High-purity PEG lipids and their uses

Abstract

The present disclosure is based, at least in part, on the discovery that highly purified PEG lipids exhibit superior physical and biological properties, particularly when used in lipid nanoparticle (LNP) formulations. Accordingly, the present disclosure provides PEG lipids at recommended purity levels for use in formulations such as LNP formulations. The present disclosure also provides LNPs containing highly purified PEG lipids and methods for using them to deliver therapeutic agents to subjects.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. USSN 62 / 733,456, filed September 19, 2018, and U.S. Provisional Patent Application No. USSN 62 / 745,164, filed October 12, 2018, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Lipids containing polyethylene glycol units (PEG lipids) have a wide range of applications, e.g., in consumer products, pharmaceutical formulations, cosmetic compositions, and drug delivery. In particular, PEG lipids are useful as components of lipid nanoparticles (LNPs) for encapsulation and delivery of drugs (e.g., therapeutic agents). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Provisional Patent Application No. USSN62 / 733,456 [Patent Document 2] U.S. Provisional Patent Application No. USSN62 / 745,164 Summary of the Invention [Problem to be solved by the invention]

[0004] Effective in vivo delivery of active agents, such as small molecule drugs, proteins, peptides, and nucleic acids, represents a continuing medical challenge. Some agents and / or particles are recognized by the immune system, reducing their efficacy. To address this issue, certain formulations incorporate polymers, such as PEG-lipids, which are believed to obscure or mask the agent, thereby reducing its antigenicity and immunogenicity. However, even these "stealth" formulations have limitations, such as the inability to be administered repeatedly and frequently over several days without losing activity. Furthermore, some agents or formulations may interact with one or more cells or factors when administered in vivo, potentially inhibiting their function and ultimately resulting in side effects. Such side effects may limit the frequency and / or dosage of administration of the agent, or may even preclude its use in vivo altogether.

[0005] Formulating LNPs to achieve ideal physical properties and biological performance is challenging, and several factors determine the properties and performance of LNPs, including the selection and ratio of lipid components, the ratio of lipid components to encapsulated drug, etc. [Means for solving the problem]

[0006] The present disclosure is based, at least in part, on the discovery that highly pure PEG-lipids exhibit superior physical and biological properties, particularly when used in lipid nanoparticle (LNP) formulations. Accordingly, the present disclosure provides PEG-lipids at recommended purity (at least 87% purity) for use in formulations such as, for example, LNP formulations.

[0007] Provided herein are a plurality of PEG lipids (i.e., mixtures, batches, solutions, etc. of PEG lipids) having a purity of 87% or greater with respect to the PEG lipid (also referred to herein as "high-purity PEG lipids"). When "PEG lipids" or "high-purity PEG lipids" are referred to herein, the PEG lipids have a proscribed purity of 87% or greater with respect to the PEG lipid. The PEG lipids can be part of a composition or formulation, and the composition or formulation may contain other ingredients. Some non-limiting examples of PEG lipids are provided herein.

[0008] For example, in certain embodiments, the plurality of PEG lipids is a plurality of compounds of formula (I), or salts thereof. Provided herein are compounds of formula (I): [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive, and the compounds have a purity of 87% or greater with respect to the compound of formula (I).

[0009] The PEG lipids provided herein may include other components, such as solvents, carriers, excipients, etc., and thus may be in the form of a solution or composition. When the PEG lipid is part of a solution or composition, it is understood that the proscribed / recommended purity (e.g., 87% or greater) refers to the PEG lipid component of the solution or composition.

[0010] The high-purity PEG-lipids provided herein have applications in pharmaceutical compositions, cosmetic compositions, and drug delivery systems, including lipid nanoparticle (LNP) formulations. Accordingly, provided herein are LNPs comprising a plurality of PEG-lipids provided herein (i.e., a plurality of PEG-lipids having a purity of 87% or greater with respect to the PEG-lipid, e.g., a plurality of compounds of Formula (I) having a purity of 87% or greater with respect to the compound of Formula (I)).

[0011] In addition to the highly purified PEG-lipid, any formulation or composition (e.g., LNP formulation) described herein may contain lipids other than PEG-lipids. In certain embodiments, the LNPs provided herein further comprise an ionic amino lipid, a helper lipid, and / or a structural lipid. In certain embodiments, the LNPs provided herein comprise a plurality of PEG-lipids provided herein (e.g., a plurality of compounds of Formula (I) provided herein), an ionic amino lipid, a helper lipid, and a structural lipid. Some non-limiting examples of PEG-lipids, ionic amino lipids, helper lipids, and structural lipids are provided herein. The LNPs provided herein are useful for delivering therapeutic agents and, therefore, may contain (e.g., encapsulate) one or more agents (e.g., therapeutic agents).

[0012] The PEG-lipids and LNPs described herein may have improved physical and biological properties compared to state-of-the-art formulations. Lipid nanoparticles (LNPs) typically induce an innate immune response after administration to a subject. In some instances, components of LNPs, such as phosphatidylcholine, may induce the production of natural IgM and / or IgG molecules, which may be mediated by the activation of B1 cells, e.g., B1a and / or B1b cells. These biological mechanisms may contribute to LNP-induced drug reactions, including accelerated blood clearance (ABC) and dose-limiting toxicities, e.g., acute phase reactions (APRs) and complement activation-associated pseudoallergy (CARPA). LNP formulations have been developed that are less susceptible to immune system recognition and, therefore, clearance (e.g., ABC). Some state-of-the-art LNP formulations have improved clearance profiles and, in some instances, improved toxicity profiles. For some examples of LNP formulations that are less susceptible to blood clearance, see International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference.

[0013] The present disclosure relates, in part, to the discovery that LNPs containing highly purified PEG-lipids have improved physical and biological properties, including reduced susceptibility to recognition and clearance by the body. For example, in certain embodiments, LNP formulations described herein (i.e., containing multiple PEG-lipids at a recommended purity of 87% or greater) are less susceptible to accelerated blood clearance (ABC) than LNP formulations containing multiple PEG-lipids with a purity of less than 87%. For example, in certain embodiments, LNP formulations containing multiple compounds of Formula (I) described herein (i.e., containing multiple compounds of Formula (I) with a recommended purity of 87% or greater) are less susceptible to accelerated blood clearance (ABC) than LNP formulations containing multiple compounds of Formula (I) with a purity of less than 87%. LNP formulations containing highly purified PEG-lipids described herein may also exhibit reduced B cell binding (e.g., B1a and / or B1b cells) and / or reduced IgM binding compared to formulations with lower purity.

[0014] The present disclosure also provides pharmaceutical and cosmetic compositions comprising the highly purified PEG-lipids described herein. In another aspect, the present disclosure provides compositions comprising a plurality of LNPs described herein.

[0015] In another aspect, the present disclosure provides a method for delivering one or more therapeutic agents to a subject, the method comprising administering to the subject an LNP provided herein, the LNP encapsulating the one or more therapeutic agents of interest.

[0016] In some instances, the method for delivering one or more therapeutic agents includes: (i) administering an initial dose of the agent to the subject; (ii) administering a second or subsequent dose of the agent to the subject, wherein the second or subsequent dose is administered within two weeks of the first or previous dose; and (iii) may involve repeating step (ii) one or more times. In certain embodiments, the drug is formulated with LNP that does not promote ABC. As described herein, such LNP can comprise the high-purity PEG lipids described herein. In certain embodiments, the half-life of the drug after the second and subsequent administrations is at least 50% or more of the half-life of the drug after the first administration. In certain embodiments, the activity or blood level of the drug after the second and subsequent administrations is at least 50% or more of the activity or blood level of the drug after the first administration.

[0017] In yet another aspect, provided herein are methods for preparing the PEG-lipids provided herein (e.g., compounds of Formula (I)).

[0018] The details of certain embodiments of the invention are set forth in the detailed description below. Other features, objects, and advantages of the invention will become apparent from the definition, examples, drawings, and claims.

[0019] The accompanying drawings, which form a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0020] [Figure 1] Structure of PEG lipid of formula (I) (also known as HO-PEG-stearate, PEG-stearate, Compound (I)) and related impurities. [Figure 2] Dynamic light scattering (DSL) profiles of LNPs formulated with PEG-lipids of various purity (Lot 5: 97.8% purity, Corden: 98.5% purity, BioVectra: 86.6% purity, Sigma: 43.8% purity). DSL shows that LNPs formulated with higher purity PEG-lipids have a sharper particle size distribution (PSD). [Figure 3] Ultra-high performance, high-resolution liquid chromatography-charged aerosol detection (UPLC-CAD) chromatogram of lipids (HO-PEG-stearate, compound (B), cholesterol, DSPC) in LNP. [Figure 4] Heparin-Sepharose column binding of LNP formulations containing PEG-lipids of various purities. [Figure 5] The PEG release for each LNP formulation was found to be the same: for each formulation, all of the PEG was released at the first time point (30 min). [Figure 6] Measurement of accessible mRNA using a fluorescent encapsulation blood escape in vitro (BEEFI) assay. Formulations with higher purity compounds of formula (I) showed greater accessible mRNA levels as detected by Ribogreen. [Figure 7] PSD of LNP formulations using highly impure PEG lipids of formula (I). LNPs were formulated according to Tables 7 and 8 (see Examples). [Figure 8] PSD of fluorescently tagged LNPs formulated with PEG-lipids of various purities. *Single addition of Compound (I) from Sigma, approximately 0.6-0.7 mol% of Compound (I) in the final formulation. **Two additions of Compound (I) from Sigma, approximately 1.3-1.4 mol% of Compound (I) in the final formulation. [Figure 9] ApoE binding assay with LNPs containing PEG-lipids of various purities. *Single addition of Compound (I) from Sigma, approximately 0.6-0.7 mol% of Compound (I) in the final formulation. **Double addition of Compound (I) from Sigma, approximately 1.3-1.4 mol% of Compound (I) in the final formulation. [Figure 10] IgM assay with LNPs containing PEG-lipids of varying purity. Signal measured with naive serum indicates native IgM binding arising from the PC epitope. *Single addition of Compound (I) from Sigma, approximately 0.6-0.7 mol% of Compound (I) in the final formulation. **Two additions of Compound (I) from Sigma, approximately 1.3-1.4 mol% of Compound (I) in the final formulation. LNPs containing higher purity PEG-lipids show significantly lower IgM binding compared to less pure formulations. [Figure 11] Exemplary synthesis of compound (I). [Figure 12A] IR spectrum of compound (I). [Figure 12B]Proton (1H) NMR spectrum of compound (I) in deuterated chloroform (CDCl3). [Figure 12C] Carbon (13C) NMR spectrum of compound (I) in deuterated chloroform (CDCl3). [Figure 12D] MALDI-TOF scan mass spectrum of compound (I). [Figure 13] 1 shows a flow chart outlining an exemplary synthesis / purification method for the preparation of the compound of formula (I) (PEG2000 stearate). DETAILED DESCRIPTION OF THE INVENTION

[0021] In part, the present disclosure provides PEG lipids of particular purity ("high-purity PEG lipids") for use in, for example, lipid nanoparticle (LNP) formulations, for use in, for example, pharmaceutical compositions, cosmetic compositions, and drug delivery systems. The present disclosure also provides LNPs comprising the high-purity PEG lipids described herein, and methods of use thereof. For example, the LNPs provided herein are useful for delivering drugs (e.g., therapeutic agents) to a subject. In certain embodiments, the LNPs provided herein are not susceptible to accelerated blood clearance (ABC) after administration to a subject, even if the subject is susceptible.

[0022] High purity PEG lipid Provided herein are a plurality of PEG lipids with a purity of 87% or more with respect to PEG lipid.When "PEG lipid" is referred to herein (also referred to herein as "high-purity PEG lipid"), it means that the mixture of PEG lipids has a purity of 87% or more, which is the prohibited / recommended purity of the PEG lipid.In certain embodiments, the PEG lipids in the plurality of PEG lipids are the same PEG lipid.In certain embodiments, the plurality of PEG lipids comprise a mixture of PEG lipids.PEG lipid is any lipid that contains a PEG (polyethylene glycol) group.Non-limiting examples of PEG lipids are provided herein.

[0023] For example, in certain embodiments, the PEG lipid is a compound of formula (I): [ka] or a salt thereof, wherein r is independently an integer of 35 to 55, inclusive.

[0024] The PEG lipids provided herein may include other components, such as solvents, carriers, excipients, etc., and thus may be in the form of a solution or composition. When the PEG lipid is part of a solution or composition, it is understood that the proscribed / recommended purity (e.g., 87% or greater) refers to the PEG lipid component of the solution or composition.

[0025] As described herein, the plurality of PEG-lipids has a purity of greater than 87% with respect to PEG-lipids. In certain embodiments, the purity is greater than 90%. In certain embodiments, the purity is greater than 91%. In certain embodiments, the purity is greater than 92%. In certain embodiments, the purity is greater than 93%. In certain embodiments, the purity is greater than 94%. In certain embodiments, the purity is greater than 95%. In certain embodiments, the purity is greater than 96%. In certain embodiments, the purity is greater than 97%. In certain embodiments, the purity is greater than 98%. In certain embodiments, the purity is greater than 98.5%. In certain embodiments, the purity is greater than 99%. In certain embodiments, the purity is greater than 99.1%. In certain embodiments, the purity is greater than 99.2%. In certain embodiments, the purity is greater than 99.3%. In certain embodiments, the purity is greater than 99.4%. In certain embodiments, the purity is greater than 99.5%. In certain embodiments, the purity is greater than 99.6%. In certain embodiments, the purity is greater than 99.7%. In certain embodiments, the purity is greater than 99.8%. In certain embodiments, the purity is greater than 99.9%. In certain embodiments, the purity is 100%.

[0026] In certain embodiments, the plurality of PEG-lipids has a purity of 87-100%, inclusive, with respect to PEG-lipids, and in certain embodiments, the purity is 87-99.9%, inclusive, with respect to PEG-lipids.

[0027] In certain embodiments, the purity is 87-90%, including 87% and 90%. In certain embodiments, the purity is 90-95%, including 90% and 95%. In certain embodiments, the purity is 95-100%, including 95% and 100%. In certain embodiments, the purity is 95-99.9%, including 95% and 99.9%.

[0028] In certain embodiments, the purity is 90-100%, including 90% and 100%. In certain embodiments, the purity is 90-99.9%, including 90% and 99.9%. In certain embodiments, the purity is 91-99.9%, including 91% and 99.9%. In certain embodiments, the purity is 92-99.9%, including 92% and 99.9%. In certain embodiments, the purity is 93-99.9%, including 93% and 99.9%. In certain embodiments, the purity is 94-99.9%, including 94% and 99.9%. In certain embodiments, the purity is 95-99.9%, including 95% and 99.9%. In certain embodiments, the purity is 96-99.9%, including 96% and 99.9%. In certain embodiments, the purity is between 97 and 99.9%, including 97% and 99.9%. In certain embodiments, the purity is between 98 and 99.9%, including 98% and 99.9%. In certain embodiments, the purity is between 99 and 99.9%, including 99% and 99.9%.

[0029] In certain embodiments, the plurality of PEG-lipids has a purity of about 87% with respect to the PEG-lipids. In certain embodiments, the purity is about 87.5%. In certain embodiments, the purity is about 88%. In certain embodiments, the purity is about 88.5%. In certain embodiments, the purity is about 89%. In certain embodiments, the purity is about 89.5%. In certain embodiments, the purity is about 90%. In certain embodiments, the purity is about 90.5%. In certain embodiments, the purity is about 91%. In certain embodiments, the purity is about 91.5%. In certain embodiments, the purity is about 92%. In certain embodiments, the purity is about 92.5%. In certain embodiments, the purity is about 93%. In certain embodiments, the purity is about 93.5%. In certain embodiments, the purity is about 94%. In certain embodiments, the purity is about 94.5%. In certain embodiments, the purity is about 95%. In certain embodiments, the purity is about 95.5%. In certain embodiments, the purity is about 96%. In certain embodiments, the purity is about 96.6%. In certain embodiments, the purity is about 97%. In certain embodiments, the purity is about 97.7%. In certain embodiments, the purity is about 98%. In certain embodiments, the purity is about 98.5%. In certain embodiments, the purity is about 99%. In certain embodiments, the purity is about 99.5%. In certain embodiments, the purity is about 99.9%. In certain embodiments, the purity is about 100%. In certain embodiments, the purity is about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, or 98.9%. In certain embodiments, the purity is about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0030] In certain embodiments, the plurality of PEG-lipids consists essentially of PEG-lipids.

[0031] In certain embodiments, the plurality of PEG lipids is substantially free of other compounds. In certain embodiments, the plurality of PEG lipids is substantially free of impurities. The term "impurities" refers to compounds other than the PEG lipids. In certain embodiments, the plurality of PEG lipids contains less than 13% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 12% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 11% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 10% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 9% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 8% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 7% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 6% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 5% impurities in total. In certain embodiments, the plurality of PEG lipids contains less than 4% impurities in total. In certain embodiments, the plurality of PEG lipids comprises less than 3% of impurities in total.In certain embodiments, the plurality of PEG lipids comprises less than 2% of impurities in total.In certain embodiments, the plurality of PEG lipids comprises less than 1% of impurities in total.

[0032] In certain embodiments, the impurities include one or more fatty acids. In certain embodiments, the impurities include one or more PEG polymers. In certain embodiments, the impurities include one or more fatty acids and one or more PEG polymers.

[0033] In certain embodiments, the plurality of PEG lipids are substantially free of PEG. In certain embodiments, the plurality of PEG lipids are substantially free of fatty acids. "Substantially free," as used herein with respect to impurities, refers to the presence of a maximum of 0-5% impurities. In certain embodiments, 0-2% impurities may be present. In certain embodiments, 0-1% impurities may be present.

[0034] In certain embodiments, the PEG lipid is a compound of the following formula: [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive; R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group.

[0035] In certain embodiments, the PEG lipid is a compound of the following formula: [ka] or a salt thereof, wherein r is independently an integer of 35 to 55, inclusive.

[0036] In certain embodiments, the impurity is: [ka] or one or more salts thereof, wherein r is independently an integer between 35 and 55, inclusive; O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group.

[0037] As described herein, the present invention relates in part to high-purity PEG lipids. Such species may alternatively be referred to as PEGylated lipids. Non-limiting examples of PEG lipids are provided herein. For other embodiments, including additional examples of PEG lipids useful in the present invention, see International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG lipids useful in the present invention can be the PEGylated lipids described in International Publication No. WO2012 / 099755, published July 26, 2012, the contents of which are incorporated herein by reference in their entirety.

[0038] PEG lipid is a lipid modified with polyethylene glycol.PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG.

[0039] Any of these exemplary PEG lipids described herein can be modified to include a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0040] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (III): Provided herein is a compound of formula (III): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer from 1 to 100, inclusive; L 1 is an arbitrarily substituted C 1-10alkylene, wherein the optionally substituted C 1-10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )-, D is a moiety obtained by click chemistry or cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] It is of L 2 each instance independently represents a bond or an optionally substituted C 1-6 alkylene, wherein the optionally substituted C 1-6 One methylene unit of the alkylene is optionally substituted with -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )-, R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, optionally where R 2one or more methylene units in the formula (I) are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, R N is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; p is 1 or 2.

[0041] In certain embodiments, the compound of formula (III) is a PEG-OH lipid (i.e., R 3 HA-OR O and R O is hydrogen). In certain embodiments, the compound of formula (III) is of formula (III-OH): [ka] or a salt thereof.

[0042] In certain embodiments, D is a moiety obtained by click chemistry (e.g., a triazole). In certain embodiments, the compound of formula (III) has formula (III-a-1) or (III-a-2): [ka] or a salt thereof.

[0043] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0044] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0045] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0046] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] [ka] or a salt thereof.

[0047] In certain embodiments, D is a moiety that is cleavable under physiological conditions (e.g., an ester, amide, carbonate, carbamate, urea). In certain embodiments, the compound of formula (III) is of formula (III-b-1) or (III-b-2): [ka] or a salt thereof.

[0048] In certain embodiments, the compound of formula (III) is of formula (III-b-1-OH) or (III-b-2-OH): [ka] or a salt thereof.

[0049] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0050] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0051] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0052] In certain embodiments, the compound of formula (III) has one of the following formulas: [ka] or a salt thereof.

[0053] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid.In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (V).Provided herein is a compound of formula (V): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl or oxygen protecting group; r is an integer from 1 to 100, inclusive; R 5 is an arbitrarily substituted C 10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl, and optionally R 5 one or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N)-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, R N Each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0054] In certain embodiments, the compound of formula (V) is of formula (V-OH): [ka] or a salt thereof.

[0055] In certain embodiments, the compound of formula (V) has one of the following formulas: [ka] or a salt thereof.

[0056] In certain embodiments, the PEG lipids useful in the present invention are PEG lipids of formula (LI): [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 is a bond, optionally substituted C 1-3 Alkylene, optionally substituted C 1-3 Heteroalkylene, optionally substituted C 2-3 Alkenylene, optionally substituted C 2-3 is alkynylene, R 1 is an arbitrarily substituted C 5-30 Alkyl, optionally substituted C 5-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer from 2 to 100, inclusive.

[0057] In certain embodiments, the PEG lipid of formula (LI) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Y 1 is a bond, -CR2-, -O-, -NR N -, or -S-, each instance of R is independently hydrogen, halogen, or optionally substituted alkyl; R Nis hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group.

[0058] In certain embodiments, the PEG lipid of formula (LI) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein Each instance of R is independently hydrogen, halogen, or optionally substituted alkyl.

[0059] In certain embodiments, the PEG lipid of formula (LI) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein s is an integer from 5 to 25, inclusive.

[0060] In certain embodiments, the PEG lipid of formula (LI) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0061] In certain embodiments, the PEG lipid of formula (LI) is: [ka] [ka] and pharmaceutically acceptable salts thereof.

[0062] In certain embodiments, the PEG lipid of formula (LI) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0063] In certain embodiments, the PEG lipids useful in the present invention are PEG lipids of formula (L-II): [ka] or a pharmaceutically acceptable salt thereof, wherein R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer from 2 to 100, inclusive; m is an integer of 5 to 15, inclusive, or an integer of 19 to 30, inclusive.

