein may have 3′ overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region. Preferably, the siRNA has 3′ overhangs of two nucleotides on each side of the double-stranded region. In certain instances, the 3′ overhang on the antisense strand has complementarity to the target sequence and the 3′ overhang on the sense strand has complementarity to a complementary strand of the target sequence. Alternatively, the 3′ overhangs do not have complementarity to the target sequence or the complementary strand thereof. In some embodiments, the 3′ overhangs comprise one, two, three, four, or more nucleotides such as 2′-deoxy (2′H) nucleotides. In certain preferred embodiments, the 3′ overhangs comprise deoxythymidine (dT) and / or uridine nucleotides. In other embodiments, one or more of the 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′-deoxy nucleotides, 2′-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, one, two, three, four, or more nucleotides in the 3′ overhangs present on the sense and / or antisense strand of the siRNA comprise 2′Ome nucleotides (e.g., 2′Ome purine and / or pyrimidine nucleotides) such as, for example, 2′Ome-guanosine nucleotides, 2′Ome-uridine nucleotides, 2′Ome-adenosine nucleotides, 2′Ome-cytosine nucleotides, and mixtures thereof.

[0145] The siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and / or modified siRNA sequences that silence target gene expression. The cocktail of siRNA may comprise sequences which are directed to the same region or domain (e.g., a “hot spot”) and / or to different regions or domains of one or more target genes. In certain instances, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA that silence target gene expression are present in a cocktail. In certain other instances, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail.

[0146] In some embodiments, the antisense strand of the siRNA molecule comprises or consists 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 consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In further embodiments, the antisense strand of the59 80125197V.1siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0147] In further embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In additional embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof. a) Selection of siRNA Sequences

[0148] Suitable siRNA sequences can be identified using any means 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 rational design rules set forth in Reynolds et al., Nature Biotech., 22(3):326-330 (2004). Generally, the nucleotide sequence 3′ of the AUG start codon of a transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N=C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotides immediately 3′ to the dinucleotide sequences are identified as potential siRNA sequences (i.e., a target sequence or a sense strand sequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3′ to the dinucleotide sequences are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence and the 19 nucleotides immediately 3′ to the AA or NA dinucleotide are identified as potential siRNA sequences. siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance silencing efficiency of the siRNA sequences, potential siRNA sequences may be analyzed to identify sites that do not contain regions of homology to other coding sequences, e.g., in the target cell or organism. For example, a suitable siRNA sequence of about 21 base pairs typically will not have more than 16-17 contiguous base pairs of homology to coding sequences in the target cell or organism. If the siRNA sequences are to be expressed from an RNA Pol III promoter, siRNA sequences lacking more than 4 contiguous A’s or T’s are selected.

[0149] Once a potential siRNA sequence has been identified, a complementary sequence (i.e., an antisense strand sequence) can be designed. A potential siRNA sequence can also be analyzed using a variety of criteria known in the art. For example, to enhance their silencing efficiency, the siRNA sequences may be analyzed by a rational design algorithm to identify60 80125197V.1sequences that have one or more of the following features: (1) G / C content of about 25% to about 60% G / C; (2) at least 3 A / Us at positions 15-19 of the sense strand; (3) no internal repeats; (4) an A at position 19 of the sense strand; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7) no G / C at position 19 of the sense strand; and (8) no G at position 13 of the sense strand. One of skill in the art will appreciate that sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential siRNA sequences.

[0150] Additionally, potential siRNA sequences with one or more of the following criteria can often be eliminated as siRNA: (1) sequences comprising a stretch of 4 or more of the same base in a row; (2) sequences comprising homopolymers of Gs (i.e., to reduce possible non- specific effects due to structural characteristics of these polymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G / Cs in a row; and (5) sequences comprising direct repeats of 4 or more bases within the candidates resulting in internal fold-back structures. However, one of skill in the art will appreciate that sequences with one or more of the foregoing characteristics may still be selected for further analysis and testing as potential siRNA sequences.

[0151] In some embodiments, potential siRNA sequences may be further analyzed based on siRNA duplex asymmetry as described in, e.g., Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115: 199-208 (2003). In other embodiments, potential siRNA sequences may be further analyzed based on secondary structure at the target site as described in, e.g., Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example, secondary structure at the target site can be modeled using the Mfold algorithm (available at www.bioinfo.rpi.edu / applications / mfold / rna / forml.cgi) to select siRNA sequences which favor accessibility at the target site where less secondary structure in the form of base-pairing and stem-loops is present.

[0152] Once a potential siRNA sequence has been identified, the sequence can be analyzed for the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo animal model. Motifs in the sense and / or antisense strand 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 may be immunostimulatory. Once an siRNA molecule is found to be immunostimulatory, it can then be modified to decrease its immunostimulatory properties as described herein. As a non-limiting example, an siRNA61 80125197V.1sequence can be contacted with a mammalian responder cell under conditions such that the cell produces a detectable immune response to determine whether the siRNA is an immunostimulatory or a non-immunostimulatory siRNA. The mammalian responder cell may be from a naive mammal (i.e., a mammal that has not previously been in contact with the gene product of the siRNA sequence). The mammalian responder cell may be, e.g., a peripheral blood mononuclear cell (PBMC), a macrophage, and the like. The detectable immune response may comprise production of a cytokine or growth factor such as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or a combination thereof. An siRNA molecule identified as being immunostimulatory can then be modified to decrease its immunostimulatory properties by replacing at least one of the nucleotides on the sense and / or antisense strand with modified nucleotides. 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 modified nucleotides such as 2′Ome nucleotides. The modified siRNA can then be contacted with a mammalian responder cell as described above to confirm that its immunostimulatory properties have been reduced or abrogated.

[0153] Suitable in vitro assays for detecting an immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Pat. 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 blot” method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays (ELISA) as described, for example, by Raines et al., J. Biol. Chem., 257:5154-5160 (1982); immunocytochemical techniques, including the use of fluorochromes (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, a number of other immunoassays are available, including those described in U.S. Pat. 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 herein incorporated by reference in their entirety for all purposes.

[0154] A non-limiting example of an in vivo model for detecting an immune response includes an in vivo mouse cytokine induction assay as described in, e.g., Judge et al., Mol. Ther., 13:494- 505 (2006). In certain embodiments, the assay that can be performed as follows: (1) siRNA can be administered by standard intravenous injection in the lateral tail vein; (2)62 80125197V.1blood can be collected by cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer’s instructions (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; San Diego, Calif); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif)).

[0155] Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from multiple sources and can be generated using methods known in the art (see, e.g., Kohler et al., Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of monoclonal antibodies has been previously described and can be accomplished 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., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection. b) Generation of siRNA Sequences

[0156] siRNA can be provided in several forms including, e.g., as one or more isolated small- interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid. siRNA can be chemically synthesized. The oligonucleotides that comprise the siRNA molecules can be synthesized using any of a variety of 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).

[0157] An RNA population can be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence can be used to make the siRNA. The RNAs can be isolated from cells or tissue, synthesized, and / or cloned according to methods well known to those of skill in the art. The RNA can be a mixed population (obtained from cells or tissue, 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 polymerase and PCR products or a cloned cDNA), or chemically synthesized.63 80125197V.1

[0158] To form a long dsRNA, for synthetic RNAs, the complement is also transcribed in vitro and hybridized to form a dsRNA. If a naturally occurring RNA population is used, the RNA complements are also provided (e.g., to form dsRNA for digestion by E.coli RNAse III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerases. The precursor RNAs are then hybridized to form double stranded RNAs for digestion. The dsRNAs can be directly administered to a subject or can be digested in vitro prior to administration.

[0159] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods of use in the provided embodiments include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nded.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are herein incorporated by reference in their entirety for all purposes.

[0160] Preferably, siRNA are chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E.coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., 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, dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain instances, siRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).

[0161] siRNA molecules can also be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or64 80125197V.1strands that hybridize to form the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siRNA can be readily adapted to both multiwell / multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like. Alternatively, siRNA molecules can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA. For example, each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and / or deprotection. In certain other instances, siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an siRNA duplex having hairpin secondary structure. c) Modification of siRNA Sequences

[0162] In certain aspects, siRNA molecules comprise a duplex having 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 a corresponding unmodified siRNA sequence, but retains the capability of silencing the expression of a target sequence. In preferred embodiments, the degree of chemical modifications introduced into the siRNA molecule strikes a balance between reduction or abrogation of the immunostimulatory properties of the siRNA and retention of RNAi activity. As a non-limiting example, an siRNA molecule that targets a gene of interest can be minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5% modified) at selective uridine and / or guanosine nucleotides within the siRNA duplex to eliminate the immune response generated by the siRNA while retaining its capability to silence target gene expression.

[0163] 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 having a Northern conformation such as those described in, e.g., Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984), are also suitable for use in siRNA molecules. Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy- 2′-chloro (2′Cl) nucleotides, and 2′-azido nucleotides. In certain instances, the siRNA65 80125197V.1molecules described herein include one or more G-clamp nucleotides. A G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3- nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into siRNA molecules.

[0164] In certain embodiments, siRNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like. Examples of terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4′,5′-methylene nucleotides, 1-(^-D-erythrofuranosyl) nucleotides, 4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L- nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5- dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties, 3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties, 5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties, 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′-phosphoramidate, 5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′- phosphate, 5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5′-mercapto moieties (see, e.g., U.S. Patent No. 5,998,203; Beaucage et al., Tetrahedron 49:1925 (1993)). Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., 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 5′-end and / or 3′-end of the sense strand, antisense strand, or both strands of the siRNA.66 80125197V.1

[0165] In some embodiments, the sense and / or antisense strand of the siRNA molecule can further comprise a 3′-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and / or any combination of modified and unmodified nucleotides. Additional examples of modified nucleotides and types of chemical modifications that can be introduced into siRNA molecules are described, e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Application Publication Nos.2004 / 0192626, 2005 / 0282188, and 2007 / 0135372.

[0166] The siRNA molecules described herein can optionally comprise one or more non- nucleotides in one or both strands of the siRNA. As used herein, the term “non-nucleotide” refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and / or phosphate substitutions, and allows the remaining bases to exhibit their activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1′-position.

[0167] In other embodiments, chemical modification of the siRNA comprises attaching a conjugate to the siRNA molecule. The conjugate can be attached at the 5′- and / or 3′-end of the sense and / or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker. The conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking group (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0074771, 2005 / 0043219, and 20050158727). In certain instances, the conjugate is a molecule that facilitates the delivery of the siRNA into a cell. Examples of conjugate molecules suitable for attachment to siRNA include, without limitation, 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 derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Application Publication Nos. 2003 / 0130186, 2004 / 0110296, and 2004 / 0249178; U.S. Patent No. 6,753,423). Other examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Application Publication Nos.2005 / 0119470 and 2005 / 0107325. Yet other examples include the 2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidinium group, cationic amino acid conjugate molecules described in67 80125197V.1U.S. Patent Application Publication No. 2005 / 0153337. Additional examples include the hydrophobic group, membrane active compound, cell penetrating compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in U.S. Patent Application Publication No.2004 / 0167090. Further examples include the conjugate molecules described in U.S. Patent Application Publication No. 2005 / 0239739. The type of conjugate used and the extent of conjugation to the siRNA molecule can be evaluated for improved pharmacokinetic profiles, bioavailability, and / or stability of the siRNA while retaining RNAi activity. As such, one skilled in the art can screen siRNA molecules having various conjugates attached thereto to identify ones having improved properties and full RNAi activity using any of a variety of well-known in vitro cell culture or in vivo animal models. 2. miRNA

[0168] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of miRNA. Generally, miRNA are single-stranded RNA molecules of about 21-23 nucleotides in length which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (non-coding RNA); instead, each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional mature miRNA. Mature miRNA molecules are either partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. The identification of miRNA molecules is described, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau et al., Science, 294:858-862; and Lee et al., Science, 294:862-864.

[0169] The genes encoding miRNA are much longer than the processed mature miRNA molecule. miRNA are first transcribed as primary transcripts or pre-miRNA with a cap and poly-A tail and processed to short, approximately 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double- stranded RNA binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These pre- miRNA are then processed to mature miRNA in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001). Either the sense strand or antisense strand of DNA can function as templates to give rise to miRNA.68 80125197V.1

[0170] When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the Argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5′ end (Preall et al., Curr. Biol., 16:530- 535 (2006)). The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate (Gregory et al., Cell, 123:631-640 (2005)). After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce target mRNA degradation and / or translational silencing.

[0171] Mammalian miRNA molecules are usually complementary to a site in the 3′ UTR of the target mRNA sequence. In certain instances, the annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other instances, the annealing of the miRNA to the target mRNA facilitates the cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNA may also target methylation of genomic sites which correspond to targeted mRNA. Generally, miRNA function in association with a complement of proteins collectively termed the miRNP.

[0172] In certain aspects, 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 are preferably about 20-24, 21-22, or 21-23 nucleotides in length. In certain other aspects, miRNA molecules may comprise one or more modified nucleotides. As a non-limiting example, miRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In a preferred embodiment, the miRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe- guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof. 3. shRNA

[0173] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of shRNA. An shRNA molecule includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA molecules of the present disclosure may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).69 80125197V.1

[0174] The shRNA molecules of the invention are typically about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (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 in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15- 25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNA duplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and / or 5′-phosphate termini on the sense strand. In some embodiments, the shRNA comprises a sense strand and / or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferably from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), more preferably from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).

[0175] Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions. In preferred embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from 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.

