Ionizable amine lipids and lipid nanoparticles

Abstract

The present disclosure describes compositions, preparations, nanoparticles (such as lipid nanoparticles), and / or nanomaterials, and methods of using the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Application No. 63 / 291,639, filed December 20, 2021, and U.S. Application No. 63 / 354,627, filed June 22, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] In the fields of chemistry, biology, and medicine, drug delivery systems have been problematic, hindering progress due to a limited understanding of how the molecular properties of the systems control tissue delivery and confer drug efficacy. Summary of the Invention

[0003] The present invention recognizes the need for compositions, preparations, nanoparticles, and / or nanomaterials, as well as methods of using the same. Among other things, the present disclosure recognizes that the structural characteristics of compositions, preparations, nanoparticles, and / or nanomaterials affect functional responses in vivo, in vitro, and ex vivo. For example, the present disclosure describes, among other things, the selection and combination of one or more components described herein affecting the functional activity of lipid nanoparticles. In some embodiments, functional activity may refer to, for example, desired tropism, stabilization, and / or drug delivery efficacy. In some embodiments, among other things, the present disclosure describes varying the ratio of one or more components to affect one or more functional activities of the compositions, preparations, nanoparticles, and / or nanomaterials described herein.

[0004] Further, among other things, the present disclosure recognizes that the chemical structure of the lipid confers improved properties compared to a reference lipid structure. For example, in some embodiments, the present disclosure provides a compound of formula I: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein L 1 , L 1’ , L 2 , L 2’ , Y 1 , Y 1’ , Y 2 , Y 2’ , R, R', X 1 , X 2 , and X 3 describes a compound or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of the following is as defined herein:

[0005] Among other things, as described herein, the present disclosure demonstrates surprising attributes (e.g., unexpected tropism, stabilization, and delivery efficacy of cargo, such as therapeutic or prophylactic agents) of ionizable lipids comprising a trivalent core (e.g., derived from a secondary triol) characteristic, and compositions, preparations, nanoparticles, and / or nanomaterials thereof (e.g., LNPs, and / or compositions, preparations, nanoparticles, and / or nanomaterials containing LNPs), and methods of using them.

[0006] Among other things, the present disclosure recognizes lipid nanoparticle (LNP) compositions comprising one or more ionizable lipids. For example, the present disclosure provides compositions and / or preparations of LNPs comprising one or more of the disclosed ionizable lipids that impart unexpected tropism.

[0007] In some embodiments, the provided compositions, preparations, nanoparticles, and / or nanomaterials are for use in methods of treating a disease or disorder, delivering or producing a polypeptide, or slowing / halting the progression of a disease or disorder.

[0008] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials provided are for use in a method of manufacturing.

[0009] In some embodiments, provided compositions, preparations, nanoparticles, and / or nanomaterials are for use in characterization methods.

[0010] Elements (eg, methods) of embodiments incorporating one aspect of the invention may be applied to embodiments incorporating other aspects of the invention, and vice versa. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows an exemplary mRNA screening system for LNP preparations according to an embodiment of the present disclosure.

[0012] [Figure 2] 1 shows an exemplary siRNA screening system for LNP preparations, according to an embodiment of the present disclosure.

[0013] definition About: As used herein, the terms "about" or "approximately," when used herein with respect to a value, refer to a value that is similar in relation to the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the relevant degree of difference encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" can encompass 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or a range of values ​​that fall in either direction (greater or less) of the referenced value (except where such number exceeds 100% of possible values), unless otherwise stated or apparent from the context.

[0014] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system. Those of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In certain embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (e.g., may be or include one or more of topical, such as dermal, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration involves administration that is intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart in the same time period) administration. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time. In some embodiments, pharmaceutical compositions comprising lipid nanoparticles may be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or by using bioerodible inserts, and may be formulated in dosage forms appropriate for each route of administration.

[0015] Aliphatic: The terms "aliphatic" or "aliphatic group," as used herein, refer to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "carbocyclic," "alicyclic," or "cycloalkyl") and has one point of attachment to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 carbon atoms. In some embodiments, an aliphatic group contains 1-4 carbon atoms. In some embodiments, an aliphatic group contains 1-3 carbon atoms, and in some embodiments, an aliphatic group contains 1-2 carbon atoms. In some embodiments, "carbocyclic" (or "alicyclic", or "carbocycle", or "cycloalkyl") refers to an optionally substituted monocyclic C3-C8 hydrocarbon or an optionally substituted C6-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. 12 "Cycloalkyl" refers to a bicyclic hydrocarbon. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0016] Alkenyl: As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds. In some embodiments, the term "alkenyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one double bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched hydrocarbon chain having an alkyl group. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

[0017] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0018] Alkyl: As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C for a straight chain). 20 , C2-C for branched chains 20 ), and alternatively having about 1-10 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure when such rings are monocyclic or bicyclic, and alternatively have about 5, 6 or 7 carbons in the ring structure. In some embodiments, alkyl groups can be lower alkyl groups having from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0019] Alkylene: The term "alkylene" or "alkylenyl" refers to a divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) that is straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the monovalent alkyl groups listed above can become an alkylene by abstracting a second hydrogen atom from the alkyl. In some embodiments, "alkylene" refers to a polymethylene group, i.e., -(CH2) n - and n is a positive integer, preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, or 4 to 8. A substituted alkylene is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0020] Alkynyl: As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds. In some embodiments, the term "alkynyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one triple bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched chain hydrocarbon group having an alkynyl group. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0021] Amino acid: In the broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids typically found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, regardless of whether it is synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy- and / or amino-terminal amino acids in a polypeptide, may contain structural modifications compared to the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing the same unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing the same unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid, and in some embodiments, to an amino acid residue of a polypeptide.

[0022] Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human of either sex and at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0023] Aptamer: As used herein, the term "aptamer" refers to a macromolecule composed of nucleic acids (e.g., RNA, DNA) that tightly binds to a specific molecular target (e.g., umbrella topology glycans). A particular aptamer can be described by its linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theory, it is contemplated that the nucleotide chains in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, allowing the aptamer to tightly bind to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the realm of all possible nucleotide sequences, aptamers can be obtained for a wide range of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers typically have very high affinity for their targets (e.g., picomolar to low nanomolar affinity for proteins). In many embodiments, aptamers are chemically stable and can be boiled or frozen without loss of activity. Because aptamers are synthetic molecules, they are amenable to a variety of modifications that can optimize their function for specific applications. For example, for in vivo applications, aptamers can be modified to dramatically reduce their susceptibility to degradation by enzymes in the blood. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.

[0024] Aryl: The term "aryl" refers to an alkyl group having a total of 6 to 14 ring members (e.g., C 6-14 "aryl" refers to monocyclic and bicyclic ring systems having a cyclic ring structure, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to aromatic ring systems, including, but not limited to, phenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Unless otherwise specified, "aryl" groups are hydrocarbons.

[0025] Associated: As used herein, two events or entities are "associated" with one another when the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism) is considered to be associated with a particular disease, disorder, or condition when its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they directly or indirectly interact with one another, such that they are in and / or maintain physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently associated, e.g., by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0026] Biocompatible: The term "biocompatible," as used herein, refers to a material that, when placed in contact with living tissue, e.g., in vivo, does not cause significant harm to such tissue. In certain embodiments, a material is "biocompatible" if it is not toxic to cells. In certain embodiments, materials are "biocompatible" if their addition to cells in vitro results in 20% or less cell death and / or if their administration in vivo does not induce significant inflammation or other such adverse effects.

[0027] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into a cell, breaks down (e.g., by cellular mechanisms, such as by enzymatic degradation, hydrolysis, and / or a combination thereof) into components that the cell can reuse or dispose of without significant toxic effects to the cell. In certain embodiments, the components produced by the degradation of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymeric materials break down into their component monomers. In some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves cleavage of urethane bonds. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including, but not limited to, poly(hydroxyl acid), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), blends and copolymers thereof. Also, polymers of hydroxy acids such as hydroxypropyl methylcellulose, hydroxypropyl ... Many natural polymers are biodegradable, including proteins such as collagen, gelatin, and prolamines, e.g., zein, and polysaccharides such as alginates, cellulose derivatives, and polyhydroxyalkanoates, e.g., polyhydroxybutyrate blends, and copolymers thereof. Those skilled in the art will know or be able to determine when such polymers are their biocompatible and / or biodegradable derivatives (e.g., when related to the parent polymer by substantially the same structure, differing only in the substitution or addition of certain chemical groups known in the art).

[0028] Biologically active: As used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed through detection of a direct or indirect product produced by the biological pathway or event of interest.

[0029] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., a tissue, or organism, or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or biological fluid. In some embodiments, the biological sample can be or include bone marrow, blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluids; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural effusion; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells derived from the individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or adding one or more agents), such as by filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0030] Divalent: As used herein, the term "divalent" refers to a chemical moiety that has two points of attachment. For example, a "divalent C 1-8 (or C 1-6) saturated or unsaturated straight or branched hydrocarbon chain" refers to straight or branched divalent alkylene, alkenylene, and alkynylene chains as defined herein.

[0031] Bridged Bicyclic: As used herein, the terms "bridged bicyclic," "bridged bicycle," "bridged bicyclic," and "bridged bicyclic ring" refer to any saturated or partially unsaturated bicyclic ring system, i.e., carbocyclic or heterocyclic, having at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or a single atom or valence bond connecting two bridgeheads, and a "bridgehead" is any skeletal atom of the ring system that is attached to three or more skeletal atoms (excluding hydrogen). In some embodiments, bridged bicyclic groups have 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include the groups described below, in which each group is attached to the remainder of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, bridged bicyclic groups are optionally substituted with one or more substituents described for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to: [ka]

[0032] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a marked loss of control of cell proliferation. In some embodiments, a tumor may be or include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancer may be characterized as a solid tumor. In some embodiments, the relevant cancer may be characterized as a hematologic tumor. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, cancer of solid tissue, squamous cell carcinoma of the mouth, throat, larynx, and lung, liver cancer, reproductive cancers such as prostate, cervical, bladder, uterine, and endometrial cancer, as well as benign lesions such as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, papilloma, etc.

[0033] Carrier: As used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, the carrier can include sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or includes one or more solid components.

[0034] Carbocyclyl: As used herein, the terms "carbocyclyl," "carbocycle," and "carbocyclic ring" refer to a saturated or partially unsaturated cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein having 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, "carbocyclyl" (or "alicyclic") refers to an optionally substituted monocyclic C3-C8 hydrocarbon or an optionally substituted C6-C8 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. 12 It refers to a bicyclic hydrocarbon. The term "cycloalkyl" refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, the cycloalkyl group has 3 to 6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0035] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them, so that a person of ordinary skill in the art would understand that conclusions could be reasonably drawn based on the observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by several substantially identical characteristics and one or a few varying characteristics. A person of ordinary skill in the art will understand what degree of identity is necessary in any given situation for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable, depending on the context. For example, a person of ordinary skill in the art will understand that sets of circumstances, individuals, or populations are comparable to one another if they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by variations in those varying characteristics, or that variations in characteristics are indicative of variations in characteristics.

[0036] Composition: Those skilled in the art will understand that the term "composition" can be used to refer to a separate physical entity that includes one or more defined components. Generally, unless otherwise specified, a composition can be in any form, e.g., gas, gel, liquid, solid, etc.

[0037] Comprising: A composition or method described herein as "comprising" one or more recited elements or steps is open-ended, meaning that the recited elements or steps are essential, but that other elements or steps may be added within the scope of the composition or method. It is also understood that, to avoid redundancy, any composition or method described as "comprising" (or "comprises") one or more recited elements or steps also describes a corresponding, more limited composition or method that "consistes essentially of" (or "consists essentially of") the same recited elements or steps, meaning that the composition or method includes the recited essential elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more recited elements or steps describes a corresponding, more limited, close-ended composition or method "consisting of" (or "consists of") the recited elements or steps, to the exclusion of any other unrecited elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any recited essential element or step may be substituted for that element or step.

[0038] "Improve," "Increase," "Inhibit," or "Reduce": As used herein, the terms "improve," "increase," "inhibit," "reduce," or their grammatical equivalents refer to a value compared to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., a single individual) under comparable conditions in the absence of a particular drug or treatment (before and / or after), or in the presence of a suitable comparable reference drug. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or expected to respond in a particular way in the presence of the relevant drug or treatment.

[0039] Determining: Many methodologies described herein include a "determining" step. Those skilled in the art will understand, upon reading this specification, that such "determining" can be utilized or accomplished through the use of any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, determining involves physical manipulation of the sample. In some embodiments, determining involves reviewing and / or manipulating data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or materials from a source. In some embodiments, determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0040] Encapsulated: The term "encapsulated" is used herein to refer to a substance that is completely surrounded by another material.

[0041] Excipient: As used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0042] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0043] Haloaliphatic: The term "haloaliphatic" refers to an aliphatic group substituted with one or more halogen atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 halo, e.g., fluoro, iodo, bromo, or chloro). In some embodiments, a haloaliphatic group contains 1-7 halogen atoms. In some embodiments, a haloaliphatic group contains 1-5 halogen atoms. In some embodiments, a haloaliphatic group contains 1-3 halogen atoms.

[0044] Haloalkyl: The term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 halo, such as fluoro, iodo, bromo, or chloro). In some embodiments, a haloalkyl group contains 1 to 7 halogen atoms. In some embodiments, a haloalkyl group contains 1 to 5 halogen atoms. In some embodiments, a haloalkyl group contains 1 to 3 halogen atoms.

[0045] Heteroalkylene: As used herein, the terms "heteroalkylene" or "heteroalkylenyl" refer to an optionally substituted straight-chain (i.e., unbranched) or branched divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) having, in addition to carbon atoms, 1 to 5 heteroatoms. The term "heteroatom" is described below. In some embodiments, a heteroalkylene group contains 2 to 10 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, a heteroalkylene group contains 2 to 8 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, a heteroalkylene group contains 4 to 8 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylene groups contain 2 to 5 carbon atoms, with 1 to 2 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroalkylene groups contain 1 to 3 carbon atoms, with 1 carbon atom optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroalkylene groups include, but are not limited to, -CHO-, -(CH)O-, -CHOCH-, -O(CH)-, -(CH)O-, -(CH)OCH-, -CHO(CH)-, -O(CH)-, -(CH)O-, -(CH)OCH-, -CHO(CH)-, -(CH)O(CH)-, and -O(CH)-. Unless otherwise specified, C x Heteroalkylene refers to a heteroalkylene having x carbon atoms before being replaced with a heteroatom.

[0046] Heteroaryl: The terms "heteroaryl," and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5-6 membered monocyclic heteroaryl or 9-10 membered bicyclic heteroaryl), sharing 6, 10, or 14 pi electrons in the cyclic array, and having 1 to 5 heteroatoms in addition to the carbon atoms. Exemplary heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and benzisoxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include optionally substituted rings.

[0047] Heteroatom: The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl).

[0048] Heterocycle: The terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably herein and refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more heteroatoms, such as 1 to 4, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and [ka] Heterocyclyl groups can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions are independently and optionally substituted. Bicyclic heterocyclic rings also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or alicyclic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocyclic rings can also be spirocyclic ring systems (e.g., 7-11 membered spirocyclic fused heterocyclic rings having, in addition to carbon atoms, one or more heteroatoms (e.g., 1, 2, 3, or 4 heteroatoms) as defined above). Bicyclic heterocyclic rings can also be bridged ring systems (e.g., 7-11 membered bridged heterocyclic rings having 1, 2, or 3 bridging atoms).

[0049] Inhibitor: As used herein, the term "inhibitor" refers to an entity, condition, or event whose presence, level, or extent correlates with a decrease in the level or activity of a target. In some embodiments, an inhibitor may act directly (where it directly affects the target, e.g., by binding to the target), while in some embodiments, an inhibitor may act indirectly (where it affects its effect by interacting with and / or altering a regulator of the target such that the level and / or activity of the target is reduced). In some embodiments, an inhibitor is one whose presence or level correlates with a target level or activity that is reduced compared to a particular reference level or activity (e.g., observed under appropriate reference conditions, such as the presence of a known inhibitor or the absence of the inhibitor in question).

[0050] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0051] Isolated: As used herein, refers to substances and / or entities that are (1) separated from at least some of the components with which they are initially produced (in nature and / or in an experimental setting) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they are initially associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by one of skill in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. For example, in some embodiments, a naturally occurring biopolymer, such as a polypeptide or polynucleotide, is considered "isolated" if: a) its origin or source is free from association with some or all of the components that accompany it in its natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed by or associated with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide.Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from a) other components with which it is associated in nature, and / or b) other components with which it was associated when originally produced.

[0052] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0053] Linker: As used herein, it is used to refer to that portion of a multi-element agent that connects different elements to one another. For example, one of skill in the art will understand that polypeptides that include two or more functional or organizational domains in their structure often include a stretch of amino acids between such domains that connects them to one another. In some embodiments, a polypeptide that includes a linker element "L" has an overall structure of the general form S1-L'-S2, where S1 and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide. A variety of different linker elements that can be suitably used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1 121-1123).

[0054] Nanoparticle: As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, a nanoparticle is a micelle in that it contains an enclosed compartment separated from the bulk solution by a micellar membrane, typically composed of amphiphilic entities that surround and encapsulate the space or compartment (e.g., to define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, the lipid nanoparticles described herein may have an average hydrodynamic diameter of about 30 to about 170 nm. In some embodiments, the lipid nanoparticles described herein can have an average hydrodynamic diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 50 nm to 100 nm.

[0055] Nanoparticle composition: As used herein, the term "nanoparticle composition" refers to a composition containing at least one nanoparticle and at least one additional agent or component. In some embodiments, the nanoparticle composition contains a population of substantially uniform nanoparticles as described herein.

[0056] Nucleic Acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, nucleic acids comprise one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in naturally occurring nucleic acids. In some embodiments, nucleic acids have a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, nucleic acids comprise one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded, hi some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide.In some embodiments, the nucleic acid has enzymatic activity.

[0057] Operably linked: As used herein, refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated with the functional element in such a manner that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest, and in some embodiments, the control element acts in trans with or on the functional element of interest.

[0058] For the purposes of this invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th Ed., the entire contents of which are incorporated herein by reference. th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: (2001).

[0059] Parenteral: As used herein, the phrases "parenteral administration" and "administered parenterally" have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intraventricular, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0060] Patient: As used herein, the term "patient" refers to any organism to which provided compositions are or can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat the disease, disorder, or condition.

[0061] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., as a sterile solution or suspension, or sustained-release formulation, e.g., subcutaneous, intramuscular, intravenous, or epidural injection; topical application, e.g., as a cream, ointment, or controlled-release patch, or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual; ophthalmic; transdermal; or compatible with the nose, lungs, and other mucosal surfaces.

[0062] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0063] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0064] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in pharmaceutical contexts, i.e., salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include non-toxic acid addition salts, which are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, 2-hydroxybenzoate, ... Representative salts of alkali or alkaline earth metals include sodium, lithium, potassium, calcium, magnesium, and the like.In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, as appropriate.

[0065] Prevent or Prevention: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition has been delayed for a predefined period of time.

[0066] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). Proteins may contain moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be processed or modified. Those of skill in the art will understand that a "protein" may be an entire polypeptide chain produced by a cell (with or without a signal sequence), or a characteristic portion thereof. Those of skill in the art will understand that a protein may sometimes comprise two or more polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0067] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will understand that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Furthermore, those of skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares one or more highly conserved regions with another polypeptide of the same genus, usually encompassing at least three to four, often up to 20 or more amino acids, including at least one region of very high identity, usually at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and even more usually often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%, is encompassed within the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0068] Prevention: As used herein, the term "prevention" refers to delaying the onset and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention can be considered complete if the onset of the disease, disorder, or condition is delayed for a predefined period of time.

[0069] Protecting group: As used herein, the phrase "protecting group" refers to a temporary substituent that protects a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. A "Si-protecting group" is a protecting group containing a Si atom, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g., t-butyldiphenylsilyl), or Si-aryl-dialkyl (e.g., Si-phenyldialkyl). Typically, Si-protecting groups are attached to an oxygen atom. The field of protecting group chemistry has been reviewed (Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Such protecting groups (and related protected moieties) are described in detail below.

[0070] Protected hydroxyl groups are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3rd Edition, incorporated herein by reference in its entirety. rdedition, John Wiley & Sons, 1999. Examples of suitable protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, propionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formates, benzoyl formates, chloroacetates, trifluoroacetates, methoxyacetates, triphenylmethoxyacetates, p-chlorophenoxyacetates, 3-phenylpropionates, 4-oxopentanoates, 4,4-(ethylenedithio)pentanoates, pivalates (trimethylacetates), crotonates, 4-methoxycrotonates, benzoates, p-benzylbenzoates, and 2,4,6-trimethylbenzoates. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ethers, or derivatives thereof. Examples of alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

[0071] Protected amines are well known in the art and include those described in detail in Greene (1999). Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected amino moieties include t-butyloxycarbonylamino (-NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxocarbonylamino (-NHCBZ), allylamino, benzylamino (-NHBn), fluorenylmethylcarbonyl (-NHFmoc), formamide, acetamide, chloroacetamide, dichloroacetamide, trichloroacetamide, phenylacetamide, trifluoroacetamide, benzamide, t-butyldiphenylsilyl, and the like. Suitable di-protected amines include amines substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides such as phthalimide, maleimide, succinimide, etc. Suitable di-protected amines also include pyrroles, 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, etc., and azides.

[0072] Protected aldehydes are well known in the art and include those detailed in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal, 1,3-dioxane, 1,3-dioxolane, semicarbazones, and derivatives thereof.

[0073] Protected carboxylic acids are well known in the art and include those detailed in Greene (1999). Suitable protected carboxylic acids include, but are not limited to, optionally substituted C 1-6 Further examples include aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, etc. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl esters, each group being optionally substituted. Additional suitable protected carboxylic acids include oxazolines and orthoesters.

[0074] Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioesters, to name just a few.

[0075] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as a separate unit. When a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form a separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When a separate functional unit is composed of two or more polypeptides that are physically associated with each other, the term "protein" may be used to refer to the multiple polypeptides that are physically associated and function together as a separate unit. In some embodiments, a protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified. One of skill in the art will understand that in some embodiments, the term "protein" may refer to the complete polypeptide chain produced by a cell (e.g., with or without a signal sequence) and / or a form that is active within the cell (e.g., a truncated or complexed form). In some embodiments in which a protein is composed of multiple polypeptide chains, such chains may be covalently associated with each other, e.g., by one or more disulfide bonds, or may be associated by other means.