[0064] In certain embodiments, the PEG lipid of formula (L-II) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0065] In certain embodiments, the PEG lipid of formula (L-II) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0066] In certain embodiments, the PEG lipid of formula (L-II) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0067] In certain embodiments, the PEG lipids useful in the present invention are PEG lipids of formula (L-III): [ka] or a pharmaceutically acceptable salt thereof, wherein Y 2 -O-, -NR N -, or -S-, R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 5-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group; r is an integer from 2 to 100, inclusive.

[0068] In certain embodiments, the PEG lipid of formula (L-III) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0069] In certain embodiments, the PEG lipid of formula (L-III) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein Each occurrence of s is independently an integer from 5 to 25, inclusive.

[0070] In certain embodiments, the PEG lipid of formula (L-III) has one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0071] In certain embodiments, the PEG lipid of formula (L-III) is: [ka] and pharmaceutically acceptable salts thereof.

[0072] In certain embodiments, the PEG lipids useful in the present invention are PEG lipids of formula (L-IV): [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 is a bond, optionally substituted C 1-3 Alkylene, optionally substituted C 1-3 Heteroalkylene, optionally substituted C 2-3 Alkenylene, optionally substituted C 2-3 is alkynylene, R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 3-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group; r is an integer from 2 to 100, inclusive.

[0073] In certain embodiments, the PEG lipid of formula (L-IV) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Y 1 is a bond, -CR2-, -O-, -NR N -, or -S-, Each instance of R is independently hydrogen, halogen, or optionally substituted alkyl.

[0074] In certain embodiments, the PEG lipid of formula (L-IV) has one of the following formulas: [ka] [ka] or a pharmaceutically acceptable salt thereof, wherein Each instance of R is independently hydrogen, halogen, or optionally substituted alkyl.

[0075] In certain embodiments, the PEG lipid of formula (L-IV) has one of the following formulas: [ka] [ka] or a pharmaceutically acceptable salt thereof, wherein Each s is independently an integer from 5 to 25, inclusive.

[0076] In certain embodiments, the PEG lipid of formula (L-IV) has one of the following formulas: [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0077] In certain embodiments, the PEG lipid of formula (L-IV) is: [ka] [ka] and pharmaceutically acceptable salts thereof.

[0078] In certain embodiments, the PEG lipid is: [ka] and pharmaceutically acceptable salts thereof.

[0079] In certain embodiments, the PEG lipid is of formula (LV): [ka] and pharmaceutically acceptable salts thereof, wherein L 2 is a bond, optionally substituted C 1-6 Alkylene, optionally substituted C 1-6 Heteroalkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6 alkynylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted heterocyclylene, or optionally substituted carbocyclylene, or a combination thereof; R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 5-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer from 2 to 100, inclusive.

[0080] In certain embodiments, the PEG lipid of formula (LV) is one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0081] In certain embodiments, the PEG lipid of formula (LV) is one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof.

[0082] In certain embodiments, the PEG lipid of formula (LV) is: [ka] and pharmaceutically acceptable salts thereof.

[0083] In certain embodiments, the PEG lipid of formula (L-VI) is: [ka] and pharmaceutically acceptable salts thereof, wherein L 2 is a bond, optionally substituted C 1-6 Alkylene, optionally substituted C 1-6 Heteroalkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6 alkynylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted heterocyclylene, or optionally substituted carbocyclylene, or a combination thereof; Ring P is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, or optionally substituted carbocyclyl; R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 3-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer from 2 to 100, inclusive.

[0084] In certain embodiments, the PEG lipid of formula (L-VI) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0085] In certain embodiments, the PEG lipid of formula (L-VI) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Each s is independently an integer from 5 to 25, inclusive.

[0086] In certain embodiments, the PEG lipid of formula (L-VI) is of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0087] High purity PEG lipid of formula (I) In certain embodiments, the PEG lipids are a plurality of compounds of formula (I), or salts thereof. Provided herein are compounds of formula (I): [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive, and the plurality of compounds of formula (I) have a purity of 87% or greater with respect to the compound of formula (I).

[0088] As defined herein, r is an integer between 35 and 55, inclusive. In certain embodiments, r is independently an integer between 36 and 54, inclusive. In certain embodiments, r is independently an integer between 37 and 53, inclusive. In certain embodiments, r is independently an integer between 38 and 52, inclusive. In certain embodiments, r is independently an integer between 39 and 51, inclusive. In certain embodiments, r is independently an integer between 40, inclusive. In certain embodiments, r is independently an integer between 40 and 50, inclusive. In certain embodiments, r is independently an integer between 41 and 49, inclusive. In certain embodiments, r is independently an integer between 42 and 48, inclusive. In certain embodiments, r is independently an integer between 43 and 57, inclusive. In certain embodiments, r is independently an integer between 44 and 46, inclusive.

[0089] In certain embodiments, r is independently 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In certain embodiments, r is independently 35. In certain embodiments, r is independently 36. In certain embodiments, r is independently 37. In certain embodiments, r is independently 38. In certain embodiments, r is independently 39. In certain embodiments, r is independently 40. In certain embodiments, r is independently 41. In certain embodiments, r is independently 42. In certain embodiments, r is independently 43. In certain embodiments, r is independently 44. In certain embodiments, r is independently 45. In certain embodiments, r is independently 46. In certain embodiments, r is independently 47. In certain embodiments, r is independently 48. In certain embodiments, r is independently 49. In certain embodiments, r is independently 50. In certain embodiments, r is independently 51. In certain embodiments, r is independently 52. ​​In certain embodiments, r is independently 53. In certain embodiments, r is independently 54. In certain embodiments, r is independently 55.

[0090] It should be understood that a "plurality" of compounds of Formula (I) can include a mixture of compounds of Formula (I) with different values ​​of r (i.e., different lengths of PEG chains). In such cases, the recommended purity (87% or greater, some embodiments provided below) refers to the sum of all compounds of Formula (I) in the mixture. For example, a plurality of compounds of Formula (I) with a purity of about 98% would contain 98% (mol%) of compounds of Formula (I), each of the compounds of Formula (I) in the mixture independently having a value of r between 35 and 55, inclusive. In certain embodiments, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the compounds have a value of r between 40 and 50, inclusive. In certain embodiments, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the compounds have a value of r between 42 and 48, inclusive. In certain embodiments, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the compounds have a value of r between 43 and 46, inclusive. In certain embodiments, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the compounds have an r=45.

[0091] As described herein, the plurality of compounds of Formula (I), or salts thereof, have a purity of greater than 87% with respect to the compounds of Formula (I), or salts thereof. In certain embodiments, the purity is greater than 90%. In certain embodiments, the purity is greater than 91%. In certain embodiments, the purity is greater than 92%. In certain embodiments, the purity is greater than 93%. In certain embodiments, the purity is greater than 94%. In certain embodiments, the purity is greater than 95%. In certain embodiments, the purity is greater than 96%. In certain embodiments, the purity is greater than 97%. In certain embodiments, the purity is greater than 98%. In certain embodiments, the purity is greater than 98.5%. In certain embodiments, the purity is greater than 99%. In certain embodiments, the purity is greater than 99.1%. In certain embodiments, the purity is greater than 99.2%. In certain embodiments, the purity is greater than 99.3%. In certain embodiments, the purity is greater than 99.4%. In certain embodiments, the purity is greater than 99.5%. In certain embodiments, the purity is greater than 99.6%. In certain embodiments, the purity is greater than 99.7%. In certain embodiments, the purity is greater than 99.8%. In certain embodiments, the purity is greater than 99.9%. In certain embodiments, the purity is 100%.

[0092] In certain embodiments, the plurality of compounds of Formula (I), or salts thereof, have a purity of 87 to 100%, inclusive, with respect to the compounds of Formula (I), or salts thereof, and in certain embodiments, the purity is 87 to 99.9%, inclusive, with respect to the compounds of Formula (I), or salts thereof.

[0093] In certain embodiments, the purity is 87-90%, including 87% and 90%. In certain embodiments, the purity is 90-95%, including 90% and 95%. In certain embodiments, the purity is 95-100%, including 95% and 100%. In certain embodiments, the purity is 95-99.9%, including 95% and 99.9%.

[0094] In certain embodiments, the purity is 90-100%, including 90% and 100%. In certain embodiments, the purity is 90-99.9%, including 90% and 99.9%. In certain embodiments, the purity is 91-99.9%, including 91% and 99.9%. In certain embodiments, the purity is 92-99.9%, including 92% and 99.9%. In certain embodiments, the purity is 93-99.9%, including 93% and 99.9%. In certain embodiments, the purity is 94-99.9%, including 94% and 99.9%. In certain embodiments, the purity is 95-99.9%, including 95% and 99.9%. In certain embodiments, the purity is 96-99.9%, including 96% and 99.9%. In certain embodiments, the purity is between 97 and 99.9%, including 97% and 99.9%. In certain embodiments, the purity is between 98 and 99.9%, including 98% and 99.9%. In certain embodiments, the purity is between 99 and 99.9%, including 99% and 99.9%.

[0095] In certain embodiments, the plurality of compounds of Formula (I), or salts thereof, have a purity of about 87% with respect to the compounds of Formula (I), or salts thereof. In certain embodiments, the purity is about 87.5%. In certain embodiments, the purity is about 88%. In certain embodiments, the purity is about 88.5%. In certain embodiments, the purity is about 89%. In certain embodiments, the purity is about 89.5%. In certain embodiments, the purity is about 90%. In certain embodiments, the purity is about 90.5%. In certain embodiments, the purity is about 91%. In certain embodiments, the purity is about 91.5%. In certain embodiments, the purity is about 92%. In certain embodiments, the purity is about 92.5%. In certain embodiments, the purity is about 93%. In certain embodiments, the purity is about 93.5%. In certain embodiments, the purity is about 94%. In certain embodiments, the purity is about 94.5%. In certain embodiments, the purity is about 95%. In certain embodiments, the purity is about 95.5%. In certain embodiments, the purity is about 96%. In certain embodiments, the purity is about 96.6%. In certain embodiments, the purity is about 97%. In certain embodiments, the purity is about 97.7%. In certain embodiments, the purity is about 98%. In certain embodiments, the purity is about 98.5%. In certain embodiments, the purity is about 99%. In certain embodiments, the purity is about 99.5%. In certain embodiments, the purity is about 99.9%. In certain embodiments, the purity is about 100%. In certain embodiments, the purity is about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, or 98.9%. In certain embodiments, the purity is about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0096] In certain embodiments, the plurality of compounds consists of compounds of formula (I), or a salt thereof. In certain embodiments, the plurality of compounds consists essentially of compounds of formula (I), or a salt thereof.

[0097] In certain embodiments, the plurality of compounds of Formula (I), or salts thereof, are substantially free of other compounds. In certain embodiments, the plurality of compounds are substantially free of impurities. The term "impurities" refers to compounds other than the compound of Formula (I) or a salt thereof. In certain embodiments, the plurality of compounds contains less than 13% impurities in total. In certain embodiments, the plurality of compounds contains less than 12% impurities in total. In certain embodiments, the plurality of compounds contains less than 11% impurities in total. In certain embodiments, the plurality of compounds contains less than 10% impurities in total. In certain embodiments, the plurality of compounds contains less than 9% impurities in total. In certain embodiments, the plurality of compounds contains less than 8% impurities in total. In certain embodiments, the plurality of compounds contains less than 7% impurities in total. In certain embodiments, the plurality of compounds contains less than 6% impurities in total. In certain embodiments, the plurality of compounds contains less than 5% impurities in total. In certain embodiments, the plurality of compounds contains less than 4% impurities in total. In certain embodiments, the plurality of compounds contains less than 3% impurities in total. In certain embodiments, the plurality of compounds contains less than 2% total impurities, hi certain embodiments, the plurality of compounds contains less than 1% total impurities.

[0098] In certain embodiments, the PEG lipids in the plurality of compounds of formula (I) are the same compound. In certain embodiments, the plurality of PEG lipids comprises a mixture of compounds of formula (I). The plurality of compounds of formula (I) provided herein may contain other components, such as solvents, carriers, excipients, etc., and therefore may be in the form of a solution or a composition. When the compound of formula (I) is part of a solution or composition, it should be understood that the prescribed / recommended purity (e.g., 87% or higher) is for the compound of formula (I) in the solution or composition.

[0099] In certain embodiments, the impurities include one or more fatty acids. In certain embodiments, the impurities include one or more PEG polymers. In certain embodiments, the impurities include one or more fatty acids and / or one or more PEG polymers. In certain embodiments, the impurities are selected from the following: [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive. In certain embodiments, the impurity is a compound of the following formula: [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive. In certain embodiments, the impurity is the following compound: [ka] In certain embodiments, the impurity comprises a polymer of the formula: [ka] or a salt thereof, wherein r is independently an integer from 35 to 55, inclusive.

[0100] In certain embodiments, the compounds are substantially free of PEG. In certain embodiments, the compounds are substantially free of fatty acids. In certain embodiments, the compounds of formula (I), or salts thereof, are selected from the group consisting of: [ka] or salts thereof, wherein r is independently an integer between 35 and 55, inclusive. In certain embodiments, the plurality of compounds of Formula (I), or salts thereof, is substantially free of one or more of the formula: [ka] or a salt thereof, wherein r is independently an integer between 35 and 55, inclusive. In certain embodiments, the plurality of compounds of Formula (I), or a salt thereof, is substantially free of a compound: [ka] In certain embodiments, the plurality of compounds of formula (I), or salts thereof, is substantially free of a compound of formula (I), or a salt thereof. [ka] or salts thereof, wherein r is independently an integer between 35 and 55, inclusive. "Substantially free," as used herein with respect to impurities, refers to the presence of a maximum of 0-5% impurities. In certain embodiments, 0-2% impurities may be present. In certain embodiments, 0-1% impurities may be present.

[0101] The polydispersity index (PDI) of the PEG lipids provided herein (e.g., compounds of formula (I)) can be measured. "Polydispersity index" refers to a measure of the heterogeneity of the size of molecules in a mixture, and is also called "heterogeneity index" or "dispersity." In other words, it is a measure of the molecular weight distribution of a given polymer sample. PDI is calculated based on the following formula: PDI=Mw / M n , where M w is the weight average molecular weight, and M n is the number average molecular weight. As the polymer chains (e.g., PEG chains) in a polymer mixture approach uniform chain length, the PDI approaches a value of 1. In certain embodiments, the PEG lipids provided herein have a PDI of 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, or less. In certain embodiments, the PEG lipids provided herein have a PDI of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or less. In certain embodiments, the PEG lipids provided herein have a PDI of 1.0 to 1.10, inclusive. In certain embodiments, the PEG lipids provided herein have a PDI of 1.02 to 1.10, inclusive. In certain embodiments, the PEG lipids provided herein have a PDI of 1.0 to 1.04, inclusive. In certain embodiments, the PEG lipids provided herein have a PDI of 1.02 to 1.04, inclusive. In certain embodiments, the PEG lipids provided herein have a PDI of 1.10 or less. In certain embodiments, the PEG lipids provided herein have a PDI of 1.04 or less. In certain embodiments, the PEG lipids provided herein have a PDI of about 1.04. In certain embodiments, the PEG lipids provided herein have a PDI of about 1.03. In certain embodiments, the PEG lipids provided herein have a PDI of about 1.02. In certain embodiments, the PEG lipids provided herein have a PDI of about 1.01.

[0102] Lipid nanoparticles (LNPs) containing highly purified PEG-lipids Provided herein is lipid nanoparticle (LNP) that comprises the high-purity PEG lipid described herein.In other words, provided herein is lipid nanoparticle (LNP) that comprises a plurality of PEG lipids, and the plurality of PEG lipids have a purity of 87% or more with respect to the PEG lipid component of the LNP.Any PEG lipid described herein can constitute the high-purity PEG lipid component of the LNP provided herein.

[0103] When "PEG lipid" or "high-purity PEG lipid" is considered a component of an LNP formulation, it means that the mixture of PEG lipids has a purity of 87% or more, as proscribed / recommended for the PEG lipid. Other embodiments (i.e., recommended purity) are provided in the above section. In addition to the PEG lipids provided herein, the LNP may contain one or more additional lipid components.

[0104] Provided herein are lipid nanoparticles (LNPs) comprising a plurality of PEG-lipids provided herein (e.g., a plurality of compounds of Formula (I) provided herein having a purity of 87% or greater). In certain embodiments, the PEG-lipid is present in the LNP at a molar ratio of 0.15-15% relative to other lipids. In certain embodiments, the PEG-lipid is present at a molar ratio of 0.15-5% relative to other lipids. In certain embodiments, the PEG-lipid is present at a molar ratio of 1-5% relative to other lipids. In certain embodiments, the PEG-lipid is present at a molar ratio of 0.15-2% relative to other lipids. In certain embodiments, the PEG-lipid is present at a molar ratio of 1-2% relative to other lipids. In certain embodiments, the PEG-lipid is present at a molar ratio of about 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% relative to other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of about 1.5% relative to other lipids.

[0105] Examples of PEG lipids are provided herein, and any of them can be used with a recommended purity of 87% or greater. In certain embodiments, the PEG lipid is a plurality of compounds of Formula (I) described herein. In certain embodiments, the plurality of compounds of Formula (I) are present in the LNP at a molar ratio of 0.15-15% relative to other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present at a molar ratio of 0.15-5% relative to other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present at a molar ratio of 1-5% relative to other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present at a molar ratio of 0.15-2% relative to other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present at a molar ratio of 1-2% relative to other lipids. In certain embodiments, the plurality of compounds of formula (I) are present in a molar ratio of about 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% relative to other lipids.In certain embodiments, the plurality of compounds of formula (I) are present in a molar ratio of about 1.5% relative to other lipids.

[0106] The LNPs provided herein can contain any lipid(s) in addition to the highly purified PEG-lipid component. As generally defined herein, the term "lipid" refers to a small molecule having hydrophobic or amphiphilic properties. Lipids can be naturally occurring or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, as well as prenol lipids. In some instances, the amphiphilic nature of some lipids allows them to form liposomes, vesicles, or membranes in aqueous media.

[0107] In certain embodiments, for example, the lipid nanoparticles may comprise a PEG lipid, an ionic amino lipid, a helper lipid, and a structural lipid.

[0108] Lipid nanoparticles (LNPs) containing highly purified PEG lipids and ionic amino lipids In certain embodiments, the LNP comprises an ionic amino lipid (ie, a cationic lipid).

[0109] Provided herein are LNPs comprising a high-purity PEG-lipid component (i.e., having a purity of 87% or greater as proscribed / recommended for the PEG-lipid component) as described herein and an ionic amino lipid. In certain embodiments, the LNPs comprise a high-purity PEG-lipid component as described herein and a compound of formula (X). In certain embodiments, the LNPs comprise a plurality of compounds of formula (I) as described herein (i.e., having a purity of 87% or greater as proscribed / recommended for the PEG-lipid component) and a compound of formula (X).

[0110] The compound of formula (X) has the following formula: [ka] or a salt or isomer thereof, wherein R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR" or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH) nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)C(O)OR; each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of Y-R″, -Y-R″, and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl, Each R * independently, C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0111] In some embodiments, the compound of formula (X) is of formula (XA): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M′; and R is an unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; R and R are independently selected from H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0112] In certain embodiments, the compound of formula (X) is of formula (XI): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M is a bond or M′; and R is an unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where n is 2, 3, or 4; Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; R and R are independently selected from H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0113] In some embodiments, the compound of formula (X) is of formula (XIa), (XIb), (XIc), or (XIe): [ka] or a salt or isomer thereof, and R4 is as defined herein.

[0114] In certain embodiments, the compound of formula (X) is of formula (XId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, each of R2 and R3 can independently be C 5-14Alkyl and C 5-14 alkenyl.

[0115] In certain embodiments, the compound of formula (X) has the following structure: [ka] or a salt thereof.

[0116] In certain embodiments, the LNPs comprise a high-purity PEG-lipid component described herein and compound (B). In certain embodiments, the LNPs comprise a plurality of compounds of formula (I) described herein and compound (B).

[0117] In certain embodiments, the compound of formula (X) has the structure: [ka] or a salt thereof.

[0118] In certain embodiments, the LNPs comprise a high-purity PEG-lipid component described herein and compound (C). In certain embodiments, the LNPs comprise a plurality of compounds of formula (I) described herein and compound (C).

[0119] In certain embodiments, the compound of formula (X) is [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the LNPs comprise a high-purity PEG-lipid component as described herein and one of the compounds of formula (X) above. In certain embodiments, the LNPs comprise a plurality of compounds of formula (I) as described herein, one of the compounds of formula (X) above.

[0120] In certain embodiments, the LNPs comprise a high purity PEG-lipid component (i.e., having a proscribed / recommended purity of 87% or greater for the PEG-lipid component) and a compound of Formula (Y) as described herein. In certain embodiments, the LNPs comprise a plurality of compounds of Formula (I) (i.e., having a proscribed / recommended purity of 87% or greater for the PEG-lipid component) and a compound of Formula (Y) as described herein.

[0121] The compound of formula (Y) has the following formula: [ka] or a salt thereof, wherein Ring A is [ka] and A1 and A2 are each independently selected from CH and N; Z is CH2 or absent, where when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is absent, both the dashed lines (1) and (2) are absent; R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 Alkenyl, -R"MR', -R * YR”, -YR”, and -R * OR" each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X1, X2, and X3 are independently selected from the group consisting of a bond, —CH2—, —(CH2)2—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH2—, —CH2C(O)—, —C(O)O—CH2—, —OC(O)—CH2—, —CH2C(O)O—, —CH2OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independently C 3-6 is a carbocyclic ring, Each R * independently, C 1-12 Alkyl and C 2-12 alkenyl, Each R is independently C 1-3 Alkyl and C 3-6 is selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 selected from the group consisting of alkenyl, and H; Each R" is independently C 3-12 Alkyl and C 3-12 alkenyl.

[0122] In certain embodiments, the compound of formula (Y) is one of formulas (Ya1), (Ya2), (Ya3), (Ya4), (Ya5), or (Ya6): [ka] or a salt or stereoisomer thereof.

[0123] In certain embodiments, the compound of formula (Y) has the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0124] In certain embodiments, the LNPs comprise a high-purity PEG-lipid component described herein and Compound (A). In certain embodiments, the LNPs comprise a plurality of compounds of Formula (I) described herein and Compound (A).

[0125] Further examples of compounds of formula (X) and (Y) can be found, for example, in International Publication No. WO2017 / 049245, published March 23, 2017, and International Publication No. WO2017 / 112865, the entire contents of each of which are incorporated herein by reference.