[0176] Suitable shRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. Additional embodiments related to the shRNA molecules of the present disclosure, as well as methods of designing and synthesizing such shRNA molecules, are described in U.S. Patent Application Publication No.2011 / 0071208, the disclosure of which is herein incorporated by reference in its entirety for all purposes.70 80125197V.14. asRNA

[0177] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of asRNA. asRNA molecules, also referred to in the art as antisense transcripts, are naturally-occurring or synthetically produced single-stranded RNA molecules that are complementary to a protein-coding messenger RNA (mRNA) with which it hybridizes and thereby blocks the translation of the mRNA into a protein. Antisense transcript are classified into short (less than 200 nucleotides) and long (greater than 200 nucleotides) non-coding RNAs (ncRNAs).

[0178] The asRNA molecules may include a sequence complementary to a genomic sequence between 100, 80, 60, 40, 20, or 10 kb upstream of the transcription initiation site of a target gene to downstream of the transcription termination site. In some embodiments, the asRNA includes a sequence complementary to a genomic sequence between 1 kb upstream of the transcription initiation 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 between 500, 250, or 100 nucleotides upstream of the transcription initiation site of the target gene to the downstream 500, 250 or 100 nucleotides of the transcription termination site of the target gene.

[0179] The asRNA molecule may include a sequence complementary to a genomic sequence comprising the coding region of the target gene. The asRNA may include a sequence complementary to a genomic sequence aligned with a promoter region of the target gene on the template strand. The gene can have a plurality of promoter regions. In this case, the asRNA can be aligned with one, two or more promoter regions. An online database of annotated gene loci can be used to identify a promoter region of a gene.

[0180] The alignment region between the asRNA molecule and the target gene promoter region may be as short as a single nucleotide in length, or can be at least 15 or at least 20 nucleotide length, or at least 25 nucleotide length, or at least 30, 35, 40, 45, or 50 nucleotide length, or at least 55, 60, 65, 70 or 75 nucleotide length, or at least 100 nucleotide length.

[0181] The primary natural function of asRNA molecules involves regulating gene expression, and synthetic versions have been used widely as research tools for gene knockdown and for therapeutic applications. asRNA molecules and their functions have been described in71 80125197V.1the art (see e.g., 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). 5. DsiRNA

[0182] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of DsiRNA. DsiRNA molecules are processed in vivo by Dicer to produce an active siRNA which is incorporated into the RISC complex for RNA interference of a target gene. In some embodiments, the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA. According to these embodiments, the DsiRNA comprises (i) a first oligonucleotide sequence (also termed the sense strand) that is between about 25 and 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 between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotide sequence (also termed the antisense strand) that anneals to the first sequence under biological conditions, such as the conditions found in the cytoplasm of a cell. The second oligonucleotide sequence may be between about 25 and 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), and is preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA has a sequence length of at least about 19 nucleotides, for example, from 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 from about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger an RNAi response.

[0183] In some embodiments, the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and has at least one of the following properties: (i) the DsiRNA is asymmetric, e.g., has a 3′-overhang on the antisense strand; and / or (ii) the DsiRNA has a modified 3′-end on the sense strand to direct orientation of Dicer binding and processing of the DsiRNA to an active siRNA. According to this latter embodiment, the sense strand comprises from about 22 to about 28 nucleotides and the antisense strand comprises from about 24 to about 30 nucleotides.

[0184] In some embodiments, the DsiRNA has an overhang on the 3′-end of the antisense strand. In some embodiments, the sense strand is modified for Dicer binding and processing by72 80125197V.1suitable modifiers located at the 3′-end of the sense strand. Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like. When nucleotide modifiers are used, they replace ribonucleotides in the DsiRNA such that the length of the DsiRNA does not change. In some embodiments, the DsiRNA has an overhang on the 3′-end of the antisense strand and the sense strand is modified for Dicer processing. In some embodiments, the 5′-end of the sense strand has a phosphate. In some embodiments, the 5′-end of the antisense strand has a phosphate. In some embodiments, the antisense strand or the sense strand or both strands have one or more 2′-O -methyl (2′OMe) modified nucleotides. In some embodiments, the antisense strand contains 2′OMe modified nucleotides. In some embodiments, the antisense stand contains a 3′-overhang that is comprised of 2′OMe modified nucleotides. The antisense strand could also include additional 2′OMe modified nucleotides. The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA has a sequence length of at least about 19 nucleotides, where these nucleotides are in the 21-nucleotide region adjacent to the 3′-end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene. Further, in accordance with these embodiments, the DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21- mer (i.e., the antisense strand includes nucleotides on the right side of the molecule when compared to the typical 21-mer); (b) the strands may not be completely complementary (i.e., the strands may contain simple mismatch pairings); and (c) base modifications such as locked nucleic acid(s) may be included in the 5′-end of the sense strand.

[0185] In some embodiments, the sense strand comprises from about 25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2 nucleotides on the 3′-end of the sense strand are deoxyribonucleotides. The sense strand contains a phosphate at the 5′-end. The antisense strand comprises from 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 nucleotides comprising the 3′- overhang are modified with 2′OMe modified ribonucleotides. The antisense strand contains alternating 2′OMe modified nucleotides beginning at the first monomer of the antisense strand adjacent to the 3′-overhang, and extending 15-19 nucleotides from the first monomer adjacent to the 3′-overhang. For example, for a 27-nucleotide antisense strand and counting the first base73 80125197V.1at the 5′-end of the antisense strand as position number 1, 2′OMe modifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, and 27.

[0186] In some embodiments, the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and (ii) the DsiRNA has a modified 3′-end on the antisense strand to direct orientation of Dicer binding and processing of the DsiRNA to an active siRNA. According to these embodiments, the sense strand comprises from about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand comprises from about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the DsiRNA has an overhang on the 3′-end of the sense strand. In some embodiments, the antisense strand is modified for Dicer binding and processing by suitable modifiers located at the 3′-end of the antisense strand. Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like. When nucleotide modifiers are used, they replace ribonucleotides in the DsiRNA such that the length of the DsiRNA does not change. In some embodiments, the DsiRNA has an overhang on the 3′-end of the sense strand and the antisense strand is modified for Dicer processing. In some embodiments, the antisense strand has a 5′-phosphate. The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3′-end of antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene. Further, in accordance with this embodiment, the DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a left shift from the typical 21-mer (i.e., the antisense strand includes nucleotides on the left side of the molecule when compared to the typical 21-mer); and (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings.

[0187] In a preferred embodiment, the DsiRNA has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2-base 3′-overhang. In certain instances, this DsiRNA having an asymmetric structure further contains 2 deoxynucleotides at the 3′-end of the sense strand in place of two of the ribonucleotides. In certain other instances, this DsiRNA having an asymmetric structure further contains 2′OMe modifications at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense74 80125197V.1strand (wherein the first base at the 5′-end of the antisense strand is position 1). In certain additional instances, this DsiRNA having an asymmetric structure further contains a 3′- overhang on the antisense strand comprising 1, 2, 3, or 42′OMe nucleotides (e.g., a 3′-overhang of 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

[0188] In another embodiment, DsiRNA may be designed by first selecting an antisense strand siRNA sequence having a length of at least 19 nucleotides. In some instances, the antisense siRNA is modified to include about 5 to about 11 ribonucleotides on the 5′-end to provide a length of about 24 to about 30 nucleotides. When the antisense strand has a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably 6 nucleotides may be added on the 5′- end. Although the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence. A sense strand is then produced that has about 22 to about 28 nucleotides. The sense strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions. In one embodiment, the sense strand is synthesized to contain a modified 3′-end to direct Dicer 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 contain a modified 3′-end for Dicer binding and processing and the antisense strand of the dsRNA has a 3′-overhang.

[0189] In a related embodiment, the antisense siRNA may be modified to include about 1 to about 9 ribonucleotides on the 5′-end to provide a length of about 22 to about 28 nucleotides. When the antisense strand has a length of 21 nucleotides, 1-7, preferably 2-5, or more preferably 4 ribonucleotides may be added on the 3′-end. The added ribonucleotides may have any sequence. Although the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence. A sense strand is then produced that has about 24 to about 30 nucleotides. The sense strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions. In one embodiment, the antisense strand is synthesized to contain a modified 3′-end to direct Dicer processing. In another embodiment, the sense strand of the dsRNA has a 3′-overhang. In a further embodiment, the antisense strand is synthesized to contain a modified 3′-end for Dicer binding and processing and the sense strand of the dsRNA has a 3′-overhang.75 80125197V.1

[0190] Additional embodiments related to the DsiRNA molecules of the present disclosure, as well as methods of designing and synthesizing such DsiRNA molecules, are described in U.S. Patent Application Publication Nos. 2005 / 0244858, 2005 / 0277610, 2007 / 0265220, and 2011 / 0071208, the disclosures of which are herein incorporated by reference in their entirety for all purposes. 6. piRNA

[0191] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of piRNA. The piRNA molecule bind to proteins of A piRNA molecule can be about 10 to 50 nucleotides in length, about 25 to 39 nucleotides in length, or about 26 to 31 nucleotides in length. See, e.g., U.S. Patent Application Pub. No.2009 / 0062228.

[0192] piRNA molecules represent the largest class of small non-coding RNA molecules. piRNAs can be substantially complementary to a target gene, and can selectively form RNA- protein complexes through interactions with the Piwi or Aubergine subclasses of Argonaute proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. They are distinct from microRNA (miRNA) in size (typically 24-31 nt rather than 21-24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, specifically the silencing of transposons. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are the piRNA target. In mammals it appears that the activity of piRNAs in transposon silencing is most important during the development of the embryo, and in both C. elegans and humans, piRNAs are necessary for spermatogenesis. piRNA has a role in RNA silencing via the formation of an RNA-induced silencing complex (RISC). 7. Antisense Oligonucleotides

[0193] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of an antisense oligonucleotide directed to a target gene or sequence of interest. The terms “antisense oligonucleotide” or “antisense” as used herein include oligonucleotides that are complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. Antisense RNA oligonucleotides prevent the translation of complementary RNA strands by binding to the RNA. Antisense DNA oligonucleotides can be used to target a specific, complementary76 80125197V.1(coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, antisense oligonucleotides comprise from about 10 to about 60 nucleotides or from about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, the lipid nanoparticles described herein can be utilized in instances where non-target specific-activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is the most preferred for a particular use. An antisense oligonucleotide can contain natural nucleotides, as well as non-natural or modified nucleotides (e.g., a modified nucleobase, modified internucleoside linkage, and / or modified sugar such as those described herein).

[0194] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, can be used to specifically inhibit protein synthesis by a targeted gene. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalacturonase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (see, U.S. Pat. Nos.5,739,119 and 5,759,829). Furthermore, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDRl), 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); Peris et al., Brain Res Mal Brain Res., 15; 57:310-20 (1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288). Moreover, antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317; and 5,783,683). The disclosures of these references are herein incorporated by reference in their entirety for all purposes.

[0195] Methods of producing antisense oligonucleotides are known in the art and can be readily adapted to produce an antisense oligonucleotide that targets any polynucleotide sequence. Selection of antisense oligonucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and77 80125197V.1those sequences that are substantially complementary to 5′ regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and / or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0196] In some embodiments, the antisense oligonucleotide component of the lipid particles described herein can be used to inhibit the expression or replication of a gene of interest. Genes of interest are set forth above in the context of siRNA molecules and include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. As a non-limiting example, the antisense oligonucleotide can hybridize to a SARS-CoV-2 gene (e.g., orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, or ORF10) and inhibit the expression or replication of the gene. 8. Morpholino Oligonucleotides

[0197] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of a morpholino oligonucleotide. Morpholino oligonucleotides are non-ionic and function by an RNase H-independent mechanism. Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and Thymine / Uracil) of the morpholino oligonucleotides is linked to a 6- membered morpholine ring. Morpholino oligonucleotides are made by joining the 4 different subunit types by, e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholino oligonucleotides have many advantages including: complete resistance to nucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica Acta. 1999. 1489:141); reliable activity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63); excellent sequence specificity (Antisense & Nucl. Acid Drug Dev.1997.7:151); minimal non- antisense activity (Biochemica Biophysica Acta.1999.1489:141); and simple osmotic or scrape delivery (Antisense & Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are also preferred because of their non-toxicity at high doses. A discussion of the preparation of morpholino oligonucleotides can be found in Antisense & Nucl. Acid Drug Dev.1997.7:187.78 80125197V.1

[0198] Morpholino oligonucleotides are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base- specific hydrogen bonding, to a base in a polynucleotide, which are linked together by phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5′ exocyclic carbon of an adjacent monomer. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337. 9. Target Genes of Downregulating Nucleic Acids

[0199] In certain embodiments, the nucleic acid component (e.g., siRNA) of the lipid nanoparticles described herein can be used to downregulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. In certain embodiments, the gene of interest is expressed in hepatic stellate cells.

[0200] In some examples, the nucleic acid component (e.g., siRNA) is configured to reduce expression of a gene in a hepatic stellate cell. For example, the siRNA can reduce expression of a gene encoding collagen type I alpha 1 chain (COL1A1), COL1A2, COL2A1, COL3A1, COL4A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL17A1, COL18A1, tissue inhibitor of metalloproteinases 1 (TIMP1), matrix metalloproteinase 2 (MMP2), MMP1, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP22, MMP23, MMP24, MMP25, MMP26, MMP28, alpha smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), heat shock protein 47 (HSP47), lysyl oxidase (LOX), epidermal growth factor receptor (EGFR), rho kinase (ROCK), integrin alpha 8 beta 1 or another integrin, yes- associated protein (YAP), a toll-like receptor pathway protein, transforming growth factor beta (TGFB), platelet-derived growth factor (PDGF), or connective tissue growth factor (CTGF)79 80125197V.1

[0201] In addition to its utility in silencing the expression of any of the above-described genes for therapeutic purposes, certain nucleic acids (e.g., siRNA) described herein are also useful in research and development applications as well as diagnostic, prophylactic, prognostic, clinical, and other healthcare applications. As a non-limiting example, certain nucleic acids (e.g., siRNA) can be used in target validation studies directed at testing whether a gene of interest has the potential to be a therapeutic target. Certain nucleic acids (e.g., siRNA) can also be used in target identification studies aimed at discovering genes as potential therapeutic targets. V. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0202] In another aspect, the disclosure provides a pharmaceutical composition that include one or more of any of the ionizable lipids described herein in Section III, or any of the lipid nanoparticles described herein in Section IV. In some embodiments, the pharmaceutical composition further includes a therapeutically effective amount of a pharmaceutically acceptable excipient. Additionally or alternatively, the pharmaceutical composition can include a pharmaceutically acceptable carrier.