[0076] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing greater than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0077] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by one of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. One of skill in the art will understand when sufficient similarity exists to justify reliance on and / or comparison to a particular possible reference or control.

[0078] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source of interest as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell, or an organism such as a microorganism, plant, or animal (e.g., human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, eye discharge, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or intercellular fluid. In some embodiments, the biological fluid may be or include phytoexudates. In some embodiments, the biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle biopsy or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or wash (e.g., bronchoalveolar, mammary ductal, nasal, ocular, oral, uterine, vaginal, or other lavage or wash). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more agents), for example, by filtration using a semi-permeable membrane. Such a "processed sample" can include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc.

[0079] Stable Nanoparticle Composition: The term "stable," as applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure (e.g., particle size range and / or distribution) over a period of time. In some embodiments, a stable nanoparticle composition is one in which the mean particle size, maximum particle size, particle size range, and / or particle size distribution (i.e., the percentage of particles above a specified size and / or outside a specified size range) are maintained under defined conditions for a period of time. In some embodiments, a stable provided composition is one in which biologically relevant activity is maintained for a period of time. In some embodiments, the period is at least about 1 hour, and in some embodiments, the period is about 5 hours, about 10 hours, about 1 (1) day, about 1 (1) week, about 2 (2) weeks, about 1 (1) month, about 2 (2) months, about 3 (3) months, about 4 (4) months, about 5 (5) months, about 6 (6) months, about 8 (8) months, about 10 (10) months, about 12 (12) months, about 24 (24) months, about 36 (36) months, or more. In some embodiments, the period is within a range of about 1 (1) day to about 24 (24) months, about 2 (2) weeks to about 12 (12) months, about 2 (2) months to about 5 (5) months, etc. For example, a nanoparticle composition is stable if a population of nanoparticles is subjected to extended storage, temperature changes, and / or pH changes, and the majority of the nanoparticles in the composition maintain diameters within the recited ranges. In some embodiments, the stable composition is stable at ambient conditions. In some embodiments, the stable composition is stable under biological conditions (i.e., in phosphate buffered saline at 37°C).

[0080] Sterolyl: The term "sterolyl," as used herein, refers to a 17-membered fused polycyclic ring moiety that is saturated or partially unsaturated, substituted with at least one hydroxyl group, and has a single point of attachment to the remainder of the molecule at any substitutable carbon or oxygen atom. In some embodiments, the sterolyl group is a cholesterolyl group, or a variant or derivative thereof. In some embodiments, the cholesterolyl group is modified. In some embodiments, the cholesterolyl group is an oxidized cholesterolyl group (e.g., oxidized at the beta ring structure or hydrocarbon tail structure). In some embodiments, the cholesterolyl group is an esterified cholesterolyl group. In some embodiments, the sterolyl group is a phytosterolyl group. Exemplary sterolyl groups include, but are not limited to, 25-hydroxycholesterolyl (25-OH), 20α-hydroxycholesterolyl (20α-OH), 27-hydroxycholesterolyl, 6-keto-5α-hydroxycholesterolyl, 7-ketocholesterolyl, 7β-hydroxycholesterolyl, 7α-hydroxycholesterolyl, 7β-25-dihydroxycholesterolyl, beta-sitosterolyl, stigmasterolyl, brassicasterolyl, and campesterolyl.

[0081] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., in some embodiments, a human, including prenatal human forms). In some embodiments, the subject is suffering from the relevant disease, disorder, or condition. In some embodiments, the subject is predisposed to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject possesses one or more characteristics that make them susceptible to, or at risk for, a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom diagnosis and / or treatment is to be and / or has been performed.

[0082] Substantially: As used herein, the term "substantially" refers to a qualitative condition indicating the total or nearly total extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go completely to completion and / or progress toward complete completion, or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0083] Substituted or Optionally Substituted: The compounds of the present disclosure described herein may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens, either explicit or implied from the structure (e.g., [ka] At least [ka] refers to, [ka] At least [ka] (refers to "optionally substituted"). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that remain substantially unchanged when subjected to conditions that enable their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as "substituted" preferably have one to four substituents, more preferably one to two substituents. Groups described as "optionally substituted" may be unsubstituted or "substituted," as described above.

[0084] Suitable monovalent substituents include halogen; -(CH2) 0-4 R°;-(CH2) 0-4 OR°;-O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 CH(OR°)2; R° can be substituted with -(CH2) 0-4 Ph; R° may be substituted with -(CH2) 0-4 O(CH2) 0-1 Ph; -CH=CHPh, which may be substituted with R°; -(CH2), which may be substituted with R° 0-4 O(CH2) 0-1 -pyridinyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;-(CH2) 0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0-4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0-4 N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0-4C(O)R°;-C(S)R°;-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 C(O)SR°;-(CH2) 0-4 C(O)OSiR°3;-(CH2) 0-4 OC(O)R°;-OC(O)(CH2) 0-4 SR°-;-SC(S)SR°;-(CH2) 0-4 SC(O)R°;-(CH2) 0-4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-SC(S)SR°;-(CH2) 0-4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0-4 SSR°;-(CH2) 0-4 S(O)2R°;-(CH2) 0-4 S(O)2OR°;-(CH2) 0-4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0-4 S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O)(OR°)2;-SiR°3;-OSiR°3;-(C 1-4 linear or branched alkylene)ON(R°)2; or -(C 1-4 and alkylene)C(O)ON(R°), where each R° can be substituted as defined below and independently represents hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (a 5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an aryl ring, or as defined above, but where two independent occurrences of R° together with their intervening atoms form a 3-12 membered saturated, partially unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an aryl monocyclic or bicyclic ring, which may be substituted as defined below.

[0085] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with the intervening atoms) are independently halogen, —(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 C(O)NH2, -(CH2) 0-2 C(O)NHR ● , -(CH2) 0-2 C(O)NR ● 2, -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● 、 -(C 1-4 Linear or branched alkylene)C(O)OR ● , or -SSR ● and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0086] Suitable divalent substituents include the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-, and each R * occurrences of C which may be substituted as defined below 1-6 A 5- to 6-membered, saturated, partially unsaturated, or aryl ring is selected from aliphatic or unsubstituted rings having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the proximal substitutable carbon of an "optionally substituted" group include -O(CR * 2) 2-3 O-, and each independent R * occurrences of C which may be substituted as defined below 1-6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or aryl rings.

[0087] R * Suitable substituents on the aliphatic group include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, and each R● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an aryl ring.

[0088] In some embodiments, suitable substituents on a substitutable nitrogen include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † are listed, and each R † are independently hydrogen, C which may be substituted as defined below 1-6 an aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an aryl ring, or as defined above but containing two independent R † together with the intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated ring or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0089] R * Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR● , -NR ● 2, or -NO2, and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an aryl ring.

[0090] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E stereoisomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen with deuterium or tritium, or 13 C or 14 Compounds having this structure including a carbon replacement with a C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as therapeutic agents according to the present invention.

[0091] Susceptible: An individual who is "susceptible" to a disease, disorder, or condition is at risk of developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has never been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with the development of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of an individual with a disease, disorder, or condition).

[0092] Systemic: As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" have their art-recognized meanings referring to the administration of a compound or composition so that it enters the system of the recipient.

[0093] Tautomeric Forms: As used herein, the phrase "tautomeric forms" is used to describe different isomeric forms of an organic compound that are readily interconvertible. Tautomers can be characterized by a geometrical shift of a hydrogen atom or proton, accompanied by a switch of a single bond and an adjacent double bond. In some embodiments, tautomers can result from prototropic tautomerism (i.e., relocation of a proton). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid reorganization of bond electrons). All such tautomeric forms are intended to be included within the scope of this disclosure. In some embodiments, tautomeric forms of a compound exist in a dispersive equilibrium with one another, such that attempts to prepare separate substances result in the formation of mixtures. In some embodiments, tautomeric forms of a compound are separable and isolatable compounds. In some embodiments of the present disclosure, chemical compositions may be provided that are pure preparations of or contain a single tautomeric form of a compound. In some embodiments, a chemical composition may be provided as a mixture of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms, and in certain embodiments, such mixtures contain unequal amounts of at least two different tautomeric forms of the compound. In some embodiments of the present disclosure, a chemical composition may contain all tautomeric forms of a compound. In some embodiments of the present disclosure, a chemical composition may contain fewer than all tautomeric forms of a compound. In some embodiments of the present disclosure, a chemical composition may contain one or more tautomeric forms of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present disclosure, the tautomer is keto-enol tautomerism. Those skilled in the art will recognize that keto-enol tautomers can be "captured" (i.e., chemically modified to remain in the "enol" form) using any suitable reagent known in the chemical arts to provide enol derivatives that can then be isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present disclosure encompasses all tautomeric forms of the relevant compounds, whether in pure form or mixed with each other.

[0094] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0095] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, inhibit, ameliorate, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be understood by one of skill in the art, the effective amount of a substance can vary depending on such factors as the desired biological endpoint, the substance being delivered, and the target cell or tissue. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. The exact dosage will vary depending on a variety of factors, including subject-specific variables (eg, age, health of the immune system, etc.), the disease, and the treatment being administered.

[0096] "Tissue" and / or "Organ": As used herein, the terms "tissue" and / or "organ," unless otherwise specified, refer to viable cellular material in aggregate form, e.g., small portions of an organ, and dispersed cells, e.g., cells dispersed, isolated, and / or grown from muscle, cardiac muscle, liver, or kidney, including bone marrow cells and progeny cells, blood-producing stem cells and progeny, and various other blood elements. In some embodiments, tissue and / or organ refers to kidney, heart liver, stomach, spleen, pancreas, lung, brain, eye, intestine, bladder, skin or skin tissue, blood vessels, veins, arteries, heart valves, sperm, and oocyte(s). As used herein, the term "organ" encompasses both solid organs, e.g., kidney, heart, liver, lung, and functional portions of organs, e.g., portions of skin, arteries, veins, transplantable liver lobes, kidneys, lung sections, etc.

[0097] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, improves, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of subjects who do not show signs of the associated disease, disorder, and / or condition and / or who show only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of subjects who show one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic, and in some embodiments, therapeutic. DETAILED DESCRIPTION OF THE INVENTION

[0098] The present disclosure describes how the selection and combination of one or more of the components of the described compositions, preparations, nanoparticles, and / or nanomaterials influences the functional activity of lipid nanoparticles, such as desired tropism, stabilization, and drug delivery efficacy. Among other things, the present disclosure provides compositions, preparations, nanoparticles, and / or nanomaterials for the delivery of therapeutic and / or prophylactic agents to target cells and / or tissues. For example, the present disclosure describes lipid compounds for use in the compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, the compositions, preparations, and / or nanomaterials comprise LNPs that carry cargo to designated target cells, tissues, and / or organs.

[0099] I. Lipid Nanoparticles (LNPs) The present invention provides compositions, preparations, and / or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise one or more components. In some embodiments, the lipid nanoparticles comprise one or more components, such as a compound, an ionizable lipid, a sterol, a conjugate-linker lipid, and a phospholipid. In particular, the present disclosure describes how the selection and combination of one or more of the components described herein affects lipid nanoparticle characteristics, such as diameter, pKa, stabilization, and ionizability.

[0100] In particular, the present disclosure describes how the selection and combination of one or more of the components described herein affects the functional activity of lipid nanoparticles, such as tropism, stabilization, and drug delivery efficacy. For example, the present disclosure describes how the combination of components may be better suited to the delivery of siRNA. As another example, the present disclosure describes how the combination of components may be better suited to the delivery of mRNA. As another example, the present disclosure describes how the combination of components may be better suited to the delivery of DNA.

[0101] In some embodiments, the lipid nanoparticles comprise one or more compounds described herein. In some embodiments, the lipid nanoparticles comprise one or more ionizable lipids described herein. In some embodiments, the lipid nanoparticles comprise one or more sterols described herein. In some embodiments, the lipid nanoparticles comprise one or more conjugate-linker lipids described herein. In some embodiments, the lipid nanoparticles comprise one or more phospholipids described herein.

[0102] A. Compound Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more compounds described herein.

[0103] In some embodiments, the present disclosure provides a compound of formula I: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein: L 1 and L 1’ Each of the 1-6 is alkylene, L 2 and L 2’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b - is replaced by Y 1 and Y 1’ each is independently —C(O)— or —C(O)O—; Y 2 and Y 2’ each is independently absent, —OC(O)—, —C(O)O—, or —OC(O)O—; Each of R and R' is independently hydrogen, [ka] or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L 3a and L 3a’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b - is replaced by R a and R a’ each independently is hydrogen or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; X 1 is absent or is -O-, -S-, or -NR b - and X 2 is absent or optionally substituted divalent saturated or unsaturated straight or branched C1- 12 A hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O-, -NR b - or -Cy A - is replaced by Cy Ais an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclylene, phenylene, a 3- to 7-membered saturated or partially unsaturated heterocyclylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroarylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 3 is hydrogen or an optionally substituted group selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R b are independently hydrogen or an optionally substituted C1-6 aliphatic group, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

[0104] In some embodiments, the present disclosure provides a compound of formula IA: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein L 1 , L 1’ , L 2 , L 2’ , Y 2 , Y 2’ , R, R', X 1 , X 2 , and X 3 Each of the following is as described above, both singly and in combination, in the classes and subclasses described herein, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

[0105] In some embodiments, the present disclosure provides a compound of formula IAa: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein L 1 , L 1’ , L 2 , L 2’ , L 3a , L 3a’ , Y 2 , R, R a , R a’ , X 1 , X 2 , and X 3 Each of the following is as described above, both singly and in combination, in the classes and subclasses described herein, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

[0106] In some embodiments, the present disclosure provides a compound of formula IAb: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein L 1 , L 1’ , L 2 , L 2’ , L 3a , L 3a’ , Y 2’ , R', R a , R a’ , X 1 , X 2 , and X 3 Each of the following is as described above, both singly and in combination, in the classes and subclasses described herein, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, the present disclosure provides a compound of formula IAc: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein L 1 , L 1’ , L 2 , L2’ , L 3a , L 3a’ , R a , R a’ , X 1 , X 2 , and X 3 Each of the following is as described above, both singly and in combination, in the classes and subclasses described herein, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

[0108] In some embodiments of any of the formulas described herein, L 1 is C 1-6 In some embodiments, L is alkylene. 1 is C 1-4 In some embodiments, L is alkylene. 1 is C 1-3 In some embodiments, L is alkylene. 1 is —(CH2)2—, —(CH2)3—, or —(CH2)4—.

[0109] In some embodiments of any of the formulas described herein, L 1’ is C 1-6 In some embodiments, L is alkylene. 1’ is C 1-4 In some embodiments, L is alkylene. 1’ is C 1-3 In some embodiments, L is alkylene. 1’ is —(CH2)2—, —(CH2)3—, or —(CH2)4—.

[0110] In some embodiments, L 1 and L 1’ are the same. In some embodiments, L 1 and L 1’ is different.

[0111] In some embodiments of any of the formulas described herein, L 2 is an absent or optionally substituted divalent saturated or unsaturated straight or branched C 1-10a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 2 is absent. 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 2-7 In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-5 In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-3 In some embodiments, L 2 is an optionally substituted divalent saturated straight-chain or branched C 1-10 In some embodiments, L 2 is an optionally substituted divalent saturated straight-chain or branched C 2-7 In some embodiments, L 2 is an optionally substituted divalent saturated straight-chain or branched C 1-5 In some embodiments, L 2 is an optionally substituted divalent saturated straight-chain or branched C 1-3 In some embodiments, L 2 is —(CH)—. In some embodiments, L 2 is —(CH2)4—, —(CH2)6—, or —(CH2)7—.

[0112] In some embodiments of any of the formulas described herein, L 2’ is an absent or optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 2’ is absent. 2’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 2’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 In some embodiments, L 2’ is an optionally substituted divalent saturated or unsaturated straight or branched C 2-7 In some embodiments, L 2’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-5 In some embodiments, L 2’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-3 In some embodiments, L 2’ is an optionally substituted divalent saturated straight-chain or branched C 1-10 In some embodiments, L 2’ is an optionally substituted divalent saturated straight-chain or branched C 2-7 In some embodiments, L 2’ is an optionally substituted divalent saturated straight-chain or branched C 1-5 In some embodiments, L 2’ is an optionally substituted divalent saturated straight-chain or branched C 1-3 In some embodiments, L 2’ is —(CH)—. In some embodiments, L2’ is —(CH2)4—, —(CH2)6—, or —(CH2)7—.

[0113] In some embodiments, L 2 and L 2’ are the same. In some embodiments, L 2 and L 2’ is different.

[0114] In some embodiments of any of the formulas described herein, Y 1 is —C(O)— or —C(O)O—. In some embodiments, Y 1 is —C(O)—. In some embodiments, Y 1 is -C(O)O-. Y 1 When is -C(O)O-, Y 1 The carbonyl of is bonded to the oxygen shown in formula I, and Y 1 The oxygen in the carbonate moiety (e.g., -OC(O)-OL) 2 -) is formed, L 2 It will be understood that the

[0115] In some embodiments of any of the formulas described herein, Y 1’ is —C(O)— or —C(O)O—. In some embodiments, Y 1’ is —C(O)—. In some embodiments, Y 1’ is -C(O)O-. Y 1’ When is -C(O)O-, Y 1’ The carbonyl of is bonded to the oxygen shown in formula I, and Y 1’ The oxygen in the carbonate moiety (e.g., -OC(O)-OL) 2’ -) is formed, L 2’ It will be understood that the

[0116] In some embodiments, Y 1 and Y 1’ are the same. In some embodiments, Y 1 and Y 1’ is different.

[0117] In some embodiments of any of the formulas described herein, Y 2 is absent, -OC(O)-, -C(O)O-, or -OC(O)O-. In some embodiments, Y 2 In some embodiments, Y is absent. 2 is —OC(O)—, —C(O)O—, or —OC(O)O—. In some embodiments, Y 2 is —OC(O)— or —C(O)O—. In some embodiments, Y 2 is -OC(O)-. In some embodiments, Y 2 is —C(O)O—. In some embodiments, Y 2 is -OC(O)O-.

[0118] In some embodiments of any of the formulas described herein, Y 2’ is absent, -OC(O)-, -C(O)O-, or -OC(O)O-. In some embodiments, Y 2’ In some embodiments, Y is absent. 2’ is —OC(O)—, —C(O)O—, or —OC(O)O—. In some embodiments, Y 2’ is —OC(O)— or —C(O)O—. In some embodiments, Y 2’ is -OC(O)-. In some embodiments, Y 2’ is —C(O)O—. In some embodiments, Y 2’ is -OC(O)O-.

[0119] In some embodiments, Y 2 and Y 2’ are the same. In some embodiments, Y 2 and Y 2’ is different.

[0120] In some embodiments of any of the formulas described herein, R is hydrogen, [ka] or C 6-20In some embodiments, R is an optionally substituted group selected from aliphatic, 3-12 membered saturated or partially unsaturated carbocyclyl, 7-12 membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl. [ka] In some embodiments, R is optionally substituted C 6-20 In some embodiments, R is an optionally substituted C 9-20 In some embodiments, R is an optionally substituted C 15-20 In some embodiments, R is C 6-20 In some embodiments, R is C 9-20 In some embodiments, R is C 15-20 In some embodiments, R is C 6-20 In some embodiments, R is C 9-20 In some embodiments, R is C 15-20 In some embodiments, R is C 6-20 In some embodiments, R is C 9-20 In some embodiments, R is C 15-20 It is alkenyl.

[0121] In some embodiments of any of the formulas described herein, R' is hydrogen, [ka] or C 6-20R' is an optionally substituted group selected from aliphatic, 3-12 membered saturated or partially unsaturated carbocyclyl, 7-12 membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl. In some embodiments, R' is [ka] In some embodiments, R' is optionally substituted C 6-20 In some embodiments, R' is an optionally substituted C 9-20 In some embodiments, R' is an optionally substituted C 15-20 In some embodiments, R' is C 6-20 In some embodiments, R' is C 9-20 In some embodiments, R' is C 15-20 In some embodiments, R' is C 6-20 In some embodiments, R' is C 9-20 In some embodiments, R' is C 15-20 In some embodiments, R' is C 6-20 In some embodiments, R' is C 9-20 In some embodiments, R' is C 15-20 It is alkenyl.

[0122] In some embodiments, R and R' are the same. In some embodiments, R and R' are different.

[0123] In some embodiments, -L 2 -Y 2 -R and -L 2’ -Y 2’ -each occurrence of R' is independently [ka] is.

[0124] In some embodiments of any of the formulas described herein, each L 3a are independently absent or optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 3a is absent. 3a is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 3a is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 It is a hydrocarbon chain.

[0125] In some embodiments of any of the formulas described herein, each L 3a’ are independently absent or optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 3a’ is absent. 3a’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b In some embodiments, L 3a’ is an optionally substituted divalent saturated or unsaturated straight or branched C 1-10 It is a hydrocarbon chain.

[0126] In some embodiments, L 3a and L 3a’ are the same. In some embodiments, L3a and L 3a’ is different.

[0127] In some embodiments of any of the formulas described herein, each R a are independently hydrogen or C 6-20 is an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl. a is an optionally substituted C 6-20 In some embodiments, R a is an optionally substituted C 6-12 In some embodiments, R a is an optionally substituted C 7-9 In some embodiments, R a is C 6-20 In some embodiments, R a is C 6-12 In some embodiments, R a is C 7-9 In some embodiments, R a is C 6-20 In some embodiments, R a is C 6-12 In some embodiments, R a is C 7-9 In some embodiments, R a is C 6-20 In some embodiments, R is alkenyl. a is C 6-12 In some embodiments, R is alkenyl. a is C 7-9 It is alkenyl.

[0128] In some embodiments of any of the formulas described herein, each R a’ are independently hydrogen or C6-20 is an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl. a’ is an optionally substituted C 6-20 In some embodiments, R a’ is an optionally substituted C 6-12 In some embodiments, R a’ is an optionally substituted C 7-9 In some embodiments, R a’ is C 6-20 In some embodiments, R a’ is C 6-12 In some embodiments, R a’ is C 7-9 In some embodiments, R a’ is C 6-20 In some embodiments, R a’ is C 6-12 In some embodiments, R a’ is C 7-9 In some embodiments, R a’ is C 6-20 In some embodiments, R is alkenyl. a’ is C 6-12 In some embodiments, R is alkenyl. a’ is C 7-9 It is alkenyl.