[0126] In certain embodiments, the ionizable amino lipid (e.g., a compound of formula (X) or (Y), or compound (A), (B), or (C)) is present in a molar ratio of about 25-65% relative to other lipids. In certain embodiments, the ionizable amino lipid is present in a molar ratio of about 25-50% relative to other lipids.

[0127] In certain embodiments, the ionic amino lipid (e.g., a compound of Formula (X) or (Y), or compound (A), (B), or (C)) is present in a molar ratio of about 40% or less relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 35% or less relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 30% or less relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 25-40% relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 25-35% relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 30-40% relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 35-45% relative to the other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 40-50% relative to the other lipids. In certain embodiments, the ionic amino lipid is present at a molar ratio of about 45-55% relative to other lipids.In certain embodiments, the ionic amino lipid is present at a molar ratio of about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% relative to other lipids.In certain embodiments, the ionic amino lipid is present at a molar ratio of about 30% relative to other lipids.In certain embodiments, the ionic amino lipid is present at a molar ratio of about 40% relative to other lipids.

[0128] The present specification provides an example of ionic amino lipid (also referred to as " cationic lipid ").Ionic / cationic lipid is generally a lipid that can have positive or partial positive charge at physiological pH.In certain embodiments, the ionic amino lipid useful in the present invention can be found in International Publication No. WO2017 / 049245, published on March 23, 2017, or International Publication No. WO2017 / 112865.The entire contents of each of these are incorporated herein by reference.

[0129] In certain embodiments, the cationic and / or ionic lipids are 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy -N,N-Dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2- Dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[ The cationic lipid may be selected from the non-limiting group consisting of (9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition, the cationic lipid may also be a lipid containing a cyclic amine group. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the present invention.

[0130] In one embodiment, the cationic lipid is selected from the group consisting of those described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, and Examples of cationic lipids include, but are not limited to, those described in WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122, and U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, and S20130225836, the contents of each of which are incorporated herein by reference in their entirety.

[0131] In another embodiment, the cationic lipid can be selected from, but is not limited to, Formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, and WO2013116126, or U.S. Patent Publication Nos. US20130178541 and US20130225836, the contents of each of which are incorporated herein by reference in their entirety. In yet another embodiment, the cationic lipid can be selected from, but is not limited to, formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of U.S. Patent No. 7,893,302, formula CLI-CLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Publication No. US20100036115, and formula I of U.S. Patent Publication No. US20130123338, each of which is incorporated herein by reference in its entirety.

[0132] Non-limiting examples of the cationic lipid include (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylheneicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17 -dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien- 8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z) -N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethyleptacosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacontriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicos-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16 -dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentyl cyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2- Decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien- 1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine 1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE, (2S)-1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(PENTYLOXY)PROPAN-2-AMINE, (2S)-1-(HEXYLOXY)-3-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE, 1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE (i) propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane- 2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, or a pharmaceutically acceptable salt or stereoisomer thereof. Each possibility represents a separate embodiment of the present invention.

[0133] In one embodiment, the lipid may be a cleavable lipid, such as those described in International Publication No. WO2012170889, the entire contents of which are incorporated herein by reference. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, Formula (I) of U.S. Patent Application No. US20130064894, the entire contents of which are incorporated herein by reference. In one embodiment, the cationic lipid may be synthesized by methods known in the art and / or the methods described in International Publication No. WO2013086354, the contents of each of which are incorporated herein by reference in their entirety. In another embodiment, the cationic lipid may be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids, as well as methods for making and using the trialkyl cationic lipids, are described in International Patent Publication No. WO2013126803, the contents of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, the LNPs comprise the lipid KL52 (an amino lipid disclosed in U.S. Patent Publication No. 2012 / 0295832, which is incorporated herein by reference in its entirety). The activity and / or safety of LNP administration (as measured by examining one or more of ALT / AST, white blood cell count, and cytokine induction) may be improved by incorporating such a lipid. KL52-containing LNPs may be administered intravenously and / or one or more times. In some embodiments, administration of KL52-containing LNPs results in comparable or improved mRNA and / or protein expression compared to LNPs containing MC3.

[0135] As non-limiting examples, the LNPs can include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, as well as the cationic peptides described in International Publication No. WO2012013326 or U.S. Patent Publication No. US20130142818, each of which is incorporated herein by reference in its entirety.

[0136] Other LNP components In certain embodiments, the LNP comprises a helper lipid. In certain embodiments, the helper lipid is present in a molar ratio of about 10-40% relative to other lipids. In certain embodiments, the helper lipid is present in a molar ratio of about 10-20% relative to other lipids. In certain embodiments, the helper lipid is present in a molar ratio of about 15-25% relative to other lipids. In certain embodiments, the helper lipid is present in a molar ratio of about 20-30% relative to other lipids. In certain embodiments, the helper lipid is present in a molar ratio of about 25-35% relative to other lipids. In certain embodiments, the helper lipid is present in a molar ratio of about 30-40% relative to other lipids. In certain embodiments, the ionic amino lipid is present in a molar ratio of about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% with respect to other lipids.In certain embodiments, the helper lipid is present in a molar ratio of about 10% with respect to other lipids.In certain embodiments, the helper lipid is present in a molar ratio of about 20% with respect to other lipids.In certain embodiments, the helper lipid is present in a molar ratio of about 30% with respect to other lipids.

[0137] The example of helper lipid is provided herein.In certain embodiments, the helper lipid is phospholipid.In certain embodiments, the helper lipid is DSPC or its analogue.In certain embodiments, the helper lipid is DSPC.In certain embodiments, the helper lipid is DOPE.In certain embodiments, the helper lipid is oleic acid or its analogue.In certain embodiments, the helper lipid is oleic acid.

[0138] The helper lipid useful in the present invention includes neutral and / or non-cationic lipid (for example, the lipid that is neutral or non-cationic lipid at physiological pH).Helper lipid can also be called "non-cationic" lipid.In certain embodiments, the helper lipid is a phospholipid.

[0139] For other embodiments, including examples of helper lipids useful in the present invention, see International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference.

[0140] As defined herein, a phospholipid is any lipid containing a phosphate group. Phospholipids are a subset of non-cationic lipids. The lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may assemble into one or more lipid bilayers. Generally, a phospholipid may include a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of 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. Non-naturally occurring species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. 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 upon exposure to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cellular recognition, or for attaching nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes). Each possibility represents a separate embodiment of the present invention.

[0141] Helper lipids, particularly phospholipids, useful in the compositions and methods include 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-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn- Glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-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-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-phosphocholine The nanoparticle composition may be selected from the non-limiting group consisting of ethanolamine, 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), and sphingomyelin. Each possibility represents a separate embodiment of the present invention. In some embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0142] In certain embodiments, the helper lipid is a fatty acid. The fatty acid may be saturated or unsaturated. Examples of unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linoelaidic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexanoic acid, or any cis / trans double bond isomer thereof. In certain embodiments, the lipid is oleic acid. In certain embodiments, the lipid is an isomer of oleic acid (e.g., its double bond is at a different position along the aliphatic chain compared to oleic acid). In certain embodiments, the lipid is an analog of oleic acid (e.g., its aliphatic chain is 1-10 carbons longer or 1-10 carbons shorter than that of oleic acid). Examples of saturated fatty acids include, but are not limited to, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid. In certain embodiments, oleic acid analogs are compounds in which the carboxylic acid moiety of oleic acid is replaced with a different group.

[0143] In certain embodiments, the helper lipid is a glycine derivative of a fatty acid (e.g., N-palitoylglycine or N-oleoglycine). In certain embodiments, the helper lipid is a glycerolipid (e.g., a monoglyceride, a diglyceride, a triglyceride). In certain embodiments, the helper lipid is a monoglyceride. In certain embodiments, the helper lipid is a diglyceride. In certain embodiments, the helper lipid is a triglyceride. In certain embodiments, the helper lipid comprises a sugar moiety (e.g., a monosaccharide, a disaccharide, a polysaccharide).

[0144] In certain embodiments, the LNP further comprises a structured lipid. In certain embodiments, the structured lipid is present in a molar ratio of about 30-50% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 30-40% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 35-45% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 40-50% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 30-35% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 35-40% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 40-45% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 45-50% relative to other lipids. In certain embodiments, the structured lipid is present in a molar ratio of about 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5% or 42% relative to other lipids.In certain embodiments, the structured lipid is present in a molar ratio of about 38.5% relative to other lipids.

[0145] Examples of structured lipids are provided herein. In certain embodiments, the structured lipid is a sterol. In certain embodiments, the structured lipid is cholesterol.

[0146] Non-limiting examples of structured lipids useful in the present invention are provided herein. Incorporation of structured lipids into the lipid nanoparticles may help reduce aggregation of other lipids within the particles. The structured lipid may be selected from the group consisting of, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol.

[0147] For other embodiments, including examples of helper lipids useful in the present invention, see International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference.

[0148] In certain embodiments, the LNP comprises a plurality of PEG-lipids provided herein (e.g., a plurality of compounds of Formula (I) provided herein), an ionic amino lipid, a helper lipid, and a structural lipid.

[0149] In certain embodiments, the LNPs comprise 25-65% ionic amino lipid, about 0.15-15% PEG lipid provided herein (e.g., a plurality of compounds of Formula (I) provided herein), about 30-50% structural lipid, and about 10-40% helper lipid.

[0150] Many other lipid and LNP formulations applicable to the present disclosure can be found, for example, in International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference.

[0151] Certain Embodiments of Lipid Nanoparticles (LNPs) Provided herein are lipid nanoparticles (LNPs) comprising an ionic amino lipid, a PEG lipid, a helper lipid (e.g., a phospholipid), and a structural lipid. In certain embodiments, the LNP comprises a drug to be delivered.

[0152] In certain embodiments, the LNPs provided herein comprise an ionizable amino lipid, Compound (A): [ka] , PEG lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (helper lipid), and cholesterol (structural lipid).

[0153] In certain embodiments, the LNPs comprise Compound (A), a high-purity PEG lipid described herein (i.e., having a purity of 87% or greater as described herein), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. In certain embodiments, the LNPs comprise Compound (A), a plurality of compounds of Formula (I) described herein (i.e., having a purity of 87% or greater as described herein), DOPE, and cholesterol.

[0154] In certain embodiments, compound (A) is present in a molar ratio of 40% or less relative to the other lipids. In certain embodiments, compound (A) is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, compound (A) is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, compound (A) is present in a molar ratio of about 30% relative to the other lipids. In certain embodiments, compound (A) is present in a molar ratio of about 40% relative to the other lipids.

[0155] In certain embodiments, the PEG lipid is present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of about 1.5% relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of about 1.5% relative to the other lipids.

[0156] In certain embodiments, DOPE is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of about 30% relative to the other lipids.

[0157] In certain embodiments, cholesterol is present in a molar ratio of 30-50%, including 30% and 50%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of 35-40%, including 35% and 40%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of about 38.5% relative to other lipids.

[0158] In certain embodiments, the LNPs provided herein comprise up to 40% molar ratio of Compound (A), 0.15-15% of a plurality of compounds of Formula (I) described herein, 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs provided herein comprise 20-40% molar ratio of Compound (A), 0.15-15% of a plurality of compounds of Formula (I) described herein, 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs comprise 25-35% molar ratio of Compound (A), 1-2% of a plurality of compounds of Formula (I), 25-35% DOPE, and 35-42% cholesterol.

[0159] In certain embodiments, the LNPs comprise Compound (A): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 30:30:38.5:1.5. In certain embodiments, the LNPs comprise Compound (A): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 40:20:38.5:1.5.

[0160] Also provided herein is Compound (B), an ionic amino lipid: [ka] , LNPs containing PEG lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (helper lipid), and cholesterol (structural lipid).

[0161] In certain embodiments, the LNPs comprise compound (B), a highly purified PEG lipid as described herein (i.e., having a purity of 87% or greater as described herein), DSPC, and cholesterol. In certain embodiments, the LNPs comprise compound (B), a plurality of compounds of formula (I) as described herein (i.e., having a purity of 87% or greater as described herein), DSPC, and cholesterol.

[0162] In certain embodiments, compound (B) is present in a molar ratio of 20-50%, including 20% ​​and 50%, relative to the other lipids. In certain embodiments, compound (B) is present in a molar ratio of 30-45%, including 30% and 45%, relative to the other lipids. In certain embodiments, compound (B) is present in a molar ratio of 40% or less relative to the other lipids. In certain embodiments, compound (B) is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, compound (B) is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, compound (B) is present in a molar ratio of about 30%. In certain embodiments, compound (B) is present in a molar ratio of about 40%.

[0163] In certain embodiments, the PEG lipid is present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of about 1.5% relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the compound of Formula (I) is present in a molar ratio of about 1.5% relative to the other lipids.

[0164] In certain embodiments, DOPE is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of about 30% relative to the other lipids.

[0165] In certain embodiments, cholesterol is present in a molar ratio of 30-50%, including 30% and 50%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of 35-40%, including 35% and 40%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of about 38.5% relative to other lipids.

[0166] In certain embodiments, the LNPs comprise up to 40% molar ratio of compound (B), 0.15-15% of a plurality of compounds of Formula (I), 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs comprise 20-40% molar ratio of compound (B), 0.15-15% of a plurality of compounds of Formula (I), 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs comprise 25-35% molar ratio of compound (B), 1-2% of a plurality of compounds of Formula (I), 25-35% DOPE, and 35-42% cholesterol.

[0167] In certain embodiments, the LNPs comprise Compound (B): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 30:30:38.5:1.5. In certain embodiments, the LNPs comprise Compound (B): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 40:20:38.5:1.5.

[0168] Also provided herein is a compound (C) which is an ionic amino lipid: [ka] , LNPs containing PEG lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (helper lipid), and cholesterol (structural lipid).

[0169] In certain embodiments, the LNPs comprise Compound (C), a highly purified PEG lipid as described herein (i.e., having a purity of 87% or greater as described herein), DSPC, and cholesterol. In certain embodiments, the LNPs comprise Compound (C), a plurality of compounds of Formula (I) as described herein (i.e., having a purity of 87% or greater as described herein), DSPC, and cholesterol.

[0170] In certain embodiments, compound (C) is present in a molar ratio of 20-50%, including 20% ​​and 50%, relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of 30-45%, including 30% and 45%, relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of 40% or less relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of about 30% relative to the other lipids. In certain embodiments, compound (C) is present in a molar ratio of about 40% relative to the other lipids.

[0171] In certain embodiments, the PEG lipid is present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the PEG lipid is present in a molar ratio of about 1.5% relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 0.15-15%, including 0.15% and 15%, relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of 1-2%, including 1% and 2%, relative to the other lipids. In certain embodiments, the plurality of compounds of Formula (I) are present in a molar ratio of about 1.5% relative to the other lipids.

[0172] In certain embodiments, DOPE is present in a molar ratio of 20-40%, including 20% ​​and 40%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of 25-35%, including 25% and 35%, relative to the other lipids. In certain embodiments, DOPE is present in a molar ratio of about 30% relative to the other lipids.

[0173] In certain embodiments, cholesterol is present in a molar ratio of 30-50%, including 30% and 50%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of 35-40%, including 35% and 40%, relative to other lipids. In certain embodiments, cholesterol is present in a molar ratio of about 38.5% relative to other lipids.

[0174] In certain embodiments, the LNPs comprise up to 40% molar ratio of compound (C), 0.15-15% of a plurality of compounds of Formula (I), 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs comprise 20-40% molar ratio of compound (C), 0.15-15% of a plurality of compounds of Formula (I), 20-40% DOPE, and 30-50% cholesterol. In certain embodiments, the LNPs comprise 25-35% molar ratio of compound (C), 1-2% of a plurality of compounds of Formula (I), 25-35% DOPE, and 35-42% cholesterol.

[0175] In certain embodiments, the LNPs comprise Compound (C): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 30:30:38.5:1.5. In certain embodiments, the LNPs comprise Compound (C): the compound of Formula (I): DOPE: cholesterol in a molar ratio of about 40:20:38.5:1.5.

[0176] Method for preparing PEG lipids Provided herein are methods for preparing PEG lipids, including compounds of formula (I). As shown in Scheme A and Scheme B, the methods include reacting a PEG component with a carboxylic acid component to obtain a PEG lipid. [ka]

[0177] Provided herein are compounds of the formula: [ka] or a salt thereof, the method comprising: [ka] or a salt thereof, [ka] or a salt thereof, wherein r is an integer between 35 and 55, inclusive; and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group.

[0178] In certain embodiments, the method comprises administering to a patient a compound of formula (I): [ka] or a salt thereof, the method comprising: [ka] or a salt thereof, [ka] or a salt thereof, wherein r is an integer between 35 and 55, inclusive.

[0179] In certain embodiments, more than 1 equivalent of the PEG component is used (relative to the carboxylic acid component). In certain embodiments, 1 to 20 equivalents of the PEG component are used. In certain embodiments, 1 to 10 equivalents of the PEG component are used. In certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents of the PEG component are used. In certain embodiments, about 2 equivalents of the PEG component are used. In certain embodiments, about 10 equivalents of the carboxylic acid component are used.

[0180] In certain embodiments, the reaction is carried out in the presence of a carboxyl-activating reagent. A carboxyl-activating reagent is a reagent capable of activating a carboxyl group for addition with a nucleophile (e.g., an alcohol, an amine). Carboxyl-activating reagents are known in the art. In certain embodiments, the carboxyl-activating reagent is a carbodiimide. In certain embodiments, the reagent is N,N'-dicyclohexylcarbodiimide (DCC). In certain embodiments, the reagent is N,N'-diisopropylcarbodiimide (DIC). In certain embodiments, the reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In certain embodiments, the reagent is the hydrochloride salt of a carbodiimide. In certain embodiments, the reagent is the hydrochloride salt of EDC (EDC·HCl). In certain embodiments, more than one equivalent (relative to the carboxylic acid moiety) of the carboxyl-activating agent (e.g., EDC or a salt thereof) is used. In certain embodiments, 1 to 2 equivalents, inclusive, are used. In certain embodiments, about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 equivalents are used. In certain embodiments, about 1.5 equivalents are used.

[0181] In certain embodiments, the reaction is carried out in the presence of a base. In certain embodiments, the base is an amine base. In certain embodiments, the base is a trialkylamine base. In certain embodiments, the base is a pyridine base. In certain embodiments, the base is 4-dimethylaminopyridine (DMAP). In certain embodiments, less than 1 equivalent (relative to the carboxylic acid component) of the base (e.g., DMAP) is used. In certain embodiments, 0.01 to 1.0 equivalents, inclusive, are used. In certain embodiments, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 equivalents are used. In certain embodiments, about 0.2 equivalents are used.

[0182] In certain embodiments, the reaction is carried out in the presence of a carboxyl-activating reagent and a base. In certain embodiments, the reaction is carried out in the presence of EDC and a base. In certain embodiments, the reaction is carried out in the presence of EDC and DMAP. In certain embodiments, the reaction is carried out in the presence of EDC·HCl and DMAP.

[0183] In certain embodiments, the reaction is carried out in a solvent. Non-limiting examples of solvents are provided below. In certain embodiments, the solvent is dichloromethane (DCM). In certain embodiments, the reaction is carried out at about room temperature. In certain embodiments, the reaction is carried out at a temperature below room temperature. In certain embodiments, the reaction is carried out at a temperature from about 0°C to about room temperature. In certain embodiments, the reaction is carried out at a temperature from about 0°C to about 20°C.

[0184] In certain embodiments, the reaction is carried out in DCM in the presence of EDC·HCl and DMAP. In certain embodiments, the reaction is carried out in DCM in the presence of EDC·HCl and DMAP at a temperature below room temperature. In certain embodiments, the reaction is carried out in DCM in the presence of EDC·HCl and DMAP at a temperature of about 0° C. to about 20° C. In certain embodiments, the reaction is carried out under the following conditions: 1 equivalent of carboxylic acid and 2.0 equivalents of PEG component, 1.5 equivalents of EDC·HCl, and 0.2 equivalents of DMAP, in DCM at a temperature of about 0° C. to about 20° C.

[0185] In certain embodiments, the PEG-lipid is purified by extraction (i.e., work-up). In certain embodiments, the PEG-lipid is purified by three-phase extraction. In certain embodiments, the PEG-lipid is purified by column chromatography following three-phase extraction. In certain embodiments, the three-phase extraction comprises partitioning the PEG-lipid mixture between acetonitrile, water (or aqueous solution), methyl tert-butyl ether (MTBE), and heptane. In certain embodiments, the column chromatography purification step is reversed-phase ultra-high performance liquid chromatography (UPLC).

[0186] General Reaction Parameters: The following embodiments apply to all synthetic methods described above and herein (i.e., methods for preparing PEG-lipids).

[0187] The reactions provided and described herein can include one or more reagents. In certain embodiments, a reagent can be present in a catalytic amount. In certain embodiments, the catalytic amount is 0-1 mol%, 0-5 mol%, 0-10 mol%, 1-5 mol%, 1-10 mol%, 5-10 mol%, 10-20 mol%, 20-30 mol%, 30-40 mol%, 40-50 mol%, 50-60 mol%, 60-70 mol%, 70-80 mol%, 80-90 mol%, or 90-99 mol%. In certain embodiments, a reagent can be present in a stoichiometric amount (i.e., about 1 equivalent). In certain embodiments, a reagent can be present in an excess amount (i.e., greater than 1 equivalent). In certain embodiments, the excess is about 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, or 20 equivalents. In certain embodiments, the excess is about 1.1 to 2, 2 to 3, 3 to 4, 4 to 5, 1.1 to 5, 5 to 10, 10 to 15, 15 to 20, or 10 to 20 equivalents. In certain embodiments, the excess is greater than 20 equivalents.

[0188] The reactions described herein can be carried out at any temperature. In certain embodiments, the reactions are carried out at or near room temperature (rt) (21° C. or 70° F.). In certain embodiments, the reactions are carried out at temperatures below room temperature (e.g., between −100° C. and 21° C.). In certain embodiments, the reactions are carried out at or near −78° C. In certain embodiments, the reactions are carried out at or near −10° C. In certain embodiments, the reactions are carried out at or near 0° C. In certain embodiments, the reactions are carried out at about −78° C. to about room temperature. In certain embodiments, the reactions are carried out at about −10° C. to about room temperature. In certain embodiments, the reactions are carried out at about 0° C. to about room temperature. In certain embodiments, the reactions are carried out above room temperature. In certain embodiments, the reactions are carried out at 30, 40, 50, 60, 70, 80, 110, 120, 130, 140, or 150° C. In certain embodiments, the reactions are carried out at temperatures above 150° C.