[0203] The pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) can be selected in accordance with the route of administration and standard pharmaceutical practice. Generally, normal buffered saline (e.g., 135-150 mM NaCl) is used as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Additional suitable carriers are described in, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition (2020). The pharmaceutically acceptable carrier is generally added following particle formation. Thus, after the particle is formed, the particle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.

[0204] Examples of suitable excipients for use with the provided pharmaceutical compositions include, but are not limited to, lactose, dextrose, sucrose, maltose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.80 80125197V.1

[0205] The provided pharmaceutical compositions can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; coloring agents; and flavoring agents. The compositions can also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0206] Pharmaceutical compositions that are liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; 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 the adjustment of tonicity such as sodium chloride or dextrose. The liquid compositions can include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0207] The provided pharmaceutical compositions for administration are preferably sterile. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and thimerosal. Compositions for administration can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic, or other material.

[0208] For oral administration, the provided pharmaceutical compositions can be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders, and sustained-release formulations. Suitable excipients for oral administration include81 80125197V.1pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0209] For topical administration, the provided pharmaceutical compositions can be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For delivery by inhalation, the composition can be delivered as a dry powder or in liquid form via a nebulizer. For parenteral administration, the compositions can be in the form of sterile injectable solutions and sterile packaged powders. Injectable solutions can be formulated at a pH of about 4.5 to about 8.5, and preferably at a pH between about 6.0 and about 7.5.

[0210] The concentration of lipid nanoparticles in the provided pharmaceutical formulations can vary widely, e.g., from less than about 0.05%, usually at or at least about 2 to about 5%, to as much as about 10 to about 90% by weight, and can be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, lipid nanoparticles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.

[0211] The provided pharmaceutical compositions can be sterilized by conventional, well- known sterilization techniques. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Additionally, the lipid nanoparticle suspension can include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free- radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable. VI. KITS

[0212] In another aspect, the disclosure provides kits to facilitate and / or standardize the use of the materials described herein (i.e., the ionizable lipids described in Section III, the lipid nanoparticles described in Section IV, and / or the pharmaceutical compositions described in82 80125197V.1Section V), as well as to facilitate the methods described herein (i.e., the methods described in Section VII). Materials and reagents to carry out these various methods can be provided in kits to facilitate execution of the methods.

[0213] The provided kits can contain chemical reagents as well as other components. In addition, the kits containing the provided lipid nanoparticles, ionizable lipids, and / or pharmaceutical compositions can include, without limitation, instructions to the kit user. Kits can also be packaged for convenient storage and safe shipping, for example, as ampules or other vials packaged in a box having a lid.

[0214] The provided kits can include one or more containers, each of which can be compartmentalized for holding the various elements of the lipid nanoparticles (e.g., the nucleic acids and the individual lipid components of the nanoparticles). In some embodiments, the kit further includes an endosomal membrane destabilizer (e.g., calcium ions). The kit typically contains the lipid nanoparticle compositions of the present disclosure, preferably in dehydrated form, with instructions for their rehydration and administration. VII. METHODS

[0215] In other aspects, the disclosure provides several methods for making and / or using the lipid nanoparticles described in Section IV, the pharmaceutical compositions described in Section V, and / or the kits described in Section VI. The methods disclosed herein benefit from the improved properties of these provided materials, e.g., properties advantageous for delivering a nucleic acid into a cell (e.g., a hepatic stellate cell). These improved properties include, for example, efficient targeting of desired cell types (e.g., hepatic stellate cells), high biocompatibility, good biodegradability, and low toxicity. A. Preparing Lipid Nanoparticles

[0216] Some methods provided by the disclosure are useful for preparing one or more lipid nanoparticles, wherein the lipid nanoparticles include those described in Section IV. The lipid nanoparticles can be prepared via, for example, a continuous mixing method and / or a direct dilution process. In some embodiments, the methods include selecting, designing, and / or preparing one or more components of the lipid nanoparticle being prepared. For example, the methods can include selecting, designing, and / or preparing a phospholipid, an ionizable lipid, a cholesterol or derivative thereof, a conjugated lipid, and / or a nucleic acid.83 80125197V.1

[0217] In some embodiments, the lipid nanoparticles described herein are produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid (e.g., any of the nucleic acids described in Section IV.E) 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 mixes with the aqueous solution so as to substantially instantaneously produce a lipid particle encapsulating the nucleic acid. Examples of this process and the apparatus for carrying out this process are described in U.S. Patent Application Publication No.2004 / 0142025.

[0218] In some examples, the organic lipid solution is an alcoholic solution that includes an alcoholic organic solvent and the lipids that will form the lipid nanoparticles. These lipids can 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, the ionizable lipid can be any of those described in Section III, the phospholipid can be any of those described in Section IV.A, the sterol can be cholesterol or any of its derivatives described in Section IV.B, and the conjugated lipid can be any of those described in Section IV.C. In some embodiments, the alcoholic organic solvent of the alcoholic solution includes or consists of ethanol. In some examples, the alcoholic solution is the product of combining the alcoholic organic solvent and a phospholipid solution, where the phospholipid solution includes or consists of a nonalcoholic organic solvent and the one or more phospholipids or pharmaceutically acceptable salts thereof. In some embodiments, the nonalcoholic organic solvent includes or consists of tetrahydrofuran.

[0219] The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid particle substantially instantaneously upon mixing. As used herein, the phrase “continuously diluting a lipid solution with a buffer solution” (and variations) generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate vesicle generation. By mixing the aqueous solution comprising a nucleic acid with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a lipid particle.

[0220] The lipid nanoparticles formed using the continuous mixing method typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 6084 80125197V.1nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0221] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution process that includes forming a lipid particle solution and immediately and directly introducing the lipid particle solution into a collection vessel containing a controlled amount of dilution buffer. In certain embodiments, the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid particle solution introduced thereto. As a non-limiting example, a lipid particle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.

[0222] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the lipid particle solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. In certain embodiments, the second mixing region includes a T-connector arranged so that the lipid particle solution and the dilution buffer flows meet as opposing 180 º flows; however, connectors providing shallower angles can be used, e.g., from about 27 º to about 180 º. A pump mechanism delivers a controllable flow of buffer to the second mixing region. In one embodiment, the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid particle solution introduced thereto from the first mixing region. This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid particle solution in the second mixing region, and therefore also the concentration of lipid particle solution in buffer throughout the second mixing process. Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations. Examples of these processes and the apparatuses for carrying out these direct dilution processes are described in U.S. Patent Application Publication No.2007 / 0042031.

[0223] The lipid particles formed using the direct dilution process typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to85 80125197V.1about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 nm to about 80 nm, from about 60 nm to about 80 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0224] If needed, the lipid particles described herein can be sized by any of the methods available to one of skill in the art. The sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes. Several techniques are available for sizing the particles to a desired size, e.g. such as described in Lam et al.2023, Adv. Mat., 35:26, 2211420. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 nm and about 80 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.

[0225] Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.

[0226] In some embodiments, the nucleic acid to lipid ratios (mass / mass ratios) in a formed lipid particle ranges from about 0.01 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08. The ratio of the starting materials can also fall within this range. In some embodiments, the lipid to nucleic acid ratio (mass / mass ratios) in a formed lipid particle ranges from about 5 to about 100, from about 8 to about 50, from about 9 to about 30, or from about 10 to about 20. In other embodiments, the lipid particle preparation includes about 12 mg total lipid per 1 mg of nucleic acid.

[0227] In other embodiments, the lipid to nucleic acid ratios (mass / mass ratios) in a formed lipid particle ranges from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about86 80125197V.13 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5 (5:1) to about 15 (15:1), from 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 the starting materials can also fall within this range. B. Introducing a Nucleic Acid into a Cell

[0228] Some methods provided by the disclosure are useful for introducing a nucleic acid into a cell. These methods generally include contacting the cell with one or more lipid nanoparticles described in Section IV. The nucleic acid introduced to the cell using the lipid nanoparticle can be, for example, any of those described in Section IV.F. Once formed, the lipid nanoparticles described herein are particularly useful for the introduction of nucleic acids such as siRNA into cells. The methods are carried out in vitro or in vivo by first forming the particles and then contacting the particles with the cells for a period of time sufficient for delivery of the nucleic acid to the cells to occur.

[0229] The lipid nanoparticles described herein can preferentially be adsorbed to hepatic stellate cells with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid.

[0230] For in vitro applications, the delivery of nucleic acids (e.g., mRNA and / or siRNA) can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In certain embodiments, the cells are animal cells, e.g., mammalian cells such as human cells.

[0231] Contact between the cells and the lipid nanoparticles, when carried out in vitro, generally takes place in a biologically compatible medium. The amount of the composition used can vary widely depending on the particular application, but is generally between about 1 μmol and about 10 mmol. Treatment of the cells with the lipid particles is generally carried out at physiological temperatures (about 37 °C) for periods of time ranging from about 1 hour to87 80125197V.1about 48 hours. In some examples, the contacting of the cells with the lipid nanoparticles is for a duration lasting from about 2 to about 4 hours.

[0232] In some embodiments, a lipid nanoparticle suspension is added to 60-80% confluent plated cells having a cell density of from about 103to about 105cells / mL, e.g., about 2×104cells / mL. The concentration of the suspension added to the cells can be from about 0.01 to 0.2 μg / mL, e.g., about 0.1 μg / mL.

[0233] Using an Endosomal Release Parameter (ERP) assay, the delivery efficiency of the lipid particle can be optimized. An ERP assay is described in U.S. Patent Application Publication No.2003 / 0077829. More particularly, the purpose of an ERP assay is to distinguish the effect of various cationic lipids and helper lipid components based on their relative effect on binding / uptake or fusion with / destabilization of the endosomal membrane. This assay allows one to determine quantitatively how each component of the lipid particle affects delivery efficiency, thereby optimizing the lipid particle. Usually, an ERP assay measures expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.), and in some instances, a lipid particle formulation optimized for an expression plasmid will also be appropriate for encapsulating other types of nucleic acid such as mRNA. In some instances, an ERP assay can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of an interfering RNA (e.g., siRNA). In other instances, an ERP assay can be adapted to measure the expression of a target protein in the presence or absence of an mRNA. By comparing the ERPs for each of the various lipid particles, one can readily determine the optimized system, e.g., the lipid particle that has the greatest uptake in the cell.

[0234] The compositions and methods described herein are used to treat a wide variety of cell types, in vivo and in vitro. Suitable cells include, e.g., hepatic stellate cells, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, immune cells, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, and the like. In some embodiments, lipid particles comprising nucleic acid (e.g., mRNA) are delivered to immune cells such as e.g., 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 embodiments, lipid particles comprising nucleic acid (e.g., siRNA) are delivered to cancer cells such as, e.g., lung cancer88 80125197V.1cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, stomach (gastric) 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, renal cancer cells, cancer cells of the central nervous system, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.

[0235] In vivo delivery of the provided lipid particles such as lipid nanoparticles encapsulating one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA) is particularly suited for targeting hepatic stellate cells. To the extent that tissue culture of cells may be required, it is well-known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., 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 a general guide to the culture of cells. Cultured cell systems often will be in the form of monolayers of cells, although cell suspensions are also used. C. Delivering a Nucleic Acid to a Subject

[0236] Some methods provided by the disclosure are useful for delivering a nucleic acid to a subject. The methods and compositions disclosed herein can be employed with cells of a wide variety of vertebrates, including mammals, such as, e.g., canines, felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans). These methods generally include administering one or more lipid nanoparticles or one or more pharmaceutical compositions to the subject, wherein the lipid nanoparticles include those described in Section IV, and the pharmaceutical compositions include those described in Section V. The nucleic acid delivered to the subject can be, for example, any of those described in Section IV.E. In some embodiments, the lipid nanoparticles described herein are administered to a subject by systemic delivery, e.g., to a distal target cell via body systems such as the circulation. In certain embodiments, the present disclosure provides fully encapsulated lipid particles that protect the nucleic acid from nuclease degradation in serum, are nonimmunogenic, are small in size, and are suitable for repeat dosing.

[0237] In some embodiments, the ionizable lipids and lipid nanoparticles provided by the current disclosure are particularly useful for efficiently delivering nucleic acids, such as RNA,89 80125197V.1to hepatic stellate cells of a subject. The specific ionizable lipids and lipid nanoparticle configurations described in Sections III and IV advantageously result in materials exhibiting improved delivery to hepatic stellate cells of a subject to whom the materials are administered. For example, in certain embodiments, the provided ionizable lipids and lipid nanoparticles can be used to deliver a nucleic acid reducing expression of a protein by hepatic stellate cells of a subject, such that the activity or secretion of the protein in the hepatic stellate cells is more than about 20-fold reduced, more than about 30-fold, more than about 40-fold, more than about 60- fold, more than about 100-fold, more than about 200-fold, more than about 300-fold, more than about 500-fold, more than about 700-fold, or more than about 1000-fold reduced relative an activity or expression of the protein in the absence of the nucleic acid delivered by the provided lipid nanoparticles.