[0129] In some embodiments, R a and R a’ are the same. In some embodiments, R a and R a’ is different.

[0130] In some embodiments, -L 3a -R a and -L 3a’ -Ra’ each of which independently [ka] is.

[0131] In some embodiments of any of the formulas described herein, X 1 is absent, -O-, -S-, or -NR b In some embodiments, X 1 In some embodiments, X is absent. 1 is -O-, -S-, or -NR b In some embodiments, X 1 is —O—. In some embodiments, X 1 is -S-. In some embodiments, X 1 is -NR b -It is.

[0132] In some embodiments of any of the formulas described herein, X 2 is an absent or optionally substituted divalent saturated or unsaturated straight or branched C 1-12 A hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O-, -NR b - or -Cy A In some embodiments, X 2 In some embodiments, X is absent. 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-12 A hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O-, -NR b - or -Cy A In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-12 a hydrocarbon chain, wherein one to three methylene units are optionally and independently —NR b In some embodiments, X 2is an optionally substituted divalent saturated or unsaturated straight or branched C 1-6 a hydrocarbon chain, wherein one methylene unit is optionally and independently selected from the group consisting of -NR b In some embodiments, X 2 is an optionally substituted divalent saturated straight-chain or branched C 1-6 a hydrocarbon chain, wherein one methylene unit is optionally and independently selected from the group consisting of -NR b In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-12 a hydrocarbon chain, wherein 1 to 3 methylene units are optionally and independently -Cy A In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-6 a hydrocarbon chain, wherein one methylene unit is optionally and independently -Cy A In some embodiments, X 2 is an optionally substituted divalent saturated straight-chain or branched C 1-6 a hydrocarbon chain, wherein one methylene unit is optionally and independently -Cy A In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-12 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated or unsaturated straight or branched C 1-6 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated straight-chain or branched C 1-12 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated straight-chain or branched C 1-6 It is a hydrocarbon chain.

[0133] In some embodiments of any of the formulas described herein, Cy Ais an optionally substituted divalent ring selected from 3-7 membered saturated or partially unsaturated carbocyclylene, phenylene, 3-7 membered saturated or partially unsaturated heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5-6 membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. A is a 3- to 7-membered saturated or partially unsaturated heterocyclylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. A is a 5-6 membered saturated or partially unsaturated heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. A is a 5-6 membered saturated or partially unsaturated heterocyclylene having 1-2 nitrogen atoms. A is a 5-6 membered saturated heterocyclylene having 1-2 nitrogen atoms. A is a pyrrolidine or piperidine ring.

[0134] In some embodiments of any of the formulas described herein, X 3 is hydrogen or an optionally substituted group selected from a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is an optionally substituted 5-6 membered saturated or partially unsaturated heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.3 is an optionally substituted 5-6 membered saturated or partially unsaturated heterocyclyl having 1-2 nitrogens. 3 is an optionally substituted 5-6 membered saturated heterocyclyl having 1-2 nitrogens (e.g., having 1-2 nitrogens and one or more C 1-6 In some embodiments, X is a 5-6 membered saturated heterocyclyl optionally substituted with alkyl. 3 is a 3-7 membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-6 membered saturated or partially unsaturated heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-6 membered saturated or partially unsaturated heterocyclyl having 1-2 nitrogens. 3 has one or two nitrogen atoms and one or more C 1-6 In some embodiments, X is a 5-6 membered saturated heterocyclyl optionally substituted with alkyl. 3 is one or more C 1-6 It is pyrrolidine or piperidine optionally substituted with alkyl (eg, methyl or ethyl).

[0135] In some embodiments, -X 2 -X 3 teeth, [ka] is.

[0136] In some embodiments of any of the formulas described herein, each R b are independently hydrogen or optionally substituted C 1-6 In some embodiments, R b is hydrogen. In some embodiments, R b is an optionally substituted C 1-6In some embodiments, R b is an optionally substituted C 1-3 In some embodiments, R b is C 1-6 In some embodiments, R b is C 1-3 In some embodiments, R b is -CH3 or -CH2CH3.

[0137] It will be understood that "[compound / formula] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof," as used herein, refers to i) the respective compound or formula, or ii) a pharmaceutically acceptable salt of an N-oxide of such compound or formula.

[0138] In some embodiments, the disclosure provides a compound selected from Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] or a pharmaceutically acceptable salt thereof.

[0139] Unless otherwise specified or prohibited by the preceding definitions of any of Formulas I, IA, IAa, IAb, and IAc, the variables L, ... 1 , L 1’ , L 2 , L 2’ , Y 1 , Y 1’ , Y 2 , Y 2’ , R, R', L 3a , L 3a’ , R a , R a’ , Cy A , R b , X 1 , X 2 , and X 3 It will be understood that the above embodiments apply to any compound of formula I, IA, IAa, IAb, and IAc, both singly and in combination.

[0140] In some embodiments, provided compounds are compounds of any of Formula I, IA, IAa, IAb, and IAc, or pharmaceutically acceptable salts thereof.

[0141] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include a reference to a salt thereof, unless otherwise indicated.

[0142] It will be understood that throughout this disclosure, unless otherwise indicated, references to compounds of formula I are intended to include any of formulas IA, IAa, IAb, and IAc, as well as species of compounds of such formulas disclosed herein.

[0143] In some embodiments, the present disclosure encompasses the recognition that provided compounds exhibit certain desirable characteristics, e.g., compared to a reference compound or other known compounds. For example, in some embodiments, provided compounds exhibit more potent delivery to various cell types in one or more experiments described herein and / or have one or more other characteristics that make them more suitable for delivery of cargo, such as therapeutic or prophylactic agents, than other known compounds. Without wishing to be bound by any particular theory, the present disclosure encompasses the recognition that provided compounds that include a trivalent core (e.g., derived from a secondary triol) feature exhibit certain more desirable characteristics (e.g., more potent delivery to various cell types in one or more experiments described herein) than corresponding compounds lacking the same trivalent core feature.

[0144] B. Preparation of the Provided Compounds The compounds provided can generally be made by the processes described in the schemes and examples that follow.

[0145] C. Ionizable lipids Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more ionizable lipids described herein.

[0146] In particular, it has been surprisingly discovered that different ratios of ionizable lipids affect one or more functional activities, such as the desired tropism, stabilization, and drug delivery efficacy, of the compositions, preparations, nanoparticles, and / or nanomaterials described herein. For example, the present disclosure demonstrates the surprising discovery that amounts of ionizable lipids different from those described in the art (see, e.g., U.S. Pat. No. 8,058,069 B2 or, e.g., U.S. Pat. No. 9,364,435, the entire contents of both of which are incorporated herein by reference) are important and / or affect one or more functional activities of the compositions, preparations, nanoparticles, and / or nanomaterials described herein. For example, in some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials having an ionizable lipid content of about 50 mol percent or less, based on the total moles of the lipid nanoparticle components, have been found to be useful and / or important for the functional activities of the lipid nanoparticles, such as the desired tropism, stabilization, and drug delivery efficacy, described herein.

[0147] In some embodiments, the ionizable lipid may comprise an amine-containing group on the head group. In some embodiments, the ionizable lipid is or comprises a compound described herein. In some embodiments, the ionizable lipid is present in the lipid nanoparticle (LNP) preparation at about 30 molar percent to about 70 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 33 molar percent to about 60 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 34 molar percent to about 55 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 33 molar percent to about 51 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 34.7 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 50 molar percent, based on the total moles of the lipid nanoparticle components.

[0148] In particular, in some embodiments, the lipid nanoparticle composition comprises an ionizable lipid. In some embodiments, the lipid nanoparticle preparation comprises an ionizable lipid, a phospholipid, a conjugate-linker lipid, and cholesterol. In some embodiments, the ionizable lipid is or comprises a structure according to a compound described herein. In some embodiments, the ionizable lipid is present in the LNP preparation at about 30 mole percent to about 70 mole percent, based on the total moles of the lipid nanoparticle components.

[0149] D. Sterols Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more sterols described herein.

[0150] In some embodiments, the sterol is cholesterol, or a variant or derivative thereof. In some embodiments, the cholesterol is modified. In some embodiments, the cholesterol is oxidized cholesterol. In some embodiments, the cholesterol is esterified cholesterol. Unmodified cholesterol may be enzymatically acted upon to form variants with oxidized side chains or rings. In some embodiments, cholesterol may be oxidized on the beta-ring structure or hydrocarbon tail structure. In some embodiments, the sterol is a phytosterol. Exemplary sterols contemplated for use in the lipid nanoparticles of the present disclosure include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, side-chain oxidized cholesterol may enhance cargo delivery compared to other cholesterol variants. In some embodiments, the cholesterol is unmodified cholesterol.

[0151] In some embodiments, the LNP composition comprises about 20 mol percent to about 50 mol percent sterol. In some embodiments, the LNP composition comprises about 38 mol percent sterol. In some embodiments, the LNP composition comprises about 38.5 mol percent sterol. In some embodiments, the LNP composition comprises about 33.8 mol percent cholesterol.

[0152] E. Conjugate-Linker Lipid Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more conjugate-linker lipids described herein.

[0153] In some embodiments, the conjugate-linker lipid is or includes a polyethylene glycol (PEG) lipid or a PEG-modified lipid. In some embodiments, PEG or a PEG-modified lipid may alternatively be referred to as a PEGylated lipid or a PEG lipid. The inclusion of a PEGylated lipid may be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible, in that the PEG moiety is gradually released into the blood circulation. Exemplary PEG lipids include, but are not limited to, PEG conjugated to a saturated or unsaturated alkyl chain having a length of C6-C20. PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, in some embodiments, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE lipid.

[0154] In some embodiments, the conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises dimyristylglycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxypolyethylene glycol (DPG-PEG), or 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, the conjugate-linker lipid has an average molecular weight of about 500 Da to about 5000 Da. In some embodiments, the conjugate-linker lipid has an average molecular weight of about 2000 Da. In some embodiments, the LNP composition comprises about 0 mol percent to about 5 mol percent of the conjugate-linker lipid. In some embodiments, the LNP composition comprises about 1.5 mol percent of the conjugate-linker lipid. In some embodiments, the LNP composition comprises about 3 mol percent of the conjugate-linker lipid.

[0155] F. Phospholipids In particular, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials comprising one or more phospholipids described herein. In some embodiments, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials comprising one or more (poly)unsaturated lipids.

[0156] In some embodiments, one or more phospholipids may be assembled into one or more lipid bilayers. In some embodiments, one or more phospholipids may comprise a phospholipid moiety. In some embodiments, one or more phospholipids may comprise one or more fatty acid moieties. In some embodiments, one or more phospholipids may comprise a phospholipid moiety and one or more fatty acid moieties. In some embodiments, the phospholipid moiety includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. In some embodiments, the fatty acid moiety includes, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also contemplated are unnatural species, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cell recognition, or for conjugating nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes).

[0157] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero -3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl l-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl In some embodiments, the phospholipid may be DSPC, DMPC, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC.

[0158] In some embodiments, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

[0159] G.Diameter Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

[0160] In some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

[0161] H. Polydispersity In particular, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI of about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0162] In some embodiments, the lipid nanoparticles described herein have a PDI of about 0.01 to about 0.3. In some embodiments, the lipid nanoparticles described herein have a PDI of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have a PDI of about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0163] I. Encapsulation Efficiency Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials, wherein the encapsulation efficiency of the provided compositions, preparations, nanoparticles, and / or nanomaterials is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles, and / or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles, and / or nanomaterials described herein is about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles, and / or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0164] In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0165] J.pKa Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having a pKa of about 5 to about 9. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a pKa of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the foregoing values. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a pKa of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the foregoing values.

[0166] In some embodiments, the lipid nanoparticles described herein have a pKa of about 5 to about 9. In some embodiments, the lipid nanoparticles described herein have a pKa of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the foregoing values. In some embodiments, the lipid nanoparticles described herein have a pKa of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the foregoing values.

[0167] II. Exemplary LNP Preparations The present invention provides compositions, preparations, nanoparticles, and / or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle preparation comprises about 30 mole percent to about 70 mole percent ionizable lipid, about 5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent cholesterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

[0168] In some embodiments, the lipid nanoparticle preparation comprises about 45 mole percent ionizable lipid, about 9 mole percent phospholipid, about 44 mole percent cholesterol, and about 2 mole percent conjugate-linker lipid. In some embodiments, the lipid nanoparticle preparation comprises about 50 mole percent ionizable lipid, about 9 mole percent phospholipid, about 38 mole percent cholesterol, and about 3 mole percent conjugate-linker lipid.

[0169] In some embodiments, the lipid nanoparticle preparation comprises about 40 mole percent to about 60 mole percent ionizable lipid of any provided compound, about 5 mole percent to about 15 mole percent 1-2-distearoyl-sn-glycero-3-phosphocholine, about 1 mole percent to about 5 mole percent C14PEG2000, and about 30 mole percent to about 47 mole percent cholesterol, based on the total moles of these four components.

[0170] In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 2:1 to 50:1. In some embodiments, the LNP preparation comprises a mass ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, ionizable lipids, cholesterol, lipid-PEG, and phospholipids:mRNA in a mass ratio of about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, or about 50:1. In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of about 11.7:1 to 19:1 (ionizable lipids, cholesterol, lipid-PEG, and phospholipids):mRNA.

[0171] In some embodiments, the lipid nanoparticle preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 2:1 to 50:1. In some embodiments, the LNP preparation comprises a mass ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67 ionizable lipids, cholesterol, lipid-PEG, and phospholipids:mRNA in a mass ratio of about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, or about 50:1. In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of about 11.7:1 to 19:1 (ionizable lipids, cholesterol, lipid-PEG, and phospholipids):mRNA.

[0172] In some embodiments, the LNP preparation comprises one or more nucleic acids, such as RNA. In some embodiments, the one or more nucleic acids (e.g., RNA), lipids, and amounts thereof in the LNP preparation may be selected to provide a particular N / P ratio. The N / P ratio can be selected from about 1 to about 30. The N / P ratio can be selected from about 2 to about 12. In some embodiments, the N / P ratio is about 0.1 to about 50. In some embodiments, the N / P ratio is about 2 to about 8. In some embodiments, the N / P ratio is about 2 to about 15, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 15, about 3 to about 10, about 3 to about 8, about 3 to about 6, about 4 to about 15, about 4 to about 10, about 4 to about 8, about 4 to about 6, or about 5 to about 7. In some embodiments, the N / P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, or about 10. In some embodiments, the N / P ratio is about 4 to about 6. In some embodiments, the N / P ratio is about 4, about 4.5, about 5, about 5.5, or about 6.

[0173] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms in a lipid (or lipids) (e.g., within a physiological pH range) to phosphate groups in a nucleic acid molecular entity (or nucleic acid molecular entities), e.g., in a nanoparticle composition comprising a lipid component and RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH ranges of different cellular compartments (e.g., organs, tissues, and cells) and bodily fluids (e.g., blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, the physiological pH range refers to the pH range of blood in mammals, e.g., about 7.35 to about 7.45. Similarly, for phosphate charge neutralizing agents having one or more ionizable nitrogen atoms, the N / P ratio can refer to the molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizing agent to phosphate groups in the nucleic acid. In some embodiments, ionizable nitrogen atoms refer to nitrogen atoms that are ionizable within a pH range of 5 to 14.

[0174] III. Pharmaceutical Compositions The present invention provides compositions, preparations, nanoparticles, and / or nanomaterials, including pharmaceutical compositions. In particular, in some embodiments, the pharmaceutical compositions comprise the lipid nanoparticles and lipid nanoparticle preparations described herein. For example, in some embodiments, the lipid nanoparticles and lipid nanoparticle preparations described herein may be formulated in whole or in part as pharmaceutical compositions.

[0175] In some embodiments, a pharmaceutical composition may comprise one or more nanoparticle compositions described herein. For example, a pharmaceutical composition may comprise one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents, including, but not limited to, different types of one or more nucleic acids, or may encode different agents. In some embodiments, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or auxiliary ingredients, including, but not limited to, a pharmaceutically acceptable carrier.

[0176] The pharmaceutical composition can be administered to a subject. In some embodiments, the pharmaceutical composition is administered as described herein. In some in vivo approaches, the nanoparticle composition disclosed herein is administered to a subject in a therapeutically effective amount as described herein.

[0177] In some embodiments, those skilled in the art will be able to devise appropriate dosage levels and administration regimens using the pharmaceutical compositions described herein for the treatment of various conditions in various patients, taking into account the recipient's therapeutic background, age, and general health. For example, in some embodiments, the selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In some embodiments, dosage levels of about 0.001 mg to about 5 mg of nucleic acid per kg of body weight are typically administered to mammals, with each dose administered. More specifically, in some embodiments, the preferred dosage of nucleic acid in the disclosed nanoparticles is about 0.1 mg / kg to about 1.0 mg / kg. The disclosed nanoparticles are typically administered to mammals at dosage levels of about 0.2 mg to about 100 mg of the four components (ionizable lipid, cholesterol, conjugate-linker conjugate, and phospholipid) per kg of body weight. More specifically, in some embodiments, the preferred dosage of the disclosed nanoparticles is about 0.5 mg / kg to about 5 mg / kg of the four components per kg of body weight.

[0178] In some embodiments, the pharmaceutical compositions described herein are administered locally, for example, by direct injection into the site to be treated. Typically, the injection causes a local increase in the concentration of the composition that is higher than that achieved by systemic administration. In some embodiments, the pharmaceutical compositions described herein can be combined with the matrices described herein to help create an increase in the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide from the site to be treated.

[0179] A. Preparations for Parenteral Administration In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein, including those containing lipid nanoparticles, are administered parenterally in aqueous solution. In some embodiments, the preparations may also be in the form of a suspension or emulsion. Generally, pharmaceutical compositions containing an effective amount of lipid nanoparticles are provided, optionally including pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally include one or more additives, such as diluents, sterile water, buffered saline solutions (e.g., Tris-HCl, acetate, phosphate) of various buffer contents, pH, and ionic strength; detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.The formulation can be lyophilized and redissolved / resuspended immediately before use.The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0180] B. Controlled Delivery Polymer Matrix In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein can also be administered in controlled-release formulations. In some embodiments, controlled-release polymeric devices can be fabricated for extended systemic release after implantation of a polymeric device (e.g., rod, cylinder, film, disk, etc.) or injection (e.g., microparticle). In some embodiments, the matrix can be in the form of microparticles, such as microspheres. In some embodiments, the drug is dispersed within a solid polymer matrix or microcapsules. In some embodiments, the core is of a different material than the polymer shell of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, the peptide is dispersed or suspended in the core, which can be liquid or solid in nature, of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. Unless specifically defined otherwise herein, microparticles, microspheres, and microcapsules are used interchangeably. In some embodiments, the polymers can be cast as thin slabs or films ranging from nanometers to 4 centimeters, as powders produced by milling or other standard techniques, or even as gels, such as hydrogels.

[0181] In some embodiments, non-biodegradable matrices are used to deliver the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, biodegradable matrices are used to deliver the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, biodegradable matrices are preferred. In some embodiments, biodegradable matrices comprise natural or synthetic polymers. In some embodiments, synthetic polymers are preferred due to better characterization of degradation and release profiles. In some embodiments, the polymer is selected based on the desired period of release. In some embodiments, linear release may be most useful, while in other embodiments, pulsed or "bulk release" may provide more effective results. In some embodiments, the polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight), optionally crosslinked with multivalent ions or polymers.

[0182] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, such as those described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988), the entire disclosures of which are incorporated herein by reference.

[0183] In some embodiments, the described compositions, preparations, nanoparticles, and / or nanomaterials can be formulated for local release to treat the area of ​​implantation or injection, and will typically deliver a much lower dose than for whole body treatment or systemic delivery. They may be implanted or injected subcutaneously, into muscle, fat, or swallowed.

[0184] C. Cargo Among other things, the present invention provides compositions, preparations, nanoparticles, and / or nanomaterials comprising the cargo described herein. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic or prophylactic agent for delivery to a subject. In some embodiments, the therapeutic or prophylactic agent is encapsulated by the lipid nanoparticles. In some embodiments, the lipid nanoparticles are loaded with one or more nucleic acids.

[0185] D. Therapeutic and / or Prophylactic Agents The cargo delivered via the LNP composition can be a biologically active agent. In some embodiments, the cargo can be an mRNA, a guide RNA (gRNA), a nucleic acid, an RNA-guided DNA binder, an expression vector, a template nucleic acid, an antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof), cholesterol, a hormone, a peptide, a protein, a chemotherapeutic agent and other types of antineoplastic agents, a small molecule drug, a vitamin, a cofactor, a nucleoside, a nucleotide, an oligonucleotide, an enzymatic nucleic acid, an antisense nucleic acid, a triplex-forming oligonucleotide, an antisense DNA or RNA composition, a chimeric DNA:RNA composition, an allozyme, an aptamer, a ribozyme, decoys and analogs thereof, a plasmid, and other types of vectors. The biologically active agent may be or include one or more biologically active agents, such as small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), "self-replicating RNA" molecules (which encode replicase enzyme activity and can induce its own replication or amplification in vivo), peptide nucleic acids (PNAs), locked nucleic acid ribonucleotides (LNAs), morpholino nucleotides, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), sisiRNAs (small internal segmented interfering RNAs), and iRNAs (asymmetric interfering RNAs). The above list of biologically active agents is illustrative only and is not intended to be limiting. Such compounds may be purified or partially purified, naturally occurring or synthetic, and chemically modified.

[0186] The cargo delivered via the LNP composition can be RNA, such as an mRNA molecule encoding a protein of interest. For example, in some embodiments, mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA binder, or a Cas nuclease is described herein. LNP compositions are provided that include a Cas nuclease mRNA, e.g., a Class 2 Cas nuclease mRNA, that enables intracellular expression of a Class 2 Cas nuclease, such as Cas9 or a Cpfl protein. Additionally, the cargo can contain one or more guide RNAs or nucleic acids encoding the guide RNAs. For example, a template nucleic acid for repair or recombination can also be included in the composition, or the template nucleic acid can be used in the methods described herein. In some embodiments, the cargo optionally includes an mRNA encoding Streptococcus pyogenes Cas9 and an S. pyogenes gRNA. In some embodiments, the cargo optionally includes an mRNA encoding Neisseria meningitidis Cas9 and an nme gRNA.