[0189] The reactions described herein can be carried out in a solvent or solvent mixture (i.e., a co-solvent). The solvent can be polar or non-polar, protic or aprotic. Any solvent can be used in the reactions described herein, and the reactions are not limited to a particular solvent or combination of solvents. Common organic solvents useful in the methods described herein include acetone, acetonitrile, benzene, benzonitrile, 1-butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide, carbon tetrachloride, chlorobenzene, 1-chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-dichlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N,N-dimethylacetamide, N,N-dimethylformamide (DMF), 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), 1,4-dioxane, 1,3-dioxane, diethyl ether, 2-ethoxyethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexane, hexamethylphosphazene, and the like. Examples of suitable amines include, but are not limited to, methyl methylpropanediol (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidinone, dimethyl sulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahydrofuran (THF), 2-methyltetrahydrofuran, toluene, trichlorobenzene, 1,1,2-trichlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N,N-diisopropylethylamine, diisopropylamine, water, o-xylene, and p-xylene.

[0190] The reactions described herein can be carried out for any length of time, hi certain embodiments, the reactions can be carried out for a few seconds, minutes, hours, or days.

[0191] The methods described herein can be used to prepare compounds with any chemical yield. In certain embodiments, compounds are produced with a yield of 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In certain embodiments, the yield is the percent yield after one synthetic step. In certain embodiments, the yield is the percent yield after two or more synthetic steps (e.g., 2, 3, 4, or 5 synthetic steps).

[0192] The methods described herein may further include one or more purification steps. For example, in certain embodiments, the compounds produced by the methods described herein may be purified by chromatography, extraction, filtration, precipitation, crystallization, or any other method known in the art. In certain embodiments, the compounds or mixtures are carried forward to the next synthetic step without purification (i.e., crude).

[0193] The synthesis methods provided herein can be carried out at any scale (i.e., to obtain any amount of product). In certain embodiments, the methods are applicable to small-scale synthesis or large-scale process manufacturing. In certain embodiments, the reactions provided herein are carried out to obtain less than 1 g of product. In certain embodiments, the reactions provided herein are carried out to obtain more than 1 g, 2 g, 5 g, 10 g, 15 g, 20 g, 25 g, 30 g, 40 g, 50 g, 100 g, 200 g, 500 g, or 1 kg of product.

[0194] Therapeutic drug delivery methods Also provided herein are methods for delivering one or more therapeutic agents to a subject, the methods comprising administering to the subject LNPs described herein, the LNPs encapsulating the one or more therapeutic agents of interest.

[0195] In certain embodiments, the LNPs described herein may be less susceptible to clearance and thus may have a long in vivo half-life. This is particularly true when the compositions are intended for repeated administration, including chronic administration, and even more particularly when such repeated administration occurs within a few days or weeks. Importantly, these compositions are less susceptible to or entirely avoid the observed accelerated blood clearance (ABC) phenomenon. ABC is the rapid clearance of certain exogenously administered drugs from the blood after second and subsequent administrations. This phenomenon has been observed in various lipid-containing compositions, including, but not limited to, lipidated drugs, liposomes, or other lipid-based delivery vehicles, and lipid-encapsulated drugs. This resistance to ABC makes these compounds and compositions particularly suitable for repeated in vivo use, including within short periods of time, including within a few days or 1-2 weeks. This increased stability and / or half-life is due, in part, to the inability of these compositions to activate B1a and / or B1b cells and / or conventional B cells, pDCs, and / or platelets. B1a cells are normally responsible for secreting natural antibodies in the form of circulating IgM. IgM is polyreactive, meaning it can bind to a variety of antigens, but with relatively low affinity for each.

[0196] Some lipidated drugs or lipid-containing formulations, such as lipid nanoparticles, administered in vivo have been shown to induce and target ABC. It has also been found that after administration of an initial dose of LNP, one or more cells involved in generating the innate immune response (referred to herein as sensors) bind to the drug, become activated, and subsequently initiate a cascade of immune factors (referred to herein as effectors) that promote ABC and toxicity. For example, B1a and B1b cells bind to LNP, become activated (alone or in the presence of other sensors, e.g., pDCs, and / or effectors, e.g., IL6), and secrete natural IgM, which binds to LNP. Pre-existing natural IgM in the subject may also recognize and bind to LNP, thereby inducing complement fixation. After administration of the initial dose, natural IgM production begins within 1–2 hours of LNP administration. Typically, natural IgM is cleared from the system by about 2–3 weeks due to the natural half-life of IgM. Natural IgG is produced approximately 96 hours after administration of LNP. When administered in a naive setting, the agent can exert its biological effects relatively unimpeded by natural IgM produced after activation of B1a or B1b cells or natural IgG. Natural IgM and natural IgG are nonspecific and therefore distinct from anti-PEG IgM and anti-PEG IgG.

[0197] For further details on the mechanisms involved in ABC, see International Publication No. WO2017 / 099823, published June 15, 2017, the entire contents of which are incorporated herein by reference.

[0198] In certain embodiments, LNPs described herein (i.e., comprising a plurality of PEG-lipids at a particular purity described herein) are less susceptible to accelerated blood clearance (ABC) than LNPs comprising a plurality of PEG-lipids with a purity of less than 87%. For example, in certain embodiments, LNP formulations comprising a plurality of compounds of Formula (I) described herein are less susceptible to ABC than LNP formulations comprising a plurality of compounds of Formula (I) with a purity of less than 87%.

[0199] As described herein, the present disclosure provides compounds and compositions that do not promote ABC. These may be further characterized as being incapable of activating B1a and / or B1b cells, platelets, and / or pDCs, and optionally, conventional B cells. These lipid nanoparticles are particularly desirable for applications requiring repeated administration, especially those administered over a short period of time (e.g., within 1-2 weeks). This is the case, for example, for nucleic acid-based therapeutics, where the agent is administered to a subject at regular, short intervals.

[0200] The LNPs provided herein may, in some instances, not elicit a CD5(+)-mediated immune response. As used herein, a CD5(+)-mediated immune response is an immune response mediated by B1a and / or B1b cells. Such responses may include ABC responses, acute phase reactions, induction of natural IgM and / or IgG, and the like.

[0201] The LNPs provided herein may, in some instances, not elicit a CD19(+)-mediated immune response. As used herein, a CD19(+)-mediated immune response refers to an immune response mediated by conventional CD19(+), CD5(-) B cells. Such responses may include anti-drug antibody (ADA) responses, including induction of IgM, induction of IgG, induction of memory B cells, ABC responses, and anti-protein responses, in which proteins may be encapsulated within the LNPs.

[0202] B1a cells are a subset of B cells involved in innate immunity. These cells are the source of circulating IgM, also known as natural antibodies or natural serum antibodies. Natural IgM antibodies are characterized by weak affinity for many antigens and are therefore termed "polyspecific" or "polyreactive," reflecting their ability to bind to multiple antigens. B1a cells cannot produce IgG. Furthermore, they do not develop into memory cells and therefore do not contribute to adaptive immune responses. However, they can secrete IgM after activation. Secreted IgM is typically cleared within approximately 2 to 3 weeks, at which point the immune system becomes relatively naive to previously administered antigens. If the same antigen is presented after this period (e.g., approximately 3 weeks after initial exposure), the antigen is not rapidly cleared. Importantly, however, if the antigen is presented within that period (e.g., within 2 weeks, 1 week, or even within a few days), the antigen is rapidly cleared. This delay between successive administrations makes certain lipid-containing therapeutic or diagnostic agents unsuitable for use.

[0203] In humans, B1a cells are CD19(+), CD20(+), CD27(+), CD43(+), CD70(-), and CD5(+). In mice, B1a cells are CD19(+), CD5(+), and the CD45 B cell isoform B220(+). Expression of CD5 typically distinguishes B1a cells from other conventional B cells. B1a cells may express high levels of CD5, which distinguishes them from other B-1 cells, such as B-1b cells, which express low or undetectable levels of CD5. CD5 is a pan-T cell surface glycoprotein. B1a cells also express CD36, also known as fatty acid translocase. CD36 is a member of the class B scavenger receptor family. CD36 can bind to many ligands, including oxidized low-density lipoprotein, native lipoprotein, oxidized phospholipids, and long-chain fatty acids.

[0204] B1b cells are another subset of B cells involved in innate immunity. These cells are another source of natural IgM in the blood. Some antigens, including PS, can induce T cell-independent immunity through B1b activation. CD27 is typically upregulated on B1b cells in response to antigen activation. Like B1a cells, B1b cells are typically located in specific body sites, such as the spleen or peritoneal cavity, and are present in very small amounts in the blood. Natural IgM secreted by B1b cells is typically eliminated within approximately 2 to 3 weeks, at which point the immune system becomes relatively naive to previously administered antigens. If the same antigen is presented after this period (e.g., approximately 3 weeks after initial exposure), the antigen is not rapidly eliminated. Importantly, however, if the antigen is presented within that period (e.g., within 2 weeks, 1 week, or even within a few days), the antigen is rapidly eliminated. This delay between successive administrations makes certain lipid-containing therapeutic or diagnostic agents unsuitable for use.

[0205] In some embodiments, it is desirable to block the activation of B1a and / or B1b cells. One method for blocking B1a and / or B1b cell activation involves identifying which components of lipid nanoparticles promote B cell activation and neutralizing those components. It has been found herein that at least PEG and phosphatidylcholine (PC) contribute to the interaction and / or activation of B1a and B1b cells with other cells. PEG may be involved in promoting aggregation between B1 cells and platelets, which may lead to activation. PC (the helper lipid in LNPs) also contributes to the activation of B1 cells, possibly via interaction with the CD36 receptor on the B cell surface. Many particles have PEG-lipid alternatives, and PEG-less and / or PC-substituted lipids (e.g., oleic acid or analogs thereof) have been designed and tested. As described herein, utilizing PEG lipids of the recommended purity provided herein is useful for preventing ABC by reducing natural IgM production and / or B cell activation by LNPs. Thus, the present invention encompasses LNPs with reduced ABC as a result of their design to eliminate B cell triggering.

[0206] Another method for blocking B1a and / or B1b cell activation involves the use of agents that inhibit LNP-induced immune responses. These types of agents are discussed in detail below. In some embodiments, these agents block the interaction between B1a / B1b cells and the LNPs, platelets, or pDCs. For example, the agent can be an antibody or other binding agent that physically blocks the interaction. An example of this is an antibody that binds to CD36 or CD6. The agent can also be a compound that prevents or abolishes B1a / B1b cell signaling after or before activation. For example, it can block one or more components in the B1a / B1b signaling cascade that results from the interaction of B cells with LNPs or other immune cells. In other embodiments, the agent can act on one or more effectors produced by B1a / B1b cells after activation. These effectors include, for example, natural IgM and cytokines.

[0207] In certain embodiments, LNPs comprising a plurality of PEG-lipids described herein exhibit reduced B cell (e.g., B1a, B1b cell) binding and / or activation compared to LNP formulations comprising a plurality of PEG-lipids with a purity of less than 87%. For example, in certain embodiments, LNPs comprising a plurality of compounds of Formula (I) described herein exhibit reduced B cell (e.g., B1a, B1b cell) binding and / or activation compared to LNP formulations comprising a plurality of compounds of Formula (I) with a purity of less than 87%.

[0208] In certain embodiments, LNPs comprising a plurality of PEG-lipids described herein exhibit reduced IgM binding compared to LNP formulations comprising a plurality of PEG-lipids with a purity of less than 87%. For example, in certain embodiments, LNPs comprising a plurality of compounds of Formula (I) described herein exhibit reduced IgM binding compared to LNP formulations comprising a plurality of compounds of Formula (I) with a purity of less than 87%.

[0209] In some instances, the method for delivering one or more therapeutic agents includes: (i) administering an initial dose of the agent to the subject; (ii) administering a second or subsequent dose of the agent to the subject, wherein the second or subsequent dose is administered within two weeks of the first or previous dose; and (iii) may involve repeating step (ii) one or more times; Here, the drug is formulated with an LNP that does not promote ABC.

[0210] Another method for delivering a drug to a subject includes: (i) administering an initial dose of the agent to the subject; (ii) administering a second or subsequent dose of the agent to the subject, wherein the second or subsequent dose is administered within two weeks of the first or previous dose; and (iii) repeating step (ii) one or more times; wherein the half-life of the agent after second and subsequent administrations is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the half-life of the agent after the first administration.

[0211] Yet another method for delivering a drug to a subject comprises: (i) administering an initial dose of the agent to the subject; (ii) administering a second or subsequent dose of the agent to the subject, wherein the second or subsequent dose is administered within two weeks of the first or previous dose; and (iii) repeating step (ii) one or more times; wherein the activity or blood level of the drug after the second and subsequent administrations is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the activity or blood level of the drug after the first administration.

[0212] The second or subsequent administration may be administered within one week, six days, five days, four days, three days, two days, or one day after the first or previous administration. The agent may be administered two or more times, three or more times, four or more times, etc. The agent administration may thus be repeated one, two, three, four, five, six, seven, eight, nine, ten, or more times. The agent may be administered chronically or acutely, depending on its intended purpose.

[0213] Second and subsequent administrations of bioactive agent may maintain at least 50% of the activity of the first administration, or at least 60% of the first administration, or at least 70% of the first administration, or at least 75% of the first administration, or at least 80% of the first administration, or at least 85% of the first administration, or at least 90% of the first administration, or at least 95% of the first administration, or more, for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the second or subsequent administration.

[0214] The interval between the first and second doses in any of the methods described herein can be less than two weeks, e.g., less than 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some examples, the subject receives any of the methods described herein once daily, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. Each possibility represents a separate embodiment of the present invention.

[0215] When the bioactive agent is mRNA (e.g., a therapeutic mRNA or an mRNA encoding a vaccine antigen), a method for reducing the ABC of LNPs encapsulating the mRNA can be achieved by using a lower dose of LNPs for the first dose and / or second dose (as well as subsequent doses). The lower dose can be 0.3 mg / kg or less, e.g., 0.2 mg / kg or 0.1 mg / kg. In some examples, the first dose, second dose, or both range from 0.1 to 0.3 mg / kg.

[0216] Also provided herein are methods of treating a subject having or at risk of having a condition that would benefit from the bioactive agent, particularly when the bioactive agent is a therapeutic agent. Alternatively, the method may be a method of diagnosing a subject, in which case the bioactive agent is a diagnostic agent.

[0217] LNP formulation Formation of the lipid nanoparticles (LNPs) described herein can be accomplished by any method known in the art, such as those described in U.S. Publication No. 2012 / 0178702, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176, Jayarama et al. (2012) Angew. Chem. Int. Ed., 51:8529-8533, and Maier et al. (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated herein by reference in their entirety.

[0218] In one embodiment, the LNP formulation can be formulated by the methods described in International Publication No. WO2011 / 127255 or WO2008 / 103276, the contents of each of which are incorporated herein by reference in their entirety. Non-limiting examples include the LNP formulations described in WO2011 / 127255 and / or WO2008 / 103276, each of which is incorporated herein by reference in its entirety. In one embodiment, the LNP formulation described herein can include a polycationic composition. Non-limiting examples include polycationic compositions selected from Formulas 1-60 of U.S. Patent Publication No. US20050222064, the contents of which are incorporated herein by reference in their entirety.

[0219] In one embodiment, the lipid nanoparticles may be formulated by the methods described in U.S. Patent Publication No. US2013 / 0156845, and International Publication No. WO2013 / 093648 or WO2012 / 024526, each of which is incorporated herein by reference in its entirety.

[0220] The lipid nanoparticles described herein can be made in a sterile environment by the systems and / or methods described in U.S. Patent Publication No. US20130164400, which is incorporated herein by reference in its entirety.

[0221] In one embodiment, the LNP formulations may be formulated into nanoparticles, such as the nucleic acid-lipid particles described in US Pat. No. 8,492,359, which is incorporated herein by reference in its entirety.

[0222] Nanoparticle composition can optionally comprise one or more coatings.For example, nanoparticle composition can be formulated into capsule, film or tablet with coating.The capsule, film or tablet comprising the composition described herein can have any useful size, tensile strength, hardness or density.

[0223] In some embodiments, such LNPs are synthesized using methods involving microfluidic mixers. Exemplary microfluidic mixers include, but are not limited to, slit interdigital micromixers and / or staggered herringbone micromixers (SHM) manufactured by Microinnova (Allerheiligen bei Wildon, Austria). (Zhigaltsev, IV et al., "Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing" has been published. (Langmuir. 2012. 28:3633-40; Belliveau, NM et al., "Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA." Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., "Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation." J Am Chem Soc. 2012. 134(16):6948-51, each of which is incorporated herein by reference in its entirety).

[0224] In some embodiments, the method for producing LNPs comprising SHMs further comprises mixing at least two input streams, wherein the mixing occurs via microstructure-induced chaotic advection (MICA). According to the method, the fluid streams flow through channels that exist in a herringbone pattern, which causes swirl and holds the fluids around each other. The method may also include a fluid mixing surface, which changes direction during fluid circulation. Methods for producing LNPs using SHMs include those disclosed in U.S. Patent Publication Nos. 2004 / 0262223 and 2012 / 0276209, each of which is incorporated herein by reference in its entirety.

[0225] In one embodiment, the lipid nanoparticles may be formulated using a micromixer, such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or a Caterpillar (CPMM) or an Impinging-jet (IJMM) manufactured by Institut fur Mikrotechnik Mainz GmbH, Mainz Germany.

[0226] In one embodiment, the lipid nanoparticles are created using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442:368-373, and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651, each of which is incorporated herein by reference in its entirety). By way of non-limiting example, controlled microfluidic formulation includes passive methods for mixing steady pressure-driven flow in microchannels at low Reynolds numbers (see, for example, Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651, each of which is incorporated herein by reference in its entirety).

[0227] In one embodiment, the mRNA of the present invention can be formulated into lipid nanoparticles created using a micromixer chip, such as, but not limited to, those manufactured by Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used for rapid mixing of two or more fluid streams by a split and recombine mechanism.

[0228] In one embodiment, the lipid nanoparticles have a diameter of about 10 to about 100 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 20 to about 90 nm The lipid nanoparticles may have a diameter of about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm. In one embodiment, the lipid nanoparticles may have a diameter of about 10 to 500 nm. In one embodiment, the lipid nanoparticles can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm. Each possibility represents a separate embodiment of the present invention.

[0229] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, or 40 to 200 nm. In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0230] In some embodiments, the lipid nanoparticles described herein have a diameter of less than 0.1 um to 100 nm, for example, but not limited to, less than 0.1 um, less than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 um, less than 450 um, less than 475 um, less than 500 um, less than 525 um, less than 550 um, less than 575 um, less than 600 um, less than 625 um, less than 650 um, less than 675 um, less than 700 um, less than 725 um, less than 750 um, less than 775 um, less than 800 um, less than 825 um, less than 850 um, less than 875 um, less than 900 um, less than 925 um, less than 950 um, or 975 um.

[0231] In another embodiment, the LNPs have a diameter of about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm m ~ about 90nm, about 5nm - about 100nm, about 5nm - about 10nm, about 5nm - about 20nm, about 5nm - about 30nm, about 5nm - about 4 0nm, approximately 5nm to approximately 50nm, approximately 5nm to approximately 60nm, approximately 5nm to approximately 70nm, approximately 5nm to approximately 80nm, approximately 5nm to approximately 90nm, approximately The range may be 10 to about 50 nM, about 20 to about 50 nm, about 30 to about 50 nm, about 40 to about 50 nm, about 20 to about 60 nm, about 30 to about 60 nm, about 40 to about 60 nm, about 20 to about 70 nm, about 30 to about 70 nm, about 40 to about 70 nm, about 50 to about 70 nm, about 60 to about 70 nm, about 20 to about 80 nm, about 30 to about 80 nm, about 40 to about 80 nm, about 50 to about 80 nm, about 60 to about 80 nm, about 20 to about 90 nm, about 30 to about 90 nm, about 40 to about 90 nm, about 50 to about 90 nm, about 60 to about 90 nm, and / or about 70 to about 90 nm. Each range represents a separate embodiment of the present invention.

[0232] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) typically indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20. Each possibility represents a separate embodiment of the present invention.

[0233] The zeta potential of a nanoparticle composition may be used to indicate the kinetic potential of the composition. For example, the zeta potential may represent the surface charge of the nanoparticle composition. Nanoparticle compositions with a relatively low positive or negative charge are generally desirable, as species with higher charges may undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV. Each possibility represents a separate embodiment of the present invention.

[0234] The encapsulation efficiency of a therapeutic agent represents the amount of therapeutic agent encapsulated in or associated with a nanoparticle composition after preparation compared to the amount initially provided. A high encapsulation efficiency (e.g., approaching 100%) is desirable. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic agent in a solution containing the nanoparticle composition before and after disruption of the nanoparticle composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., nucleic acid) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. Each possibility represents a separate embodiment of the present invention.

[0235] In some embodiments, the lipid nanoparticles have a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH. In some embodiments, the lipid nanoparticles have a mean diameter of 50-200 nm.

[0236] The properties of lipid nanoparticle formulations can be influenced by factors including, but not limited to, the selection of the cationic lipid component, the degree of saturation of the cationic lipid, the selection of the non-cationic lipid component, the degree of saturation of the non-cationic lipid, the selection of the structural lipid component, the nature of the PEGylation, and biophysical parameters such as the ratio and size of all components. As described herein, the purity of the PEG-lipid component is also important to the properties and performance of LNPs.