[0238] For in vivo administration, administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration can be accomplished via single or divided doses. The pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Patent No.5,286,634). Intracellular nucleic acid delivery has also been 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). Still other methods of administering lipid-based therapeutics are described in, for example, U.S. Patent Nos.3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid nanoparticles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp. 70- 71(1994)).

[0239] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In particular embodiments, the compositions are administered intravenously90 80125197V.1(e.g., by intravenous infusion), intramuscularly, pulmonarily, orally, topically, intranasally, intracerebrally, intraperitoneally, intravesically, or intrathecally.

[0240] Generally, when administered intravenously, the lipid particle formulations are formulated with a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers may be employed in the compositions and methods described herein. Suitable formulations for use are found, for example, in Adejare, A. (Ed.). (2020). Remington: The Science and Practice of Pharmacy (23rd ed.). Elsevier. A variety of aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Generally, normal buffered saline (135-150 mM NaCl) is used as the pharmaceutically acceptable carrier, but other suitable carriers will suffice. These compositions can be sterilized by conventional liposomal sterilization techniques, such as filtration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions can be sterilized using the techniques referred to above or, alternatively, they can be produced under sterile conditions. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[0241] When preparing pharmaceutical preparations of the lipid particles described herein, it is preferable to use quantities of the particles which have been purified to reduce or eliminate empty particles or particles with nucleic acid associated with the external surface.

[0242] The methods described herein may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees as well as other nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and swine.

[0243] The amount of particles administered will depend upon the ratio of nucleic acid (e.g., siRNA) to lipid, the particular nucleic acid used, the disease or disorder being treated, the age, weight, and condition of the subject, and the judgment of the clinician, but will generally be between about 0.01 mg / kg and about 50 mg / kg of body weight, between about 0.1 mg / kg and about 5 mg / kg of body weight, or about 108-1010particles per administration (e.g., injection).91 80125197V.1D. Preventing or Treating a Disease or Disorder

[0244] Some methods provided by the disclosure are useful for preventing or treating a disease or a disorder of a subject. These methods generally include administering one or more lipid nanoparticles or one or more pharmaceutical compositions to the subject, where the lipid nanoparticles include those described in Section IV and the pharmaceutical compositions include those described in Section V. In some embodiments, the treating of the disease in the subject includes decreasing or eliminating one or more signs or symptoms of the disease.

[0245] In some embodiments, the prevented or treated disease is a liver disease or disorder. The prevented or treated liver disease or disorder can be, for example, a disease or disorder associated with hepatic stellate cells. Non-limiting examples of liver diseases or disorders that are associated with hepatic stellate cells and that can be prevented or treated with the provided lipid nanoparticles and / or pharmaceutical compositions include liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), nonalcoholic fatty liver disease (NASH), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), alcoholic liver disease (ALD), immune-induced hepatitis (e.g., Concanavalin A (ConA)-induced hepatitis and lipopolysaccharide / D-galactosamine (LPS / GalN)-induced hepatitis), ischemia and reperfusion (I / R) liver injury, and schistosome and / or chronic viral hepatitis.

[0246] In some embodiments, the disease or disorder is a cancer and treatment with the provided lipid nanoparticles and / or pharmaceutical compositions results in a stable disease, partial remission, or complete remission in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and / or pharmaceutical compositions that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject). In some embodiments, treatment with the provided lipid nanoparticles and / or pharmaceutical compositions results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and / or pharmaceutical compositions that reduces metastases of the cancer in the subject). In some embodiments, treatment with the provided lipid nanoparticles and / or pharmaceutical compositions results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and / or pharmaceutical compositions that reduces the volume, size, or growth of a tumor in the subject). In some embodiments, treatment with the provided lipid92 80125197V.1nanoparticles and / or pharmaceutical compositions results in an increased responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and / or pharmaceutical compositions that increases responsiveness of the cancer to a subsequently administered anti-cancer agent).

[0247] In some embodiments, the provided method further includes obtaining a test sample from the subject. The test sample can 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. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.

[0248] In examples where the provided lipid nanoparticle or pharmaceutical composition includes a nucleic acid encoding a protein of interest or a fragment thereof, the measured biomarker can be an indicator of an expression level of the protein or fragment by a target cell or target organism, e.g., the cell or organism to which the lipid nanoparticle or pharmaceutical composition is administered. For example, the biomarker can be the protein or fragment itself, such that a measured level of the protein or fragment indicates the level of its expression. When the protein of interest is an antigen, the measured biomarker can include or consist of one or more components of the immune system of the target cell or target organism. For example, the biomarker can include or consist of one or more species of T cells, e.g., CD8+T cells. In these ways, the determination of a level of a biomarker in a test sample can provide information related to the efficiency and / or effectiveness of the administration of the lipid nanoparticle in preventing or treating a disease or disorder.

[0249] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample. The reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials. In this way, the reference sample can provide information about baseline levels of the biomarkers in the sample before the treatment, and the test sample can provide information about levels of the biomarkers after the treatment.93 80125197V.1

[0250] Alternatively, the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment. The reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method. In this way, the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.

[0251] The reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0252] Depending on the biomarker, an increase or decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease. In some embodiments, an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., an infectious disease or cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a control. In other embodiments, a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a control.94 80125197V.1

[0253] The biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads. In some instances, nucleic acid sequencing is employed.

[0254] In certain embodiments, the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR. This detectable signal can be compared to the signal from a reference sample or to a threshold value.

[0255] In some embodiments, the results of the biomarker level determinations are recorded in a tangible medium. For example, the results of diagnostic assays, e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., an infectious disease or cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.

[0256] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and / or the results of treatment. VIII. EXEMPLARY EMBODIMENTS

[0257] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0258] Embodiment 1: A compound having the formula:or the formula of a salt thereof, wherein: R1, R2, R3, and R4are each independently C1-40alkyl, C2-40 alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40-membered heteroalkynyl, each of which is optionally substituted with one or more R7groups; R5is C1-8alkyl, C2-8alkenyl, or C2-8alkynyl, each of which is optionally substituted with one or more R7groups, or R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R795 80125197V.1groups; R6is hydrogen or is C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups; each R7is independently C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, C1-6 haloalkyl, hydroxy, halogen, oxo, -NH2, nitro, or cyano, or is combined with another R7and the carbon to which they are attached to form cyclopropyl or cyclobutyl; X1is C1-12 alkylene, C3-12cycloalkylene, or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups, or X1or is combined with R5and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups; X2is C1-12alkylene, 2- to 12-membered heteroalkylene, or 3- to 15- membered heterocyclylene; and m and n are each independently an integer from 1 to 12.

[0259] Embodiment 2: An embodiment of embodiment 1, wherein R1, R2, R3, and R4are each independently C2-40 alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, 2- to 40- membered heteroalkenyl, 2- to 40-membered heteroalkynyl, or linear C1-40alkyl, each of which is optionally substituted with one or more R7groups.

[0260] Embodiment 3: An embodiment of embodiment 1 or 2, wherein at least one of R1, R2, R3, and R4is C2-40alkenyl optionally substituted with one or more R7groups.

[0261] Embodiment 4: An embodiment of embodiment 3, wherein at least one of R1, R2, R3, and R4is (Z)-C2-40 alkenyl optionally substituted with one or more R7groups.

[0262] Embodiment 5: An embodiment of embodiment 4, wherein at least one of R1, R2, R3, and R4is (Z)-dec-4-enyl, (Z)-undec-5-enyl, (Z)-non-4-enyl, or (Z)-undec-8-enyl.

[0263] Embodiment 6: An embodiment of any one of embodiments 1-5, wherein at least one of R1, R2, R3, and R4is C2-40 alkyl optionally substituted with one or more R7groups.

[0264] Embodiment 7: An embodiment of embodiment 6, wherein at least one of R1, R2, R3, and R4is decyl.

[0265] Embodiment 8: An embodiment of any one of embodiments 1-7, wherein R1, R2, R3, and R4are identical.

[0266] Embodiment 9: An embodiment of any one of embodiments 1-8, wherein R5is C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, each of which is optionally substituted with one or more R7groups, or R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 5- membered monocylclic heterocyclylene group, a 5- to 15-membered spiro or bridged96 80125197V.1polycyclic heterocyclylene group, or a 5- to 15-membered fused heterocyclylene diastereomer group, each of which is optionally substituted with one or more R7groups.

[0267] Embodiment 10: An embodiment of any one of embodiments 1-9, wherein R5is C1-8alkyl, optionally substiuted with one or more R7groups.

[0268] Embodiment 11: An embodiment of embodiment 10, wherein R5is methyl or hydroxyethyl.

[0269] Embodiment 12: An embodiment of any one of embodiments 1-9, wherein R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 5-membered monocylclic heterocyclylene group, a 5- to 15-membered spiro or bridged polycyclic heterocyclylene group, or a 5- to 15-membered fused heterocyclylene diastereomer group, each of which is optionally substituted with one or more R7groups.

[0270] Embodiment 13: An embodiment of embodiment 12, wherein R5is combined with X1and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl, azetidindiyl, or hexahydro-2H-pyrrolo[3,4-b]pyridindiyl, each of which is optionally substituted with one or more R7groups.

[0271] Embodiment 14: An embodiment of any one of embodiments 1-13, wherein R6is C1-8 alkyl, optionally substituted with one or more R7groups.

[0272] Embodiment 15: An embodiment of embodiment 14, wherein R6is methyl or hydroxyethyl.

[0273] Embodiment 16: An embodiment of any one of embodiments 1-15, wherein R5and R6are identical.

[0274] Embodiment 17: An embodiment of any one of embodiments 1-13, wherein R6is hydrogen.

[0275] Embodiment 18: An embodiment of any one of embodiments 1-17, wherein X1is C2- 12 alkylene optionally substituted with one or more R7groups.

[0276] Embodiment 19: An embodiment of embodiment 18, wherein X1is ethylene.

[0277] Embodiment 20: An embodiment of any one of embodiments 1-17, wherein X1is C3- 12 cycloalkylene optionally substituted with one or more R7groups.

[0278] Embodiment 21: An embodiment of embodiment 20, wherein X1is cyclohexylene.97 80125197V.1

[0279] Embodiment 22: An embodiment of any one of embodiments 1-17, wherein X1is 2- to 12-membered heteroalkylene optionally substituted with one or more R7groups.

[0280] Embodiment 23: An embodiment of embodiment 22, wherein X1is piperidin-1,4- diyl.

[0281] Embodiment 24: An embodiment of any one of embodiments 1-23, wherein X2is 2- to 12-membered heteroalkylene.

[0282] Embodiment 25: An embodiment of embodiment 24, wherein X2is or.

[0283] Embodiment 26: An embodiment of any one of embodiments 1-25, wherein m and n are each independently 3, 4, 5, 6, or 7.

[0284] Embodiment 27: An embodiment of any one of embodiments 1-26, wherein m and n are not identical.

[0285] Embodiment 28: An embodiment of any one of embodiments 1-26, wherein m and n are identical.

[0286] Embodiment 29: An embodiment of any one of embodiments 1-28, wherein the salt comprises a pharmaceutically acceptable salt.

[0287] Embodiment 30: An embodiment of any one of embodiments 1-29, wherein the salt comprises a bromide salt, a chloride salt, an iodide salt, or a combination thereof.

[0288] Embodiment 31: An embodiment of embodiment 1, wherein the compound has the formula:or the formula of a salt thereof.

[0289] Embodiment 32: An embodiment of embodiment 31, wherein X1is C1-12 alkylene optionally substituted with one or more R7groups.98 80125197V.1

[0290] Embodiment 33: An embodiment of embodiment 32, wherein the compound has the formula:(I)(b), or the formula of a salt thereof.

[0291] Embodiment 34: An embodiment of embodiment 33 wherein the compound has the99 80125197V.1,100 80125197V.1, .

[0292] Embodiment 35: An embodiment of 31, wherein X1is C3-12 cycloalkylene optionally substituted with one or more R7groups.

[0293] Embodiment 36: An embodiment of embodiment 35, wherein the compound has the formula:(I)(c), or the formula of a salt thereof.

[0294] Embodiment 37: An embodiment of embodiment 36, wherein the compound has the structure:101 80125197V.1102 80125197V.1,

[0295] Embodiment 38: An embodiment of embodiment 31, wherein X1is 2- to 12- membered heteroalkylene optionally substituted with one or more R7groups.

[0296] Embodiment 39: An embodiment of embodiment 38, wherein the compound has the formula:(I)(d), or the formula of a salt thereof.

[0297] Embodiment 40: An embodiment of embodiment 39, wherein the compound has the structure:103 80125197V.1.

[0298] Embodiment 41: An embodiment of embodiment 1, wherein the compound has the structure:104 80125197V.1105 80125197V.1.

[0299] Embodiment 42: A lipid nanoparticle comprising: an ionizable lipid or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid is the compound of any one of embodiments 1-41; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; and a conjugated lipid.

[0300] Embodiment 43: An embodiment of embodiment 42, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.

[0301] Embodiment 44: An embodiment of embodiment 42 or 43, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.

[0302] Embodiment 45: An embodiment of any one of embodiments 42-44, wherein the cholesterol or the derivative thereof comprises between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.106 80125197V.1

[0303] Embodiment 46: An embodiment of any one of embodiments 42-45, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

[0304] Embodiment 47: An embodiment of any one of embodiments 42-46, wherein the conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle.

[0305] Embodiment 48: An embodiment of any one of embodiments 42-47, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 45 mol% and 55 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 36.5 mol% and 40.5 mol% of the total lipid of the lipid nanoparticle; and the conjugated lipid comprising between 0.85 mol% and 2.6 mol% of the total lipid of the lipid nanoparticle.