[0187] "mRNA" refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and amino-acylated tRNAs). mRNA can contain a phosphate-sugar backbone that includes ribose residues or analogs thereof, e.g., 2'-methoxyribose residues. In some embodiments, the sugars of the mRNA phosphate sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, mRNA does not contain substantial amounts of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA can contain modified uridines at some or all of its uridine positions.

[0188] E. CRISPR / Cas cargo In some embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise mRNA encoding an RNA-guided DNA binder, such as a Cas nuclease. In certain embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9.

[0189] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavase / nickases and their inactivated forms (dCas DNA binders). "Cas nuclease," as used herein, encompasses Cas cleavage enzymes, Cas nickases, and dCas DNA binders. Cas cleavage enzymes / nickases and dCas DNA binders include the Csm or Cmr complex of a type III CRISPR system, its Cas1O, Csml, or Cmr2 subunit, the Cascade complex of a type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-chain polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavages / nickases that also have RNA-guided DNA cleavage or nickase activity (e.g., H840A, D10A, or N863A variants), and Class 2 dCas DNA binders in which the cleavage / nickase activity has been inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. The Cpfl protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpfl sequences of Zetsche are incorporated herein by reference in their entirety, see, e.g., Tables S1 and S3 of Zetsche.See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015), the contents of which are incorporated herein by reference in their entirety.

[0190] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA comprising an RNA-guided DNA-binding agent, e.g., a Cas nuclease, e.g., a Cas cleavase, a Cas nickase, or a dCas DNA-binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA-binding agent, such as Cas9, to a target sequence, the guide RNA hybridizes to the target sequence, and an agent binds to the target sequence, where the agent is a cleavase or nickase and can perform cleavage or nicking after binding.

[0191] In some embodiments, the cargo of the LNP composition comprises at least one guide RNA comprising a guide sequence that guides an RNA-guided DNA-binding agent, which may be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. The gRNA may guide the Cas nuclease or a Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a Class 2 Cas nuclease, thereby providing specificity for cleavage. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type V CRISPR / Cas complex, such as a Cpfl / guide RNA complex. The Cas nuclease and cognate gRNA may be paired. The gRNA scaffold structure that pairs with each Class 2 Cas nuclease varies depending on the specific CRISPR / Cas system.

[0192] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). Guide RNA may include modified RNA as described herein. CrRNA and trRNA may associate as a single-stranded RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence with modifications or mutations compared to the naturally occurring sequence.

[0193] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence may be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can also be used as a guide. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain one, two, three, or four mismatches, and the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain one to four mismatches, and the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, and the guide sequence comprises 20 nucleotides.

[0194] The target sequence of an RNA-guided DNA-binding protein, such as a Cas protein, includes both the positive and negative strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence) because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide RNA can be directed to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence without the PAM) except for the substitution of U for T in the guide sequence.

[0195] The length of the targeting sequence can depend on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have varying optimal targeting sequences. Thus, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring nucleotide sequence.

[0196] In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0197] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a "Cpfl sgRNA" capable of mediating RNA-guided DNA cleavage by a Cpfl protein. In some embodiments, the gRNA comprises sufficient crRNA and tracrRNA to form an active complex with a Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA comprises sufficient crRNA to form an active complex with a Cpfl protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0198] Certain embodiments of the present invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes, which are nucleic acids that encode one or more guide RNAs.

[0199] Certain embodiments of the present disclosure also provide for delivery of adenine base editors ("ABEs") using the LNP compositions, preparations, nanoparticles, and / or nanomaterials described herein. ABEs and methods of using same are described, for example, in U.S. Patent No. 10,113,163 and U.S. Patent Publication No. 2021 / 0130805, the contents of each of which are incorporated herein by reference in their entirety.

[0200] Certain embodiments of the present disclosure also provide for delivery of cytosine base editors ("CBEs") using the LNP compositions, preparations, nanoparticles, and / or nanomaterials described herein. CBEs and methods of using same are described, for example, in U.S. Patent Nos. 10,167,457 and 9,840,699, the contents of each of which are incorporated herein by reference in their entirety.

[0201] The term "base editor (BE)" or "nucleic acid base editor (NBE)" refers to an agent comprising a polypeptide capable of making modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base in a nucleic acid. In some embodiments, the base editor is capable of deaminating a base in a DNA molecule. In some embodiments, the base editor is capable of deaminating adenine (A) in DNA. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair (e.g., a UGI domain or a dISN domain). In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair (e.g., a UGI domain or a dISN domain). The term "nucleic acid-programmed DNA-binding protein" or "napDNAbp" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, that guides the napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that has complementarity to the guide RNA. In some embodiments, the napDNAbp is a Class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, e.g., a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9).Examples of nucleic acid-programmed DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. However, it should be understood that nucleic acid-programmed DNA-binding proteins can also include nucleic acid-programmed proteins that bind to RNA. For example, napDNAbp can associate with a nucleic acid that guides the napDNAbp to RNA. Other nucleic acid-programmed DNA-binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure.

[0202] F. Modified RNA In certain embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise modified nucleic acids, including modified RNA.

[0203] Modified nucleosides or nucleotides can be present in an RNA, such as a gRNA or mRNA. For example, a gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA, accounting for the presence of one or more non-natural and / or naturally occurring components or arrangements that are used in place of or in addition to the standard A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with non-standard nucleosides or nucleotides and is referred to herein as "modified."

[0204] Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of one or more of the unlinked and / or linked phosphate oxygens in the phosphodiester backbone linkages (exemplary backbone modifications); (ii) alteration, e.g., replacement, of a component of the ribose sugar, e.g., the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (iii) wholesale replacement of a phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) modification or replacement of a naturally occurring nucleobase, including a non-canonical nucleobase (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) modification or replacement of a sugar (exemplary sugar modifications). Certain embodiments include 5'-end modifications to the mRNA, gRNA, or nucleic acid. Certain embodiments include 3'-end modifications to the mRNA, gRNA, or nucleic acid. The modified RNA may contain 5'-end and 3'-end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

[0205] Unmodified nucleic acids may be susceptible to degradation, for example, by intracellular nucleases or those found in serum. For example, nucleases can hydrolyze phosphodiester bonds of nucleic acids. Thus, in one aspect, RNAs (e.g., mRNAs, gRNAs) described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population, both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, including induction of cytokine (particularly interferon) expression and release and cell death.

[0206] Thus, in some embodiments, the RNA or nucleic acid in the compositions, preparations, nanoparticles, and / or nanomaterials of the present disclosure comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modification" and "modified," when such terms relate to the nucleic acids provided herein, preferably include at least one alteration that enhances stability and makes the RNA or nucleic acid more stable (e.g., resistant to nuclease digestion) than a wild-type or naturally occurring version of the RNA or nucleic acid. As used herein, the terms "stable" and "stability," when such terms relate to the nucleic acids of the present invention, particularly with respect to RNA, refer to, for example, increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that would normally degrade such RNA. Increased stability can include, for example, reduced susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within target cells or tissues, thereby increasing or enhancing the presence of such RNA in the target cells, tissues, subjects, and / or cytoplasm. The stabilized RNA molecules provided herein exhibit a longer half-life compared to their naturally occurring, unmodified counterparts (e.g., wild-type versions of mRNA). Also contemplated by the terms "modification" and "modified," when such terms relate to the mRNA of the LNP compositions disclosed herein, are modifications that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of a sequence that functions in the initiation of protein translation (e.g., a Kozac consensus sequence). (Kozak, M., Nucleic Acids Res 15(20):8125-48 (1987)), the entire contents of which are incorporated herein by reference.)

[0207] In some embodiments, the RNA or nucleic acid of the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein is chemically or biologically modified to make it more stable. Exemplary modifications to RNA include base depletion (e.g., by deletion or by substituting one nucleotide for another) or base modification, e.g., chemical modification of the base. As used herein, the phrase "chemical modification" includes modifications that introduce chemistry different from that found in naturally occurring RNA, e.g., covalent modifications such as the introduction of modified nucleotides (e.g., nucleotide analogs or pendant groups not naturally found in such RNA molecules).

[0208] In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more oxygens with different substituents. Furthermore, modified residues, such as those present in modified nucleic acids, may include extensive replacement of unmodified phosphate moieties with modified phosphate groups, as described herein. In some embodiments, backbone modifications of the phosphate backbone may include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphate atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the atoms or groups of atoms described above can make the phosphorus atom chiral. The stereogenic phosphorus atom may have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). The backbone can also be modified by replacing bridging oxygens (i.e., oxygens linking the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at either bridging oxygen or both bridging oxygens. The phosphate group can be replaced by a non-phosphorus-containing linking group in some backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group can include, but are not limited to, for example, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino.

[0209] G. mRNA In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials of the present disclosure comprise an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. The mRNA may comprise one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may comprise an open reading frame modified, for example, to encode a nuclear localization sequence or to use alternative codons to encode a protein.

[0210] The mRNA in the disclosed compositions, preparations, nanoparticles, and / or nanomaterials can encode, for example, a normally secreted hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest. In one embodiment of the present invention, the mRNA can optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA or improve or enhance protein production.

[0211] Additionally, suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse relationship between RNA stability and a higher number of cytidine (C) and / or uridine (U) residues has been demonstrated, with RNA lacking C and U residues being found to be stable against most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994), the entire disclosure of which is incorporated herein by reference). In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In other embodiments, the number of C and / or U residues is reduced by substituting one codon encoding a particular amino acid with another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridine. Incorporation of pseudouridine into the mRNA nucleic acid of the present invention can enhance stability and translation ability, as well as reduce immunogenicity in vivo. See, for example, Kariko, K., et al. Molecular Therapy 16(11):1833-1840 (2008), the entire contents of which are incorporated herein by reference. Substitutions and modifications to the mRNA of the present invention can be made by methods readily known to those skilled in the art.

[0212] Constraints on reducing the number of C and U residues in a sequence will likely be greater in the coding region of an mRNA compared to untranslated regions (i.e., it will likely not be possible to eliminate all of the C and U residues present in a message while still retaining the capacity of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code presents opportunities to reduce the number of C and / or U residues present in a sequence while maintaining the same coding capacity (i.e., depending on which amino acids are encoded by the codons, several different possibilities for modification of an RNA sequence may be possible).

[0213] The term modified also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the invention (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), alteration of the 3' or 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and the inclusion of elements that alter the structure of the mRNA molecule (e.g., form secondary structures).

[0214] Poly-A tails are believed to stabilize natural messengers. Thus, in one embodiment, long poly-A tails can be added to mRNA molecules, thus making the mRNA more stable. Poly-A tails can be added using a variety of art-recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed mRNA using poly-A polymerase (Yokoe, et al. Nature 1999, 11, 141-143, the entire contents of which are incorporated herein by reference). Biotechnology. 1996;14:(1252-1256). Transcription vectors can also encode long polyA tails. In addition, polyA tails can be added by direct transcription from PCR products. In one embodiment, the length of the polyA tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In one embodiment, the length of the polyA tail is adjusted to control the stability of the modified mRNA molecules of the present invention and, therefore, protein transcription. For example, since the length of the polyA tail can affect the half-life of the mRNA molecule, the length of the polyA tail can be adjusted to alter the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in cells. In one embodiment, the stabilized mRNA molecule is sufficiently resistant to in vivo degradation (e.g., by nucleases) that it can be delivered to target cells without the use of a transfer vehicle.

[0215] In some embodiments, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences not naturally found in wild-type mRNA. In one embodiment, for modification, 3' and / or 5' flanking sequences naturally adjacent to the mRNA and encoding a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein. For example, to increase the stability of a sense mRNA molecule, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or a citric acid cycle enzyme) can be incorporated into the 3' and / or 5' region of the sense mRNA nucleic acid molecule. See, e.g., US2003 / 0083272, the entire contents of which are incorporated herein by reference. A more detailed description of mRNA modifications can be found in US2017 / 0210698A1, pages 57-68, the entire contents of which are incorporated herein by reference.

[0216] H. Template Nucleic Acid The compositions, preparations, nanoparticles, and / or nanomaterials, and methods disclosed herein may include a template nucleic acid. The template may be used to modify or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein, such as a Cas nuclease, e.g., a class 2 Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In some embodiments, two or more templates may be provided to enable editing at two or more target sites. For example, different templates may be provided for editing a single gene in a cell or two different genes in a cell.

[0217] In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination may result in the integration of the template sequence or a portion of the template sequence into a target nucleic acid molecule. In some embodiments, the template may be used in homology-directed repair, which involves DNA strand invasion at the nucleic acid cleavage site. In some embodiments, homology-directed repair may result in the inclusion of the template sequence in an edited target nucleic acid molecule. In some embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence does not share similarity with the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is integrated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.

[0218] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. The template sequence may also, or alternatively, correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or a sequence that is at a location different from its native location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell.

[0219] In some embodiments, the endogenous sequence may be a chromosomal sequence or an extrachromosomal sequence, hi some embodiments, the endogenous sequence may be a plasmid sequence of the cell.

[0220] In some embodiments, the template contains ssDNA or dsDNA containing flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0221] In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, nucleic acids are purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein). In some embodiments, nucleic acids are purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, nucleic acids are purified by tangential flow filtration (TFF).

[0222] IV. Methods of Producing LNPs Methods for producing lipid nanoparticles are known in the art. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described are produced using microfluidics. For example, exemplary methods for forming lipid nanoparticles using microfluidics are described by Leung, AKK, et al., J Phys Chem, 116: 18440-18450 (2012); Chen, D., et al., J Am Chem Soc, 134: 6947-6951 (2012); and Belliveau, NM, et al., Molecular Therapy-Nucleic Acids, 1: e37 (2012), the entire disclosures of which are incorporated herein by reference.

[0223] Briefly, a cargo, such as those described herein, is prepared in a first buffer solution. Other lipid nanoparticle components (such as ionizable lipids, conjugate-linker lipids, cholesterol, and phospholipids) are prepared in a second buffer solution. In some embodiments, the two solutions are introduced into a microfluidic device by a syringe pump. The two solutions contact within the microfluidic device to form lipid nanoparticles that encapsulate the cargo.

[0224] Methods for screening disclosed lipid nanoparticles are described in International Patent Application No. PCT / US2018 / 058171, which is incorporated herein by reference in its entirety. In some embodiments, the screening method characterizes vehicle delivery preparations to identify preparations that have the desired tropism and deliver functional cargo to the cytoplasm of specific cells. In some embodiments, the screening method uses a functional reporter that can be detected upon delivery to a cell. For example, detecting a functional reporter within a cell indicates that the LNP preparation delivers functional cargo to the cell. Notably, in some embodiments, each different delivery vehicle formulation contains a chemical composition identifier that keeps track of the chemical composition specific to each different delivery vehicle formulation. In some embodiments, the chemical composition identifier is a nucleic acid barcode. In some embodiments, sequencing the nucleic acid barcode identifies the chemical composition of the delivery vehicle that delivered the barcode, as the sequence of the nucleic acid barcode is paired with the chemical components used to formulate the loaded LNP preparation. Representative barcodes include, but are not limited to, those described in Sago, 2018 PNAS and Sago, JACS 2018, the entire disclosures of which are incorporated herein by reference. Representative reporters include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotypic modifiers. DNA (genome and DNA barcode) can be isolated using QuickExtract (Lucigen) and sequenced using Illumina MiniSeq as described by Sago et al. PNAS 2018, Sago et al. JACS 2018, and Sago, Lokugamage et al. Nano Letters 2018, the entire disclosures of which are incorporated herein by reference.

[0225] V. How to Use Among other things, the present disclosure describes methods of using the compositions, preparations, nanoparticles, and / or nanomaterials described herein. For example, in some embodiments, the present disclosure describes methods of delivering cargo to specific cells, tissues, or organs using the compositions, preparations, nanoparticles, and / or nanomaterials described herein. As another example, in some embodiments, the present disclosure describes methods of treating and / or slowing and / or halting the progression of a disease or disorder using the compositions, preparations, nanoparticles, and / or nanomaterials described herein. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein are for use in medicine.

[0226] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein deliver a therapeutic or prophylactic agent to specific cells or organs in a subject in need thereof. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials deliver a therapeutic or prophylactic agent to specific cells or organs in a subject in need thereof in the absence of a targeting ligand. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials are useful for treating or preventing disease in a subject in need thereof.

[0227] A. Methods for Delivering Cargo to Cells, Tissues, or Organs In particular, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are targeted to a particular type or class of cell (e.g., cells of a particular organ or system), tissue, cell population, or organ. In some embodiments, the present disclosure provides methods of delivering one or more cargoes described herein to a subject in need thereof. In some embodiments, such methods include in vivo and / or in vitro delivery. In some embodiments, such methods include in vivo delivery. In some embodiments, such methods include in vitro delivery. In some embodiments, the present disclosure provides methods of delivering one or more therapeutic and / or prophylactic nucleic acids to a subject in need thereof, as described herein.

[0228] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to liver cells in a subject. Exemplary liver cells include, but are not limited to, hepatocytes.

[0229] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to lung cells in a subject.

[0230] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to spleen cells in a subject. Exemplary spleen cells include, but are not limited to, spleen monocytes, spleen T cells, spleen memory B cells, or spleen B cells.

[0231] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to bone marrow cells in a subject. Exemplary bone marrow cells include, but are not limited to, myelomonocytic cells, bone marrow B cells, bone marrow memory B cells, or bone marrow T cells.

[0232] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to immune cells in a subject. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells.

[0233] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to cells of the central nervous system in a subject.

[0234] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic and / or prophylactic agent of interest that can be delivered specifically to hematopoietic stem cells in a subject. Unless otherwise specified, it is understood that the terms "hematopoietic stem cells (HSCs)" and "hematopoietic stem and progenitor cells (HSPCs)" are used interchangeably in this disclosure.

[0235] In some embodiments, lipid nanoparticles can be formulated to be delivered to mammalian liver hepatocytes, liver immune cells, splenic T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery to a particular class or type of cell indicates that a higher percentage of lipid nanoparticles are delivered to the target type or class of cells. In some embodiments, specific delivery can be 2-fold, 5-fold, 10-fold, 15-fold, or even 20-fold greater than delivery using conventional nanoparticle systems (e.g., MC3-containing LNPs).

[0236] B. Methods for Producing Polypeptides In particular, in some embodiments, methods using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used in methods for producing polypeptides. In particular, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells in subjects in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells in subjects in need thereof. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein comprise one or more nucleic acid sequences to be delivered to cells.

[0237] In some embodiments, one or more nucleic acids are expressed in a cell. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0238] C. Methods of Gene Regulation In particular, in some embodiments, methods using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used for gene regulation. In particular, in some embodiments, the lipid nanoparticles described herein can be used to reduce and / or increase gene expression in target cells in a subject in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can deliver one or more nucleic acids to target cells in a subject without a targeting ligand. In some embodiments, the nucleic acid is an inhibitor nucleic acid. In some embodiments, the inhibitory nucleic acid is an siRNA. In some embodiments, the nucleic acid is a nucleic acid described herein. As another example, in some embodiments, the lipid nanoparticles described herein can deliver a cargo to target cells in a subject without a targeting ligand. In some embodiments, the cargo is any cargo described herein.

[0239] In particular, in some embodiments, methods of using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein to edit genes in cells in a subject in need thereof.

[0240] In some embodiments, the cells targeted for gene regulation are immune cells. The immune cells can be T cells, such as CD8+ T cells, CD4+ T cells, or T regulatory cells. Other exemplary immune cells for gene editing include, but are not limited to, macrophages, dendritic cells, B cells, or natural killer cells. In some embodiments, the cells targeted for gene regulation are hepatocytes.

[0241] Exemplary genes that may be targeted include, but are not limited to, T cell receptor, B cell receptor, CTLA4, PD1, FOXO1, FOXO3, AKTs, CCR5, CXCR4, LAG3, TIM3, killer immunoglobulin-like receptor, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNFR-II, IL-7 R, OX-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK-R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NFAT, IL2R, and IL7. Other exemplary genes that can be targeted include, but are not limited to, OCT, G6Pase, Mut, PCCA, PCCB, PCSK9, ALAS1, and PAH. Exemplary tumor-associated antigens recognized by T cells and contemplated for targeting include, but are not limited to, MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1 / Melan A, MC1R, GP100, tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2 / Neu, hTERT, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN, and β-catenin.

[0242] D. Subject to Treatment In some embodiments, the subject being treated is a mammal experiencing cancer, an autoimmune disease, an infectious disease, an organ transplant, organ failure, a protein deficiency, or a combination thereof. In some embodiments, the subject is human. In some embodiments, the methods described herein can cause hepatocytes to translate certain proteins. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to hepatocytes. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic T cells. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic B cells. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic monocytes. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to bone marrow cells. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to lung cells.