[0237] In certain embodiments, when a lipid nanoparticle (LNP) comprises a phospholipid and encapsulates an oligonucleotide, polynucleotide, or nucleic acid, the LNP has an N / P ratio. "N / P ratio" refers to the ratio of positively charged polymeric amines (e.g., nitrogens from phosphatidylcholine groups on a phospholipid) ("N") in the nanoparticle to phosphate groups ("P") in the oligonucleotide, polynucleotide, or nucleic acid agent. The N / P ratio of an LNP can affect many properties, including its net surface charge, size, stability, and in vivo activity. In certain embodiments, the LNPs described herein have an N / P ratio between 1.0 and 10.0, inclusive. In certain embodiments, the LNPs described herein have an N / P ratio of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. In certain embodiments, the N / P ratio is between 1.0 and 5.0, inclusive. In certain embodiments, the N / P ratio is between 2.0 and 5.0, inclusive. In certain embodiments, the N / P ratio is between 2.0 and 4.0, inclusive. In certain embodiments, the N / P ratio is about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. In certain embodiments, the N / P ratio is between 2.5 and 3.5, inclusive. In certain embodiments, the N / P ratio is about 2.0. In certain embodiments, the N / P ratio is about 3.0. In certain embodiments, the N / P ratio is about 4.0. In certain embodiments, the N / P ratio is about 5.0.

[0238] Drugs and Therapeutic Agents The present disclosure contemplates that the LNPs provided herein and / or the various combination therapies provided herein can be used to deliver a variety of agents to a subject. Such agents are typically bioactive agents. Bioactive agents are agents that exert an effect in vivo, preferably an agent that exerts a beneficial effect, such as a desired immunomodulatory, immunostimulatory, immunosuppressive, cell killing, cell preservation, altered gene expression, protein replacement, etc. Bioactive agents include, but are not limited to, prophylactic, therapeutic, and diagnostic agents. Bioactive agents include immunomodulatory agents, e.g., immunostimulatory or immunosuppressive agents, antigens, antibodies and antibody fragments, e.g., antigen-binding antibody fragments, adjuvants, cytokines, e.g., interleukins, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, anticancer agents, anti-inflammatory agents, etc.

[0239] Such agents may be, but are not limited to, nucleic acids, proteins or peptides, small organic compounds, carbohydrates and / or polysaccharides, etc. They may be used to express nucleic acids and / or proteins in cells, particularly cells that are deficient in or have a mutant form of such nucleic acid or protein. They may also be used to introduce and express nucleic acids or proteins that are not native to a cell or organism, for example, in the context of immunization or vaccination protocols. In this regard, the nucleic acid or protein may be foreign to the subject to which it is administered (e.g., not naturally occurring in the subject, or not naturally occurring at all), and it is administered to the subject to induce and / or enhance an immune response against such nucleic acid or protein. The nucleic acids provided herein may be used for such purposes.

[0240] Other bioactive agents may be used alone or together with such nucleic acids or proteins, including formulated together with such nucleic acids or proteins, including formulated into the LNPs of the present disclosure.

[0241] As used herein, the term "nucleic acid" refers to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are sometimes used interchangeably and refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise specified.

[0242] Nucleic acids include any compound and / or substance containing a polymer of nucleotides. These polymers are called polynucleotides. Nucleic acids can be or include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, LNA with a β-D-ribonucleotide structure, α-LNA with an α-L-ribonucleotide structure (a diastereomer of LNA), 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0243] A nucleic acid can be naturally occurring, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule can be a non-naturally occurring molecule, for example, a recombinant DNA or RNA, an artificial chromosome, a modified genome, or a fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or contain non-naturally occurring nucleotides or nucleosides. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.

[0244] Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.

[0245] Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, e.g., analogs with chemically modified bases or sugars, and backbone modifications. In some embodiments, nucleic acids include natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyl ... The base may be or contain a base such as cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine, chemically modified bases, biologically modified bases (e.g., methylated bases), inserted bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0246] "Nucleoside" refers to a compound comprising a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). "Nucleotide" refers to a nucleoside comprising a phosphate group. Modified nucleotides can be synthesized in any useful manner, for example, chemically, enzymatically, or recombinantly, to include one or more modified or unnatural nucleosides. A polynucleotide can comprise a region(s) of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide comprises a region of nucleotides.

[0247] Base pairing of modified nucleotides encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing and / or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard base and a standard base, or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into the polynucleotides of the present disclosure.

[0248] Those of skill in the art will understand that unless otherwise indicated, the polynucleotide sequences set forth in this application recite a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0249] Modifications of polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides) useful in the therapeutic agents described herein include, but are not limited to, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, 1,2'-O-dimethyladenosine, 1-methyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2-methyladenosine, 2-methylthio-N6 -Isopentenyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6,N6-dimethyladenosine, N6-acetyladenosine, N6-hydroxynorvalylcarbamoyl adenosine, N6-methyl-N6-threonylcarbamoyl adenosine, 2-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 7-deaza-adenosine, N1-methyl-adenosine, N6,N6-(dimethyl)adenine, N6-cis-hydroxy-isopentenyl-adenosine, α-thio-adenosine, 2-(amino)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 2-(alkyl)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(halo)adenine, 2-(halo)adenine, 2-(propyl)adenine, 2'-amino-2'-deoxy-ATP, 2'-Azido-2'-deoxy-ATP, 2'-deoxy-2'-α-aminoadenosine TP, 2'-deoxy-2'-α-azidoadenosine TP, 6-(alkyl)adenine, 6-(methyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(thioalkyl)adenine, 8-(alkenyl)adenine, 8-(alkyl) Adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, 8-azido-adenosine, azaadenine, deazaadenine, N6-(methyl)adenine, N6-(isopentyl)adenine, 7-deaza-8-aza-adenosine, 7-methyladenine, 1-deazaadenosine TP, 2'-fluoro-N6-Bz-deoxyadenosine TP , 2'-OMe-2-amino-ATP, 2'-O-methyl-N6-Bz-deoxyadenosine TP, 2'-α-ethynyl adenosine TP, 2-aminoadenine, 2-amino adenosine TP, 2-amino-ATP, 2'-α-trifluoromethyl adenosine TP, 2-azidoadenosine TP, 2'-β-ethynyl adenosine TP, 2-bromoadenosine TP, 2'-β-trifluoromethyl adenosine TP, 2-chloroadenosine TP, 2'-deoxy-2',2'-Difluoroadenosine TP, 2'-deoxy-2'-α-mercaptoadenosine TP, 2'-deoxy-2'-α-thiomethoxyadenosine TP, 2'-deoxy-2'-β-aminoadenosine TP, 2'-deoxy-2'-β-azidoadenosine TP, 2'-deoxy-2'-β-bromoadenosine TP, 2'-deoxy-2'-β-chloroadenosine TP, 2'-deoxy-2'-β-fluoroadenosine TP, 2'-deoxy-2'-β-iodoadenosine TP, 2 '-Deoxy-2'-b-mercaptoadenosine TP, 2'-deoxy-2'-b-thiomethoxyadenosine TP, 2-fluoroadenosine TP, 2-iodoadenosine TP, 2-mercaptoadenosine TP, 2-methoxy-adenine, 2-methylthio-adenine, 2-trifluoromethyladenosine TP, 3-deaza-3-bromoadenosine TP, 3-deaza-3-chloroadenosine TP, 3-deaza-3-fluoroadenosine TP, 3-deaza-3-iodoadenosine TP, 3-Deazaadenosine TP, 4'-Azidoadenosine TP, 4'-Carbocyclic Adenosine TP, 4'-Ethynyladenosine TP, 5'-Homo-Adenosine TP, 8-Aza-ATP, 8-Bromo-Adenosine TP, 8-Trifluoromethyladenosine TP, 9-Deazaadenosine TP, 2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 7-Deaza-8-Aza-2-Aminopurine, 2,6-Diaminopurine, 7- Deaza-8-aza-adenine, 7-deaza-2-aminopurine, 2-thiocytidine, 3-methylcytidine, 5-formylcytidine, 5-hydroxymethylcytidine, 5-methylcytidine, N4-acetylcytidine, 2'-O-methylcytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, lysidine, N4,2'-O-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N4-methylcytidine, N4,N4-dimethyl-2'-OMe-cytidine TP, 4-methylcytidine, 5-aza-cytidine, pseudo-iso-cytidine, pyrrolo-cytidine, α-thio-cytidine, 2-(thio)cytosine, 2'-amino-2'-deoxy-CTP, 2'-azido-2'-deoxy-CTP, 2'-deoxy-2'-α-aminocytidine TP, 2'-deoxy-2'-α-azidocytidine TP, 3-(deaza)-5-(aza)cytosine, 3-(methyl)cytosine, 3-(alkyl)cytosine, 3-(deaza)-5-(aza)cytosine , 3-(methyl)cytidine, 4,2'-O-dimethylcytidine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-bromo-cytidine, 5-iodo-cytidine, 5-propynylcytosine, 6-(azo)cytosine, 6-aza-cytidine, azacytosine, deazacytosine, N4-(acetyl ) Cytosine, 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-pseudoisocytidine, 2-methoxy-5-methyl-cytidine, 2-methoxy-cytidine, 2-thio-5-methyl-cytidine, 4-methoxy-1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-pseudoisocytidine, 5-aza-zebularine, 5-methyl-zebularine, pyrrolo-pseudoisocytidine Socytidine, Zebularine, (E)-5-(2-bromo-vinyl)cytidine TP, 2,2'-anhydro-cytidine TP hydrochloride, 2'-fluoro-N4-Bz-cytidine TP, 2'-fluoro-N4-acetyl-cytidine TP, 2'-O-methyl-N4-acetyl-cytidine TP, 2'-O-methyl-N4-Bz-cytidine TP, 2'-a-ethynylcytidine TP, 2'-a-trifluoromethylcytidine TP, 2'-b-ethynylcytidine TP, 2'-b-trifluoromethylcytidine TP, 2'-deoxy-2',2'-Difluorocytidine TP, 2'-deoxy-2'-α-mercaptocytidine TP, 2'-deoxy-2'-α-thiomethoxycytidine TP, 2'-deoxy-2'-β-aminocytidine TP, 2'-deoxy-2'-β-azidocytidine TP, 2'-deoxy-2'-β-bromocytidine TP, 2'-deoxy-2'-β-chlorocytidine TP, 2'-deoxy-2'-β-fluorocytidine TP, 2'-deoxy-2'-β-iodocytidine TP, 2' -Deoxy-2'-b-mercaptocytidine TP, 2'-deoxy-2'-b-thiomethoxycytidine TP, 2'-O-methyl-5-(1-propynyl)cytidine TP, 3'-ethynylcytidine TP, 4'-azidocytidine TP, 4'-carbocyclic cytidine TP, 4'-ethynylcytidine TP, 5-(1-propynyl)ara-cytidine TP, 5-(2-chloro-phenyl)-2-thiocytidine TP, 5-(4-amino-phenyl)-2-thiocytidine T P, 5-aminoallyl-CTP, 5-cyanocytidine TP, 5-ethynylala-cytidine TP, 5-ethynylcytidine TP, 5'-homo-cytidine TP, 5-methoxycytidine TP, 5-trifluoromethyl-cytidine TP, N4-amino-cytidine TP, N4-benzoyl-cytidine TP, pseudoisocytidine, 7-methylguanosine, N2,2'-O-dimethylguanosine, N2-methylguanosine, 1,2'-O-dimethyl ... -methylguanosine, 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, archaeosine, methylwiosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, 6-thio-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N1-methyl-guanosine, α-thio-guanosine, 2-(propyl)guanine, 2-(alkyl)guanine, 2'-amino-2'-deoxy-GTP, 2'-azido-2'-deoxy-GTP, 2'-deoxy-2'-α-aminoguanosine TP, 2'-deoxy-2'-α-azidoguanosine TP, 6-(methyl)guanosine, 6-(alkanol)guanine (alkyl)guanine, 6-(methyl)guanine, 6-methyl-guanosine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(halo)guanine, 8-(thioalkyl)guanine, 8-(alkenyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(amino) N-guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, azaguanine, deazaguanine, N-(methyl)guanine, N-(methyl)guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7- Methyl-8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, 1-Me-GTP, 2'-fluoro-N2-isobutyl-guanosine TP, 2'-O-methyl-N2-isobutyl-guanosine TP, 2'-a-ethynylguanosine TP, 2'-a-trifluoromethylguanosine TP, 2'-b-ethynylguanosine TP, 2'-b-trifluoromethylguanosine TP, 2'-deoxy-2',2'-Difluoroguanosine TP, 2'-deoxy-2'-α-mercaptoguanosine TP, 2'-deoxy-2'-α-thiomethoxyguanosine TP, 2'-deoxy-2'-β-aminoguanosine TP, 2'-deoxy-2'-β-azidoguanosine TP, 2'-deoxy-2'-β-bromoguanosine TP, 2'-deoxy-2'-β-chloroguanosine TP, 2'-deoxy-2'-β-fluoroguanosine TP, 2'-deoxy-2'-β-iodoguanosine TP, 2, '-Deoxy-2'-b-mercaptoguanosine TP, 2'-deoxy-2'-b-thiomethoxyguanosine TP, 4'-azidoguanosine TP, 4'-carbocyclic guanosine TP, 4'-ethynylguanosine TP, 5'-homoguanosine TP, 8-bromoguanosine TP, 9-deazaguanosine TP, N2-isobutylguanosine TP, 1-methylinosine, inosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 7-methylinosine, 2'-O-methylinosine, epoxyqueuosine, galactosylqueuosine, mannosylqueuosine, queuosine, allylamino-thymidine, azathymidine, deazathymidine, deoxythymidine, 2'-O-methyluridine, 2-thiouridine, 3-methyluridine, 5-carboxymethyluridine, 5-hydrogen Xyluridine, 5-methyluridine, 5-taurinomethyl-2-thiouridine, 5-taurinomethyluridine, dihydrouridine, pseudouridine, (3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, 1-methylpseudouridine, 1-methyl-pseudouridine, 2'-O-methyluridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, 2-thio-2'-O-methyluridine, 3-(3-amino-3-carboxypropyl)uridine, 3,2'-O-dimethyluridine, 3-methyl-pseudouridine TP, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine, 5,6-Dihydro-uridine, 5-aminomethyl-2-thiouridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, Rubamoylmethyluridine TP, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methyluridine), 5-methoxyuridine, 5-methyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methyldihydrouridine, 5-oxyacetic acid-uridine TP, 5-oxyacetic acid-methylester-uridine TP, N1-methyl-pseudo- Uridine, Uridine-5-oxyacetic acid, Uridine-5-oxyacetic acid methyl ester, 3-(3-amino-3-carboxypropyl)-uridine TP, 5-(iso-pentenylaminomethyl)-2-thiouridine TP, 5-(iso-pentenylaminomethyl)-2'-O-methyluridine TP, 5-(iso-pentenylaminomethyl)uridine TP, 5-propynyluracil, α-thio-uridine, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)- 2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2-(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-substituted 2-(thio)-pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP, 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP, 1-methyl-pseudo-UTP, 2-(thio)pseudouracil, 2'-deoxyuridine, 2'-fluorouridine, 2-(thio)pseudouracil ) uracil, 2,4-(dithio)pseudouracil, 2'-methyl,2'-amino,2'-azido,2'-fluoro-guanosine, 2'-amino-2'-deoxy-UTP, 2'-azido-2'-deoxy-UTP, 2'-azido-deoxyuridine TP, 2'-O-methylpseudouridine, 2'-deoxyuridine, 2'-fluorouridine, 2'-deoxy-2'-α-aminouridine TP, 2'-deoxy-2'-α-azidouridine TP, 2-methylpseudouridine, 3-(3- Amino-3-carboxypropyl)uracil, 4-(thio)pseudouracil, 4-(thio)pseudouracil, 4-(thio)uracil, 4-thiouracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(2-aminopropyl)uracil, 5-(aminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidinium alkyl)uracil, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5 -(methyl)-2-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(alkyl)-2,4(dithio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(alkyl)pseudouracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(halo)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(methoxy)uracil, 5-(methoxy carbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(methyl)-2-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methyl)-2-(thio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(methyl)pseudouracil, 5-(methylaminomethyl)-2-(thio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-aminoallyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-uracil, 6-(azo)uracil, 6-(azo)uracil, 6-aza-uridine, allylamino-uracil, azauracil, deazauracil, N3-(methyl)uracil, pseudo-UTP-1-2-ethanoic acid, pseudouracil, 4-thio-pseudo-UTP, 1-caprylic acid carboxymethyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 1-propynyl-uridine, 1-taurinomethyl-1-methyl-uridine, 1-taurinomethyl-4-thio-uridine, 1-taurinomethyl-pseudouridine, 2-methoxy-4-thio-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio Opseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, (±)1-(2-hydroxypropyl)pseudouridine TP, (2R)-1-(2-hydroxypropyl)pseudouridine TP, (2S)-1-(2-hydroxypropyl)pseudouridine TP, (E)-5-(2-bromo-vinyl)ara-uridine TP, (E)-5 -(2-Bromo-vinyl)uridine TP, (Z)-5-(2-Bromo-vinyl)ara-uridine TP, (Z)-5-(2-Bromo-vinyl)uridine TP, 1-(2,2,2-trifluoroethyl)-pseudo-UTP, 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP, 1-(2,2-diethoxyethyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudo-UTP, 1-(2,4,6-trimethyl-phenyl)pseudo-UTP, 1-(2-amino-2-carboxyethyl)pseudo-UTP, 1-(2-amino-ethyl)pseudo-UTP, 1-(2-hydroxyethyl)pseudouridine TP, 1-(2-methoxyethyl)pseudouridine TP, 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP, 1-(3,4-dimethoxybenzyl)pseudouridine TP, 1-(3-amino-3-carboxypropyl)pseudo-UTP -UTP, 1-(3-amino-propyl)pseudo-UTP, 1-(3-cyclopropyl-prop-2-ynyl)pseudouridine TP, 1-(4-amino-4-carboxybutyl)pseudo-UTP, 1-(4-amino-benzyl)pseudo-UTP, 1-(4-amino-butyl)pseudo-UTP, 1-(4-amino-phenyl)pseudo-UTP, 1-(4-azidobenzyl)pseudouridine TP, 1-(4-bromobenzyl)pseudouridine TP, 1-(4- 1-(chlorobenzyl)pseudouridine TP, 1-(4-fluorobenzyl)pseudouridine TP, 1-(4-iodobenzyl)pseudouridine TP, 1-(4-methanesulfonylbenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudo-UTP, 1-(4-methoxyphenyl)pseudo-UTP, 1-(4-methylbenzyl)pseudouridine TP, 1-(4-methylbenzyl)pseudouridine TP do-UTP, 1-(4-nitrobenzyl)pseudo-UTP, 1-(4-nitro-benzyl)pseudo-UTP, 1-(4-nitro-phenyl)pseudo-UTP, 1-(4-thiomethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethylbenzyl)pseudouridine TP, 1-(5-amino-pentyl)pseudo-UTP, 1-(6-amino-hexyl)pseudo-UTP, 1,6-dimethyl-pseudo-UTP, 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP, 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP, 1-acetylpseudouridine TP, 1-alkyl-6-(1-propynyl)-pseudo-UTP, 1-alkyl-6-(2-propynyl)-pseudo-UTP, 1-alkyl-6-allyl-pseudo-UTP, 1-alkyl-6-ethynyl-pseudo-UTP, 1-alkyl-6-homoallyl-pseudo-UTP, 1-alkyl-6-vinyl-pseudo-UTP, 1-allylpseudouridine TP, 1-aminomethyl-pseudo, -UTP, 1-benzoylpseudouridine TP, 1-benzyloxymethylpseudouridine TP, 1-benzyl-pseudo-UTP, 1-biotinyl-PEG2-pseudouridine TP, 1-biotinylpseudouridine TP, 1-butyl-pseudo-UTP, 1-cyanomethylpseudouridine TP, 1-cyclobutylmethyl-pseudo-UTP, 1-cyclobutyl-pseudo-UTP, 1-cycloheptylmethyl-pseudo-UTP, 1-cycloheptyl-pseudo-UTP, 1-cyclohexylmethyl-pseudo-UTP, 1-cyclo Hexyl-pseudo-UTP, 1-cyclooctylmethyl-pseudo-UTP, 1-cyclooctyl-pseudo-UTP, 1-cyclopentylmethyl-pseudo-UTP, 1-cyclopentyl-pseudo-UTP, 1-cyclopropylmethyl-pseudo-UTP, 1-cyclopropyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudouridine TP, 1-hydroxymethylpseudouridine TP, 1-isopropyl-pseudo-UTP, 1-Me-2-thio-pseudo-UTP, 1 -Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP, 1-methanesulfonylmethylpseudouridine TP, 1-methoxymethylpseudouridine TP, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP, 1-methyl-6-(4-morpholino)-pseudo-UTP, 1-methyl-6-(4-thiomorpholino)-pseudo-UTP, 1-methyl-6-(substituted phenyl)pseudo-UTP, 1-methyl-6-amino-pseudo-UTP, 1-methyl-6-azido-pseudo-UTP, 1-methyl -6-bromo-pseudo-UTP, 1-methyl-6-butyl-pseudo-UTP, 1-methyl-6-chloro-pseudo-UTP, 1-methyl-6-cyano-pseudo-UTP, 1-methyl-6-dimethylamino-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-ethylcarboxylate-pseudo-UTP, 1-methyl-6-ethyl-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-formyl-pseudo-UTP, 1-methyl-6-hydroxyamino-pseudo-UTP,1-methyl-6-hydroxy-pseudo-UTP, 1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-iso-propyl-pseudo-UTP, 1-methyl-6-methoxy-pseudo-UTP, 1-methyl-6-methylamino-pseudo-UTP, 1-methyl-6-phenyl-pseudo-UTP, 1-methyl-6-propyl-pseudo-UTP, 1-methyl-6-tert-butyl-pseudo-UTP, 1-methyl-6-trifluoromethoxy-pseudo-UTP, 1-methyl-6-trifluoromethyl-pseudo-UTP, 1-morpholinometabolite Pseudouridine TP, 1-pentyl-pseudo-UTP, 1-phenyl-pseudo-UTP, 1-pivaloylpseudouridine TP, 1-propargylpseudouridine TP, 1-propyl-pseudo-UTP, 1-propynyl-pseudouridine, 1-p-tolyl-pseudo-UTP, 1-tert-butyl-pseudo-UTP, 1-thiomethoxymethylpseudouridine TP, 1-thiomorpholinomethylpseudouridine TP, 1-trifluoroacetylpseudouridine TP, 1-trifluoromethyl-pseudo-UTP, 1-vinylpseudouridine TP Idouridine TP, 2,2'-anhydro-uridine TP, 2'-bromo-deoxyuridine TP, 2'-F-5-methyl-2'-deoxy-UTP, 2'-OMe-5-Me-UTP, 2'-OMe-pseudo-UTP, 2'-α-ethynyluridine TP, 2'-α-trifluoromethyluridine TP, 2'-b-ethynyluridine TP, 2'-b-trifluoromethyluridine TP, 2'-deoxy-2',2'-difluorouridine TP, 2'-deoxy-2'-α-mercaptouridine TP, 2'-deoxy-2'-α-thiomethoxyuridine TP, 2'-deoxy-2'-b-aminouridine TP, 2'-deoxy-2'-b-azidouridine TP, 2'-deoxy-2'-b-bromouridine TP, 2'-deoxy-2'-b-chlorouridine TP, 2'-deoxy-2'-b-fluorouridine TP, 2'-deoxy-2'-b-iodouridine TP, 2'-deoxy-2'-b-mercaptouridine TP, 2'-deoxy-2'-b-thiomethoxyuridine TP, 2-methoxy-4-thiouridine, 2-methoxyuridine, 2'-O-methyl-5-(1-propynyl)uridine TP,3-Alkyl-pseudo-UTP, 4'-azidouridine TP, 4'-carbocyclic uridine TP, 4'-ethynyluridine TP, 5-(1-propynyl)ara-uridine TP, 5-(2-furanyl)uridine TP, 5-cyanouridine TP, 5-dimethylaminouridine TP, 5'-homo-uridine TP, 5-iodo-2'-fluoro-deoxyuridine TP, 5-phenylethynyluridine TP, 5-trideuteromethyl-6-deuterouridine TP, 5-trifluoromethyl-uridine TP, 5-vinylarauridine TP, 6-(2, 2,2-trifluoroethyl)-pseudo-UTP, 6-(4-morpholino)-pseudo-UTP, 6-(4-thiomorpholino)-pseudo-UTP, 6-(substituted-phenyl)-pseudo-UTP, 6-amino-pseudo-UTP, 6-azido-pseudo-UTP, 6-bromo-pseudo-UTP, 6-butyl-pseudo-UTP, 6-chloro-pseudo-UTP, 6-cyano-pseudo-UTP, 6-dimethylamino-pseudo-UTP, 6-ethoxy-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP TP, 6-fluoro-pseudo-UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-isopropyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-propyl-pseudo-UTP, 6-tert-butyl-pseudo-UTP, 6-trifluoromethoxy-pseudo-UTP, 6-trifluoro-pseudo-UTP, Trifluoromethyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine-1-(4-methylbenzenesulfonic acid) TP, pseudouridine-1-(4-methylbenzoic acid) TP, pseudouridine TP-1-[3-(2-ethoxy)]propionic acid, pseudouridine TP-1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP-1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid,Pseudouridine TP-1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid, Pseudouridine TP-1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid, Pseudouridine TP-1-methylphosphonic acid, Pseudouridine TP-1-methylphosphonic acid diethyl ester, Pseudo-UTP-N1-3-propionic acid, Pseudo-UTP-N1-4-butanoic acid, Pseudo-UTP-N1-5-pentanoic acid, Pseudo-UTP-N1-6-hexanoic acid, Pseudo-UTP-N1-7-heptanoic acid, Pseudo- UTP-N1-methyl-p-benzoic acid, pseudo-UTP-N1-p-benzoic acid, wybutosin, hydroxywybutosin, isowyosin, peroxywybutosin, undermodified hydroxywybutosin, 4-demethylwybutosin, 2,6-(diamino)purine, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-( (dioxa)-naphthalene, 2-(amino)purine, 2,4,5-(trimethyl)phenyl, 2'-methyl,2'-amino,2'-azido,2'-fluoro-cytidine, 2'-methyl,2'-amino,2'-azido,2'-fluoro-adenine, 2'-methyl,2'-amino,2'-azido,2'-fluoro-uridine, 2'-amino-2'-deoxyribose, 2-amino-6-chloro-purine, 2-aza-inosinyl, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-fluoro-modified base, 2'-O-methyl ribose, 2-oxo-7-aminopyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridinone, 3-nitropyrrole, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 3-(methyl)isocarbostyrilyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, 5-nitroindole, 5-substituted pyrimidines, 5-(methyl)isocarbostyrilyl, 5-nitroindole, 6-(aza)pyrimidines,6-(Azo)thymine, 6-(methyl)-7-(aza)indolyl, 6-chloro-purine, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2- (oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aza)indolyl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-( Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(propynyl)isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 7-deaza-inosinyl, 7-substituted 1-(aza)-2-(thio)-3-(aza) 1,3-(diaza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 9-(methyl)-imidizopyridinyl, aminoindolyl, anthracenyl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, difluorotolyl, hypoxanthine, imidizopyridinyl, inosinyl, isocarbostyrilyl, isoguanisine, N2-substituted purines,N6-Methyl-2-amino-purine, N6-substituted purines, N-alkylated derivatives, naphthalenyl, nitrobenzimidazolyl, nitroimidazolyl, nitroindazolyl, nitropyrazolyl, nubularine, O6-substituted purines, O-alkylated derivatives, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho, Para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxoformicin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pentacenyl, phenanthracenyl, phenyl, propynyl-7-(aza)indolyl, pyrenyl, pyridopyrimidin-3-yl, pyridopyrimidin-3-yl, 2-oxo-7- Amino-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyridinyl, stilbenzyl, substituted 1,2,4-triazoles, tetracenyl, tubercidin, xanthine, xanthosine-5'-TP, 2-thio-zebularine, 5-aza-2-thio-zebularine, 7-deaza-2-amino-purine, pyridin-4-one ribonucleoside, 2-amino-riboside-TP, formycin ATP, formycin B TP, pyrrolosine TP, 2'-OH-ara-adenosine TP, 2'-OH-ara-cytidine TP, 2'-OH-ara-uridine TP, 2'-OH-ara-guanosine TP, 5-(2-carbomethoxyvinyl)uridine TP, and N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0250] In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases.