[0306] Embodiment 49: An embodiment of any one of embodiments 42-48, wherein the lipid nanoparticle further comprises a permanently charged lipid or a pharmaceutically acceptable salt thereof.

[0307] Embodiment 50: An embodiment of embodiment 51, wherein the permanently charged lipid or the pharmaceutically acceptable salt thereof comprises between 0.5 mol% and 30 mol% of the total lipid of the lipid nanoparticle.

[0308] Embodiment 51: An embodiment of any one of embodiments 42-50, wherein the lipid nanoparticle further comprises a nucleic acid.

[0309] Embodiment 52: An embodiment of embodiment 51, wherein the nucleic acid comprises RNA.

[0310] Embodiment 53: An embodiment of embodiment 52, wherein the RNA comprises siRNA, miRNA, shRNA, asRNA, DsiRNA, piRNA, or a combination thereof.

[0311] Embodiment 54: An embodiment of embodiment 53, wherein the RNA comprises siRNA.

[0312] Embodiment 55: An embodiment of embodiment 51, wherein the nucleic acid comprises an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.107 80125197V.1

[0313] Embodiment 56: An embodiment of any one of embodiments 51-55, wherein the nucleic acid is configured to reduce expression of a gene in a cell.

[0314] Embodiment 57: An embodiment of embodiment 56, wherein the cell is a hepatic stellate cell.

[0315] Embodiment 58: An embodiment of embodiment 56 or 57, wherein the gene encodes collagen type I alpha 1 chain (COL1A1), tissue inhibitor of metalloproteinases 1 (TIMP1), matrix metalloproteinase 2 (MMP2 ), alpha smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), heat shock protein 47 (HSP47), lysyl oxidase (LOX), epidermal growth factor receptor (EGFR), rho kinase (ROCK), integrin alpha 8 beta 1 or another integrin, yes- associated protein (YAP), a toll-like receptor pathway protein, transforming growth factor beta (TGFB), platelet-derived growth factor (PDGF), or connective tissue growth factor (CTGF).

[0316] Embodiment 59: A pharmaceutical composition comprising: the lipid nanoparticle of any one of embodiments 42-58, or the compound of any one of embodiments 1-41; and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.

[0317] Embodiment 60: A method for introducing the nucleic acid of any one of embodiments 51-58 into a cell, the method comprising contacting the cell with the lipid nanoparticle of any one of embodiments 51-58.

[0318] Embodiment 61: An embodiment of embodiment 60, wherein the nucleic acid is configured to reduce expression of a gene in the cell.

[0319] Embodiment 62: An embodiment of embodiment 61, wherein the method further comprises reducing expression of the gene by the cell.

[0320] Embodiment 63: An embodiment of any one of embodiments 60-62, wherein the cell is a hepatic stellate cell.

[0321] Embodiment 64: An embodiment of any one of embodiments 61-63, wherein the gene encodes COL1A1, TIMP1, MMP2, α-SMA, CTGF, HSP47, LOX, EGFR, ROCK, integrin alpha 8 beta 1 or another integrin, YAP, a toll-like receptor pathway protein, TGFB, PDGF, or CTGF.

[0322] Embodiment 65: The lipid nanoparticle of any one of embodiments 56-58 for use in reducing expression of the gene in the hepatic stellate cell.108 80125197V.1

[0323] Embodiment 66: The use of the lipid nanoparticle of any one of embodiments 56-58 to prepare a medicament for reducing expression of the gene in the hepatic stellate cell.

[0324] Embodiment 67: A method for the in vivo delivery of the nucleic acid of any one of embodiments 51-58, the method comprising administering to a subject the lipid nanoparticle of any one of embodiments 51-58.

[0325] Embodiment 68: A lipid nanoparticle of any one of embodiments 51-58 for use in the in vivo delivery of the nucleic acid of any one of embodiments 51-58 to a subject.

[0326] Embodiment 69: The use of the lipid nanoparticle of any one of embodiments 51-58 to prepare a medicament for the in vivo delivery of the nucleic acid of any one of embodiments 51-58 to a subject.

[0327] Embodiment 70: A method for preventing or treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the lipid nanoparticle of any one of any one of embodiments 42-58, the compound of any one of embodiments 1-41, or the pharmaceutical composition of embodiment 59.

[0328] Embodiment 71: An embodiment of embodiment 70, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

[0329] Embodiment 72: An embodiment of embodiment 71, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.

[0330] Embodiment 73: An embodiment of embodiment 71 or 72, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), nonalcoholic fatty liver disease (NASH), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), alcoholic liver disease (ALD), immune-induced hepatitis, ischemia and reperfusion (I / R) liver injury, schistosome viral hepatitis, or chronic viral hepatitis.

[0331] Embodiment 74: A lipid nanoparticle of any one of embodiments 42-58, compound of any one of embodiments 1-41, or pharmaceutical composition of embodiment 59, for use in preventing or treating a disease or disorder in a subject.

[0332] Embodiment 75: An embodiment of embodiment 74, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

[0333] Embodiment 76: An embodiment of embodiment 75, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.109 80125197V.1

[0334] Embodiment 77: An embodiment of embodiment 75 or 76, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I / R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.

[0335] Embodiment 78: The use of a lipid nanoparticle of any one of embodiments 42-58, compound of any one of embodiments 1-41, or pharmaceutical composition of embodiment 59, to prepare a medicament for preventing or treating a disease or disorder in a subject.

[0336] Embodiment 79: An embodiment of embodiment 78, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

[0337] Embodiment 80: An embodiment of embodiment 79, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.

[0338] Embodiment 81: An embodiment of embodiment 79 or 80, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I / R liver injury, schistosome viral hepatitis, or chronic viral hepatitis. EXAMPLES

[0339] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Example 1. Preparation of ionizable lipids.

[0340] The provided compounds can be synthesized by a variety of methods known to one of skill in the art (see, e.g., Comprehensive Organic Transformations, R.C. Larock, 1989) or by an appropriate combination of generally well known synthetic methods. Techniques useful in synthesizing the disclosed compounds are both readily apparent and accessible to those of skill in the relevant art. The examples below are offered to illustrate certain of the diverse methods available for use in assembling the provided compounds. However, the examples are not intended to define the scope of reactions or reaction sequences that are useful in preparing the compounds. Exemplary structures below are named according to standard IUPAC nomenclature using the CambridgeSoft ChemDraw naming package.110 80125197V.1

[0341] In these Examples, certain ionizable lipids are referred to as “Lipid X,” with X being a number, and certain (other) compounds (synthetic intermediates and reagents) are referred to as “Compound Y” or “cmpd. Y,” with Y being a number. Table 1 below and the following Examples include ionizable lipids in accordance with provided embodiments, e.g., ionizable lipids having the formula:where R1, R2, R3, R4, R5, R6, X1, X2, m, and n of formula (I) are as defined in Section III. Table 1 Lipid Number Chemical Structure111 80125197V.1O ONON N O N112 80125197V.1012 3113 80125197V.14 5 O N O O6NNN O 7114 80125197V.18 90 1 2115 80125197V.13 4 5 6 7 A. Scheme 1: Synthesis of N1,N3-bis(6-((di((Z)-undec-5-en-1-yl)amino)oxy)-6- oxohexyl)-N1,N3-dimethylpropane-1,3-diamine (Lipid 1). 1. Synthesis of (Z)-dec-4-en-1-yl methanesulfonate (2)116 80125197V.1

[0342] A solution of (4Z)-4-decenol (1) (60 g, 384 mmol) and triethylamine (TEA) (70 mL, 500 mmol) in dichloromethane (DCM) (600 mL) was cooled to 0 °C and treated dropwise with methanesulfonyl chloride (48.4 g, 422 mmol). The mixture was reacted at room temperature (RT) for 2 h, then quenched with saturated NaHCO3 and extracted with DCM. The organic layer was washed with brine, dried (MgSO4), filtered, and concentrated to give crude (Z)-dec- 4-en-1-yl methanesulfonate (2) (89.9 g, quantitative), which was used without further purification. 2. Synthesis of (Z)-1-bromodec-4-ene (3)

[0343] (z)-Dec-4-en-1-yl methanesulfonate (2) (83 g, 354 mmol) was taken up in 2- methyltetrahydrofuran (400 mL) and treated with tetrabutylammonium bromide (143 g, 443 mmol) at 80 °C. After stirring for 2 h, the reaction was cooled to RT, diluted with hexane, and washed with brine. The aqueous layer was back extracted with hexane and the combined organics were dried (MgSO4), filtered, and concentrated. The crude material was purified by flushing through a silica plug with 100% hexane to yield (Z)-1-bromodec-4-ene (3) (77.6 g, quantitative). 3. Synthesis of (Z)-undec-5-enal (4)

[0344] Magnesium (9.5 g, 390 mmol) was submersed in anhydrous THF (200 mL) and vigorously stirred under N2 and (z)-1-bromodec-4-ene (3) (77.6 g, 354 mmol) in anhydrous tetrahydrofuran (THF) was added dropwise. The reaction was stirred for 1 h at RT then cooled to 0 °C. Dimethylformamide (DMF) (77.7 g, 1063 mmol) was added dropwise under N2. The reaction was warmed to RT over 15 h and then quenched with water. NaHSO3 saturated solution was added and the aqueous layer washed with Et2O, and then basified with 1 M NaOH to pH 14. The aqueous layer was extracted with Et2O, the organics were dried (MgSO4) and filtered. and the filtrate was concentrated under vacuum to afford (Z)-undec-5-enal (4) (18.9 g, 31.7%), which was used without further purification.117 80125197V.14. Synthesis of N,N-di((Z)-undec-5-en-1-yl)hydroxylamine (5)

[0345] (z)-Undec-5-enal (4) (18.9 g, 112 mmol) was dissolved in DCM (350 mL). Hydroxylamine hydrochloride (2.7 g, 39.3 mmol) and TEA (15.8 mL, 112 mmol) were added, the mixture was stirred at RT for 30 min, NaBH3CN (23.7 g, 112 mmol) was added to the reaction mixture, and stirring was continued for 15 h. The reaction was quenched with 10 M NaOH, MgSO4was added, and the slurry was filtered and washed with hexane prior to concentration of the organic phase. The crude material was purified by flash chromatography (0-100% EtOAc / Hexane) to afford N,N-di((Z)-undec-5-en-1-yl)hydroxylamine (5) (9.2 g, Yield 69.3%). 5. Synthesis of O-(6-bromohexanoyl)-N,N-di((Z)-undec-5-en-1- yl)hydroxylamine (7)

[0346] N,N-Di((Z)-undec-5-en-1-yl)hydroxylamine (5) (3 g, 8.9 mmol) was dissolved in DCM (30 mL). N,N′-Dicyclohexylcarbodiimide (DCC) (3.7 g, 17.8 mmol) and 4- dimethylaminopyridine (DMAP) (0.054 g, 0.4 mmol) were added to the reaction mixture. After 30 m, 6-bromohexanoic acid (6) (2.6 g, 13.3 mmol) was added to the reaction mixture and the reaction was stirred for 15 h. Solvent was removed under vacuum and the residue was taken up in hexane and filtered. The filtrate was concentrated under vacuum and purified by flash chromatography (0-50% Et2O / Hexane) to afford O-(6-bromohexanoyl)-N,N-di((Z)-undec-5- en-1-yl)hydroxylamine (7) (4.2 g, 93.4%).118 80125197V.16. Synthesis of N1,N3-bis(6-((di((Z)-undec-5-en-1-yl)amino)oxy)-6- oxohexyl)-N1,N3-dimethylpropane-1,3-diamine (Lipid 1)

[0347] O-(6-bromohexanoyl)-N,N-di((Z)-undec-5-en-1-yl)hydroxylamine (7) (2 g, 3.9 mmol) was dissolved in DMF (1.5 mL), followed by addition of TEA (0.8 mL, 5.8 mmol). Methyl[3-(methylamino)propyl]amine (8) (0.2 g, 1.8 mmol) was added and the reaction was stirred at RT for 3 d. The solution was diluted with EtOAc and washed with saturated NaHCO3 and brine. The organics were dried (MgSO4), filtered, and concentrated under vacuum. Crude was purified by flash chromatography 0-15% MeOH / DCM to afford N1,N3-bis(6-((di((Z)- undec-5-en-1-yl)amino)oxy)-6-oxohexyl)-N1,N3-dimethylpropane-1,3-diamine (Lipid 1) (380 mg, 21.2%).1H NMR (400 MHz, CDCl3) δ 5.43 – 5.28 (m, 8H), 2.83 (t, J = 7.6 Hz, 8H), 2.52 – 2.27 (m, 12H), 2.27 – 2.16 (m, 6H), 2.03 (dq, J = 13.7, 6.7 Hz, 16H), 1.68 (p, J = 7.6 Hz, 6H), 1.61 – 1.46 (m, 12H), 1.45 – 1.22 (m, 36H), 0.91 (t, J = 6.9 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C61H116N4O4+968.9; Found 970.6. B. Synthesis of N1,N3-bis(6-((di((Z)-dec-4-en-1-yl)amino)oxy)-6-oxohexyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 2).

[0348] Lipid 2 was synthesized in an analogous fashion to Lipid 1.1H NMR (400 MHz, CDCl3) δ 5.37 (tdd, J = 18.0, 11.3, 6.9 Hz, 8H), 2.88 – 2.79 (m, 8H), 2.30 (t, J = 7.6 Hz, 12H), 2.23 (s, 5H), 2.06 (dq, J = 27.6, 7.2 Hz, 16H), 1.74 – 1.53 (m, 18H), 1.32 (ddq, J = 11.2, 8.7, 5.6 Hz, 28H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C57H108N4O4+921.8; Found 923.0.119 80125197V.1C. Synthesis of (1r,4r)-N1,N4-bis(6-((di((Z)-dec-4-en-1-yl)amino)oxy)-6- oxohexyl)-N1,N4-dimethylcyclohexane-1,4-diamine (Lipid 3).