[0243] It should be understood that the order or sequence of steps for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0244] While the present invention has been particularly shown and described with reference to certain preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0245] Illustrative Embodiments The following numbered embodiments are non-limiting examples of certain aspects of the present disclosure. 1. A compound of the formula: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein: L 1 and L 1’ Each of the 1-6 is alkylene, L 2 and L 2’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b - is replaced by Y 1 and Y 1’ each is independently —C(O)— or —C(O)O—; Y 2 and Y 2’ each is independently absent, —OC(O)—, —C(O)O—, or —OC(O)O—; Each of R and R' is independently hydrogen, [ka] or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L 3a and L 3a’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-10 a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O- or -NR b - is replaced by Ra and R a’ each independently is hydrogen or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; X 1 is absent or is -O-, -S-, or -NR b - and X 2 is absent or optionally substituted divalent saturated or unsaturated straight or branched C1- 12 A hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O-, -NR b - or -Cy A - is replaced by Cy A is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclylene, phenylene, a 3- to 7-membered saturated or partially unsaturated heterocyclylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroarylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 3 is hydrogen or an optionally substituted group selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R b are independently hydrogen or an optionally substituted C1-6 aliphatic group, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. 2. The compound is a compound of formula IA: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. 3. The compound is a compound of formula IAa: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. 4. The compound is a compound of formula IAb: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. 5. The compound is a compound of formula IAc: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. 6. The compound of any one of the preceding embodiments, wherein the compound is a compound of any of Formulas I, IA, IAa, IAb, and IAc, or a pharmaceutically acceptable salt thereof. 7.L 1’ But C 1-3 The compound of any one of embodiments 1-6, wherein the compound is alkylene. 8.L 1 The compound of any one of embodiments 1-7, wherein is —(CH 2 ) 2 — or —(CH 2 ) 3 —. 9.L 1’ But C 1-3 The compound of any one of embodiments 1-8, wherein the compound is alkylene. 10.L 1’ The compound of any one of embodiments 1-9, wherein is —(CH 2 ) 2 — or —(CH 2 ) 3 —. 11.L 2The compound of any one of embodiments 1-10, wherein: 12.L 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-10 The compound of any one of embodiments 1-10, which is a hydrocarbon chain. 13.L 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-5 The compound of any one of embodiments 1 to 10 and 12, which is a hydrocarbon chain. 14.L 2 The compound of any one of embodiments 1 to 10, 12, and 13, wherein is —(CH 2 ) 2 —. 15.L 2’ The compound of any one of embodiments 1-14, wherein: 16.L 2’ optionally substituted divalent saturated or unsaturated straight or branched C 1-10 The compound of any one of embodiments 1-14, which is a hydrocarbon chain. 17.L 2’ optionally substituted divalent saturated or unsaturated straight or branched C 1-5 The compound of any one of embodiments 1 to 14 and 16, which is a hydrocarbon chain. 18.L 2’ The compound of any one of embodiments 1-14, 16, and 17, wherein is —(CH 2 ) 2 —. 19.Y 1 The compound of any one of embodiments 1-18, wherein is —C(O)—. 20.Y 1’ The compound of any one of embodiments 1-19, wherein is —C(O)—. 21.Y 2 The compound of any one of embodiments 1-20, wherein: 22.Y 2 The compound of any one of embodiments 1-21, wherein is —C(O)O—. 23.Y 2’ The compound of any one of embodiments 1-22, wherein 24.Y 2’ The compound of any one of embodiments 1-22, wherein is —C(O)O—. 25.R is, [ka] 25. The compound of any one of embodiments 1-24, wherein: 26. R is optionally substituted C 6-20 The compound of any one of embodiments 1-24, which is aliphatic. 27. R is optionally substituted C 9-20 The compound of any one of embodiments 1 to 24 and 26, which is aliphatic. 28.R' is [ka] The compound of any one of embodiments 1-27, wherein 29. R' is optionally substituted C 6-20 The compound of any one of embodiments 1-27, which is aliphatic. 30. R' is optionally substituted C 9-20 The compound of any one of embodiments 1 to 27 and 29, which is aliphatic. 31.-L 2 -Y 2 -R and -L 2’ -Y 2’ -each R' is independently [ka] The compound of any one of the preceding embodiments, wherein 32.Each L 3a The compound of any one of embodiments 1-31, wherein: 33.Each L 3a’ The compound of any one of embodiments 1-32, wherein: 34.Each R a optionally substituted C 6-20 The compound of any one of embodiments 1-33, which is aliphatic. 35.Each R a optionally substituted C 6-12 The compound of any one of embodiments 1-34, which is aliphatic. 36.Each R a’ are independently optionally substituted C 6-20 The compound of any one of embodiments 1-35, which is aliphatic. 37.Each R a’ optionally substituted C 6-12 The compound of any one of embodiments 1-36, which is aliphatic. 38.-L 3a -R a and -L 3a’ -R a’ Each of the following may independently: [ka] The compound of any one of embodiments 1-37, wherein 39.X 1 The compound of any one of embodiments 1-38, wherein: 40.X 1 The compound of any one of embodiments 1-38, wherein is —O—. 41.X 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-12 a hydrocarbon chain, wherein one to three methylene units are optionally and independently —NR b The compound of any one of embodiments 1-40, wherein - is replaced by -. 42.X 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-6 a hydrocarbon chain, wherein one methylene unit is optionally and independently selected from the group consisting of -NR b The compound of any one of embodiments 1-41, wherein - is replaced by -. 43.X 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-12 The compound of any one of embodiments 1-41, which is a hydrocarbon chain. 44.X 2 optionally substituted divalent saturated or unsaturated straight or branched C 1-6 The compound of any one of embodiments 1-43, which is a hydrocarbon chain. 45.X 3 is a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 46.X 3 The compound of any one of embodiments 1-45, wherein is a 5-6 membered saturated or partially unsaturated heterocyclyl having 1-2 nitrogens. 47.-X 2 -X 3 but, [ka] The compound of any one of the preceding embodiments, wherein 48.Each R b are independently optionally substituted C 1-6 The compound of any one of embodiments 1-47, which is aliphatic. 49.Each R b is independently -CH3 or -CH2CH3. 50. The compound of embodiment 1, wherein the compound is selected from Table 1, or a pharmaceutically acceptable salt thereof. 51. A lipid nanoparticle (LNP) preparation comprising an ionizable lipid according to any one of embodiments 1 to 50. 52. An ionizable lipid according to any one of embodiments 1 to 50; Phospholipids and Sterols and A lipid nanoparticle (LNP) preparation comprising: a conjugate-linker lipid (e.g., a polyethylene glycol lipid). 53. The LNP preparation of embodiment 51, further comprising a therapeutic and / or prophylactic agent. 54. The LNP preparation of embodiment 53, wherein the therapeutic and / or prophylactic agent is or comprises one or more nucleic acids. 55. The LNP preparation of embodiment 54, wherein the one or more nucleic acids are or comprise RNA. 56. The LNP preparation of embodiment 54, wherein the one or more nucleic acids are or comprise DNA. 57. The LNP preparation of any one of embodiments 53-56, wherein the LNP preparation is formulated for delivering a therapeutic and / or prophylactic agent to target cells. 58. The LNP preparation of embodiment 57, wherein the target cell is or comprises a spleen cell (e.g., a splenic B cell, a splenic T cell, a splenic monocyte), a liver cell (e.g., a hepatocyte), a bone marrow cell (e.g., a bone marrow monocyte), an immune cell, a kidney cell, a muscle cell, a cardiac cell, or a cell in the central nervous system. 59. The LNP preparation of embodiment 58, wherein the target cells are or comprise hematopoietic stem cells (HSCs). 60. A pharmaceutical composition comprising the LNP preparation of embodiment 51 and a pharmaceutically acceptable excipient. 61. A method for administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising administering to the subject an LNP preparation described in embodiment 51 or a pharmaceutical composition described in embodiment 60. 62. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an LNP preparation described in embodiment 51 or a pharmaceutical composition described in embodiment 60, wherein the therapeutic and / or prophylactic agent is effective in treating the disease. 63. A method for slowing and / or halting the progression of a disease or disorder in a subject in need thereof, the method comprising administering to the subject an LNP preparation described in embodiment 51 or a pharmaceutical composition described in embodiment 60, wherein the therapeutic and / or prophylactic agent is effective in treating the disease. 64. A method for delivering a therapeutic and / or prophylactic agent to mammalian cells derived from a subject, the method comprising contacting cells of a subject that has been administered an LNP preparation described in embodiment 51 or a pharmaceutical composition described in embodiment 60. 65. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the LNP preparation of embodiment 51 or the pharmaceutical composition of embodiment 60, wherein the therapeutic and / or prophylactic agent is or comprises mRNA, and the mRNA encodes the polypeptide of interest, such that the mRNA is capable of being translated in the cell to produce the polypeptide of interest. 66. A method for inhibiting production of a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the LNP preparation of embodiment 51 or the pharmaceutical composition of embodiment 60, wherein the therapeutic and / or prophylactic agent is or comprises RNA, whereby the RNA is capable of inhibiting production of the polypeptide of interest. 67. A method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising contacting the mammalian organ with an LNP preparation described in embodiment 51 or a pharmaceutical composition described in embodiment 60, whereby the therapeutic and / or prophylactic agent is delivered to the organ. 68. The method of embodiment 67, comprising administering to the subject the LNP preparation of embodiment 51 or the pharmaceutical composition of embodiment 60. 69. A method of vaccination by administering an LNP preparation according to embodiment 51 or a pharmaceutical composition according to embodiment 60. 70. A method for inducing an adaptive immune response in a subject, comprising administering to the subject an effective amount of a composition comprising at least one RNA, wherein the composition comprises an LNP preparation comprising the compound of embodiment 1, or a pharmaceutically acceptable salt thereof. [Example]

[0246] Example This disclosure exemplifies the compositions, preparations, formulations, nanoparticles, and / or nanomaterials described herein. This disclosure also exemplifies methods of preparing, characterizing, and validating the compositions, preparations, formulations, nanoparticles, and / or nanomaterials described herein.

[0247] Example 1: Materials and Methods This example provides exemplary materials and methods for preparing, characterizing, and validating the compositions, formulations, nanoparticles, and / or nanomaterials described herein.

[0248] LNP preparation Among other things, this example provides exemplary LNP preparations.

[0249] Lipid nanoparticle components are dissolved in 100% ethanol at a defined lipid component molar ratio. Nucleic acid (NA) cargo is dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of approximately 0.22 mg / mL NA cargo. In some embodiments, the NA cargo comprises both functional NA and reporter DNA barcodes mixed at a functional NA to barcode mass ratio of 1:10 to 10:1. As described herein, NA can be siRNA, antisense, expressed DNA, or mRNA.

[0250] LNPs are prepared at a total lipid-to-NA mass ratio of 11.7. LNPs are formed by microfluidic mixing of lipid and NA solutions using Precision Nanosystems NanoAssemblr Spark or Benchtop series instruments according to the manufacturer's protocol. An aqueous-to-organic solvent ratio of approximately 2:1 or 3:1 is maintained during mixing using different flow rates. After mixing, LNPs are collected and diluted in PBS (approximately 1:1 v / v). Further buffer exchange is performed using dialysis against a 20 kDa filter in PBS at 4°C for 4 to 24 hours. After this initial dialysis, each individual LNP preparation is characterized via dynamic light scattering (DLS) to measure size (e.g., diameter) and polydispersity. Additionally, the pKa of LNP subpopulations is measured via a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay. LNPs that fall within the specific diameter and polydispersity range are pooled and further dialyzed against phosphate-buffered saline (PBS) for 1-4 h at 4 °C against a 100 kDa dialysis cassette. After the second dialysis, sterile-filter the LNPs using a 0.22 µM filter and store at 4 °C for further use.

[0251] LNP characterization DLS - High-throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt) is used to measure LNP hydrodynamic diameter and polydispersity index (PDI). LNPs are diluted in 1x PBS to the appropriate concentration and analyzed.

[0252] Concentration and encapsulation efficiency Determine the concentration of nucleic acids using the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Determine the encapsulation efficiency by measuring the nucleic acid concentration in the undissolved and dissolved LNPs.

[0253] pKa Prepare a stock solution of 10 mM HEPES (Sigma-Aldrich), 10 mM MES (Sigma-Aldrich), 10 mM sodium acetate (Sigma-Aldrich), and 140 nM sodium chloride (Sigma-Aldrich). Adjust the pH to approximately pH 4-10 using hydrogen chloride and sodium hydroxide. Add 140 μL of pH-adjusted buffer to a 96-well plate, using four replicates at each pH value, followed by 5 μL of 2-(p-toluidino)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After a 5-minute incubation with gentle shaking, measure fluorescence using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0254] LNP administration For the study described in this example, male and female mice approximately 8-12 weeks old were used. Each mouse was temporarily restrained, and pooled LNP was administered intravenously (IV) via tail vein injection to up to five animals per experiment. Age-matched mice were also used, and up to three animals per experiment were administered vehicle (1x PBS) via tail vein injection. At 72 hours post-administration, tissues including liver, spleen, bone marrow, kidney, lung, muscle, and blood were collected for analysis.

[0255] flow Liver, kidney, lung, and muscle tissues were mechanically and then enzymatically digested using a mixture of proteinases and then passed through a 70 μM filter to generate a single-cell suspension. Spleen tissue was mechanically digested to generate a single-cell suspension. All tissues were treated with (ammonium-chloride-potassium) ACK buffer to lyse red blood cells and then stained with fluorescently labeled antibodies for flow cytometry and fluorescence-activated cell sorting (FACS). Commercially available antibodies were used. Samples were acquired via flow cytometry using a BD FACSMelody (Becton Dickinson) and gates were generated before sorting. The gating structure was generally size → single cells → live cells → cells of interest. T cells were defined as CD45+CD3+, monocytes as CD45+CD11b+, and B cells as CD45+CD19+. Endothelial cells are defined as CD31+, monocytes and Kupffer cells as CD45+CD11b+, and hepatocytes as CD31- / CD45-. For siRNA studies, downregulation of the target gene is gated. For mRNA studies, upregulation of the target gene is gated. Tissue from vehicle-treated mice is used to set the gates for sorting. Up to 1 million cells of each cell subset with the correct phenotype are sorted in PBS. After sorting, cells are pelleted via centrifugation, and DNA is extracted using Quick Extract DNA Extraction Solution (Lucigen) according to the manufacturer's protocol. Following DNA extraction, the DNA is stored at -20°C.

[0256] Barcode sequencing DNA (genomic and DNA barcode) was isolated using QuickExtract (Lucigen) and sequenced using Illumina MiniSeq as described herein, and the frequency of DNA barcode numbers in the FACS-isolated samples was normalized to the frequency in the injected dose. These data are plotted as "Normalized Fold Above Input" (data not shown).

[0257] confirmation The structural and functional characteristics of the provided LNPs are confirmed based on the protocols described herein.

[0258] LNP preparation Lipid nanoparticle components are dissolved in 100% ethanol at a defined lipid component molar ratio. Nucleic acid (NA) cargo is dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of approximately 0.22 mg / mL NA cargo. In some embodiments, the NA cargo contains both functional NA and reporter DNA barcodes, mixed at a functional NA to barcode mass ratio of 1:10 to 10:1. LNPs are formulated at a total lipid to NA mass ratio of 11.7. LNPs are formed by microfluidic mixing of lipid and NA solutions using Precision Nanosystems NanoAssemblr Spark or Benchtop series instruments according to the manufacturer's protocol. A 2:1 or 3:1 aqueous to organic solvent ratio is maintained during mixing using different flow rates. After mixing, the LNPs are collected and diluted in PBS (approximately 1:1 v / v) and further buffer exchange is performed using dialysis against a 20 kDa filter in PBS at 4 °C for 8-24 h. After this initial dialysis, each individual LNP formulation is characterized via DLS to measure size and polydispersity, and via TNS assay to measure the pKa of LNP subpopulations. After dialysis, the LNPs are sterile filtered using a 0.22 micron sterile filter and stored at 4 °C for further use.

[0259] LNP characterization DLS High-throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt) is used to measure LNP hydrodynamic diameter and polydispersity index (PDI). LNPs are diluted in 1x PBS to the appropriate concentration and analyzed.

[0260] Concentration and encapsulation efficiency Determine the concentration of NAs using the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Determine the encapsulation efficiency by measuring the undissolved and dissolved LNPs.

[0261] pKa Prepare a stock solution of 10 mM HEPES (Sigma-Aldrich), 10 mM MES (Sigma-Aldrich), 10 mM sodium acetate (Sigma-Aldrich), and 140 nM sodium chloride (Sigma-Aldrich). Adjust the pH to approximately pH 4-10 using hydrogen chloride and sodium hydroxide. Add 140 μL of pH-adjusted buffer to a 96-well plate, using four replicates at each pH, ​​followed by 5 μL of 2-(p-toluidino)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After a 5-minute incubation with gentle shaking, measure fluorescence using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0262] LNP administration For the study described in this example, male and female mice approximately 8-12 weeks old are used. Each mouse is temporarily restrained, and pooled LNP is administered intravenously via tail vein injection to up to five animals per experiment. Age-matched mice are also used, and vehicle (1x PBS) is administered via tail vein injection to up to three animals per experiment. Additional administration routes may also be used, including intravenous (ICV), intracisternal manganese (ICM), intrathecal (IT), intramuscular (IM), aerosol, intranasal (IN), subcutaneous (SC), intraarticular, and intradermal (ID). At 72 hours post-administration, tissues including liver, spleen, bone marrow, and blood are collected for analysis.

[0263] flow Liver, kidney, lung, and muscle (e.g., skeletal and cardiac) tissues were mechanically and then enzymatically digested using a mixture of proteinases and then passed through a 70 μM filter to generate a single-cell suspension. Spleen tissue was mechanically digested to generate a single-cell suspension. The tissues were treated with ACK buffer to lyse red blood cells and then stained with fluorescently labeled antibodies for flow cytometry and fluorescence-activated cell sorting (FACS). Commercially available antibodies were used in this example. Samples were acquired via flow cytometry using a BD FACSMelody (Becton Dickinson) to generate gates before sorting. The gating structure was generally size → single cells → live cells → cells of interest. T cells were defined as CD45+CD3+, monocytes as CD45+CD11b+, and B cells as CD45+CD19+. Endothelial cells are defined as CD31+, monocytes and Kupffer cells as CD45+CD11b+, and hepatocytes and myocytes in liver and muscle as CD31- / CD45-, respectively. Tissue from vehicle-treated mice was used to set the gates for sorting.

[0264] hEPO expression For human EPO (hEPO) protein expression, mice are briefly restrained and bled (via the tail vein) 6 hours after dosing. Blood is collected in heparin tubes, processed to plasma, and stored at -80°C until ready for use. hEPO protein is measured using appropriate dilutions of plasma using an R&D Systems ELISA kit (DuoSet; DY286-05) according to the manufacturer's instructions.

[0265] Tolerability ALT / AST quantification For quantification of rat aspartate transaminase (AST) and alanine transaminase (ALT), rats are briefly restrained and blood is drawn at 2, 4, 6, 24, 48, and 72 hours post-dose. Blood is collected in heparin tubes, processed to plasma, and stored at -80°C until ready for use. AST is quantified using the AST / GOT reagent (ThermoFisher, TR70121), and ALT is quantified using the ALT / GPT reagent (ThermoFisher, TR71121) according to the manufacturer's instructions.

[0266] Rat MCP-1 ELISA For rat monocyte chemoattractant protein-1 (MCP-1) protein expression, rats are temporarily restrained and blood is collected at 2, 4, 6, 24, 48, and 72 hours post-dose. Blood is collected in heparin tubes, processed to plasma, and stored at -80°C until ready for use. MCP-1 protein is measured using appropriate dilutions of plasma using an R&D Systems ELISA kit (DuoSet; DY3144-05) according to the manufacturer's instructions.

[0267] Screening experiments Multiple LNPs described herein (e.g., more than 300 LNP preparations) can be tested simultaneously in a single screening experiment. In some embodiments, more than 300 LNPs are screened in a single mouse. In some embodiments, more than 850 LNPs are screened in a single mouse (see Figures 1 and 2). Screening experiments are used to measure mRNA or siRNA delivery to cells and tissues, as described herein.

[0268] For mRNA delivery, each LNP preparation is formulated to carry Cre mRNA and a barcode as described herein. Each LNP preparation is administered to LSL-tdTom mice (Ai14) according to the methods described herein (see FIG. 1 ) (see also FIG. 1 ). Referring to FIG. 1 , a library of LNP preparations, each containing one or more components, a barcode sequence, and Cre mRNA, is administered to Cre-LoxP reporter mice. Mouse cells are sorted using FACS based on whether they are tdTom- or tdTom+, as described herein. The sorted tdTom+ cells are then sequenced as described herein.

[0269] For siRNA delivery, each LNP preparation is formulated to carry siGFP and a barcode as described herein. Each LNP preparation is administered to GFP mice (see also FIG. 2) according to the method described herein (see FIG. 2). Referring to FIG. 2, a library of LNP preparations, each containing one or more components, a barcode sequence, and siGFP, is administered to GFP reporter mice. Cells are sorted using FACS based on GFP expression as described herein. The sorted cells are then sequenced as described herein.

[0270] LNP preparations are formulated using compounds described herein or comparative compounds. LNP preparations are formulated using MC3 as a control. The following measurements are performed: diameter of the LNP preparation, polydispersity of the LNP preparation, "normalized delivery efficiency" for any combination of cell type and tissue type, pKA of the LNP preparation (related to, but not the same as, lipid pKA), lipid pKA, and ionizability of the LNP preparation. Encapsulation efficiency and delivery potency are also measured for each pool of LNP preparations. hEPO expression and Cre expression measurements are performed as described herein.

[0271] Example 2: Potency per Screen This example provides exemplary compositions, preparations, nanoparticles, and / or nanomaterials, as well as materials and methods for screening the efficacy of such compositions, preparations, nanoparticles, and / or nanomaterials described herein.

[0272] Using the exemplary LNP screen described in Example 1, it can be shown that each pool of LNPs is highly potent across many tissues.

[0273] Example 3: Exemplary LNP preparations with potent delivery to various cell types This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials for potent delivery to various cell types described herein. In some embodiments, this example can be used to demonstrate that the provided lipids exhibit potent delivery to various cell types.

[0274] Based on the results from the screening in Example 1, exemplary LNP preparations are identified for use in spleen delivery, particularly for use in B cell delivery.These identified lipids are formulated into LNP preparations and screened using the Cre reporter system described herein.Three Ai14 mice are used per group.The payload contains 0.3 mg / kg Cre mRNA.Data are collected 168 hours after injection.Results are compared with MC3-LNP preparation as a control.

[0275] Example 4: Delivery of exemplary LNP preparations to various cell types. This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials that have potent delivery to various cell types as described herein. This example can be used to demonstrate that the provided lipids exhibit potent delivery across various cell types.

[0276] LNP preparations are selected and efficacy results are confirmed using the Cre reporter system and Ai14 mouse model described herein. Three Ai14 mice are used per group in each experiment. Data are collected 72 hours after injection. The screening platform described herein can identify several highly potent LNP preparations to determine which type of LNP preparation is most potent for a particular cell type.

[0277] Example 5: Exemplary LNP preparations deliver functional mRNA. This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials that deliver functional mRNA to various cell types. This example can demonstrate that lipids are capable of delivering functional mRNA in mice.

[0278] LNP preparations were selected to determine their ability to intravenously deliver functional mRNA in mice. Each LNP preparation carried 0.15 mg / kg of hEPO mRNA and was administered at a mass ratio of 11.7 and 19 in two to three C57BL6 mice. hEPO expression in plasma was measured 6 hours after injection of the LNP preparation.

[0279] Example 6: Tolerability and Efficacy Studies This example provides exemplary materials and methods for preparing, characterizing, and validating the compositions, preparations, nanoparticles, and / or nanomaterials described herein. This example can be used to demonstrate that the provided lipids are capable of delivering functional mRNA in mice.