[0251] In some embodiments, the modified nucleobase in the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5- ...1-methyl-pseudouridine In some embodiments, the modified nucleobases are selected from the group consisting of 2-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases. Each possibility represents a separate embodiment of the present invention.

[0252] In some embodiments, the modified nucleobases in the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the polynucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the present invention.

[0253] In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (mC). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ) and 5-methyl-cytidine (mC). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (mC). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C). Each possibility represents a separate embodiment of the present invention.

[0254] In some embodiments, the polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) is uniformly modified (e.g., fully modified, i.e., modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C). That is, all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified with respect to any type of nucleoside residue present in the sequence by replacing it with any of the modified residues described above. Each possibility represents a separate embodiment of the present invention.

[0255] In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0256] In some embodiments, the modified nucleobase is a modified uridine. Examples of nucleobases and nucleosides having modified uridine include 5-cyanouridine or 4'-thiouridine. Each possibility represents a separate embodiment of the present invention.

[0257] In some embodiments, the modified nucleobase is a modified adenine. Examples of nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine. Each possibility represents a separate embodiment of the present invention.

[0258] In some embodiments, the modified nucleobase is a modified guanine. Examples of nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine. Each possibility represents a separate embodiment of the present invention.

[0259] In some embodiments, a polynucleotide functions as a messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (a natural, non-natural, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. The major components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. A polynucleotide that can function as an mRNA may be distinguished from wild-type mRNA in functional and / or structural design features that help overcome existing problems with effective polypeptide expression using nucleic acid-based therapeutics.

[0260] The mRNAs provided herein comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one polypeptide of interest. In some embodiments, the RNA polynucleotides of the mRNA encode 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 100 or at least 200 polypeptides.

[0261] In some embodiments, the nucleic acid is a therapeutic mRNA. As used herein, the term "therapeutic mRNA" refers to an mRNA that encodes a therapeutic protein. Therapeutic proteins mediate various effects in a host cell or subject to treat a disease or ameliorate the signs or symptoms of a disease. For example, a therapeutic protein may replace a missing or abnormal protein, enhance the function of an endogenous protein, confer a new function to a cell (e.g., inhibit or activate an endogenous cellular activity), or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNAs may be useful in treating the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune diseases.

[0262] Thus, the structures of the present invention may be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, mRNA of the structures described herein can be administered to a subject, where the polynucleotide is translated in vivo to produce a therapeutic peptide. Compositions, methods, kits, and reagents are provided for the diagnosis, treatment, or prevention of diseases or conditions in humans and other mammals. Active therapeutic agents of the present invention include the structures, cells containing the structures, or polypeptides translated from the polynucleotide contained in the structures.

[0263] The constructs can be induced for translation in a cell, tissue, or organism. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue, or organism is contacted with an effective amount of a composition containing constructs comprising mRNA polynucleotides, each having at least one translatable region encoding a peptide.

[0264] An "effective amount" of the construct is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and degree of nucleoside modification) and other components of the nucleic acid, as well as other determinants. Generally, an effective amount of the nucleic acid induces or enhances peptide production in a cell.

[0265] The mRNA of the invention can be designed to encode a polypeptide of interest selected from any of several target categories, including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell-penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-associated proteins, nuclear proteins, proteins associated with human disease, targeting moieties, or proteins encoded by the human genome that have no identified therapeutic indication but have utility in the fields of research and discovery. A "therapeutic protein" refers to a protein that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a cell.

[0266] The mRNA disclosed herein may encode one or more biologics. As used herein, a "biologic" is a polypeptide-based molecule produced by the methods provided herein and may be used to treat, cure, mitigate, prevent, or diagnose serious or life-threatening diseases or conditions. Biologics, according to the present invention, include, but are not limited to, allergen extracts (e.g., for allergy injections and testing), blood components, gene therapy drugs, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytic agents, and immunomodulatory agents, among others.

[0267] According to the present invention, one or more biologics currently on the market or in development may be encoded by the mRNA of the present invention. Without wishing to be bound by theory, incorporating the encoding polynucleotide of a known biologic into the mRNA of the present invention improves therapeutic efficacy, at least in part, due to the specificity, purity, and / or selectivity of the design of the construct.

[0268] The mRNA disclosed herein may encode one or more antibodies or fragments thereof. The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, single-chain molecules), and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site.

[0269] The monoclonal antibodies of the present invention specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include, but are not limited to, "primatized" antibodies comprising antigen-binding sequences of variable domains derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0270] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, nanobodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0271] Any of the five classes of immunoglobulins, namely, IgA, IgD, IgE, IgG, and IgM, may be encoded by the mRNA of the present invention, and include heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. Also included are polynucleotide sequences encoding the subclasses gamma and mu. Thus, any of the antibody subclasses may be encoded in part or in whole, including the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. According to the present invention, one or more antibodies or fragments currently on the market or in development may be encoded by the mRNA of the present invention.

[0272] Antibodies encoded in the mRNA of the present invention may be used to treat conditions or diseases in many therapeutic areas, including but not limited to, hematological, cardiovascular, CNS, poisoning (including antitoxins), dermatological, endocrine, gastrointestinal, medical imaging, musculoskeletal, oncology, immunological, respiratory, sensory, and anti-infective.

[0273] In one embodiment, the mRNA disclosed herein may encode a monoclonal antibody and / or a variant thereof. Antibody variants may include, but are not limited to, substitutional variants, conservative amino acid substitutions, insertional variants, deletion variants, and / or covalent derivatives. In one embodiment, the mRNA disclosed herein may encode an immunoglobulin Fc region. In another embodiment, the mRNA may encode a variant immunoglobulin Fc region.

[0274] The mRNA disclosed herein can encode one or more vaccine antigens. As used herein, a "vaccine antigen" is a biological product that improves immunity against a specific disease or infectious agent. According to the present invention, one or more vaccine antigens currently on the market or under development can be encoded by the mRNA of the present invention. The vaccine antigens encoded in the mRNA of the present invention can be used to treat conditions or diseases in many therapeutic areas, including, but not limited to, cancer, allergies, and infectious diseases.

[0275] The mRNA of the present invention can be designed to encode one or more antimicrobial peptides (AMPs) or antiviral peptides (AVPs). AMPs and AVPs have been isolated and characterized from a wide range of animals, including, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals. The antimicrobial polypeptides described herein can block cell fusion and / or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the antimicrobial polypeptides can comprise or consist of a synthetic peptide corresponding to a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a region, e.g., a transmembrane subunit of a viral envelope protein, e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described, for example, in Kuiken et al., (2008) "HIV Sequence Compendium," Los Alamos National Laboratory.

[0276] In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence identity to the corresponding viral protein sequence. In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding viral protein sequence.

[0277] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids, of the binding domain of a capsid-binding protein. In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding sequence of the capsid-binding protein.

[0278] The antimicrobial polypeptides described herein may block protease dimerization and inhibit cleavage of viral proproteins into functional proteins (e.g., HIV Gag-pol processing), thereby preventing the release of one or more enveloped viruses (e.g., HIV, HCV). In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding viral protein sequence.

[0279] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids, of a binding domain of a protease-binding protein. In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding sequence of the protease-binding protein.

[0280] A non-limiting list of infectious diseases that the mRNA vaccine antigen or antimicrobial polypeptide may treat is as follows: human immunodeficiency virus (HIV), HIV leading to mycobacterial infection, AIDS-associated cachexia, AIDS-associated cytomegalovirus infection, HIV-associated nephropathy, lipodystrophy, AIDS-associated cryptococcal meningitis, AIDS-associated neutropenia, Pneumocystis jiroveci (Pneumocystis carinii infection, AIDS-related toxoplasmosis, hepatitis A, B, C, D, or E, herpes, shingles (chickenpox), rubella (rubella virus), yellow fever, dengue fever, etc. (flaviviruses), flu (influenza virus), hemorrhagic infections (Marburg or Ebola viruses), bacterial infections such as Legionnaires' disease (Legionella), stomach ulcers (Helicobacter), cholera (Vibrio), E. coli infection, staphylococcal infection, salmonellosis, or streptococcal infection, tetanus (Clostridium tetani), protozoal infections (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e. infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma), diphtheria, leprosy, measles, whooping cough, rabies, tetanus, tuberculosis, typhoid, chickenpox, diarrhoeal infections such as amebiasis, Clostridium difficile associated diarrhea (CDAD), cryptosporidiosis, giardiasis, cyclosporiasis and rotavirus gastroenteritis, encephalitis such as Japanese encephalitis, western equine encephalitis and tick-borne encephalitis (TBE), fungal skin diseases such as candidiasis, onychomycosis, tinea capitis / tinea of ​​the scalp, tinea corporis / tinea corporis, jock itch / jock itch, sporotrichosis and tinea pedis / athlete's foot, meningitis such as Haemophilus influenza Type b (Hib), meningitis, viruses, meningococcal and pneumococcal diseases, neglected tropical diseases such as Argentine hemorrhagic fever, leishmaniasis, nematode / roundworm infections, Ross River virus and West Nile virus (WNV) diseases, non-HIVSTDs such as trichomoniasis, human papillomavirus (HPV) infections, sexually transmitted chlamydiosis, chancroid and syphilis, non-septic bacterial infections such as cellulitis, Lyme disease, MRSA infections, pseudomonas, staphylococcal infections, boutonnellitis, leptospirosis, rheumatic fever, botulism, rickettsiosis and mastoiditis, parasitic infections such as cysticercosis, echinococcosis, fluke / distoma infections, trichinosis, babesiosis, hypodermatosis, diphyllobothriasis and trypanosomiasis, respiratory infections such as adenovirus infections, aspergillosis, avian (H5N1) influenza influenza, RSV infection, Severe Acute Respiratory Syndrome (SARS), sinusitis, Legionnaires' disease, coccidioidomycosis and swine (H1N1) influenza, sepsis, e.g. bacteremia, sepsis / septic shock, sepsis in premature infants, urinary tract infections, e.g. vaginal infections (bacterial), vaginal infections (fungal) and gonorrhea, viral skin diseases, e.g. B19 parvovirus infection, warts, genital herpes, orofacial herpes, shingles, inner ear infections, fetal cytomegalovirus syndrome, foodborne diseases, e.g. brucellosis (Brucella spp.), Clostridium perfringens (epsilon toxin), E. coli O157:H7 (Escherichia coli), salmonellosis (Salmonella spp.), shigellosis (Shingella), vibriosis and listeriosis, bioterrorism and potential pandemic diseases such as Ebola hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, plague, anthrax-Nipah virus disease, hantavirus, smallpox, glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), tularemia (Fancisella tularensis), rubella, mumps and polio.

[0281] The mRNAs disclosed herein may encode one or more validated or "investigational" therapeutic proteins or peptides. According to the present invention, one or more therapeutic proteins or peptides currently on the market or in development may be encoded by the mRNAs of the present invention. The therapeutic proteins and peptides encoded in the mRNAs of the present invention may be used to treat conditions or diseases in many therapeutic areas, including, but not limited to, hematological, cardiovascular, CNS, poisoning (including antitoxins), dermatological, endocrine, genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunological, respiratory, sensory, and anti-infective.

[0282] The mRNA disclosed herein may encode one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide that can facilitate cellular uptake of a molecule. The cell-penetrating polypeptide of the present invention may contain one or more detectable labels. The polypeptide may be partially labeled or completely labeled throughout. The mRNA may encode the detectable label completely, partially, or not at all. The cell-penetrating peptide may also contain a signal sequence. As used herein, "signal sequence" refers to a sequence of amino acid residues that is attached to the amino terminus of a nascent protein during protein translation. The signal sequence can be used to signal the secretion of the cell-penetrating polypeptide.

[0283] In one embodiment, the mRNA may also encode a fusion protein. The fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked" refers to the therapeutic protein and the charged protein being connected in a manner that allows for expression of the complex when introduced into a cell. As used herein, a "charged protein" refers to a protein that has a positive, negative, or overall neutral charge. Preferably, the therapeutic protein may be covalently linked to the charged protein in the formation of a fusion protein. The ratio of total surface charge or surface amino acid charge may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0284] The cell-penetrating polypeptide encoded by the mRNA may form a complex after translation, which may include a charged protein linked, for example, covalently bound, to the cell-penetrating polypeptide.

[0285] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner" includes, but is not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be secreted from cells into which the mRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating the first cell.

[0286] In one embodiment, the mRNA can encode a cell-penetrating polypeptide that can contain a protein-binding partner. The protein-binding partner can include, but is not limited to, an antibody, a supercharged antibody, or a functional fragment. The mRNA can be introduced into cells into which the cell-penetrating polypeptide containing the protein-binding partner is introduced.

[0287] Some embodiments of the present disclosure provide a therapeutic mRNA comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide, wherein the RNA polynucleotide comprises at least one chemical modification. In some embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. Each possibility represents a separate embodiment of the present invention.

[0288] Any of the aforementioned polynucleotides of this disclosure may, in some embodiments, be codon-optimized. Codon optimization methods are known in the art and may be used as described herein. Codon optimization may, in some embodiments, be used to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structure, minimize tandem repeat codons or runs of bases that may impair gene organization or expression, customize transcriptional and translational regulatory regions, insert or remove protein trafficking sequences, remove / add sites for post-translational modification of the encoded protein (e.g., glycosylation sites), add, remove, or swap protein domains, insert or delete restriction enzyme recognition sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rate, correctly fold various domains of a protein, or reduce or remove problematic secondary structure within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0289] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0290] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity to the naturally occurring or wild-type sequence (e.g., the naturally occurring or wild-type mRNA sequence encoding the polypeptide or protein of interest (e.g., the antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares 65% to 75 or about 80% sequence identity to the naturally occurring or wild-type sequence (e.g., the naturally occurring or wild-type mRNA sequence encoding the polypeptide or protein of interest (e.g., the antigenic protein or polypeptide)).

[0291] In some embodiments, the codon-optimized RNA may have, for example, an increased G / C level. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO 02 / 098443 discloses a pharmaceutical composition comprising an mRNA stabilized by sequence modification in the coding region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with ones that further enhance the stability of the RNA without changing the resulting amino acid. The method is limited to the coding region of the RNA.

[0292] As used herein, with respect to amino acid-based embodiments when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain." As used herein, with respect to nucleotide-based embodiments when referring to polynucleotides, the term "site" is used synonymously with "nucleotide." A site represents a position within a peptide, or polypeptide, or polynucleotide that can be modified, manipulated, altered, derivatized, or changed within the polypeptide or polynucleotide-based molecule.

[0293] As used herein, the terms "terminus" and "terminus" when referring to a polypeptide or polynucleotide refer to the end of the polypeptide or polynucleotide, respectively. Such ends are not limited to the beginning or end of the polypeptide or polynucleotide, but may also include additional amino acids or nucleotides in the terminal region. A polypeptide molecule may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are sometimes composed of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or non-covalent forces. These proteins have multiple N- and C-termini. Alternatively, the ends of polypeptides may optionally be modified to begin or end with non-polypeptide moieties, such as organic conjugates.

[0294] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide of interest. For example, provided herein are any protein fragments (meaning polypeptide sequences that are at least one amino acid residue shorter than the reference polypeptide sequence but are otherwise identical) of a reference protein that are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length. In another example, any protein containing a stretch of 20, 30, 40, 50, or 100 amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be used in accordance with the present disclosure. In some embodiments, a polypeptide contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another example, any protein containing a stretch of 20, 30, 40, 50, or 100 amino acids that is more than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, and having a stretch of 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, 75%, 70%, 65%, or 60% identical to any of the sequences described herein can be used in accordance with the present disclosure.

[0295] A polypeptide or polynucleotide molecule useful in the present disclosure may share a certain degree of sequence homology or identity with a comparison molecule (e.g., a comparison polypeptide or comparison polynucleotide), for example, with a molecule described in the art (e.g., an artificially created or designed molecule or a wild-type molecule). The term "identity," as known in the art, refers to the relationship between the sequences of two or more polypeptides or polynucleotides and is determined by comparing the sequences. In the art, identity also refers to the degree of sequence correlation between them and is determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of perfect matches between the smaller of two or more sequences with gap alignment (if any), as handled by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be easily calculated using known methods. "Percent identity," as applied to a polypeptide or polynucleotide sequence, is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for such alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but may vary depending on the gaps and penalties introduced into the calculation. Generally, a particular polynucleotide or polypeptide variant will have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity to a particular comparison polynucleotide or polypeptide, as determined by sequence alignment programs and parameters known to those skilled in the art and described herein.Tools for such alignment include those in the BLAST suite (Stephen F. Altschul, et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another well-known local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences", J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol. 48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which purportedly produces global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0296] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity as determined by alignment of matching residues are called homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that determines the degree of sequence match between two compared sequences. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical over at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least 20 amino acids.