[0349] Lipid 3 was synthesized in an analogous fashion to Lipid 1 usingdimethylcyclohexane-1,4-diamine.1H NMR (400 MHz, CDCl3) δ 5.37 (tdd, J = 18.1, 11.1, 7.0 Hz, 8H), 2.88 – 2.79 (m, 8H), 2.41 (t, J = 7.5 Hz, 4H), 2.34 – 2.22 (m, 11H), 2.06 (dq, J = 27.4, 7.2 Hz, 16H), 1.87 (d, J = 7.6 Hz, 4H), 1.68 (p, J = 7.6 Hz, 4H), 1.64 – 1.54 (m, 12H), 1.47 (q, J = 7.3 Hz, 4H), 1.32 (ddd, J = 15.3, 11.9, 7.1 Hz, 30H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C60H112N4O4+952.9; Found 954.6. D. Synthesis of N1,N3-bis(6-((didecylamino)oxy)-6-oxohexyl)-N1,N3- dimethylpropane-1,3-diamine (Lipid 4).

[0350] Lipid 4 was synthesized in an analogous fashion to Lipid 1 using 1-decanol.1H NMR (400 MHz, CDCl3) δ 2.86 – 2.78 (m, 8H), 2.31 (t, J = 7.6 Hz, 7H), 2.24 (s, 4H), 1.75 – 1.63 (m, 12H), 1.58 – 1.46 (m, 12H), 1.42 – 1.21 (m, 60H), 0.90 (t, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C57H116N4O4+920.9; Found 922.4.120 80125197V.1E. Synthesis of N1-(6-((di((Z)-dec-4-en-1-yl)amino)oxy)-6-oxohexyl)-N2-(2-((6- ((di((Z)-dec-4-en-1-yl)amino)oxy)-6-oxohexyl)(methyl)amino)ethyl)-N1,N2- dimethylethane-1,2-diamine (Lipid 5).

[0351] Lipid 5 was synthesized in an analogous fashion to Lipid 1 using N,N',N''- trimethyldiethylenetriamine.1H NMR (400 MHz, CDCl3) δ 5.37 (tdd, J = 18.1, 10.5, 6.8 Hz, 9H), 2.88 – 2.77 (m, 9H), 2.57 – 2.50 (m, 3H), 2.31 (t, J = 7.5 Hz, 9H), 2.06 (dq, J = 28.0, 7.2 Hz, 17H), 1.92 (s, 8H), 1.68 (p, J = 7.6 Hz, 5H), 1.58 (p, J = 7.5 Hz, 10H), 1.33 (dtd, J = 15.3, 9.2, 4.5 Hz, 29H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+)m / z: [M + H]+Calculated for C59H113N5O4+955.9; Found 956.8. F. Synthesis of N1,N3-bis(8-((di((Z)-dec-4-en-1-yl)amino)oxy)-8-oxooctyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 6).

[0352] Lipid 6 was synthesized in an analogous fashion to Lipid 1 using 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.27 (tdd, J = 18.0, 11.1, 6.9 Hz, 8H), 2.83 – 2.67 (m, 8H), 2.30 – 2.17 (m, 10H), 1.97 (dq, J = 27.4, 7.1 Hz, 14H), 1.64 – 1.45 (m, 18H), 1.38 (t, J = 7.5 Hz, 4H), 1.23 (ddt, J = 17.2, 8.7, 4.2 Hz, 36H), 0.81 (t, J = 6.8 Hz, 12H). LRMS (ESI+)m / z: [M + H]+Calculated for C61H116N4O4+968.9; Found 970.4. G. Synthesis of (Z)-15-((Z)-dec-4-en-1-yl)-3-(6-((di((Z)-dec-4-en-1- yl)amino)oxy)-6-oxohexyl)-7-(2-hydroxyethyl)-13-oxo-14-oxa-3,7,15- triazapentacos-19-en-1-ol (Lipid 7).121 80125197V.1

[0353] Lipid 7 was synthesized in an analogous fashion to Lipid 1 using N,N'-bis(2- hydroxyethyl)-1,3-propanediamine.1H NMR (400 MHz, CDCl3) δ 5.43 – 5.31 (m, 9H), 3.63 (s, 4H), 2.88 – 2.79 (m, 8H), 2.61 (M, 12H), 2.31 (t, J = 7.5 Hz, 4H), 2.06 (dq, J = 28.3, 7.2 Hz, 16H), 1.63 (dp, J = 41.2, 7.5 Hz, 18H), 1.32 (ddt, J = 18.9, 10.2, 5.2 Hz, 28H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C59H112N4O6+972.9; Found 973.8. H. Synthesis of O,O'-(6,6'-(3,9-diazaspiro[5.5]undecane-3,9- diyl)bis(hexanoyl))bis(N,N-di((Z)-dec-4-en-1-yl)hydroxylamine) (Lipid 8).

[0354] Lipid 8 was synthesized in an analogous fashion to Lipid 1 using 3,9- diazaspiro[5.5]undecane.1H NMR (400 MHz, CDCl3) δ 5.41 – 5.31 (m, 8H), 2.90 – 2.78 (m, 8H), 2.37 (s, 6H), 2.31 (q, J = 7.6 Hz, 8H), 2.06 (dq, J = 27.1, 7.2 Hz, 16H), 1.66 (ddd, J = 24.0, 15.8, 7.4 Hz, 16H), 1.54 (d, J = 16.5 Hz, 13H), 1.32 (ddd, J = 15.4, 12.1, 6.9 Hz, 27H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C61H112N4O4+964.8; Found 966.0.122 80125197V.1I. Synthesis of O,O'-(6,6'-([3,3'-biazetidine]-1,1'-diyl)bis(hexanoyl))bis(N,N- di((Z)-dec-4-en-1-yl)hydroxylamine) (Lipid 9).

[0355] Lipid 9 was synthesized in an analogous fashion to Lipid 1 using biazetidin-3-yl.1H NMR (400 MHz, CDCl3) δ 5.41 – 5.31 (m, 8H), 3.34 (d, J = 6.7 Hz, 4H), 2.89 – 2.79 (m, 8H), 2.75 (d, J = 7.2 Hz, 4H), 2.67 (s, 2H), 2.38 (s, 4H), 2.29 (t, J = 7.6 Hz, 4H), 2.06 (dq, J = 26.8, 7.2 Hz, 16H), 1.73 – 1.53 (m, 20H), 1.32 (tdd, J = 11.2, 9.7, 5.1 Hz, 30H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C58H106N4O4+922.8; Found 923.8. J. Synthesis of N1-(8-((di((Z)-dec-4-en-1-yl)amino)oxy)-8-oxooctyl)-N2-(2-((8- ((di((Z)-dec-4-en-1-yl)amino)oxy)-8-oxooctyl)(methyl)amino)ethyl)-N1,N2- dimethylethane-1,2-diamine (Lipid 10).

[0356] Lipid 10 was synthesized in an analogous fashion to Lipid 1 using N,N',N''- trimethyldiethylenetriamine and 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.41 – 5.31 (m, 8H), 2.88 – 2.79 (m, 8H), 2.51 (s, 7H), 2.35 (s, 3H), 2.33 – 2.23 (m, 13H), 2.06 (dq, J = 27.5, 7.2 Hz, 16H), 1.60 (tt, J = 15.0, 7.3 Hz, 14H), 1.48 (s, 4H), 1.32 (ddq, J = 11.2, 8.3, 5.3 Hz, 36H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C63H121N5O4+1011.9; Found 1013.0.123 80125197V.1K. Synthesis of N1,N3-bis(4-((di((Z)-dec-4-en-1-yl)amino)oxy)-4-oxobutyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 11).

[0357] Lipid 11 was synthesized in an analogous fashion to Lipid 1 using 4-bromobutyric acid.1H NMR (400 MHz, CDCl3) δ 5.36 (tdd, J = 17.9, 10.6, 6.8 Hz, 8H), 2.88 – 2.79 (m, 8H), 2.35 (dt, J = 15.8, 7.5 Hz, 12H), 2.22 (s, 6H), 2.06 (dq, J = 27.6, 7.2 Hz, 16H), 1.82 (p, J = 7.5 Hz, 4H), 1.59 (p, J = 7.3 Hz, 12H), 1.41 – 1.24 (m, 24H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C53H100N4O4+856.8; Found 858.2. L. Synthesis of N1-(4-((di((Z)-dec-4-en-1-yl)amino)oxy)-4-oxobutyl)-N2-(2-((4- ((di((Z)-dec-4-en-1-yl)amino)oxy)-4-oxobutyl)(methyl)amino)ethyl)-N1,N2- dimethylethane-1,2-diamine (Lipid 12).

[0358] Lipid 12 was synthesized in an analogous fashion to Lipid 1 using N,N',N''- trimethyldiethylenetriamine and 4-bromobutyric acid.1H NMR (400 MHz, CDCl3) δ 5.42 – 5.30 (m, 8H), 2.90 – 2.79 (m, 8H), 2.50 (s, 6H), 2.40 (t, J = 7.2 Hz, 4H), 2.33 (t, J = 7.5 Hz, 4H), 2.26 (d, J = 10.5 Hz, 8H), 2.06 (dq, J = 27.6, 7.1 Hz, 16H), 1.82 (p, J = 7.5 Hz, 4H), 1.65 – 1.50 (m, 9H), 1.41 – 1.22 (m, 24H), 0.91 (t, J = 6.8 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C55H105N5O4+899.8; Found 901.6. M. Synthesis of N1,N3-bis(4-((di((Z)-undec-5-en-1-yl)amino)oxy)-4-oxobutyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 13).

[0359] Lipid 13 was synthesized in an analogous fashion to Lipid 1 using 4-bromobutyric acid.1H NMR (400 MHz, CDCl3) δ 5.34 (dtt, J = 17.8, 11.3, 5.8 Hz, 8H), 2.81 (t, J = 7.5 Hz,124 80125197V.18H), 2.51 – 2.26 (m, 12H), 2.26 – 2.13 (m, 6H), 2.10 – 1.90 (m, 16H), 1.90 – 1.69 (m, 8H), 1.57 – 1.43 (m, 8H), 1.43 – 1.16 (m, 31H), 0.88 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C57H108N4O4+912.8; Found 914.0. N. Synthesis of N1,N3-bis(8-((di((Z)-undec-5-en-1-yl)amino)oxy)-8-oxooctyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 14).

[0360] Lipid 14 was synthesized in an analogous fashion to Lipid 1 using 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.36 (dtt, J = 17.8, 11.3, 5.8 Hz, 8H), 2.89 – 2.77 (m, 8H), 2.31 (tt, J = 14.4, 7.6 Hz, 12H), 2.22 (s, 6H), 2.10 – 1.93 (m, 16H), 1.65 (q, J = 7.4 Hz, 6H), 1.60 – 1.21 (m, 58H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C65H124N4O4+1025.0; Found 1026.4. O. Synthesis of (1r,4r)-N1,N4-bis(6-((di((Z)-undec-5-en-1-yl)amino)oxy)-6- oxohexyl)-N1,N4-dimethylcyclohexane-1,4-diamine (Lipid 15).

[0361] Lipid 15 was synthesized in an analogous fashion to Lipid 1 using trans-N1,N4- dimethylcyclohexane-1,4-diamine.1H NMR (400 MHz, CDCl3) δ 5.44 – 5.25 (m, 8H), 2.88 – 2.72 (m, 8H), 2.46 – 2.35 (m, 4H), 2.31 – 2.25 (m, 5H), 2.09 – 1.91 (m, 16H), 1.91 – 1.79 (m, 4H), 1.76 – 1.59 (m, 6H), 1.59 – 1.42 (m, 12H), 1.42 – 1.16 (m, 39H), 0.88 (t, J = 6.8 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C64H120N4O4+1008.9; Found 1010.0. P. Synthesis of N,1-bis(6-((di((Z)-dec-4-en-1-yl)amino)oxy)-6-oxohexyl)-N- methylpiperidin-4-amine (Lipid 16).125 80125197V.1

[0362] Lipid 16 was synthesized in an analogous fashion to Lipid 1 using 4- (methylamino)piperidine.1H NMR (400 MHz, CDCl3) δ 5.44 – 5.29 (m, 8H), 2.90 – 2.74 (m, 8H), 2.44 (s, 3H), 2.30 (t, J = 7.6 Hz, 7H), 2.06 (dq, J = 27.5, 7.2 Hz, 18H), 1.68 (p, J = 7.6 Hz, 7H), 1.57 (d, J = 7.4 Hz, 14H), 1.32 (ddt, J = 18.5, 8.5, 5.6 Hz, 26H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C58H108N4O4+924.8; Found 926.6. Q. Synthesis of O,O'-(6,6'-((4aR,7aR)-hexahydro-1H-pyrrolo[3,4-b]pyridine- 1,6(2H)-diyl)bis(hexanoyl))bis(N,N-di((Z)-dec-4-en-1-yl)hydroxylamine) (Lipid 17).

[0363] Lipid 17 was synthesized in an analogous fashion to Lipid 1 using [R,R]-2,8- diazabicyclo[4.3.0]nonane.1H NMR (400 MHz, CDCl3) δ 5.45 – 5.28 (m, 8H), 3.02 – 2.95 (m, 1H), 2.82 (q, J = 9.8 Hz, 9H), 2.69 – 2.61 (m, 2H), 2.56 – 2.38 (m, 4H), 2.30 (t, J = 7.6 Hz, 4H), 2.26 – 2.17 (m, 3H), 2.06 (dq, J = 27.5, 7.2 Hz, 15H), 1.75 – 1.45 (m, 23H), 1.41 – 1.19 (m, 26H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C59H108N4O4+936.8; Found 938.6. R. Synthesis of (1r,4r)-N1,N4-bis(8-((di((Z)-undec-5-en-1-yl)amino)oxy)-8- oxooctyl)-N1,N4-dimethylcyclohexane-1,4-diamine (Lipid 18).