[0280] As described herein, exemplary LNP preparations are selected to determine the tolerability and efficacy of hEPO mRNA delivery in rats. Each LNP preparation containing 1.0 mg / kg of hEPO mRNA is injected into Sprague-Dawley rats (N=2). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are collected from the rats' plasma 24 hours after injection of each exemplary LNP preparation (U / L). Monocyte chemoattractant protein-1 (MCP-1) is collected from the rats' plasma 6 hours after injection of each LNP preparation (ng / mL). Saline is used as a control. After administration of vehicle (control) and exemplary LNP preparations, the amount of hEPO is collected from the rats' plasma (ng / mL) at various time points (0, 2, 4, 6, 24, 48, and 96 hours) after injection.

[0281] Example 7: Synthesis of ionizable lipids This example provides exemplary materials and methods for preparing, characterizing, and validating the ionizable lipids described herein.As described in the following examples, in certain exemplary embodiments, compounds are prepared according to the following general procedures.Although the general methods illustrate the synthesis of certain specific compounds of the present disclosure, it will be understood that the following general methods and other methods known to those skilled in the art can be applied to all compounds described herein and each subclass and species of these compounds.

[0282] General notes: Unless otherwise stated, all reactions were carried out using anhydrous-grade solvents under a nitrogen atmosphere in flasks or vials with magnetic stirring. Anhydrous solvents were purchased from Sigma-Aldrich and used as received. Flash column chromatography was performed using a Biotage Selekt or Teledyne-Isco Combiflash Nextgen300+ with pre-packed silica gel cartridges. Thin-layer chromatography was performed using Merck silica gel 60 plates, and compounds were visualized using iodine. Nuclear magnetic resonance (NMR) spectroscopy was performed using either a Varian INOVA 500 MHz or a Bruker AVANCE 400 MHz spectrometer; chemical shifts are reported in δ parts per million (ppm) referenced to tetramethylsilane at δ = 0.00 ppm for CDCl3 samples and the residual solvent peak (δ = 2.50 ppm) for DMSO samples. QDa detector (ESI) was used using one of the following methods: + Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using a Waters Acquity UPLC H-class Plus equipped with:

[0283] Method A: 5 min run. Column: XTERRA RP 18 (4.6 × 50 mm), 5 μm. Mobile phase: starting with 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], then 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] for 2.65 min, held at this mobile phase composition for up to 3.75 min, and finally returned to the starting condition, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] for 4.90 min, held at this mobile phase composition for up to 5.10 min. Flow rate = 1.2 mL / min.

[0284] Method B: Run for 12 minutes, Column: XTERRA RP 18 (4.6 x 50 mm), 5 μm, Mobile phase: Initially 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF], held at this initial condition for 0.75 minutes, then 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH in (70:30) ACN:THF] for 3.0 minutes, then 2% [0.1% HCOOH in water]. This mobile phase composition was maintained for a maximum of 9.0 min, followed by 6.0 min of elution with 98% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF], and finally the starting condition, i.e., 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF], was maintained for a maximum of 11.00 min of elution with 12.10 min of elution with 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF]. Flow rate = 1.2 ml / min.

[0285] List of abbreviations ACN: acetonitrile d: double line CDI: 1,1'-carbonyldiimidazole DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane DIBAL-H: Diisobutylaluminum hydride DIPEA: N,N-diisopropylethylamine DMAP: 4-(dimethylamino)pyridine DMSO: dimethyl sulfoxide EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride Equiv: Equivalent Et: Ethyl i-Pr: Isopropyl m: multiplet Me: Methyl p:quintet PPTS: Pyridinium p-toluenesulfonate q:Quarter Rt: retention time s: single line t: Mie line TBAF: Tetrabutylammonium fluoride TBDMSCl: tert-butyldimethylsilyl chloride TEA: Triethylamine THF: tetrahydrofuran

[0286] [ka] Example 7-1: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Step 1: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile General Procedure A: To a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 equiv.) were added 4,4-diethoxybutanenitrile (1.5 g, 9.5 mmol, 1 equiv.) and cis-5-octen-1-ol (3.7 g, 29 mmol, 3 equiv.). The vial was tightly capped and the resulting mixture was heated at 105 °C for 72 h. After this time, the mixture was cooled to room temperature. The crude material was purified by flash column chromatography (100 g silica, 0 to 100% dichloromethane in hexanes for 20 min). 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (1.14 g, 37%) was isolated as a colorless oil. 1 H NMR(500MHz,chloroform-d)δ 5.43-5.27(m,4H),4.56(t,J=5.3Hz,1H),3.61(dt,J=9.3,6.6Hz,2H),3.44(dt,J=9.3,6.6Hz,2H),2 .42(t,J=7.4Hz,2H),2.12-1.91(m,9H),1.66-1.54(m,5H),1.49-1.36(m,4H),0.96(t,J=7.6Hz,6H).

[0287] [ka] Step 2: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid General Procedure B: To a vial containing 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (1.14 g, 3.54 mmol, 1 equiv.) was added potassium hydroxide (0.60 g, 10.6 mmol, 3 equiv.), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was tightly capped, and the reaction mixture was heated to 110° C. for 18 h. After this time, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL), and the pH was adjusted to approximately 5 by adding 1 M HCl. The resulting biphasic mixture was separated, and the aqueous phase was extracted twice more with ethyl acetate (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated to afford 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid as a sticky white solid (1.16 g, 96% yield). 1 H NMR(400MHz,chloroform-d)δ 5.41-5.24(m,4H),4.45(t,J=5.6Hz,1H),3.51(dt,J=9.0,6.7Hz,2H),3.39(dt,J=9.0,6.7Hz,2H),2.17(t,J=7. 6Hz,2H),2.08-1.98(m,8H),1.81(q,J=7.3Hz,2H),1.59-1.52(m,4H),1.44-1.32(m,4H),0.94(t,J=7.5Hz,6H).

[0288] [ka] Step 3: Diethyl 3-((tert-butyldimethylsilyl)oxy)pentanedioate To a stirred solution of diethyl 3-hydroxypentanedioate (2 g, 9.80 mmol) in DCM (20 mL) was added TBDMSCl (1.77 g, 11.75 mmol) and imidazole (1.33 g, 19.58 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 17 h. Upon completion, the reaction mixture was filtered and concentrated. The crude material thus obtained was purified by CombiFlash column chromatography, eluting with 10% ethyl acetate in hexane, to give diethyl 3-((tert-butyldimethylsilyl)oxy)pentanedioate (1.9 g, 61%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 0.05 (s, 6H), 0.83 (s, 9H), 1.23-1.27 (m, 6H), 2.52-2.56 (m, 4H), 4.07-4.15 (m, 4H), 4.52-4.56 (m, 1H)

[0289] [ka] Step 4: 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol To a stirred solution of diethyl 3-((tert-butyldimethylsilyl)oxy)pentanedioate (1.9 g, 6.96 mmol) in DCM (20 mL) was added a 1 M solution of DIBAL-H in THF (5.4 mL, 5.4 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 17 h. Upon completion, the reaction mixture was quenched with MeOH and the temperature was maintained at 0 °C. The solid material was filtered off. The filtrate was concentrated. The crude material thus obtained was purified by CombiFlash column chromatography, eluting with 10% MeOH-DCM to give 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol (600 mg, 43%) as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ 0.03(s,6H),0.85(s,9H),1.50-1.60(m,4H),3.40-3.46(m,4H),3.88-3.91(m,1H),4.28(t,J=4.9Hz,2H).

[0290] [ka] Step 5: 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate General Procedure C: To a stirred solution of 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (240 mg, 0.70 mmol) in DCM (5 mL) was added DCC (218 mg, 1.05 mmol) and DMAP (26 mg, 0.21 mmol) at 0° C. The reaction mixture was stirred for 30 minutes. Then, 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol (165.2 mg, 0.70 mmol) was added to the reaction mixture. The reaction mixture was stirred at 25° C. for 8 hours. The reaction mixture was filtered and evaporated under reduced pressure to give the crude compound. The crude material was purified by Combiflash column chromatography eluting with 10% ethyl acetate-hexane as eluent to afford 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate (200 mg, 51%) as a pale yellow liquid. 1 H NMR (400 MHz, chloroform-d) δ 0.06-0.08 (m, 6H), 0.88-0.96 (m, 14H), 1.30-2.10 (m, 24H), 2.36 (t, J = 7.3 Hz, 2H), 3.36-3.80 (m, 6H), 4.02-4.15 (m, 3H), 4.48 (t, J = 5.5 Hz, 1H), 5.20-5.40 (m, 4H).

[0291] [ka] Step 6: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate General Procedure D: To a stirred solution of linoleic acid (85 mg, 0.30 mmol) in DCM (10 mL) was added EDC·HCl (75.56 mg, 0.39 mmol), DMAP (7.8 mg, 0.06 mmol), and 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate (185 mg, 0.33 mmol) and anhydrous DIPEA (0.21 mL, 1.2 mmol). The reaction mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was concentrated, diluted with DCM (25 mL), and extracted with saturated NaHCO solution (2 × 25 mL). The organic layer was washed with water (2 × 25 mL) and brine (2 × 15 mL). The organic portion was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by Combiflash column chromatography, eluting with 10% ethyl acetate-hexanes to give 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (135 mg, 54%) as a colorless liquid. 1 H NMR(400MHz,chloroform-d)δ 0.10(s,6H),0.87-0.96(m,19H),1.24-1.43(m,23H),1.58-2.10(m,18H),2.20-2.40(m,4H),2 .70-2.80(m,2H),3.30-3.60(m,4H),3.90-4.20(m,5H),4.40-4.50(m,1H),5.20-5.40(m,8H).

[0292] [ka] Step 7: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate General Procedure E: To a stirred solution of 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (135 mg, 0.16 mmol) was added a 1 M solution of TBAF in THF (0.2 mL, 0.2 mmol). The reaction mixture was stirred at 25° C. for 6 hours. Upon completion, the reaction mixture was concentrated and diluted with DCM (15 mL). The reaction mixture was extracted with ethyl acetate (3×10 mL). The combined organic portions were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to afford 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate (145 mg, crude) as a colorless oil, which was used directly in the next step. 1 H NMR (400 MHz, chloroform-d) δ 0.85-0.96 (m, 16H), 1.29-2.10 (m, 37H), 2.20-2.40 (m, 3H), 2.70-2.80 (m, 2H), 3.30-3.80 (m, 5H), 4.10-4.50 (m, 5H), 5.20-5.40 (m, 8H)

[0293] [ka] Step 8: 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate General Procedure F: To a stirred solution of 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate (50 mg, 0.07 mmol) in toluene (1 mL) was added CDI (45.95 mg, 0.28 mmol) and KOH (0.23 mg, 0.004 mmol) and stirred at 70° C. for 4 h. Upon completion, the reaction mixture was concentrated under reduced pressure, diluted with DCM (10 mL), and washed with water (3×10 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate (55 mg, crude), which was used in the next step. 1 H NMR(400MHz,chloroform-d)δ 0.81-0.95(m,9H),1.24-1.85(m,23H),1.85-2.40(m,16H),2.70-2.80(m,1H),3.30-3.60(m,3H),3.90-3. 50(m,4H),5.20-5.40(m,5H),7.06-7.10(m,4H),7.40(d,J=9.2Hz,1H),7.67(s,2H),8.12(d,J=7.2Hz,1H).

[0294] Step 9: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (Example 7-1) General Procedure G: To a stirred solution of compound 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate (50 mg, 0.062 mmol, 1.0 equiv) in dry toluene (2 mL) was added KOH (1 mg, 0.016 mmol, 0.25 equiv), followed by (1-ethylpiperidin-3-yl)methanol (11 mg, 0.074 mmol, 1.2 equiv) under an inert atmosphere. The reaction mixture was heated at 70° C. for 4 hours. Upon completion, the reaction mixture was concentrated under reduced pressure, diluted with DCM (30 mL), and washed with water (3×10 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by Combiflash column purification, eluting with 10% MeOH-DCM to give 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (37%, 3 steps) as a colorless liquid. UPLC-MS (Method A): retention time 2.35 min, m / z calculated [M+H]: 874.7, found 874.8.

[0295] [ka] Example 7-2: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 26 mg, 30%. UPLC-MS (Method A): Retention time 2.35 min, m / z calculated [M+H]: 862.7, found 862.8.

[0296] [ka] Example 7-3: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 21 mg, 33%. UPLC-MS (Method A): Retention time 2.18 min, m / z calculated [M+H]: 848.7, found 848.7.

[0297] [ka] Example 7-4: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 38 mg, 37%. 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.8 Hz, 3H), 0.94 (t, J = 7.4 Hz, 6H), 1.19-1.50 (m, 14H), 1.50-1.67 (m, 6H), 1.82-2.16 (m, 24H), 2.28 (t, J = 7.6 Hz, 2H), 2.32-2.45 (m, 8H), 2.62 (s, 2H), 2.76 (t, J = 6.5 Hz, 2H) ),3.34-3.46(m,2H),3.50-3.63(m,2H),4.00-4.25(m,6H),4.47(t,J=5.4Hz,1H),4.82-4 .98(m,1H),5.24-5.43(m,8H).UPLC-MS (Method A): Retention time 2.12 minutes, m / z calculated value [M+H]: 834.6, actual value 834.7.

[0298] [ka] Example 7-5: 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((4-(pyrrolidin-1-yl)butanoyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate General Procedure H: To a stirred solution of 4-(pyrrolidin-1-yl)butanoic acid (15 mg, 0.078 mmol) in DCM (2 mL) was added EDC·HCl (19.31 mg, 0.10 mmol), DMAP (2 mg, 0.02 mmol), DIPEA (0.054 mL, 0.31 mmol), and 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate (60.12 mg, 0.08 mmol). The reaction mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was concentrated, diluted with DCM (25 mL), and extracted with saturated NaHCO solution (2 × 25 mL). The organic layer was washed with water (2 × 25 mL) and brine (2 × 15 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by CombiFlash column purification, eluting with 10% MeOH-DCM to give 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((4-(pyrrolidin-1-yl)butanoyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (48 mg, 73%) as a colorless liquid. UPLC-MS (Method A): retention time 2.12 min, m / z calculated [M+H]: 834.6, found 834.7.

[0299] [ka] Example 7-6: 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) [ka] Step 1: 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) General Procedure J: To a stirred solution of 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (240 mg, 0.70 mmol) in DCM (5 mL) was added DCC (218 mg, 1.05 mmol) and DMAP (26 mg, 0.21 mmol) at 0° C. The reaction mixture was stirred at 25° C. for 1 h. Then, 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol (165.2 mg, 0.70 mmol) was added and stirred at 25° C. for an additional 8 h. The reaction mixture was filtered and evaporated under reduced pressure to give the crude compound. The crude material was purified by Combiflash column chromatography, eluting with 10% ethyl acetate in hexanes, to give 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (30 mg, 11%) as a pale yellow liquid. 1 H NMR (400 MHz, chloroform-d) δ 0.03 (s, 6H), 0.87 (s, 9H), 0.92-1.05 (m, 11H), 1.24-1.43 (m, 8H), 1.76-2.06 (m, 24H), 2.34-2.38 (m, 4H), 3.36-3.42 (m, 5H), 3.53-3.59 (m, 5H), 3.80-4.15 (m, 6H), 4.48 (t, J = 5.6 Hz, 1H), 5.28-5.37 (m, 7H).

[0300] [ka] Step 2: 3-Hydroxypentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure E, substituting 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 230 mg, used crude directly in next step. 1 H NMR(400MHz,chloroform-d)δ 0.90-1.00(m,12H),1.24-2.10(m,41H),2.30-2.40(m,4H),3.36-3.80(m,9H),4.10-4.40(m,4H),4.48(t,J=5.5Hz,2H),5.28-5.37(m,8H).

[0301] [ka] Step 3: 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure F, substituting 3-hydroxypentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 170 mg, used crude directly in next step. 1 H NMR(400MHz,chloroform-d)δ 0.85-0.96(m,13H),1.24-1.42(m,12H),1.57-1.59(m,2H),1.85-2.10(m,21H),2.28-2.32(m,4H),3.35-3.57(m ,8H),4.17(t,J=6.3Hz,3H),4.43-4.46(m,2H),5.28-5.37(m,8H),7.00-7.10(m,2H),7.39(s,1H),8.10(s,1H).

[0302] Step 4: 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (Example 7-6) Prepared according to general procedure G, substituting 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 20 mg, 30%. UPLC-MS (Method A): retention time 2.27 min, m / z calculated [M+H]: 934.7, found 934.7.

[0303] [ka] Example 7-7: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for (1-ethylpiperidin-3-yl)methanol. Isolated 16 mg, 15%. UPLC-MS (Method A): Retention time 2.05 min, m / z calculated [M+H]: 922.7, found 922.6.

[0304] [ka] Examples 7-8: 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for (1-ethylpiperidin-3-yl)methanol. Isolated 21 mg, 20%. UPLC-MS (Method A): Retention time 2.08 min, m / z calculated [M+H]: 908.7, found 908.6.

[0305] [ka] Examples 7-9: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for (1-ethylpiperidin-3-yl)methanol. Isolated 14 mg, 17%. UPLC-MS (Method A): Retention time 2.07 min, m / z calculated [M+H]: 894.7, found 894.6.

[0306] [ka] Examples 7-10: 3-((4-(pyrrolidin-1-yl)butanoyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure H, substituting 3-hydroxypentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 31 mg, 55%. UPLC-MS (Method A): retention time 2.26 min, m / z calculated [M+H]: 904.7, found 904.8.

[0307] [ka] Examples 7-11: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Step 1: 4,4-bis(octyloxy)butanenitrile Prepared according to general procedure A, substituting 1-octanol for cis-5-octen-1-ol. Isolated 1.45g, 39%. 1 H NMR(400MHz,CDCl3)δ 0.87(d,J=6.9Hz,6H),1.21-1.30(m,20H),1.53-1.58(m,4H),1.90(q,J=7.2Hz,2H) ,2.41(t,J=7.3Hz,2H),3.39-3.44(m,2H),3.56-3.61(m,2H),4.54(t,J=5.2Hz,1H).

[0308] [ka] Step 2: 4,4-bis(octyloxy)butanoic acid Prepared according to general procedure B, substituting 4,4-bis(octyloxy)butanenitrile for 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile. Isolated 1.8 g, 84%. 1 H NMR(400MHz,DMSO-d6)δ 0.85(d,J=6.8Hz,6H),1.21-1.30(m,20H),1.43-1.48(m,4H),1.69(q,J=6.8Hz,2H),2.21(t ,J=7.2Hz,2H),3.35-3.39(m,2H),3.44-3.50(m,2H),4.44(t,J=5.5Hz,1H),12.06(bs,1H).

[0309] [ka] Step 3: 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(octyloxy)butanoate Prepared according to general procedure C, substituting 4,4-bis(octyloxy)butanoic acid for 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid. Isolated 130 mg, 40%. 1 H NMR(400MHz,DMSO-d6)δ 0.01-0.04(m,6H),0.83-0.87(m,12H),1.17-1.24(m,16H),1.44-1.76(m,10H ),2.27-2.32(m,2H),3.34-3.50(m,6H),3.89-4.09(m,3H),4.33-4.50(m,2H)

[0310] [ka] Step 4: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure D, substituting 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(octyloxy)butanoate. Isolated 625 mg, 53%. 1 H NMR(400MHz,chloroform-d)δ 0.10(s,6H),0.85-0.87(m,15H),1.19-1.29(m,36H),1.76-2.04(m,9H),2.21-2.38(m,5H),2.75(t,J=5.9 Hz,2H),3.36-3.58(m,6H),3.90-4.14(m,5H),4.10-4.14(m,5H),4.47(t,J=5.3Hz,1H),5.32-5.35(m,4H).

[0311] [ka] Step 5: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure E, substituting 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate for 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 540 mg, used crude directly in next step. 1 H NMR(400MHz,chloroform-d)δ 0.81-0.87(m,9H),1.26-1.29(m,34H),1.52-2.40(m,22H),2.70-2.80(m,2H),3.30-3.40(m,4H),4.0-4.50(m,5H),5.20-5.40(m,4H)

[0312] [ka] Step 6: 1-((4,4-bis(octyloxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate Prepared according to general procedure F, substituting 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate for 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 110 mg, used crude directly in next step. 1 H NMR(400MHz,chloroform-d)δ 0.84-0.87(m,9H),1.26-1.30(m,32H),1.53-1.61(m,6H),1.85-2.40(m,14H),2.75(t,J=6.0Hz,2H),3.3 6-3.54(m,4H),4.10-4.18(m,3H),4.43-4.44(m,1H),5.28-5.37(m,4H),7.05-7.39(m,3H),8.10(s,1H).

[0313] Step 7: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate (Examples 7-11) Prepared according to general procedure G, substituting 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate with 1-((4,4-bis(octyloxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 30 mg, 30%. UPLC-MS (Method A): retention time 2.41 min, m / z calculated [M+H]: 878.7, found 878.8.

[0314] [ka] Examples 7-12: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(octyloxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 29 mg, 30%. UPLC-MS (Method A): Retention time 2.42 min, m / z calculated [M+H]: 866.7, found 866.7.

[0315] [ka] Example 7-13: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(octyloxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 24 mg, 32%. UPLC-MS (Method A): Retention time 2.38 min, m / z calculated [M+H]: 852.7, found 852.8.

[0316] [ka] Example 7-14: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(octyloxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 93 mg, 45%. UPLC-MS (Method B): Retention time 5.49 min, m / z calculated [M+H]: 838.7, found 838.7.

[0317] [ka] Example 7-15: 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-((4-(pyrrolidin-1-yl)butanoyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure H, substituting 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate for 5-((4,4-bis(octyloxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 31 mg, 55%. UPLC-MS (Method A): retention time 2.32 min, m / z calculated [M+H]: 848.7, found 848.7.

[0318] [ka] Example 7-16: 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) [ka] Step 1: 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure J, substituting 4,4-bis(octyloxy)butanoic acid for 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid. Isolated 600 mg, 12%. 1 H NMR (400 MHz, chloroform-d) δ 0.10 (s, 6H), 0.80-0.90 (m, 21H), 1.12-1.38 (m, 41H), 1.50-1.55 (m, 6H), 1.73-2.03 (m, 8H), 2.34-2.38 (m, 4H), 3.36-3.58 (m, 8H), 3.89-3.92 (m, 1H), 4.06-4.18 (m, 4H), 4.47 (t, J = 5.5 Hz, 2H).