[0297] Homology implies that the compared sequences have diverged during evolution from a common origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related to a second amino acid or nucleic acid sequence by descent from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins that have been separated by a speciation event or between genes and / or proteins that have been separated by a gene duplication event. An "ortholog" is a gene (or protein) in different species that has evolved from a common ancestral gene (or protein) by speciation. Orthologs typically retain the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs evolve new functions, even if related to the original function.

[0298] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity between two polynucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the compared sequences. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0299] Immunomodulatory agents may be either immunostimulatory or immunosuppressive agents. Immunostimulatory agents, whether administered alone or in combination with other agents, stimulate an immune response (including enhancing an existing immune response) in a subject to which they are administered. Examples include antigens, adjuvants (e.g., TLR ligands, e.g., imiquimod, imidazoquinoline, nucleic acids containing unmethylated CpG dinucleotides, monophosphoryl lipid A or other lipopolysaccharide derivatives, single- or double-stranded RNA, flagellin, muramyl dipeptide), cytokines including interleukins (e.g., IL-2, IL-7, IL-15 (or superagonists / mutants of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3 ligand, etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single-chain / antibody fragments of these molecules), etc.

[0300] Immunosuppressants are drugs that inhibit the immune response in a subject to which they are administered, whether alone or in combination with other drugs. Examples include steroids, retinoic acid, dexamethasone, cyclophosphamide, anti-CD3 antibodies or antibody fragments, and other immunosuppressants.

[0301] Adjuvants are agents that enhance immune responses. Examples of adjuvants include, but are not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate), saponin purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid eluting in the 21st peak by HPLC fractionation, Antigenics, Inc., Worcester, Mass.), poly[di(carboxylatophenoxy)phosphazene] (PCPP polymer, Virus Research Institute, USA), Flt3 ligand, leishmanial elongation factor (a purified leishmanial protein, Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes containing mixed saponins and lipids that form virus-sized particles with pores capable of retaining antigens, CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (a SmithKline product containing alum and MPL), and the like. Beecham Adjuvant System #4, SBB, Belgium), micelle-forming nonionic block copolymers such as CRL1005 (which contain linear chains of hydrophobic polyoxypropylene flanked on both sides by polyoxyethylene chains, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS 1312, aqueous nanoparticles mixed with a soluble immunostimulant, Seppic).

[0302] The adjuvant may be a TLR ligand. Adjuvants acting through TLR3 include, without limitation, double-stranded RNA. Adjuvants acting through TLR4 include, without limitation, lipopolysaccharide derivatives, such as monophosphoryl lipid A (MPLA, Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP, Ribi), and threonylmuramyl dipeptide (t-MDP, Ribi), and OM-174 (a glucosamine disaccharide related to lipid A, OM Pharma SA, Meyrin, Switzerland). Adjuvants acting through TLR5 include, without limitation, flagellin. Adjuvants acting through TLR7 and / or TLR8 include, without limitation, single-stranded RNA, oligoribonucleotides (ORN), and synthetic low-molecular-weight compounds, such as imidazoquinoline amines (e.g., imiquimod, resiquimod). Adjuvants that act through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another class of adjuvants is phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.

[0303] The antigen may be, without limitation, a cancer antigen, an autoantigen, a microbial antigen, an allergen, or an environmental antigen. The antigen may also be peptide, lipid, or carbohydrate in nature, but is not so limited.

[0304] Cancer antigens are antigens that are preferentially expressed by cancer cells (i.e., they are expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, they are expressed only by cancer cells. The cancer antigen can be expressed within cancer cells or on the surface of the cancer cells. The cancer antigen can be MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-related antigen (CRC)--C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, and CD20. The cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, and MAGE-C5. The cancer antigen may be selected from the group consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, and GAGE-9.The cancer antigens include BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, and I The antibody may be selected from the group consisting of g-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus proteins, the Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, and c-erbB-2. Each possibility represents a separate embodiment of the present invention.

[0305] Microbial antigens are antigens derived from microbial species, including, but not limited to, bacteria, viruses, fungi, parasites, and mycobacteria species. Thus, microbial antigens include bacterial antigens, viral antigens, fungal antigens, parasite antigens, and mycobacteria antigens. Examples of bacterial, viral, fungal, parasite, and mycobacteria species are provided herein. The microbial antigen may be a portion of a microbial species or the whole microorganism.

[0306] Anticancer drugs are agents that at least partially inhibit the development or progression of cancer, including completely or partially inhibiting symptoms associated with cancer, even if only for a short period of time. Some anticancer drugs can be classified as DNA damaging agents, including topoisomerase inhibitors (e.g., etoposide, lamprothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, columbucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducers (e.g., cyclophosphamide, cyclophosphamide, ifosfamide, melphalan, columbucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), and DNA strand break inducers (e.g., cyclophosphamide, cyclophosphamide, ifosfamide, melphalan, columbucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine). , bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), microtubule inhibitors (e.g., vincristine, vinblastine), antimetabolites (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, or epipodophyllotoxins.

[0307] Examples of anticancer drugs include, without limitation, acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, bortezomib (VELCADE), brequinar sodium, Bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin (platinum-containing regimen), carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin (platinum-containing regimen), cladribine, crisnatol mesilate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexorumaplatin, dezaguanine, diaziquone, docetaxel (TAXOTERE), doxorubicin, droloxifene, Dromostanolone, zuazomycin, edatrexate, eflornithine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, elbrozole, erlotinib (TARCEVA), esorubicin, estramustine, etanidazole, etoposide, etopurine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, 5-fluorouracil, flurocitabine, foskidone, fostriecin, gefitinib (IRESSA), gemcitabine, hydroxyurea, idarubicin, ifosf amide, ilmofosine, imatinib mesylate (GLEEVAC), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan, lanreotide, lenalidomide (REVLIMID, REVIMID), letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine, megestrol,Melengestrol, melphalan, menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, methindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone, mycophenolate, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pemetrexed (ALIMTA), pegaspargase, perimycin Syn, pentamstine, pentomone, peplomycin, perfosfamide, pipobroman, piposulfan, piritrexim isethionate, piroxantrone, plicamycin, promestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, ribopurin, rogletimide, safingol, semustine, simtrazene, sitogluside, sparfosate, sparsomycin thalidomide, thiamin, thiamin, thiamin derivatives ... Examples of antihistamines include zofrin, tirapazamine, topotecan, toremifene, trestron, triciribine, tremetrexate, triptorelin, tubrozole, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine, vindesine, vinepidine, vinglisinate, vinleurosine, vinorelbine, vinrocidine, vinzolidine, vorozole, zeniplatin, zinostatin, and zorubicin.

[0308] The anti-cancer agent may be an enzyme inhibitor, including, without limitation, a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be, without limitation, genistein (4',5,7-trihydroxyisoflavone), tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, herbimycin A, daidzein (4',7-dihydroxyisoflavone), AG-126, trans-1-(3'-carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2-hydroxy-5-(2,5-dihydroxybenzylamino)-2-hydroxybenzoic acid). The CDK inhibitor may be, without limitation, p21, p27, p57, p15, p16, p18, or p19. The MAP kinase inhibitor may be, without limitation, KY12420 (C 23 H 24 O8), CNI-1493, PD98059, or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole. The EGFR inhibitor can be, without limitation, erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.

[0309] The anti-cancer agent may be a VEGF inhibitor, including, without limitation, bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorins.

[0310] The anti-cancer agent may be an antibody or antibody fragment, including, without limitation, bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for the treatment of B-cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for the treatment of leukemias such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for the treatment of B-cell malignancies), MDX-210 (anti-HER-2 / neu oncogene protein product and immunoglobulin G (IgG)), and others. bispecific antibody that simultaneously binds to type I Fc receptors (Fc gamma RI) for ovarian cancer, oregovomab (OVAREX, indicated for the treatment of ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for the treatment of respiratory diseases such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for the treatment of non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR Antibodies or antibody fragments include, but are not limited to, EGF / R3, cerogovab (ONCOSCINT OV103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and gliomab-H (indicated for the treatment of brain cancer, melanoma).

[0311] A diagnostic agent, sometimes referred to herein as a contrast agent, is an agent that directly or indirectly emits a signal, thereby enabling its detection in vivo. Diagnostic agents, such as contrast agents and radiopharmaceuticals, can be detected using medical imaging techniques, such as nuclear medicine and magnetic resonance imaging (MRI). Contrast agents for magnetic resonance imaging (MRI) include Gd(DOTA), iron oxide, or gold nanoparticles, and contrast agents for nuclear medicine include: 201Imaging agents for positron emission tomography (PET) include the positron-emitting isotope, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64, gadoamide, and radioisotopes of Pb(II), e.g., 203Pb, and 11In; contrast agents for in vivo fluorescence imaging, e.g., fluorescent dyes or dye-conjugated nanoparticles. In other embodiments, the agent to be delivered is conjugated, fused, mixed, or bound to a diagnostic agent.

[0312] The compounds and compositions can be administered to virtually any type of subject that is likely to benefit from the delivery of the drugs contemplated herein.Human subjects are preferred subjects in some embodiments of the present invention.Subjects also include animals, such as pets (e.g., dogs, cats, rabbits, ferrets, etc.), livestock or farm animals (e.g., cows, pigs, sheep, chickens and other poultry), horses, such as thoroughbred horses, laboratory animals (e.g., mice, rats, rabbits, etc.), etc.Subjects also include fish and other aquatic animal species.

[0313] The subject to whom the drug is delivered may be a normal subject. Alternatively, the subject may have a condition that can be diagnosed or may benefit from the local delivery of one or more specific drugs, or may be at risk of progression. Such conditions include cancer (e.g., solid tumor cancer), infectious diseases (especially infectious diseases that are limited to specific regions or tissues in the body), autoimmune diseases, allergies or allergic conditions, asthma, transplant rejection, etc. In some embodiments, the subject has been diagnosed with a genetic defect and has been administered a nucleic acid-based therapeutic agent.

[0314] The drug may be administered systemically or locally. The drug may be administered in an effective amount. An effective amount is a dose of the drug sufficient to provide a medically desirable result. The effective amount will vary depending on the specific condition being treated, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concomitant or mixed therapy (if any), the specific route of administration, and similar factors within the knowledge and opinion of a medical professional. Generally, it is preferred to use a maximum dosage, i.e., the maximum safe dose according to sound medical judgment.

[0315] The present invention provides pharmaceutical compositions. Pharmaceutical compositions are sterile compositions that contain a drug and may include delivery vehicles, nanoparticles, etc., preferably in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other subjects contemplated by the present invention. The term "carrier" refers to an organic or inorganic, natural, or synthetic component with which cells, nanoparticles, and drug(s) are mixed to facilitate administration. The components of the pharmaceutical composition are mixed in a manner such that there is no interaction which would substantially impair their desired pharmaceutical efficiency.

[0316] When it is desired to deliver the compounds and compositions systemically, they can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations can be contained in unit dosage forms, for example, in ampoules or multi-dose containers. Pharmaceutical parenteral preparations include aqueous solutions of the components. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Alternatively, suspensions of the components can be prepared as oily suspensions, for example, as known in the art or as would be readily apparent to those skilled in the art based on the present disclosure.

[0317] The present disclosure further contemplates the use of LNPs in conjunction with one or more second agents, including agents normally indicated for the treatment of the subject.

[0318] In some examples, the LNPs can be administered substantially simultaneously with the second agent, meaning that the LNPs are administered to the subject close in time to the administration of the second agent, e.g., within 1 hour, within 30 minutes, within 10 minutes, or within 5 minutes.

[0319] In some examples, the second agent(s) can be administered before the LNP. For example, the second agent(s) can be administered within 24 hours, or within 18 hours, or within 12 hours, or within 6 hours, or within 3 hours, or within 2 hours before administration of the LNP. The second agent(s) can be administered 18 to 24 hours, or 12 to 18 hours, or 6 to 12 hours, or 2 to 6 hours before administration of the LNP.

[0320] A subject who receives one or more second agents two or more hours prior to LNP administration may be referred to as being premedicated with such agent(s). A subject who receives one or more second agents within one hour prior to LNP administration may be referred to as being co-medicated with such agent(s).

[0321] In some examples, the second agent(s) may be administered to the subject continuously, as needed, or on a fixed schedule (eg, daily, once every two days, etc.).

[0322] In other examples, the second agent may be administered before or after administration of the LNP.

[0323] Such second agents can include, but are not limited to, antihistamines, antiplatelet agents, and antisteroidal anti-inflammatory agents. In certain embodiments, the LNPs are not formulated with, and the subject is not pre- or co-medicated with, a corticosteroid, such as, but not limited to, dexamethasone.

[0324] In certain embodiments, a single second drug is used that has anti-inflammatory and antiplatelet properties, one example of such a drug is aspirin.

[0325] In certain embodiments, a combination of aspirin, clopidogrel (Plavix®), and an antihistamine, such as, but not limited to, diphenhydramine (Benadryl), fexofenadine (Allegra), loratadine (Claritin), or cetirizine, is used. One or more second agents may be administered once per administration of LNP, while others may be administered more frequently. For example, clopidogrel (Plavix®) may be administered once per administration of LNP, while aspirin and / or the antihistamine may be administered daily.

[0326] Antihistamines include H1 receptor antagonists and H1 receptor inverse agonists. Examples of H1 receptor antagonists include acrivastine, alimemazine, alimemazine tartrate, antazoline, astemizole, azatadine, azatadine maleate, azelastine, bamipine, benzquinamide, bepotastine, bepotastine besylate, bilastine, bromazine, brompheniramine, buclizine, carbinoxamine, chlorphenoxamine, chlorcyclizine, cinnopentazone, histapirozine, and chlordif. Cenhydramine, chlorpyramine, chlorphenamine, chlorpromazine, cinnarizine, clemastine, clemizole, clocinidine, cyclizine, cyproheptadine, desloratadine, deptropine, dexchlorpheniramine, dexbrompheniramine, dimenhydrinate, dimethindene, dimethothiazin, diphenhydramine (Benadryl), diphenylpyraline, doxepin, doxylamine, ebastine, efletiri Zinc, embramin, emedastine, epinastine, fexofenadine (Allegra), flunarizine, homochlorcyclizine, hydroxyzine, isothipendyl, ketotifen, levocabastine (2nd generation), loratadine (Claritin), mebhydroline, meclozine, mepyramine, mequitazine, methdilazine, mirtazapine, mizolastine, niaprazine, olopatadine, orphenadrine, oxatomide, oxomemazine, These include, but are not limited to, pemirolast, phenindamine, pheniramine, phenyltoloxaquin, pimethixene, piprine hydrinate, promethazine, propiomazine, pyrobutamine, quetiapine, quifenadine, rupatadine, setastine, terfenadine, thenyldiamine, thiethylperazine, thonzylamine, tolpropamine, trimethobenzamine, tripelennamine, triprolidine, and tritoqualine.

[0327] Examples of H1 receptor inverse agonists include, but are not limited to, pyrilamine, cetirizine, levocetirizine, and desloratadine.

[0328] Antiplatelet agents include, but are not limited to, activation inhibitors, aggregation inhibitors, adhesion antagonists, anticoagulants (which do not directly target platelets), and agents that reduce platelet count or number.

[0329] Examples of activation inhibitors include: (1) thrombin receptor PAR-1 inhibitors, such as SCH 530348 (vorapaxar), E-5555 (atopaxar), SCH79797, FR171113, RWJ56110, BMS-200661, RWJ-58259, SCH205831, Pipal-7 pepducin, and P1pal-12 pepducin; (2) thrombin receptor PAR-4 inhibitors, such as ML 354, tcY-NH2, P4pal-10 pepducin, and P4pal-i1 pepducin; (3) FSLLRY-NH2 (PAR-2 ​​peptide antagonist); and (4) TxA2 receptor antagonists, such as AH 23,848, SQ 29,548, or R 68,070, S-1452, iosartan, seratrodast, (5) thromboxane receptor antagonists, such as terutroban, (6) ADP P2Y12 receptor inhibitors, such as ticlopidine, clopidogrel, prasugrel, ticagrelor, cangrelor, elinogrel, AZD6140, AR-C69931, CoA, (7) ADP P2Y1 receptor inhibitors, such as A2P5P, A3P5P, MRS2179, MRS2279, MRS2500, palmitoyl-CoA (also acts on P2Y12), and Thalji et al. Other compounds from the SAR study by et al. 2010, (8) 5-HT2A antagonists, e.g., R-1012444, naftidrofuryl, sarpogrelate, AT-1015, (9) thromboxane synthase inhibitors, e.g., dazoxiben, CS-518 (TXA2 synthase inhibitor), SB 203580, U63557A, imidazo(1,5-2)pyridine-5-hexanoic acid, (10) COX-1 inhibitors such as aspirin, NCX-4016, ridogrel, S18886, picotamide, ramatroban (also a TXA2 receptor antagonist), SC-560, FR122047, mofezolac, P6, TFAP, ibuprofen and naproxen (also a Cox-2 inhibitor), (11) COX-2 inhibitors such as triflusal (also a COX-1 and PDE inhibitor), etoricoxib, rofecoxib, celecoxib, meloxicam, and (12) PI3K inhibitors such as AZD6482.

[0330] Examples of aggregation inhibitors include, but are not limited to: (1) GPIa / IIa inhibitors, such as EMS16; (2) GPVI inhibitors, such as monoclonal antibodies and Fab fragments of mAb 12A5; (3) GPIIb / IIIa inhibitors, such as abciximab, eptifibatide, and tirofiban; (4) PDE inhibitors, such as dipyridamole (also an adenosine reuptake inhibitor), cilostazol (a PDE3 inhibitor that increases cAMP and activates PKA); and (5) ADP receptor antagonists. Other platelet aggregation inhibitors include aspirin, clopidogrel (Plavix®), aspirin / pravastatin, cilostazol, prasugrel, aspirin / dipyridamole, ticagrelor, cangrelor, elinogrel, dipyridamole, and ticlopidine.

[0331] Examples of adhesion antagonists (against fibrinogen) include, but are not limited to, C1qTNF-related protein-1, DZ-697b, RG12986.

[0332] Examples of non-platelet anticoagulants include, but are not limited to, warfarin, factor Xa inhibitors such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, and otamixaban, thrombin inhibitors such as bivalirudin, hirudin, dabigatran, lepirudin, desirudin, argatroban, melagatran, dabigatran, CDSO3, FDSO3, and SDSO3, and the additional benzofuran sulfate allosteric inhibitors reported by Sidhu et al.

[0333] Examples of agents that reduce platelet count or number include: (1) cAMP phosphodiesterase inhibitors (e.g., anagrelide), 6,7-dichloro-1,5-dihydroimidazo-[2,1-b]quinazolin-2(3H)-one or 6,7-dichloro-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one (U.S. Pat. Nos. 3,932,407, 4,146,718, RE31,617, Haematologica 1992 77:40-3); (2) antibodies against cell surface receptors specifically expressed by platelets or megakaryocytes, e.g., glycoprotein IIb / IIIa receptor antibodies; and (3) most chemotherapeutic anticancer agents, e.g., busulfan (Br. J. Haematol. 1986 62:229-37), hydroxyurea (N Engl J Med 1995 332:1132-6), hepsulfan, phosphorus 32 (Br J Radiol 1997 70:1169-73), pipobroman (Scand J. Haematol 1986 37:306-9), cyclophosphamide (J Cell Physiol 1982 112:222-8), certain alkylating agents and certain antimetabolites, (4) cytokines, growth factors and interleukins, such as alpha-interferon (Cancer Immunol Immunother 1987 25:266-73), gamma interferon, transforming growth factor-beta, neutrophil-activating peptide-2 and its analogs (U.S. Pat. No. 5,472,944), macrophage inflammatory protein and its analogs (U.S. Pat. No. 5,306,709), (5) compounds secreted by either platelets or megakaryocytes, such as platelet factor 4 (U.S. Pat. No. 5,185,323), transforming growth factor-beta, 12-17 kD glycoproteins produced by megakaryocytes, thrombin and thrombospondin, and amino (1-174 amino acids) terminal fragments thereof (J Lab Clin Med 1997 129:231-8), and (6) anti-keloid agents, such as tranilast (Rizaben) (J Dermatol 1998 25:706-9), forskolin and spleen anti-maturation factor (US Pat. No. 4,088,753).

[0334] Antiplatelet agents may also be characterized as antithrombotic agents, thrombolytic agents, direct thrombin inhibitors, glycoprotein IIb / IIIa receptor inhibitors, agents that bind to cell adhesion molecules and inhibit the ability of leukocytes to adhere to such molecules, calcium channel blockers, beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors, and angiotensin system inhibitors.

[0335] Antithrombotic agents are defined as agents that prevent thrombus formation through several potential mechanisms, including thrombolytic agents, anticoagulants, and inhibitors of platelet function.

[0336] Thrombolytic agents are defined as agents that dissolve thrombi (e.g., blood clots), usually through the dissolution of fibrin by enzymatic action. Examples of thrombolytic agents include, but are not limited to, ancrod, anistreplase, viscobrinolase lactate, brinolase, Hageman factor (i.e., factor XII) fragments, molsidomine, plasminogen activators such as streptokinase, tissue plasminogen activator (TPA) and urokinase, as well as plasmin and plasminogen. Anticoagulants also include inhibitors of factor Xa, TFPI, factor VIIa, factor IXc, factor Va, factor VIIIa, and other coagulation factor inhibitors.

[0337] Anticoagulants are drugs that inhibit the coagulation pathway by adversely affecting the production, deposition, cleavage, and / or activation of factors essential for clot formation. Anticoagulants include, but are not limited to, vitamin K antagonists, such as coumarin and coumarin derivatives (e.g., warfarin sodium), glycosaminoglycans, such as heparin in both unfractionated and low molecular weight forms, ardeparin sodium, bivalirudin, bromindione, coumarin, dalteparin sodium, desirudin, dicoumarol, liaporate sodium, nafamostat mesylate, phenprocoumon, sulfatides, and tinzaparin sodium.

[0338] Other "anticoagulant" and / or "thrombolytic" drugs include plasminogen, streptokinase, urokinase, anisoylated plasminogen-streptokinase activator complex, pro-urokinase (Pro-UK), rTPA (alteplase or activase, r stands for recombinant), rPro-UK, abokinase, eminase, streptase, anagrelide hydrochloride, bivalirudin, dalteparin sodium, danaparoid sodium, dazoxiben hydrochloride, efegatran sulfate, enoxaparin sodium, ifetroban, ifetroban sodium, tinzaparin sodium, retaplase, trifenagrel, warfarin, and dextran.