[0364] Lipid 18 was synthesized in an analogous fashion to Lipid 1 using trans-N1,N4- dimethylcyclohexane-1,4-diamine and 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ126 80125197V.15.47 – 5.28 (m, 8H), 2.83 (t, J = 7.5 Hz, 8H), 2.29 (t, J = 7.5 Hz, 5H), 2.12 – 1.90 (m, 17H), 1.87 – 1.13 (m, 72H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C68H128N4O4+1065.0; Found 1066.6. S. Synthesis of N1-(8-((di((Z)-undec-5-en-1-yl)amino)oxy)-8-oxooctyl)-N2-(2- ((8-((di((Z)-undec-5-en-1-yl)amino)oxy)-8-oxooctyl)(methyl)amino)ethyl)- N1,N2-dimethylethane-1,2-diamine (Lipid 19).

[0365] Lipid 19 was synthesized in an analogous fashion to Lipid 1 using N,N',N''- trimethyldiethylenetriamine and 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.42 – 5.26 (m, 8H), 2.87 – 2.73 (m, 8H), 2.55 – 2.41 (m, 8H), 2.37 – 2.29 (m, 4H), 2.29 – 2.24 (m, 7H), 2.22 (s, 6H), 2.12 – 1.89 (m, 16H), 1.57 – 1.17 (m, 58H), 0.88 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C67H129N5O4+1068.0; Found 1069.6. T. Synthesis of N1,N3-bis(5-((di((Z)-dec-4-en-1-yl)amino)oxy)-5-oxopentyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 20).

[0366] Lipid 20 was synthesized in an analogous fashion to Lipid 1 using 5-bromopentanoic acid.1H NMR (400 MHz, CDCl3) δ 5.37 (tdd, J = 18.1, 11.1, 7.1 Hz, 8H), 2.88 – 2.79 (m, 7H), 2.47 – 2.29 (m, 11H), 2.25 (s, 5H), 2.06 (dq, J = 27.7, 7.2 Hz, 16H), 1.94 – 1.75 (m, 4H), 1.63 (dp, J = 36.6, 7.4 Hz, 18H), 1.32 (ddt, J = 18.7, 10.3, 5.2 Hz, 24H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C55H104N4O4+884.8; Found 886.4. U. Synthesis of N1,N3-bis(7-((di((Z)-dec-4-en-1-yl)amino)oxy)-7-oxoheptyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 21).127 80125197V.1

[0367] Lipid 21 was synthesized in an analogous fashion to Lipid 1 using 7-bromoheptanoic acid.1H NMR (400 MHz, CDCl3) δ 5.37 (tdd, J = 18.0, 11.0, 7.0 Hz, 8H), 2.89 – 2.78 (m, 8H), 2.46 – 2.26 (m, 12H), 2.25 (s, 4H), 2.06 (dq, J = 27.7, 7.2 Hz, 16H), 1.78 – 1.43 (m, 20H), 1.41 – 1.25 (m, 32H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C59H112N4O4+940.9; Found 942.2. V. Synthesis of N1,N3-bis(6-((di((Z)-undec-8-en-1-yl)amino)oxy)-6-oxohexyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 22).

[0368] Lipid 22 was synthesized in an analogous fashion to Lipid 1 using (Z)-undec-8-en- 1-ol.1H NMR (400 MHz, CDCl3) δ 5.45 – 5.26 (m, 8H), 2.89 – 2.73 (m, 8H), 2.32 (ddd, J = 14.9, 8.7, 5.3 Hz, 12H), 2.22 (s, 6H), 2.04 (h, J = 7.2 Hz, 16H), 1.74 – 1.59 (m, 10H), 1.51 (q, J = 7.4 Hz, 13H), 1.33 (d, J = 12.7 Hz, 37H), 0.97 (t, J = 7.5 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C61H116N4O4+968.9; Found 970.0. W. Synthesis of (1r,4r)-N1,N4-bis(8-((di((Z)-dec-4-en-1-yl)amino)oxy)-8- oxooctyl)-N1,N4-dimethylcyclohexane-1,4-diamine (Lipid 23).

[0369] Lipid 23 was synthesized in an analogous fashion to Lipid 1 using trans-N1,N4- dimethylcyclohexane-1,4-diamine and 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.37 (ddt, J = 20.9, 11.1, 5.6 Hz, 8H), 2.88 – 2.79 (m, 8H), 2.41 (s, 4H), 2.28 (dd, J = 15.4, 7.8 Hz, 11H), 2.06 (dq, J = 27.4, 7.2 Hz, 16H), 1.88 (d, J = 7.4 Hz, 4H), 1.62 (dt, J = 28.9, 7.3 Hz, 20H), 1.45 (s, 3H), 1.41 – 1.24 (m, 39H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C64H120N4O4+1008.9; Found 1010.0.128 80125197V.1X. Synthesis of (1r,4r)-N1,N4-bis(7-((di((Z)-dec-4-en-1-yl)amino)oxy)-7- oxoheptyl)-N1,N4-dimethylcyclohexane-1,4-diamine (Lipid 24).

[0370] Lipid 24 was synthesized in an analogous fashion to Lipid 1 using trans-N1,N4- dimethylcyclohexane-1,4-diamine and 7-bromoheptanoic acid.1H NMR (400 MHz, CDCl3) δ 5.44 – 5.29 (m, 8H), 2.89 – 2.79 (m, 8H), 2.41 (t, J = 7.5 Hz, 4H), 2.34 – 2.23 (m, 10H), 2.06 (dq, J = 27.4, 7.2 Hz, 16H), 1.88 (d, J = 6.0 Hz, 4H), 1.73 – 1.54 (m, 19H), 1.46 (s, 5H), 1.32 (dddd, J = 19.3, 15.0, 11.4, 6.1 Hz, 34H), 0.91 (t, J = 6.7 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C62H116N4O4+980.9; Found 982.6. Y. Synthesis of N1,N3-bis(6-((di((Z)-non-4-en-1-yl)amino)oxy)-6-oxohexyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 25).

[0371] Lipid 25 was synthesized in an analogous fashion to Lipid 1 using (Z)-non-4-en-1- ol.1H NMR (400 MHz, CDCl3) δ 5.45 – 5.27 (m, 9H), 2.87 – 2.78 (m, 8H), 2.32 (dt, J = 17.3, 7.6 Hz, 12H), 2.23 (s, 6H), 2.14 – 1.96 (m, 17H), 1.60 (ddp, J = 38.0, 23.0, 7.5 Hz, 19H), 1.34 (qd, J = 6.4, 2.6 Hz, 21H), 0.91 (h, J = 3.1 Hz, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C53H100N4O4+856.8; Found 858.2. Z. Synthesis of N1,N3-bis(7-((di((Z)-non-4-en-1-yl)amino)oxy)-7-oxoheptyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 26).

[0372] Lipid 26 was synthesized in an analogous fashion to Lipid 1 using (Z)-non-4-en-1- ol and 7-bromoheptanoic acid.1H NMR (400 MHz, CDCl3) δ 5.36 (tdd, J = 18.0, 11.0, 7.0 Hz, 8H), 2.87 – 2.78 (m, 8H), 2.48 (dd, J = 19.0, 11.4 Hz, 7H), 2.30 (dd, J = 15.4, 7.9 Hz, 10H), 2.09 (q, J = 7.2 Hz, 8H), 2.06 – 1.98 (m, 8H), 1.82 (s, 2H), 1.62 (dp, J = 33.6, 7.4 Hz, 16H),129 80125197V.11.33 (tt, J = 7.4, 3.7 Hz, 25H), 0.96 – 0.87 (m, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C55H104N4O4+884.8; Found 886.4. AA. Synthesis of N1,N3-bis(8-((di((Z)-non-4-en-1-yl)amino)oxy)-8-oxooctyl)- N1,N3-dimethylpropane-1,3-diamine (Lipid 27).

[0373] Lipid 27 was synthesized in an analogous fashion to Lipid 1 using (Z)-non-4-en-1- ol and 8-bromooctanoic acid.1H NMR (400 MHz, CDCl3) δ 5.44 – 5.26 (m, 8H), 2.89 – 2.77 (m, 12H), 2.75 – 2.65 (m, 4H), 2.51 (s, 6H), 2.29 (t, J = 7.6 Hz, 4H), 2.15 – 1.95 (m, 18H), 1.61 (dp, J = 30.0, 7.2 Hz, 16H), 1.34 (dh, J = 11.1, 3.8 Hz, 29H), 0.96 – 0.85 (m, 12H). LRMS (ESI+) m / z: [M + H]+Calculated for C57H108N4O4+912.8; Found 914.2. Example 2. Preparation of lipid nanoparticles.

[0374] Lipid nanoparticles were prepared from lipid ethanolic stock solutions containing four components: a PEG-conjugated lipid, an ionizable lipid, cholesterol, and a phospholipid (e.g., DSPC) in the molar ratios of 1.5:50:38.5:10. The combined lipid stocks had a concentration of approximately 8-8.5 mg / mL total lipid in 90% (v / v) ethanol and 10% (v / v) water.

[0375] A nucleic acid stock solution was created by diluting hepatic stellate cell-specific Reelin (RELN) siRNA (Biosynthesis, custom design) to reach a target concentration of 0.608 mg / mL siRNA in 100 mM acetate buffer, pH 5. Equal volumes of the lipid and nucleic acid solutions were blended at a flow rate of 400 mL / min through a T-connector, and diluter with approximately 4 volumes of phosphate buffered saline (PBS), pH 7.4. Formulations were placed in Slide-A-Lyzer dialysis units (ThermoFisher, MWCO 10,000) and dialyzed overnight against 10 mM Tris, 500 mM NaCl, pH 8 buffer. Following dialysis, the formulations were concentrated using VivaSpin concentrator units (Cytiva, MWCO 100,000) and dialyzed overnight against 5 mM Tris, 10% sucrose, pH 8 buffer. Formulations were filtered through a 0.2 µm syringe filter (polyethersulfone membrane) and stored at -80 °C until use. Nucleic acid concentration and percent encapsulation were determined by the RiboGreen assay. Particle size (Z-average) and polydispersity index (PDI) were determined by dynamic light scattering (DLS) using a Malvern Nano Series Zetasizer.130 80125197V.1Example 3. Gene silencing activity of RELN siRNA-LNPs following in vivo administration in mice.

[0376] LNP formulations encapsulating RELN (a hepatic stellate cell gene target) siRNA were injected intravenously at 0.01 mg / kg to female BALB / c mice (6-8 weeks old). On the day of injection, the LNP stocks were thawed, filtered, and diluted to the required dosing concentration with PBS, pH 7.4. At 48 hours post-dose the mice were anaesthetized with a lethal dosage of ketamine / xylazine. Liver samples (left lateral lobe) were collected into RNAlaterTMStabilization Solution (Invitrogen, AM7024) and stored at 2-8 °C until analysis. Knockdown of RELN mRNA was assessed by QuantiGene assay (ThermoFisher) following the manufacturer’s instructions, with RELN mRNA expression normalized to the housekeeping gene GAPDH. A decrease in the relative mRNA signal (RELN:GAPDH) compared to that of the PBS-treated group indicates an increase in % reelin knockdown and improved gene silencing in hepatic stellate cells.

[0377] Table 2, Table 3. and Table 4 presents results from multiple experiments determining RELN:GAPDH mRNA ratios following administration of LNP compositions from Example 2 or PBS controls. The data in the tables is presented as an average of three or four replicates, and also include the apparent pKa of the ionizable lipid present in each tested LNP composition. The results from these experiments demonstrate that LNP formulations containing a provided N,N-substituted hydroxylamine ester ionizable lipid provide significantly more gene silencing activity than the PBS control. Table 2. In vivo gene silencing in mice. Average of n=3 with standard deviation. Ionizable Lipid LNP Apparent % Knockdown of reelin mRNA pKa relative to PBS-treated animals Lipid 2 6.70 38.6% Lipid 3 6.95 74.4% Lipid 5 7.00 13.1%131 80125197V.1Table 3. In vivo gene silencing in mice. Average of n=4 with standard deviation. Ionizable Lipid LNP Apparent % Knockdown of reelin mRNA pKa relative to PBS-treated animals Lipid 2 6.70 44.4% Lipid 4 6.40 36.6% Lipid 6 6.65 65.7% Lipid 7 7.30 20.5% Lipid 8 7.00 71.7% Lipid 9 approx.7 68.7% Lipid 10 6.95 72.0% Lipid 11 5.70 11.4% Lipid 12 6.30 35.9% Lipid 13 5.75 12.4% Lipid 1 6.30 74.7% Lipid 14 6.40 39.6% Lipid 15 6.75 78.9% Lipid 16 6.3 49.8% Lipid 17 6.40 59.0% Lipid 18 6.60 73.8% Lipid 19 6.75 67.0% Table 4. In vivo gene silencing in mice. Average of n=4 with standard deviation. Ionizable Lipid Ionizable Lipid % Knockdown of reelin mRNA Apparent pKa relative to PBS-treated animals Lipid 2 6.70 40.0% Lipid 20 6.35 27.1% Lipid 21 6.50 72.8% Lipid 22 6.40 26.8% Lipid 23 7.20 70.8% Lipid 24 6.80 73.5% Lipid 24 HBr Salt 7.15 71.7% Lipid 25 6.85 39.8% Lipid 26 6.75 69.3% Lipid 27 6.65 68.4% Example 4. Tolerability of RELN siRNA-LNPs following in vivo administration in mice.