[0319] [ka] Step 2: 3-Hydroxypentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure E, substituting 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-((tert-butyldimethylsilyl)oxy)pentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 50 mg, used crude directly in next step. 1 H NMR(400MHz,chloroform-d)δ 0.85-0.92(m,14H),1.26-1.28(m,40H),1.50-1.94(m,20H),2.36-2.41(m,4H),3.36-3.4 1(m,4H),3.52-3.57(m,4H),3.70-3.80(m,1H),4.10-4.32(m,3H),4.47(t,J=5.5Hz,2H).

[0320] [ka] Step 3: 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure F, substituting 3-hydroxypentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 45 mg, used crude directly in next step. 1 H NMR (400 MHz, chloroform-d) δ 0.84-0.86 (m, 1H), 1.20-1.30 (m, 5H), 1.80-2.40 (m, 1H), 3.20-3.50 (m, 1H), 4.02 (s, 3H), 7.06 (s, 1H), 7.40-7.50 (m, 1H), 8.12 (s, 1H)

[0321] Step 4: 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (Examples 7-16) Prepared according to general procedure G, substituting 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 37 mg, 35%. UPLC-MS (Method A): retention time 2.52 min, m / z calculated [M+H]: 942.8, found 942.8.

[0322] [ka] Example 7-17: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 16 mg, 33%. UPLC-MS (Method A): Retention time 2.43 min, m / z calculated [M+H]: 930.8, found 930.8.

[0323] [ka] Examples 7-18: 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure G, substituting 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 67 mg, 38%. UPLC-MS (Method A): Retention time 2.50 min, m / z calculated [M+H]: 916.7, found 916.8.

[0324] [ka] Example 7-19: 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure G, substituting 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 3-((1H-imidazole-1-carbonyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 68 mg, 45%. UPLC-MS (Method B): Retention time 5.49 min, m / z calculated [M+H]: 902.7, found 902.8.

[0325] [ka] Example 7-20: 3-((4-(pyrrolidin-1-yl)butanoyl)oxy)pentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) Prepared according to general procedure H, substituting 3-hydroxypentane-1,5-diyl bis(4,4-bis(octyloxy)butanoate) for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 62 mg, 65%. UPLC-MS (Method A): retention time 2.47 min, m / z calculated [M+H]: 912.7, found 912.8.

[0326] [ka] Example 7-21: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Step 1: Heptane-1,4,7-triol To a stirred solution of diethyl 4-oxoheptanedioate (4 g, 17.37 mmol) in THF (40 mL) was added dropwise 2.4 (M) LAH (36.18 mL, 86.85 mmol) in THF at 0 °C. The reaction mixture was stirred at 25 °C for 2 h. Upon completion, it was quenched with NaSO 10H O, and the resulting precipitate was filtered and washed with MeOH. The organic layer was concentrated under reduced pressure to give heptane-1,4,7-triol (2.1 g, 70%) as a semi-solid compound. 1 H NMR (400 MHz, methanol-d₄) δ 1.39-1.68 (m, 7H), 3.30-3.33 (m, 2H), 3.47-3.57 (m, 4H).

[0327] [ka] Step 2: 4,7-Dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate Prepared according to general procedure C, substituting heptane-1,4,7-triol for 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol. Isolated 500 mg, 23%. 1 H NMR(400MHz,chloroform-d)δ 0.90-1.0(m,6H),1.20-1.50(m,9H),1.60-1.70(m,3H),1.90-2.10(m,11H),2.37(t,J=7.4H z,2H),3.36-3.69(m,10H),4.09(t,J=6.4Hz,2H),4.48(t,J=5.5Hz,2H),5.26-5.36(m,5H).

[0328] [ka] Step 3: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure D, substituting 4,7-dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate. Isolated 300 mg, 32%. 1 H NMR (400 MHz, chloroform-d) δ 0.86-0.96 (m, 9H), 1.24-2.10 (m, 48H), 2.20-2.40 (m, 4H), 2.70-2.80 (m, 2H), 3.50-3.70 (m, 3H), 4.08 (t, J = 6.4 Hz, 4H), 4.40-4.50 (m, 1H), 5.28-5.39 (m, 9H).

[0329] [ka] Step 4: 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-7-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)heptan-4-yl 1H-imidazole-1-carboxylate Prepared according to general procedure F, substituting 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl(9Z,12Z)-octadeca-9,12-dienoate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated 110 mg, used crude directly in next step. 1 H NMR (400 MHz, chloroform-d) δ 0.70-1.00 (m, 11H), 1.20-2.10 (m, 44H), 2.20-2.50 (m, 4H), 2.74-2.75 (m, 2H), 3.30-3.60 (m, 4H), 4.00-4.20 (m, 5H), 4.40-4.50 (m, 1H), 5.00-5.40 (m, 10H), 7.07 (s, 1H), 7.40 (s, 1H), 8.11 (s, 1H).

[0330] Step 5: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate (Example 7-21) Prepared according to general procedure G, substituting 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate with 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-7-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)heptan-4-yl 1H-imidazole-1-carboxylate. Isolated 22 mg, 29%. UPLC-MS (Method A): retention time 2.17 min, m / z calculated [M+H]: 902.7, found 902.7.

[0331] [ka] Example 7-22: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-7-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)heptan-4-yl 1H-imidazole-1-carboxylate. Isolated 26 mg, 30%. UPLC-MS (Method A): Retention time 2.12 min, m / z calculated [M+H]: 890.7, found 890.6.

[0332] [ka] Example 7-23: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-7-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)heptan-4-yl 1H-imidazole-1-carboxylate. Isolated 58 mg, 40%. UPLC-MS (Method A): Retention time 2.13 min, m / z calculated [M+H]: 876.7, found 876.7.

[0333] [ka] Example 7-24: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure G, substituting 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol and 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-7-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)heptan-4-yl 1H-imidazole-1-carboxylate. Isolated 74 mg, 46%. 1 H NMR(400MHz,chloroform-d)δ 0.88(t,J=6.9Hz,3H),0.94(t,J=7.5Hz,6H),1.18-1.46(m,14H),1.46-1.77(m,19H),1.80-1.96 (m,4H),1.96-2.09(m,11H),2.21-2.31(m,8H),2.33-2.45(m,4H),2.76(t,J=6.5Hz,2H),3.34-3 .45(m,2H),3.51-3.62(m,2H),4.01-4.09(m,4H),4.17(t,J=6.5Hz,2H),4.48(t,J=5.6Hz,1H),4 .66-4.83(m,1H),5.24-5.42(m,8H).UPLC-MS (Method A): Retention time 2.13 minutes, calculated m / z value [M+H]: 862.7, measured value 862.6.

[0334] [ka] Example 7-25: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)heptyl (9Z,12Z)-octadeca-9,12-dienoate Prepared according to general procedure H, substituting 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl(9Z,12Z)-octadeca-9,12-dienoate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated 22 mg, 55%. UPLC-MS (Method A): retention time 2.19 min, m / z calculated [M+H]: 872.7, found 872.7.

[0335] [ka] Example 7-26: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)heptyl((Z)-non-2-en-1-yl)succinate [ka] Step 1: (Z)-4-(non-2-en-1-yloxy)-4-oxobutanoic acid To a stirred solution of succinic anhydride (1.5 g, 10.55 mmol) in DCM (12 mL) and THF (5 mL), DMAP (11.861 g, 97.09 mmol) was added, followed by (Z)-non-2-en-1-ol (2.11 g, 21.10 mmol). The reaction mixture was stirred at 25 °C for 2 h. After the reaction was complete, the reaction mixture was washed with 30% aqueous HCl (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were evaporated under reduced pressure to give the crude compound. The crude material was purified by CombiFlash column chromatography, eluting with 15% ethyl acetate-hexane, to give (Z)-4-(non-2-en-1-yloxy)-4-oxobutanoic acid (1.17 g, 34%) as a colorless liquid. 1H NMR(400MHz,DMSO-d6)δ 0.84-0.85(m,3H),1.15-1.30(m,8H),1.98-2.06(m,2H),2.40-2.49(m,4H),4.57(d,J=6.3Hz,2H),5.44-5.75(m,2H),12.19(s,1H).

[0336] [ka] Step 2: (Z)-4,7-dihydroxyheptyl non-2-en-1-yl succinate Prepared according to general procedure C, substituting heptane-1,4,7-triol for 3-((tert-butyldimethylsilyl)oxy)pentane-1,5-diol and (Z)-4-(non-2-en-1-yloxy)-4-oxobutanoic acid for 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid. Isolated 235 mg, 15%. 1 H NMR(400MHz,chloroform-d)δ 0.85-0.88(m,3H),1.25-1.80(m,17H),2.00-2.10(m,2H),2.50-2.70(m,4H),2.80-2.90 (m,1H),3.60-3.70(m,3H),4.10-4.20(m,2H),4.63(d,J=6.8Hz,2H),5.51-5.65(m,2H).

[0337] [ka] Step 3: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl((Z)-non-2-en-1-yl)succinate Prepared according to general procedure D, substituting (Z)-4,7-dihydroxyheptyl non-2-en-1-ylsuccinate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for linoleic acid. Isolated 225 mg, 45%. 1 H NMR(400MHz,chloroform-d)δ 0.85-0.96(m,9H),1.26-1.41(m,14H),1.54-2.08(m,21H),2.35-2.38(m,2H),2.59-2. 62(m,4H),3.38-3.66(m,6H),4.07-4.13(m,4H),4.48-4.64(m,4H),5.31-5.70(m,6H).

[0338] Step 4: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)heptyl((Z)-non-2-en-1-yl)succinate (Example 7-26) Prepared according to general procedure H, substituting 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl((Z)-non-2-en-1-yl)succinate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated 62 mg, 65%. 1H NMR(400MHz,chloroform-d)δ 0.87(t,J=7.0Hz,3H),0.94(t,J=7.5Hz,6H),1.17-1.46(m,19H),1.48-1.59(m,8H),1.71-1.82(m ,4H),1.79-1.96(m,4H),1.96-2.13(m,10H),2.36(q,J=7.3Hz,4H),2.42-2.56(m,5H),2.61(s,4H ),3.34-3.45(m,2H),3.51-3.61(m,2H),3.93-4.19(m,4H),4.48(t,J=5.6Hz,1H),4.63(d,J=6.8H z,2H),4.91(s,1H),5.24-5.42(m,4H).UPLC-MS (Method A): Retention time 1.92 minutes, m / z calculated value [M+H]: 834.6, actual value 834.7.

[0339] [ka] Example 7-27: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl(3-pentyloctyl)adipate [ka] Step 1: Methyl 3-pentyloctanoate Copper(I) bromide (0.1 equiv.) and lithium chloride (0.2 equiv.) were added to a flame-dried flask under an argon atmosphere. Dry THF (20 mL) was then added, and the mixture was stirred for 10 minutes, during which time the solids dissolved. The reaction mixture was placed in an ice bath and stirred for 5 minutes. Subsequently, (E)-methyl oct-2-enoate (1.0 equiv.) and chlorotrimethylsilane (1.1 equiv.) were added, and the mixture was stirred for 15 minutes. Pentylmagnesium bromide (18% w / v in THF) (1.4 equiv. v) was then added dropwise, and the reaction was stirred at 0°C for an additional hour. The solution was poured into saturated NH4Cl solution (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash column chromatography, eluting with 1-2% ethyl acetate-hexane to give methyl 3-pentyloctanoate as a yellow oil (1.0 g, 68%). 1 H NMR(400MHz,chloroform-d)δ 0.87(t,J=6.8Hz,6H),1.06-1.46(m,19H),1.82(s,1H),2.22(d,J=6.9Hz,2H),3.64(s,2H).

[0340] [ka] Step 2: 3-Pentyloctan-1-ol To a stirred solution of methyl 3-pentyloctanoate (1 equiv.) in THF (10 mL) was added lithium aluminum hydride (2 M in THF) (3.0 equiv.) at 0° C. and stirred at 25° C. for 2 h. The reaction was then quenched with sodium sulfate decahydrate at 0° C. and filtered through Celite®. The filtrate was concentrated under reduced pressure and purified by CombiFlash column chromatography, eluting with 3-5% ethyl acetate-hexane, to give 3-pentyloctan-1-ol as a colorless oil (580 mg, 65%). 1H NMR (400 MHz, chloroform-d) δ 0.87 (t, J = 6.9 Hz, 6H), 1.13 (t, J = 5.4 Hz, 1H), 1.17-1.35 (m, 15H), 1.38-1.44 (m, 1H), 1.51 (t, J = 6.8 Hz, 2H), 3.65 (q, J = 6.5 Hz, 2H), 5.29 (s, 1H).

[0341] [ka] Step 3: 6-oxo-6-((3-pentyloctyl)oxy)hexanoic acid General Procedure K: To a stirred solution of adipic acid (365 mg, 2.5 mmol, 5.0 equiv) in DCM (20 mL) was added EDC (144 mg, 0.75 mmol, 1.5 equiv), DMAP (31 mg, 0.25 mmol, 0.5 equiv), and DIPEA (194 mg, 1.5 mmol, 3.0 equiv). The reaction mixture was stirred at 25 °C for 30 min. 3-Pentyloctan-1-ol (100 mg, 0.50 mmol, 1.0 equiv) was then added and further stirred at 25 °C for 16 h. Water (50 mL) was added and extracted with DCM (2 × 100 mL). The organic layer was dried over anhydrous NaSO and evaporated under reduced pressure. The crude material thus obtained was purified by flash chromatography, eluting with 30-50% EtOAc-hexane to give 6-oxo-6-((3-pentyloctyl)oxy)hexanoic acid (87 mg, 53%). 1 H NMR(400MHz,DMSO-d6)δ 0.85(t,J=7.0Hz,6H),1.13-1.33(m,16H),1.32-1.42(m,1H),1.44-1.58(m,6H), 2.20(t,J=6.8Hz,2H),2.27(t,J=6.8Hz,2H),4.02(t,J=6.6Hz,2H),12.00(s,1H).

[0342] [ka] Step 4: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl(3-pentyloctyl)adipate Prepared according to general procedure D, substituting 4,7-dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 6-oxo-6-((3-pentyloctyl)oxy)hexanoic acid for linoleic acid. Isolated 255 mg, 43%. 1 H NMR(400MHz,CDCl3)δ 0.87(t,J=6.9Hz,6H),0.94(t,J=7.5Hz,6H),1.24-1.28(m,11H),1.37-1.46(m,6H),1. 52-1.58(m,14H),1.63-1.69(m,6H),1.74-1.83(m,2H),1.89-1.95(m,2H),2.00-2.09( m,8H),2.31(d,J=4.7Hz,4H),2.37(t,J=7.6Hz,2H),3.35-3.44(m,2H),3.52-3.60(m,2 H),3.62-3.66(m,2H),4.08(q,J=7.2Hz,6H),4.48(t,J=5.4Hz,1H),5.29-5.39(m,4H).

[0343] [ka] Step 5: 4-((1H-imidazole-1-carbonyl)oxy)-7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)heptyl(3-pentyloctyl)adipate Prepared according to general procedure F, substituting 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl(3-pentyloctyl)adipate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 280 mg, used directly crude in next step. 1 H NMR(400MHz,CDCl3)δ 0.87(t,J=6.9Hz,7H),0.94(t,J=7.5Hz,6H),1.18-1.30(m,20H),1.36-1.42(m,4H),1 .53-1.58(m,6H),1.61-1.65(m,4H),1.88-1.92(m,7H),2.00-2.07(m,8H),2.30(s,4H) ),2.37(t,J=7.4Hz,2H),3.36-3.43(m,2H),3.53-3.59(m,2H),3.60-3.66(m,1H),4.0 1-4.11(m,6H),4.43-4.52(m,1H),5.22-5.41(m,4H),7.05-7.13(m,2H),7.71(s,1H).

[0344] Step 6: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl(3-pentyloctyl)adipate (Example 7-27) Prepared according to general procedure G, substituting 4-((1H-imidazole-1-carbonyl)oxy)-7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)heptyl(3-pentyloctyl)adipate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)heptyl(3-pentyloctyl)adipate. Isolated 110 mg, 35% (2 steps). UPLC-MS (Method A): retention time 2.13 min, m / z calculated [M+H]: 950.7, found 951.2.

[0345] [ka] Example 7-28: 1-(7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl)8-(3-pentyloctyl)octanedioate [ka] Step 1: 8-oxo-8-((3-pentyloctyl)oxy)octanoic acid Prepared according to general procedure K, substituting suberic acid for adipic acid. Isolated 990 mg, 56%. 1 H NMR(400MHz,DMSO)δ 0.85(t,J=6.9Hz,6H),1.18-1.29(m,20H),1.35-1.40(m,1H),1.44-1.53(m,6H),2.1 8(t,J=7.4Hz,2H),2.25(t,J=7.1Hz,2H),4.02(t,J=6.7Hz,2H),11.84-12.07(m,1H).

[0346] [ka] Step 2: 1-(7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl)8-(3-pentyloctyl)octanedioate Prepared according to general procedure D, substituting 4,7-dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 8-oxo-8-((3-pentyloctyl)oxy)octanoic acid for linoleic acid. Isolated 310 mg, 43%. 1H NMR (400 MHz, CDCl3) δ 0.87(t,J=6.9Hz,6H),0.92-0.97(m,6H),1.22-1.26(m,14H),1.28-1.34(m,9 H),1.50-1.62(m,19H),1.74-1.93(m,5H),2.00-2.07(m,8H),2.24-2.29(m,4 H),2.34-2.39(m,1H),2.57-2.65(m,1H),2.76-2.82(m,1H),3.35-3.42(m,1H ),3.53-3.58(m,1H),3.61-3.65(m,2H),4.04-4.11(m,6H),5.26-5.36(m,4H).

[0347] [ka] Step 3: 1-(4-((1H-imidazole-1-carbonyl)oxy)-7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)heptyl)8-(3-pentyloctyl)octanedioate Prepared according to general procedure F, substituting 1-(7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl)8-(3-pentyloctyl)octanedioate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 340 mg, used directly crude in next step. 1H NMR(400MHz,CDCl3)δ 0.87(t,J=6.7Hz,6H),0.94(t,J=7.6Hz,6H),1.22-1.26(m,11H),1.30-1.42(m, 9H),1.53-1.62(m,9H),1.68-1.92(m,12H),2.01-2.08(m,12H),2.25-2.30(m,4H) ),2.33-2.39(m,2H),3.37-3.42(m,2H),3.51-3.58(m,2H),4.01-4.13(m,6H),4. 47(s,1H),5.09-5.16(m,1H),5.26-5.37(m,4H),7.11-7.15(m,2H),8.12(s,1H).

[0348] Step 4: 1-(7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl)8-(3-pentyloctyl)octanedioate (Example 7-28) Prepared according to general procedure G, substituting 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate with 1-(4-((1H-imidazole-1-carbonyl)oxy)-7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)heptyl)8-(3-pentyloctyl)octanedioate. Isolated 112 mg, 31% (2 steps). 1H NMR(400MHz,CDCl3)δ 0.87(t,J=6.9Hz,6H),0.94(t,J=7.5Hz,6H),1.21-1.25(m,14H),1.29-1.43(m,13H),1.53-1.69(m, 26H),1.86-1.96(m,3H),1.97-2.07(m,9H),2.23-2.32(m,5H),2.36(t,J=7.6Hz,3H),2.49-2.54(m, 1H),2.84-3.15(m,2H),3.34-3.44(m,2H),3.51-3.61(m,2H),3.94-3.99(m,1H),4.48(t,J=5.5Hz,1 H),4.71-4.76(m,1H),5.24-5.41(m,4H).UPLC-MS (Method A): Retention time 2.13 minutes, calculated m / z value [M+H]: 978.8, measured value 979.3.

[0349] [ka] Example 7-29: 7-((7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl)oxy)-7-oxoheptyl 2-butyloctanoate [ka] Step 1: 7-Hydroxyheptyl 2-butyloctanoate To a stirred solution of 2-butyloctanoic acid (1 equiv.) in DCM (5 mL / 0.5 mmol) was added DIPEA (3 equiv.), EDC (1.5 equiv.), and DMAP (0.5 equiv.) at 25 °C. The reaction mixture was stirred for 15 min, then 1,7-heptanediol (3.0 equiv.) was added and stirred for an additional 16 h at 25 °C. Upon completion, the reaction mixture was diluted with DCM (2 × 50 mL) and washed with saturated NaHCO3 solution (2 × 25 mL), followed by water and brine (25 mL). The organic layer was separated, passed through anhydrous Na2SO4, and dried on a rotary evaporator. The crude material thus obtained was purified by Combiflash chromatography, eluting with 15–20% EtOAc-hexane to give 7-hydroxyheptyl 2-butyloctanoate (1.5 g, 49%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 0.82-0.91 (m, 6H), 1.18-1.33 (m, 12H), 1.36-1.74 (m, 15H), 2.24-2.36 (m, 1H), 3.64 (t, J = 6.4 Hz, 2H), 4.08 (t, J = 6.5 Hz, 2H).

[0350] [ka] Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid A stirred solution of 7-hydroxyheptyl 2-butyloctanoate (1.0 equiv.) in acetone (30 mL) was cooled to 0° C., and to this was added Jones reagent (2 M solution in HSO, 1.5 equiv.) dropwise. The reaction mixture was stirred at 25° C. for 17 hours. Upon completion, isopropanol was added, and the mixture was filtered through Celite® and washed with acetone. The filtrate was concentrated under reduced pressure. The crude mass was diluted with water, the pH of the aqueous layer was adjusted to about 6, and the mixture was extracted with DCM (3×150 mL). The combined organic layers were dried over anhydrous NaSO and then concentrated under reduced pressure to give 7-((2-butyloctanoyl)oxy)heptanoic acid (1.45 g, 92%) as a pale yellow gum. 1H NMR(400MHz,DMSO)δ 0.82-0.86(m,6H),1.20-1.30(m,16H),1.40-1.53(m,8H),2.16-2.21(m,2H),2.25-2.31(m,1H),3.99-4.03(m,2H),11.96(s,1H).

[0351] [ka] Step 3: 7-((7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl)oxy)-7-oxoheptyl 2-butyloctanoate Prepared according to general procedure D, substituting 4,7-dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 7-((2-butyloctanoyl)oxy)heptanoic acid for linoleic acid. Isolated 730 mg, 49%. 1 H NMR (400 MHz, CDCl3) δ 0.83-0.89(m,6H),0.94(t,J=7.5Hz,6H),1.18-1.27(m,16H),1.34-1.43(m ,10H),1.53-1.61(m,14H),1.74-1.79(m,1H),1.89-1.95(m,2H),1.98-2.0 6(m,8H),2.25-2.33(m,3H),2.37(t,J=7.6Hz,2H),3.36-3.43(m,2H),3.51 -3.64(m,3H),4.01-4.12(m,6H),4.48(t,J=5.4Hz,1H),5.25-5.40(m,4H).

[0352] [ka] Step 4: (Z)-32-Butyl-10-(((Z)-oct-5-en-1-yl)oxy)-13,23,31-trioxo-9,14,22,30-tetraoxaoctatriacont-3-en-18-yl 1H-imidazole-1-carboxylate Prepared according to general procedure F, substituting 7-((7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl)oxy)-7-oxoheptyl 2-butyloctanoate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 350 mg, used crude directly in next step. 1 H NMR(400MHz,CDCl3)δ 0.84-0.88(m,6H),0.94(t,J=7.5Hz,6H),1.23-1.26(m,8H),1.33-1.43(m,10H),1.53-1.62( m,10H),1.72-1.82(m,11H),1.91(q,J=7.4Hz,3H),2.00-2.06(m,8H),2.24-2.32(m,3H),2.34 -2.40(m,2H),3.34-3.44(m,2H),3.53-3.60(m,2H),4.02-4.10(m,6H),4.47(t,J=5.5Hz,1H) ,5.07-5.17(m,1H),5.24-5.41(m,4H),7.04-7.14(m,1H),7.41(d,J=1.3Hz,1H),8.12(s,1H).

[0353] Step 5: 7-((7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl)oxy)-7-oxoheptyl 2-butyloctanoate (Example 7-29) Prepared according to general procedure G, substituting (Z)-32-butyl-10-(((Z)-oct-5-en-1-yl)oxy)-13,23,31-trioxo-9,14,22,30-tetraoxaoctatriacont-3-en-18-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 130 mg, 35% (2 steps). 1 H NMR(400MHz,CDCl3)δ 0.84-0.89(m,6H),0.94(t,J=7.5Hz,6H),1.06-1.28(m,34H),1.34-1.44(m,10H),1.61-1.68(m,9H) ,1.76-1.80(m,1H),1.88-1.94(m,2H),1.99-2.06(m,7H),2.28(t,J=7.5Hz,3H),2.36(t,J=7.6Hz,2 H),3.37-3.42(m,2H),3.51-3.61(m,3H),3.65-3.73(m,1H),3.99-4.10(m,7H),4.48(t,J=5.6Hz,1H ),4.68-4.76(m,1H),5.27-5.39(m,4H).UPLC-MS (Method A): Retention time 2.08 minutes, calculated m / z value [M+H]: 950.7, measured value 951.2.

[0354] [ka] Example 7-30: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl 8-((2-butyloctanoyl)oxy)octanoate [ka] Step 1: 8-hydroxyoctyl 2-butyloctanoate To a stirred solution of 2-butyloctanoic acid (1 equiv.) in DCM (5 mL / 0.5 mmol) was added DIPEA (3 equiv.), EDC (1.5 equiv.), and DMAP (0.5 equiv.) at 25 °C. The reaction mixture was stirred for 15 min, then 1,8-octanediol (3.0 equiv.) was added and stirred for an additional 16 h at 25 °C. Upon completion, the reaction mixture was diluted with DCM (2 × 50 mL) and washed with saturated NaHCO3 solution (2 × 25 mL), followed by water and brine (25 mL). The organic layer was separated, passed through anhydrous Na2SO4, and dried on a rotary evaporator. The crude material thus obtained was purified by Combiflash chromatography, eluting with 15–20% EtOAc–hexane to give 7-hydroxyheptyl 2-butyloctanoate (1.6 g, 49%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 0.82-0.91 (m, 6H), 1.13-1.47 (m, 25H), 1.49-1.69 (m, 4H), 2.24-2.36 (m, 1H), 3.63 (q, J = 5.8 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H).

[0355] [ka] Step 2: 8-((2-butyloctanoyl)oxy)octanoic acid A stirred solution of 8-hydroxyoctyl 2-butyloctanoate (1.0 equiv.) in acetone (30 mL) was cooled to 0° C., and to it was added Jones reagent (2 M solution in HSO, 1.5 equiv.) dropwise. The reaction mixture was stirred at 25° C. for 17 hours. Upon completion, isopropanol was added, and the mixture was filtered through Celite® and washed with acetone. The filtrate was concentrated under reduced pressure. The crude mass was diluted with water, the pH of the aqueous layer was adjusted to approximately 6, and the mixture was extracted with DCM (3×150 mL). The combined organic layers were dried over anhydrous NaSO and then concentrated under reduced pressure to give 8-((2-butyloctanoyl)oxy)octanoic acid (1.42 g, 96%) as a pale yellow gum. 1H NMR(400MHz,DMSO-d6)δ 0.84(t,J=6.9Hz,6H),1.17-1.32(m,18H),1.33-1.56(m,8H),2.18(t,J=7.4Hz,2H),2.22-2.34(m,1H),4.01(t,J=6.4Hz,2H),11.94(s,1H).

[0356] [ka] Step 3: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl 8-((2-butyloctanoyl)oxy)octanoate Prepared according to general procedure D, substituting 4,7-dihydroxyheptyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-((tert-butyldimethylsilyl)oxy)-5-hydroxypentyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 8-((2-butyloctanoyl)oxy)octanoic acid for linoleic acid. Isolated 720 mg, 48%. 1 H NMR (400 MHz, CDCl3) δ 0.83-0.90(m,6H),0.94(t,J=7.5Hz,6H),1.15-1.28(m,13H),1.29-1.35( m,7H),1.35-1.55(m,18H),1.66-1.93(m,6H),1.98-2.06(m,8H),2.24-2.3 2(m,3H),2.37(t,J=7.5Hz,2H),3.34-3.44(m,2H),3.53-3.59(m,2H),3.62 -3.67(m,1H),4.02-4.10(m,6H),4.48(t,J=5.6Hz,1H),5.22-5.38(m,4H).

[0357] [ka] Step 4: (Z)-33-Butyl-10-(((Z)-oct-5-en-1-yl)oxy)-13,23,32-trioxo-9,14,22,31-tetraoxanonatriaconta-3-en-18-yl 1H-imidazole-1-carboxylate Prepared according to general procedure F, substituting 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-hydroxyheptyl 8-((2-butyloctanoyl)oxy)octanoate for 5-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-3-hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated 355 mg, used crude directly in next step. 1 H NMR(400MHz,CDCl3)δ 0.84-0.89(m,6H),0.94(t,J=7.5Hz,6H),1.18-1.28(m,14H),1.29-1.33(m,6H),1.38- 1.42(m,4H),1.52-1.62(m,10H),1.66-1.80(m,8H),1.98-2.03(m,10H),2.23-2.32(m,3 H),2.36(t,J=7.6Hz,2H),3.36-3.42(m,2H),3.52-3.59(m,2H),4.02-4.11(m,6H),4.47 (t,J=5.5Hz,1H),5.08-5.19(m,1H),5.24-5.41(m,4H),7.11-7.14(m,2H),8.12(s,1H).

[0358] Step 5: 7-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)heptyl 8-((2-butyloctanoyl)oxy)octanoate (Example 7-30) Prepared according to general procedure G, substituting (Z)-33-butyl-10-(((Z)-oct-5-en-1-yl)oxy)-13,23,32-trioxo-9,14,22,31-tetraoxanonatriacont-3-en-18-yl 1H-imidazole-1-carboxylate for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 168 mg, 41% (2 steps). UPLC-MS (Method A): retention time 2.10 min, m / z calculated [M+H]: 964.7, found 965.3.

[0359] [ka] Example 7-31: 5-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) [ka] Step 1: Ethyl 3-(2,6-dioxocyclohexyl)propanoate To a stirred solution of cyclohexane-1,3-dione (10 g, 89.24 mmol) in ethanol (100 mL) and HO (50 mL) were added KCO (12.3 g, 89.24 mmol) and ethyl acrylate (10.4 mL, 98.17 mmol). The reaction mixture was refluxed for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was partitioned between water (20 mL) and EtOAc (250 mL). The aqueous layer was extracted with EtOAc (8 × 100 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous NaSO, and concentrated under reduced pressure to give the crude compound. The crude product was purified by column chromatography using ethyl acetate-hexane (30%) as the eluent to give ethyl 3-(2,6-dioxocyclohexyl)propanoate (6.5 g, 46%) as a crystalline colorless solid. 1H NMR (400 MHz, chloroform-d) δ 1.25 (t, J = 7.2 Hz, 3H), 1.80-2.02 (m, 2H), 2.31 (t, J = 6.8 Hz, 2H), 2.37-2.66 (m, 6H), 4.17 (q, J = 7.0 Hz, 2H), 9.53 (s, 1H).

[0360] [ka] Step 2: 5-oxononanedioic acid A stirred solution of ethyl 3-(2,6-dioxocyclohexyl)propanoate (2.8 g, 13.20 mmol) in 6 N aqueous HCl (50 mL) was stirred under reflux for 16 h. All volatiles were evaporated under reduced pressure to give 5-oxononanedioic acid (2.3 g, 92%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 1.53-1.73(m,4H),2.17(t,J=7.0Hz,4H),2.42(t,J=7.0Hz,4H),12.02(s,2H).

[0361] [ka] Step 3: Dimethyl 5-oxononanedioate To a stirred solution of 5-oxononanedioic acid (2.7 g, 13.36 mmol) in MeOH (50 mL) was added H2SO4 (0.4 mL, 6.68 mmol). The reaction mixture was heated at reflux for 16 h. Upon completion, all volatiles were evaporated under reduced pressure. The crude compound was dissolved in DCM (100 mL) and washed with bicarbonate solution (50 mL), followed by brine (50 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude compound. The crude compound was purified by flash chromatography, eluting with 30% ethyl acetate-hexane to give dimethyl 5-oxononanedioate (1.8 g, 53%) as a brown liquid. 1 H NMR (400 MHz, chloroform-d) δ 1.81-1.94 (m, 4H), 2.31 (t, J = 7.1 Hz, 4H), 2.46 (t, J = 7.2 Hz, 4H), 3.61-3.71 (m, 6H).

[0362] [ka] Step 4: Nonane-1,5,9-triol To a stirred solution of dimethyl 5-oxononanedioate (0.3 g, 1.30 mmol) in dry THF (5 mL) was added LAH (2.4 M solution in THF) (3.2 mL, 7.82 mmol) under ice-cooled conditions. The reaction mixture was stirred at 25 °C for 1 h. After Fischer workup, the filtrate was evaporated under reduced pressure to give nonane-1,5,9-triol (210 mg, 72%) as a colorless liquid. 1 H NMR (400MHz, deuterium oxide) δ 1.25-1.80 (m, 12H), 3.64 (t, J = 6.4Hz, 4H), 3.68-3.86 (m, 1H).

[0363] [ka] Step 5: 5-Hydroxynonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) To a stirred solution of nonane-1,5,9-triol (1.2 g, 3.62 mmol) in a mixture of DCM (20 mL) and DMF (2 mL) was added EDC·HCl (1.1 g, 5.76 mmol), DMAP (81 mg, 0.65 mmol), and 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (290 mg, 1.64 mmol) and anhydrous DIPEA (1.7 mL, 9.87 mmol). The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with DCM (100 mL) and washed with water (50 mL). The aqueous layer was extracted with DCM (2 × 50 mL). The organic layer was washed with brine (2 × 15 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash column chromatography, eluting with 30% ethyl acetate-hexane to give 5-hydroxynonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (563 mg, 39%) as a colorless liquid. 1 H NMR(400MHz,chloroform-d)δ 0.94(t,J=7.5Hz,12H),1.25-1.78(m,30H),1.81-2.14(m,20H),2.37(t,J=7.6Hz,4H),3.34-3 .45(m,4H),3.49-3.65(m,4H),4.06(t,J=6.5Hz,4H),4.48(t,J=5.6Hz,2H),5.12-5.54(m,8H).

[0364] [ka] Step 6: 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) To a stirred solution of 5-hydroxynonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (232 mg, 0.28 mmol) in toluene (2 mL) was added 1,1′-carbonyldiimidazole (138 mg, 0.84 mmol) and KOH (1 mg, 0.14 mmol) and heated at 70° C. for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure, diluted with DCM (10 mL), and washed with water (3×10 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to afford 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (178 mg, crude), which was used in the next step. 1 H NMR(400MHz,chloroform-d)δ 0.94(t,J=7.4Hz,12H),1.33-1.50(m,12H),1.52-1.73(m,17H),1.85-1.97(m,4H),1.96-2.09(m,16H),2.36(q,J=7.2Hz,4H),3.34-3 .45(m,4H),3.51-3.62(m,4H),4.06(q,J=6.1Hz,4H),4.48(q,J=5.2Hz,2H),5.24-5.42(m,8H),7.08(s,1H),7.41(s,1H),8.14(s,1H).

[0365] Step 7: 5-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (Example 7-31) Prepared according to general procedure G, substituting 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) and 3-(dimethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 40 mg, 22% (2 steps). UPLC-MS (Method A): retention time 2.12 min, m / z calculated [M+H]: 950.7, found 951.0.

[0366] [ka] Example 7-32: 5-(((3-(diethylamino)propoxy)carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate and 3-(diethylamino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 53 mg, 29% (2 steps). 1H NMR(400MHz,chloroform-d)δ 0.94(t,J=7.6Hz,12H),0.98-1.22(m,4H),1.33-1.46(m,12H),1.48-1.69(m,26H),1.91 (q,J=7.3Hz,4H),1.96-2.09(m,16H),2.36(t,J=7.6Hz,4H),3.34-3.45(m,4H),3.51-3. 61(m,4H),4.03(t,J=6.7Hz,4H),4.18(t,J=6.2Hz,2H),4.48(t,J=5.6Hz,2H),4.61-4.7 5(m,1H),5.24-5.42(m,8H).UPLC-MS (Method A): Retention time 2.14 minutes, calculated m / z value [M+H]: 978.8, measured value 979.0.

[0367] [ka] Example 7-33: 5-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) and 3-(ethyl(methyl)amino)propan-1-ol for (1-ethylpiperidin-3-yl)methanol. Isolated 43 mg, 23% (2 steps). UPLC-MS (Method A): retention time 2.13 min, m / z calculated [M+H]: 964.7, found 965.0.

[0368] [ka] Example 7-34: 5-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure G, substituting 5-((1H-imidazole-1-carbonyl)oxy)nonane-1,9-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 1-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)pentan-3-yl 1H-imidazole-1-carboxylate. Isolated 143 mg, 43% (2 steps). 1 H NMR(400MHz,chloroform-d)δ 0.94(t,J=7.5Hz,12H),1.01-1.22(m,4H),1.31-1.45(m,12H),1.48-1.69(m,14H),1.68-1 .83(m,4H),1.92(q,J=7.4Hz,7H),1.96-2.13(m,18H),2.36(t,J=7.6Hz,4H),2.74-3.17(m, 2H),3.34-3.45(m,4H),3.51-3.61(m,4H),3.90-4.15(m,7H),4.48(t,J=5.6Hz,2H),4.61- 4.74(m,1H),5.24-5.42(m,8H).UPLC-MS (Method A): Retention time 2.14 minutes, m / z calculated value [M+H]: 990.8, actual value 991.0.

[0369] [ka] Example 7-35: 5-((4-(pyrrolidin-1-yl)but...

Claims

1. A compound of the formula: 【Chemistry 1】 or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein: L １ and L １’ Each of the groups independently represents C １－６ is alkylene, L ２ and L ２’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C １－１０ a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently —O— or —NR ｂ is replaced by -, Y ２ and Y ２’ each is independently absent, —OC(O)—, —C(O)O—, or —OC(O)O—; Each of R and R' is independently hydrogen, 【Chemistry 2】 or C ６－２０ an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L ３ａ and L ３ａ’ each independently being absent or an optionally substituted divalent saturated or unsaturated straight or branched chain C １－１０ a hydrocarbon chain in which 1 to 3 methylene units are optionally and independently —O— or —NR ｂ is replaced by -, R ａ and R ａ’ Each of is independently hydrogen or C ６－２０ an optionally substituted group selected from aliphatic, 3- to 12-membered saturated or partially unsaturated carbocyclyl, 7- to 12-membered saturated or partially unsaturated bridged bicyclyl having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; X １ is absent or is —O—, —S—, or —NR ｂ - and X ２ is absent or optionally substituted divalent saturated or unsaturated straight or branched chain C １ - １２ A hydrocarbon chain in which 1 to 3 methylene units are optionally and independently -O-, -NR ｂ - or -Cy Ａ is replaced by -, Cy Ａ is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclylene, phenylene, 3- to 7-membered saturated or partially unsaturated heterocyclylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroarylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X ３ is hydrogen or an optionally substituted group selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R ｂ are independently hydrogen or optionally substituted C １ - ６ A compound, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, which is an aliphatic group.

2. The compound is a compound of formula IAa: 【Transformation 3】 Or a compound of formula IAb: 【Chemistry 4】 Or a compound of formula I-Ac: 【Transformation 5】 or an N-oxide of said compound, or a pharmaceutically acceptable salt thereof.

3. L １ But C １－４ alkylene, and / or L １’ But C １－４ alkylene, and / or L ２ is absent or optionally substituted divalent saturated or unsaturated straight or branched chain C １－１０ a hydrocarbon chain, and / or L ２’ is absent or optionally substituted divalent saturated or unsaturated straight or branched chain C １－１０ a hydrocarbon chain, and / or Y １ is —C(O)—, and / or Y １’ is —C(O)—, and / or Y ２ is absent, is —C(O)O—, or is —OC(O)—, and / or Y ２’ is absent, is —C(O)O—, or is —OC(O)—, and / or R is, 【Transformation 6】 or optionally substituted C ９－２０ aliphatic, and / or R' is 【Transformation 7】 or optionally substituted C ９－２０ is aliphatic, The compound of claim 1.

4. -L ２ -Y ２ -R and -L ２’ -Y ２’ - each R' is independently 【Transformation 8】 2. The compound of claim 1, wherein:

5. Each L ３ａ does not exist, and / or Each L ３ａ’ does not exist, and / or Each R ａ is optionally substituted C ６－１２ aliphatic, and / or Each R ａ’ is optionally substituted C ６－１２ aliphatic, and / or -L ３ａ -R ａ and -L ３ａ’ -R ａ’ Each of the following may independently: 【Chemistry 9】 and / or X １ is absent or is —O—, and / or X ２ optionally substituted divalent saturated or unsaturated straight or branched C １－６ a hydrocarbon chain, wherein one methylene unit is optionally and independently —NR ｂ -substituted or optionally substituted divalent saturated or unsaturated straight or branched C １－６ a hydrocarbon chain, wherein one methylene unit is optionally and independently -Cy Ａ - and / or Cy Ａ is a 3- to 7-membered saturated or partially unsaturated heterocyclylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and / or X ３ is a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or is an optionally substituted 5- to 6-membered saturated or partially unsaturated heterocyclyl having 1 to 2 nitrogens; The compound of claim 1.

6. -X ２ -X ３ but, 【Chemistry 10】 2. The compound of claim 1, wherein:

7. Each R ｂ independently optionally substituted C １－６ The compound of claim 1 which is aliphatic.

8. A compound selected from the following, or a pharmaceutically acceptable salt thereof:

9. A lipid nanoparticle (LNP) preparation comprising an ionizable lipid according to any one of claims 1 to 8.

10. An ionizable lipid according to any one of claims 1 to 8; Phospholipids and Sterols and A lipid nanoparticle (LNP) preparation comprising: a conjugate; and a linker lipid (e.g., a polyethylene glycol lipid).

11. further comprising a therapeutic and / or prophylactic agent; In particular, the therapeutic and / or prophylactic agent is or comprises one or more nucleic acids, In particular, the one or more nucleic acids are or comprise RNA, or the one or more nucleic acids are or comprise DNA, In particular, the LNP preparation is formulated to deliver the therapeutic and / or prophylactic agent to target cells; In particular, the target cell is or comprises a spleen cell (e.g., a splenic B cell, a splenic T cell, a splenic monocyte), a liver cell (e.g., a hepatocyte), a bone marrow cell (e.g., a myelomonocytic cell), an immune cell, a kidney cell, a muscle cell, a heart cell, a lung cell, or a cell in the central nervous system; In particular, the target cells are or comprise hematopoietic stem cells (HSCs), The LNP preparation of claim 9.

12. A pharmaceutical composition comprising the LNP preparation of claim 9 and a pharmaceutically acceptable excipient.

13. For use in a method for administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising administering to the subject the LNP preparation of claim 9; or For use in a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the LNP preparation of claim 9, wherein the therapeutic and / or prophylactic agent is effective in treating the disease; or For use in a method for slowing and / or halting the progression of a disease or disorder in a subject in need thereof, said method comprising administering to said subject the LNP preparation of claim 9, wherein said therapeutic and / or prophylactic agent is effective in treating said disease; or or for use in a method of producing a polypeptide of interest in a mammalian cell, said method comprising contacting said cell with the LNP preparation of claim 9, wherein said therapeutic and / or prophylactic agent is or comprises mRNA, said mRNA encoding said polypeptide of interest, whereby said mRNA is capable of being translated in said cell to produce said polypeptide of interest; or or for use in a method of inhibiting the production of a polypeptide of interest in a mammalian cell, said method comprising contacting said cell with the LNP preparation of claim 9, wherein said therapeutic and / or prophylactic agent is or comprises RNA, whereby said RNA is capable of inhibiting production of said polypeptide of interest; 10. For use in a method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, tissue, cell, or cell population, said method comprising contacting a mammalian organ, tissue, cell, or cell population with the LNP preparation of claim 9, whereby said therapeutic and / or prophylactic agent is delivered to said organ, tissue, cell, or cell population. In particular, for use in a method comprising administering to said subject the LNP preparation of claim 9, or For use in a method of vaccination by administering the LNP preparation of claim 9, The LNP preparation of claim 9.

14. An LNP preparation comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in a method of inducing an adaptive immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising at least one RNA, the composition comprising an LNP preparation comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

15. An LNP preparation for use in a method of delivering a therapeutic and / or prophylactic agent to mammalian cells derived from a subject, said method comprising contacting said cells of said subject that has been administered the LNP preparation of claim 9.

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