[0378] LNP formulations were dosed as in Example 3 to female BALB / c mice (6-8 weeks old) at 3.0 mg / kg. Plasma was collected 6 hours after dosing to assess MCP-1 cytokine levels. The levels of liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were also determined by IDEXX (Delta, BC) 24 hours after injection. ALT is an enzyme that is primarily found in the liver, and that plays a crucial role in amino acid metabolism. Elevated ALT levels can indicate liver injury or stress, which can be the result of liver damage caused by an administrated agent such as a population of LNPs. Like ALT, AST is also an enzyme involved in amino acid metabolism, and can also indicate liver damage when the enzyme is detected at elevated levels. However, because AST is found not only in the liver, but also in132 80125197V.1various other tissues, measurement of AST levels alongside ALT levels can provide a more specific indication of liver effects such as the toxicity and tolerability of an administered agent such as an LNP.

[0379] Table 5 presents data showing minimal to no increase in MCP-1, ALT, and AST levels following administration of LNP formulations containing a provided N,N-substituted hydroxylamine ester ionizable lipid. Together, these results demonstrate the advantageous tolerability of the compounds and lipid nanoparticles disclosed herein. Table 5. In vivo tolerability assessment in mice. Average of n=3 with standard deviation. Ionizable Lipid MCP-1 ALT AST (pg / mL) (U / L) (U / L) N / A – PBS Control 61 ± 5 23 ± 3 53 ± 5 23-B134 233 ± 28 18 ± 2 45 ± 3 24-B26 293 ± 210 28 ± 7 84 ± 38

[0380] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.133 80125197V.1

Claims

WHAT IS CLAIMED IS:

1. A compound having the formula:or the formula of a salt thereof, wherein: R1, R2, R3, and R4are each independently C1-40 alkyl, C2-40 alkenyl, C2-40 alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, or 2- to 40- membered heteroalkynyl, each of which is optionally substituted with one or more R7groups; R5is C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, each of which is optionally substituted with one or more R7groups, or R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups; R6is hydrogen or is C1-8alkyl, C2-8alkenyl, C2-8alkynyl, 2- to 40-membered heteroalkyl, or 2- to 40-membered heteroalkenyl, each of which is optionally substituted with one or more R7groups; each R7is independently C1-6 alkyl, C1-6 alkoxy, C1-6 hydroxyalkyl, C1-6 haloalkyl, hydroxy, halogen, oxo, -NH2, nitro, or cyano, or is combined with another R7and the carbon to which they are attached to form cyclopropyl or cyclobutyl; X1is C1-12 alkylene, C3-12 cycloalkylene, or 2- to 12-membered heteroalkylene, each of which is optionally substituted with one or more R7groups, or X1or is combined with R5and the nitrogen to which R5is attached to form a 3- to 15-membered heterocyclylene group optionally substituted with one or more R7groups; X2is C1-12alkylene, 2- to 12-membered heteroalkylene, or 3- to 15-membered heterocyclylene; and m and n are each independently an integer from 1 to 12.

2. The compound of claim 1, wherein R1, R2, R3, and R4are each independently C2-40alkenyl, C2-40alkynyl, 2- to 40-membered heteroalkyl, 2- to 40-membered heteroalkenyl, 2- to 40-membered heteroalkynyl, or linear C1-40 alkyl, each of which is optionally substituted with one or more R7groups.134 80125197V.

13. The compound of claim 1 or 2, wherein at least one of R1, R2, R3, and R4is C2-40 alkenyl optionally substituted with one or more R7groups.

4. The compound of claim 3, wherein at least one of R1, R2, R3, and R4is (Z)-C2-40 alkenyl optionally substituted with one or more R7groups.

5. The compound of claim 4, wherein at least one of R1, R2, R3, and R4is (Z)-dec-4-enyl, (Z)-undec-5-enyl, (Z)-non-4-enyl, or (Z)-undec-8-enyl.

6. The compound of any one of claims 1-5, wherein at least one of R1, R2, R3, and R4is C2-40 alkyl optionally substituted with one or more R7groups.

7. The compound of claim 6, wherein at least one of R1, R2, R3, and R4is decyl.

8. The compound of any one of claims 1-7, wherein R1, R2, R3, and R4are identical.

9. The compound of any one of claims 1-8, wherein R5is C1-8alkyl, C2-8alkenyl, or C2-8 alkynyl, each of which is optionally substituted with one or more R7groups, or R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 5-membered monocylclic heterocyclylene group, a 5- to 15-membered spiro or bridged polycyclic heterocyclylene group, or a 5- to 15-membered fused heterocyclylene diastereomer group, each of which is optionally substituted with one or more R7groups.

10. The compound of any one of claims 1-9, wherein R5is C1-8alkyl, optionally substituted with one or more R7groups.

11. The compound of claim 10, wherein R5is methyl or hydroxyethyl.

12. The compound of any one of claims 1-9, wherein R5is combined with X1and the nitrogen to which R5is attached to form a 3- to 5-membered monocylclic heterocyclylene group, a 5- to 15-membered spiro or bridged polycyclic heterocyclylene group, or a 5- to 15-membered fused heterocyclylene diastereomer group, each of which is optionally substituted with one or more R7groups.

13. The compound of claim 12, wherein R5is combined with X1and the nitrogen to which R5is attached to form diazaspiro[5.5]undecandiyl, azetidindiyl, or135 80125197V.1hexahydro-2H-pyrrolo[3,4-b]pyridindiyl, each of which is optionally substituted with one or more R7groups.

14. The compound of any one of claims 1-13, wherein R6is C1-8alkyl, optionally substituted with one or more R7groups.

15. The compound of claim 14, wherein R6is methyl or hydroxyethyl.

16. The compound of any one of clams 1-15, wherein R5and R6are identical.

17. The compound of any one of claims 1-13, wherein R6is hydrogen.

18. The compound of any one of claims 1-17, wherein X1is C2-12alkylene optionally substituted with one or more R7groups.

19. The compound of claim 18, wherein X1is ethylene.

20. The compound of any one of claims 1-17, wherein X1is C3-12 cycloalkylene optionally substituted with one or more R7groups.

21. The compound of claim 20, wherein X1is cyclohexylene.

22. The compound of any one of claims 1-17, wherein X1is 2- to 12- membered heteroalkylene optionally substituted with one or more R7groups.

23. The compound of claim 22, wherein X1is piperidin-1,4-diyl.

24. The compound of any one of claims 1-23, wherein X2is 2- to 12- membered heteroalkylene.

25. The compound of claim 24, wherein X2is.

26. The compound of any one of claims 1-25, wherein m and n are each independently 3, 4, 5, 6, or 7.

27. The compound of any one of claims 1-26, wherein m and n are not identical.136 80125197V.

128. The compound of any one of claims 1-26, wherein m and n are identical.

29. The compound of any one of claims 1-28, wherein the salt comprises a pharmaceutically acceptable salt.

30. The compound of any one of claims 1-29, wherein the salt comprises a bromide salt, a chloride salt, an iodide salt, or a combination thereof.

31. The compound of claim 1, wherein the compound has the formula:(I)(a), or the formula of a salt thereof.

32. The compound of claim 31, wherein X1is C1-12alkylene optionally substituted with one or more R7groups.

33. The compound of claim 32, wherein the compound has the formula:(I)(b), or the formula of a salt thereof.137 80125197V.1138 80125197V.1, , .

35. The compound of claim 31, wherein X1is C3-12 cycloalkylene optionally substituted with one or more R7groups.

36. The compound of claim 35, wherein the compound has the formula:139 80125197V.1(I)(c), or the formula of a salt thereof.

37. The compound of claim 36, wherein the compound has the structure:140 80125197V.1, 38. The compound of claim 31, wherein X1is 2- to 12-membered heteroalkylene optionally substituted with one or more R7groups.

39. The compound of claim 38, wherein the compound has the formula:141 80125197V.1(I)(d), or the formula of a salt thereof.

40. The compound of claim 39, wherein the compound has the structure:.

41. The compound of claim 1, wherein the compound has the structure:142 80125197V.1143 80125197V.

1.

42. A lipid nanoparticle comprising: an ionizable lipid or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid is the compound of any one of claims 1-41; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; and a conjugated lipid.

43. The lipid nanoparticle of claim 42, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.

44. The lipid nanoparticle of claim 42 or 43, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.144 80125197V.

145. The lipid nanoparticle of any one of claims 42-44, wherein the cholesterol or the derivative thereof comprises between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.

46. The lipid nanoparticle of any one of claims 42-45, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

47. The lipid nanoparticle of any one of claims 42-46, wherein the conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle.

48. The lipid nanoparticle of any one of claims 42-47, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 45 mol% and 55 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 36.5 mol% and 40.5 mol% of the total lipid of the lipid nanoparticle; and the conjugated lipid comprising between 0.85 mol% and 2.6 mol% of the total lipid of the lipid nanoparticle.

49. The lipid nanoparticle of any one of claims 42-48, wherein the lipid nanoparticle further comprises a permanently charged lipid or a pharmaceutically acceptable salt thereof.

50. The lipid nanoparticle of claim 51, wherein the permanently charged lipid or the pharmaceutically acceptable salt thereof comprises between 0.5 mol% and 30 mol% of the total lipid of the lipid nanoparticle.

51. The lipid nanoparticle of any one of claims 42-50, wherein the lipid nanoparticle further comprises a nucleic acid.

52. The lipid nanoparticle of claim 51, wherein the nucleic acid comprises RNA.

53. The lipid nanoparticle of claim 52, wherein the RNA comprises siRNA, miRNA, shRNA, asRNA, DsiRNA, piRNA, or a combination thereof.145 80125197V.

154. The lipid nanoparticle of claim 53, wherein the RNA comprises siRNA.

55. The lipid nanoparticle of claim 51, wherein the nucleic acid comprises an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.

56. The lipid nanoparticle of any one of claims 51-55, wherein the nucleic acid is configured to reduce expression of a gene in a cell.

57. The lipid nanoparticle of claim 56, wherein the cell is a hepatic stellate cell.

58. The lipid nanoparticle of claim 56 or 57, wherein the gene encodes collagen type I alpha 1 chain (COL1A1), tissue inhibitor of metalloproteinases 1 (TIMP1), matrix metalloproteinase 2 (MMP2 ), alpha smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), heat shock protein 47 (HSP47), lysyl oxidase (LOX), epidermal growth factor receptor (EGFR), rho kinase (ROCK), integrin alpha 8 beta 1 or another integrin, yes- associated protein (YAP), a toll-like receptor pathway protein, transforming growth factor beta (TGFB), platelet-derived growth factor (PDGF), or connective tissue growth factor (CTGF).

59. A pharmaceutical composition comprising: the lipid nanoparticle of any one of claims 42-58, or the compound of any one of claims 1-41; and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.

60. A method for introducing the nucleic acid of any one of claims 51-58 into a cell, the method comprising contacting the cell with the lipid nanoparticle of any one of claims 51-58.

61. The method of claim 60, wherein the nucleic acid is configured to reduce expression of a gene in the cell.

62. The method of claim 61, wherein the method further comprises reducing expression of the gene by the cell.

63. The method of any one of claims 60-62, wherein the cell is a hepatic stellate cell.146 80125197V.

164. The method of any one of claims 61-63, wherein the gene encodes COL1A1, TIMP1, MMP2, α-SMA, CTGF, HSP47, LOX, EGFR, ROCK, integrin alpha 8 beta 1 or another integrin, YAP, a toll-like receptor pathway protein, TGFB, PDGF, or CTGF.

65. The lipid nanoparticle of any one of claims 56-58 for use in reducing expression of the gene in the hepatic stellate cell.

66. The use of the lipid nanoparticle of any one of claims 56-58 to prepare a medicament for reducing expression of the gene in the hepatic stellate cell.

67. A method for the in vivo delivery of the nucleic acid of any one of claims 51-58, the method comprising administering to a subject the lipid nanoparticle of any one of claims 51-58.

68. A lipid nanoparticle of any one of claims 51-58 for use in the in vivo delivery of the nucleic acid of any one of claims 51-58 to a subject.

69. The use of the lipid nanoparticle of any one of claims 51-58 to prepare a medicament for the in vivo delivery of the nucleic acid of any one of claims 51-58 to a subject.

70. A method for preventing or treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the lipid nanoparticle of any one of any one of claims 42-58, the compound of any one of claims 1-41, or the pharmaceutical composition of claim 59.

71. The method of claim 70, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

72. The method of claim 71, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.

73. The method of claim 71 or 72, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), nonalcoholic fatty liver disease (NASH), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), alcoholic liver disease (ALD), immune-induced hepatitis, ischemia and reperfusion (I / R) liver injury, schistosome viral hepatitis, or chronic viral hepatitis.147 80125197V.

174. A lipid nanoparticle of any one of claims 42-58, compound of any one of claims 1-41, or pharmaceutical composition of claim 59, for use in preventing or treating a disease or disorder in a subject.

75. The lipid nanoparticle, compound, or pharmaceutical composition of claim 74, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

76. The lipid nanoparticle, compound, or pharmaceutical composition of claim 75, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.

77. The lipid nanoparticle, compound, or pharmaceutical composition of claim 75 or 76, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I / R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.

78. The use of a lipid nanoparticle of any one of claims 42-58, compound of any one of claims 1-41, or pharmaceutical composition of claim 59, to prepare a medicament for preventing or treating a disease or disorder in a subject.

79. The use of claim 78, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.

80. The use of claim 79, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.

81. The use of claim 79 or 80, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I / R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.148 80125197V.1

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