[0339] Still other anticoagulants include, but are not limited to, ancrod, anticoagulant citrate dextrose solution, anticoagulant citrate phosphate dextrose adenine solution, anticoagulant citrate phosphate dextrose solution, anticoagulant heparin solution, anticoagulant sodium citrate solution, ardeparin sodium, bromindione, desirudin, dicumarol, heparin calcium, heparin sodium, liaborate sodium, nafamostat mesylate, and phenprocoumon.

[0340] Clot dissolving agents include, but are not limited to, tissue plasminogen activator, streptokinase, and nimodipine.

[0341] Platelet function inhibitors are agents that impair the ability of mature platelets to perform their normal physiological roles (i.e., their normal functions). Platelets are normally involved in multiple physiological processes, such as adhesion, e.g., to cellular and non-cellular entities, aggregation, e.g., to form blood clots, and release of factors such as growth factors (e.g., platelet-derived growth factor (PDGF)) and platelet particulate components. One subcategory of platelet function inhibitors is inhibitors of platelet aggregation, which are compounds that reduce or disrupt the ability of platelets to physically associate with themselves or with other cellular and non-cellular components, thereby eliminating the ability of platelets to form thrombi.

[0342] Examples of useful inhibitors of platelet function include acadesine, anagrelide, anipamil, argatroban, aspirin, clopidogrel, cyclooxygenase inhibitors, such as nonsteroidal anti-inflammatory drugs and the synthetic compound FR-122047, danaparoid sodium, dazoxiben hydrochloride, diadenosine 5',5''-P1,P4-tetraphosphate (Ap4A) analogs, difibrotide, dilazep hydrochloride, 1,2- and 1,3-glyceryl diphosphate, nitrate, dipyridamole, dopamine and 3-methoxytyramine, efegatran sulfate, enoxaparin sodium, glucagon, glycoprotein IIb / IIIa antagonists such as Ro-43-8857 and L-700,462, ifetroban, ifetroban sodium, iloprost, isocarbacycline methyl ester, isosorbide-5-mononitrate, itazigrel, ketanserin, and BM-13.177, lamifiban, rifariidine, molsidomine, nifedipine, oxagrelate, PGE, platelet-activating factor antagonists, e.g., lexipafant, prostacyclin (PGI2), pyrazine, pyridinol carbamate, ReoPro (i.e., abciximab), sulfinpyrazone, synthetic compounds BN-50727, BN-52021 , CV-4151, E-5510, FK-409, GU-7, KB-2796, KBT-3022, KC-404, KF-4939, OP-41483, TRK-100, TA-3090, TFC-612 and ZK-36374, 2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane 2,2-dioxide, 2,4,5-trithiahexane , theophylline, pentoxifylline, thromboxane and thromboxane synthetase inhibitors such as picotamide and sulotroban, ticlopidine, tirofiban, trapidil and ticlopidine, tripenagrel, trilinolein, 3-substituted 5,6-bis(4-methoxyphenyl)-1,2,4-triazines, and antibodies to glycoprotein IIb / IIIa, and those disclosed in U.S. Pat. No. 5,440,020, as well as antiserotonin drugs, clopidogrel, sulfinpyrazone, aspirin, dipyridamole, clofibrate, pyridinol carbamate, PGE, glucagon, antiserotonin drugs, caffeine, theophylline, pentoxifylline, ticlopidine.

[0343] "Direct thrombin inhibitors" include hirudin, hirugen, hirulog, argatroban, PPACK, and thrombin aptamers.

[0344] "Glycoprotein IIb / IIIa receptor inhibitors" are both antibodies and non-antibodies, and include, but are not limited to, ReoPro (abciximab), lamifiban, and tirofiban.

[0345] "Calcium channel blockers" are a chemically diverse class of compounds with significant therapeutic value in the prevention of various diseases (Fleckenstein, Cir. Res., v. 52, (suppl. 1), pp. 13-16 (1983); Fleckenstein, Experimental Facts and Therapeutic Prospects, John Wiley, New York (1983); McCall, D., Curr Pract Cardiol, v. 10, pp. 1-11 (1985)). Calcium channel blockers are a heterogeneous group of drugs that block or slow the entry of calcium into cells by modulating cellular calcium channels (Remington, The Science and Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company, Eaton, PA, p. 963 (1995)). Most currently available calcium channel blockers useful in the context of the present invention belong to one of three major chemical groups of drugs: dihydropyridines, e.g., nifedipine; phenylalkylamines, e.g., verapamil; and benzothiazepines, e.g., diltiazem. Other calcium channel blockers useful in the context of the present invention include, but are not limited to, amrinone, amlodipine, bencyclane, felodipine, fendiline, flunarizine, isradipine, nicardipine, nimodipine, perhexylene, gallopamil, tiapamil and tiapamil analogs (e.g., 1993RO-11-2933), phenytoin, barbiturates, and peptides such as dynorphins, omega-conotoxins, and omega-agatoxins, and / or pharmaceutically acceptable salts thereof.

[0346] "Beta-adrenergic receptor blockers" are a class of drugs that antagonize the cardiovascular effects of catecholamines in angina, hypertension, and cardiac arrhythmias. Beta-adrenergic receptor blockers include atenolol, acebutolol, alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol, hedroxalol, indenolol, labetalol, levobunolol, mepindolol, metipranol, metindol, metoprolol, metrizolanol, oxprenolol, pindolol, propranolol, practolol, sotalol, nadolol, tiprenolol, tomalol, timolol, bupranolol, penbutolol, trimepranolol, 2-(3-(1,1-dimethylethyl)-amino-2-hydroxybenzoate, benzocaine, benzodi ... Examples of isomeric compounds include, but are not limited to, 1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol, 3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol, 2-(3-t-butylamino-2-hydroxypropylthio)-4-(5-carbamoyl-2-thienyl)thiazole, and 7-(2-hydroxy-3-t-butylaminepropoxy)phthalide. The compounds listed above can be used as isomeric mixtures or in their respective levorotatory or dextrorotatory forms.

[0347] Cyclooxygenase-2 (COX-2) is a recently identified form of cyclooxygenase. "Cyclooxygenase" is an enzyme complex present in most tissues that produces various prostaglandins and thromboxanes from arachidonic acid. Nonsteroidal anti-inflammatory drugs exert most of their anti-inflammatory, analgesic, and antipyretic effects, as well as inhibit hormone-induced uterine contractions and the growth of certain types of cancer, through inhibition of cyclooxygenase (also known as prostaglandin G / H synthase and / or prostaglandin endoperoxide synthase). Initially, only one form of cyclooxygenase was known: the "constitutive enzyme" or cyclooxygenase-1 (COX-1). It was first identified in bovine seminal vesicles.

[0348] Cyclooxygenase-2 (COX-2) was originally cloned, sequenced, and characterized from chicken, mouse, and human sources (see, e.g., U.S. Pat. No. 5,543,297, issued August 6, 1996, Cromlish et al., Merck Frosst Canada, Inc., Kirkland, CA, entitled "Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity").

[0349] Several selective "COX-2 inhibitors" are known in the art. These include U.S. Patent No. 5,474,995 entitled "Phenyl heterocycles as COX-2 inhibitors," U.S. Patent No. 5,521,213 entitled "Diaryl bicyclic heterocycles as inhibitors of cyclooxygenase-2," U.S. Patent No. 5,536,752 entitled "Phenyl heterocycles as COX-2 inhibitors," U.S. Patent No. 5,550,142 entitled "Phenyl heterocycles as COX-2 inhibitors," U.S. Patent No. 5,552,422 entitled "Aryl substituted 5,5 fused aromatic nitrogen compounds as anti-inflammatory agents," U.S. Patent No. 5,604,253 entitled "N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase inhibitors," and U.S. Patent No. 5,604,260 entitled "5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”, U.S. Patent No. 5,639,780 “N-benzyl indol-3-yl butanoic acid derivatives as cyclooxygenase inhibitors”, U.S. Patent No. 5,677,318 “Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”, U.S. Patent No. 5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”, U.S. Patent No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-dihydrofurans as prodrugs to COX-2 inhibitors”, U.S. Patent No. 5,710,No. 140 "Phenyl heterocycles as COX-2 inhibitors", U.S. Patent No. 5,733,909 "Diphenyl stilbenes as prodrugs to COX-2 inhibitors", U.S. Patent No. 5,789,413 "Alkylated styrenes as prodrugs to COX-2 inhibitors", U.S. Patent No. 5,817,700 "Bisaryl cyclobutenes derivatives as cyclooxygenase inhibitors", U.S. Patent No. 5,849,943 "Stilbene derivatives useful as cyclooxygenase-2 inhibitors", U.S. Patent No. 5,861,419 "Substituted pyridines as selective cyclooxygenase-2 inhibitors", U.S. Patent No. 5,922,742 "Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2 inhibitors", U.S. Patent No. 5,925,631 "Alkylated styrenes as prodrugs to COX-2 inhibitors include, but are not limited to, those described in "COX-2 inhibitors." Additional COX-2 inhibitors are also described in U.S. Pat. No. 5,643,933, entitled "Substituted sulfonylphenylheterocycles as cyclooxygenase-2 and 5-lipoxygenase inhibitors," assigned to G.D. Searle & Co. (Skokie, IL). Aspirin is an example of a COX-2 inhibitor.

[0350] The COX-2 inhibitors listed above are prodrugs of selective COX-2 inhibitors, and exert their effects by conversion in vivo to active and selective COX-2 inhibitors. Active and selective COX-2 inhibitors formed from the COX-2 inhibitor prodrugs listed above are described in detail in WO 95 / 00501, published January 5, 1995, WO 95 / 18799, published July 13, 1995, and U.S. Patent No. 5,474,995, issued December 12, 1995. Given the teachings of U.S. Patent No. 5,543,297, entitled "Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity," one skilled in the art can determine whether a drug is a selective COX-2 inhibitor or a precursor of a COX-2 inhibitor, and thus part of the present invention.

[0351] Nonsteroidal anti-inflammatory drugs include, but are not limited to, naproxen sodium, diclofenac, sulindac, oxaprozin, diflunisal, aspirin, piroxicam, indomethacin, etodolac, ibuprofen, fenoprofen, ketoprofen, mefenamic acid, nabumetone, tolmetin sodium, and ketorolac tromethamine.

[0352] In certain embodiments, the LNPs may contain one or more second agents that inhibit the immune response elicited by the LNPs, for example, by inhibiting binding to the sensor or inhibiting the activity of the sensor, such as inhibiting the production of natural IgM, the production of natural IgG, the activation of B1a cells, the activation of B1b cells, and / or the activation of platelets and / or dendritic cells, or the activity or production of any effector. The second agents may alternatively be referred to as agents that inhibit the immune response elicited by the LNPs. In some instances, the second agents may inhibit the production of natural IgM that binds to the LNPs or neutralize such natural IgM. In other instances, the second agents may inhibit the activation of B1a cells or remove B1a cells. For example, such second agents may inhibit a surface receptor on B1a cells, including, but not limited to, CD36. Alternatively or additionally, the second agents may interfere with the binding of IgM to its target. In other embodiments, the second agent may inhibit the production of natural IgG that binds to the LNP, may neutralize such natural IgG, or may prevent IgG from binding to its target. In other examples, the second agent may inhibit the activation of B1b cells or may eliminate B1b cells.

[0353] In some embodiments, the second agent can be an agent that inhibits natural IgM, IgG production by the LNP, and / or activation of B1a and / or B1b cells. Such an agent can be an antagonist of a surface receptor on B1a cells (e.g., CD36 and C5a) or B1b cells, e.g., an antibody or small molecule inhibitor that binds to the surface receptor and prevents its binding to its cognate ligand (e.g., a lipid component such as phosphatidylcholine in certain LNPs).

[0354] In other embodiments, the second agent may be an agent that inhibits LNP-induced activation of platelets and / or the complement system (classical or alternative pathway). Such an agent may be a CD36 antagonist, a TLR antagonist, or an antagonist of any component in the complement cascade. Such an antagonist may be an antagonist antibody specific for one of the targets. In some examples, the antagonist may be a protease inhibitor that targets one or more serine protease components of the complement system. Other CD36 antagonists include, but are not limited to, salvianolic acid or its metabolites (e.g., RA and DSS), 3-cinnamoylindole, 13-pentylberberine, hexarelin, or certain fatty acids such as DHA.

[0355] It should be understood that the present disclosure contemplates the use of one or more of the aforementioned second agents with any of the LNPs provided herein.

[0356] chemical definition Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined as described therein. Further, general principles of organic chemistry, as well as specific functional groups and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 thEdition, John Wiley&Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, and Carruthers, Some Modern Methods of Organic Synthesis, 3. rd Edition, Cambridge University Press, Cambridge, 1987.

[0357] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of an individual enantiomer, diastereomer, or geometric isomer, or in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be isolated from mixtures by methods known in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0358] During the ceremony, [ka] is a single bond to which the stereochemistry of the moiety directly attached is unspecified, [ka] is absent or a single bond, [ka] or [ka] is a single bond or a double bond.

[0359] 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. 19 F 18 Substitution by F, or 12 C 13 C or 14 Compounds having this structure except for substitution by C are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0360] When a range of values ​​is recited, it is intended to encompass each value and sub-range within that range. For example, "C 1-6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6Alkyl is intended to be included.

[0361] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Similarly, the term "heteroaliphatic" refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

[0362] The term "alkyl" refers to the radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms ("C 1-10 In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C 1-9 In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C 1-7 In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C 1-5 In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C 1-3 In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C 1-2 In some embodiments, the alkyl group has 1 carbon atom ("C alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C 2-6 alkyl). C 1-6Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C6) (e.g., n-hexyl). Further examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (e.g., halogen, such as F) ("substituted alkyl"). In certain embodiments, the alkyl group is an unsubstituted C 1-10 Alkyl (e.g., unsubstituted C 1-6 Alkyl, for example, -CH3(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, for example, unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, for example, unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C 1-10 Alkyl (e.g., substituted C 1-6 alkyl, for example, -CF3, Bn).

[0363] The term "haloalkyl" refers to a substituted alkyl group in which one or more hydrogen atoms are independently replaced with a halogen, such as fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms ("C1-3 In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms ("C 1-2 Examples of haloalkyl groups include -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, -CCl3, -CFCl2, -CF2Cl, and the like.

[0364] The term "heteroalkyl" refers to an alkyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within the parent chain (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal position(s). In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 10 carbon atoms and one or more heteroatoms within the parent chain ("heteroC 1-10 In some embodiments, heteroalkyl groups are saturated groups having 1 to 9 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-9 In some embodiments, heteroalkyl groups are saturated groups having 1 to 8 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-8 In some embodiments, heteroalkyl groups are saturated groups having 1 to 7 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-7 In some embodiments, heteroalkyl groups are saturated groups having 1 to 6 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-6 In some embodiments, heteroalkyl groups are saturated groups having 1 to 5 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1-5 In some embodiments, heteroalkyl groups are saturated groups having 1 to 4 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1-4 In some embodiments, heteroalkyl groups are saturated groups having 1 to 3 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1-3In some embodiments, heteroalkyl groups are saturated groups having 1 to 2 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1-2 In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom ("heteroC1 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 2-6 Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an "unsubstituted heteroalkyl") or substituted (a "substituted heteroalkyl") with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC 1-10 In certain embodiments, the heteroalkyl group is a substituted heteroC 1-10 It is alkyl.

[0365] The term "alkenyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C 2-9 In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C 2-3In some embodiments, the alkenyl group has two carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). C 2-4 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, the alkenyl group is an unsubstituted C 2-10 In certain embodiments, the alkenyl group is a substituted C 2-10 Alkenyl groups are those with a C=C double bond where the stereochemistry is not specified (e.g., -CH=CHCH3 or [ka] ) may be an (E)-double bond or a (Z)-double bond.

[0366] The term "heteroalkenyl" refers to an alkenyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within the parent chain (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal position(s). In certain embodiments, a heteroalkenyl group refers to a group having 2 to 10 carbon atoms, at least one double bond, and one or more heteroatoms within the parent chain ("heteroC 2-10In some embodiments, heteroalkenyl groups have 2 to 9 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-9 In some embodiments, heteroalkenyl groups have 2 to 8 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-8 In some embodiments, heteroalkenyl groups have 2 to 7 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-7 In some embodiments, heteroalkenyl groups have 2 to 6 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-6 In some embodiments, heteroalkenyl groups have 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-5 In some embodiments, heteroalkenyl groups have 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-4 In some embodiments, heteroalkenyl groups have 2 to 3 carbon atoms, at least one double bond, and one heteroatom in the parent chain ("heteroC 2-3 In some embodiments, heteroalkenyl groups have 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-6 Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an "unsubstituted heteroalkenyl") or substituted (a "substituted heteroalkenyl") with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC 2-10 In certain embodiments, the heteroalkenyl group is a substituted heteroC 2-10 It is alkenyl.

[0367] The term "alkynyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C 2-10 In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C 2-9 In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C 2-3 In some embodiments, the alkynyl group has two carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). C 2-4 Examples of alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkynyl groups include pentynyl (C5), hexynyl (C6), and the like. Further examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, the alkynyl group is an unsubstituted C 2-10 In certain embodiments, the alkynyl group is a substituted C 2-10 It is alkynyl.

[0368] The term "heteroalkynyl" refers to an alkynyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within the parent chain (i.e., inserted between adjacent carbon atoms) and / or at one or more terminal position(s). In certain embodiments, a heteroalkynyl group refers to a group having 2 to 10 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain ("heteroalkynyl"). 2-10 In some embodiments, heteroalkynyl groups have 2 to 9 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-9 In some embodiments, heteroalkynyl groups have 2 to 8 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-8 In some embodiments, heteroalkynyl groups have 2 to 7 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-7 In some embodiments, heteroalkynyl groups have 2 to 6 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-6 In some embodiments, heteroalkynyl groups have 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-5 In some embodiments, heteroalkynyl groups have 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-4 In some embodiments, heteroalkynyl groups have 2 to 3 carbon atoms, at least one triple bond, and one heteroatom in the parent chain ("heteroC 2-3 In some embodiments, heteroalkynyl groups have 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-6Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an "unsubstituted heteroalkynyl") or substituted (a "substituted heteroalkynyl") with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC 2-10 In certain embodiments, the heteroalkynyl group is a substituted heteroC 2-10 It is alkynyl.

[0369] The term "carbocyclyl" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon group having 3 to 14 ring carbon atoms ("C 3-14 In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms ("C 3-10 In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C 3-8 In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C 3-7 In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C 3-6 In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C 4-6 In some embodiments, the carbocyclyl group has 5 to 6 ring carbon atoms ("C 5-6 In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C 5-10 carbocyclyl). Exemplary C 3-6 Carbocyclyl groups include, without limitation, cyclopropyl (C), cyclopropenyl (C), cyclobutyl (C), cyclobutenyl (C), cyclopentyl (C), cyclopentenyl (C), cyclohexyl (C), cyclohexenyl (C), cyclohexadienyl (C), and the like. 3-8 The carbocyclyl group is not limited to the above-mentioned C 3-6Examples include carbocyclyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C 3-10 The carbocyclyl group is not limited to the above-mentioned C 3-8 Carbocyclyl groups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ) and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., including fused, bridged, or spiro ring systems, such as bicyclic ("bicyclic carbocyclyl") or tricyclic ("tricyclic carbocyclyl") ring systems), and may be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups at the point of attachment on the carbocyclyl ring; in such cases, the number of carbons still designates the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is an unsubstituted C 3-14 In certain embodiments, the carbocyclyl group is a substituted C 3-14 It is a carbocyclyl.

[0370] In some embodiments, a "carbocyclyl" is a monocyclic saturated carbocyclyl group having 3 to 14 ring carbon atoms ("C 3-14 In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms ("C 3-10In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3-8 In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C 3-6 In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C 4-6 In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C 5-6 In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C 5-10 Cycloalkyl). C 5-6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). 3-6 Examples of cycloalkyl groups include the above-mentioned C 5-6 Cycloalkyl groups include cyclopropyl (C3) and cyclobutyl (C4). 3-8 Examples of cycloalkyl groups include the above-mentioned C 3-6 Cycloalkyl groups include cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is an unsubstituted C 3-14 In certain embodiments, the cycloalkyl group is a substituted C 3-14 It is cycloalkyl.

[0371] The terms "heterocyclyl" or "heterocyclic" refer to the radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be at a carbon atom or a nitrogen atom, depending on valency. Heterocyclyl groups can be either monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems, such as bicyclic ("bicyclic heterocyclyl") or tricyclic ("tricyclic heterocyclyl") ring systems), and can be saturated or contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also refers to ring systems in which a heterocyclyl ring, as defined above, is fused to one or more carbocyclyl groups at the point of attachment either on the carbocyclyl or heterocyclyl ring, or Heterocyclyl rings defined by also include ring systems in which the point of attachment on the heterocyclyl ring is fused to one or more aryl or heteroaryl groups; in such cases, the number of ring members still designates the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 14-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 14-membered heterocyclyl.

[0372] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"). In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each possibility represents a separate embodiment of the present invention.

[0373] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepin ... Examples thereof include 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, and 1,2,3,4-tetrahydro-1,6-naphthyridinyl.

[0374] The term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared by the cyclic array) radical having 6 to 14 ring carbon atoms, with no heteroatoms in the aromatic ring system ("C 6-14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl", e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl. In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl" (e.g., anthracyl). "Aryl" also includes ring systems in which the aryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups at the bonding radical or point on the aryl ring; in such cases, the number of carbon atoms still designates the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is an unsubstituted C 6-14 In certain embodiments, the aryl group is a substituted C 6-14 It is aryl.

[0375] The term "heteroaryl" refers to a radical of a 5-14-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π-electrons shared by the cyclic array) having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be at either a carbon atom or a nitrogen atom, depending on valence. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which the heteroaryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups on the heteroaryl ring; in such cases, the number of ring members still designates the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, the point of attachment being either on the aryl or heteroaryl ring; in such cases, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. The point of attachment of polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) can be in either ring, i.e., in a ring containing a heteroatom (e.g., 2-indolyl) or in a ring without a heteroatom (e.g., 5-indolyl).

[0376] In some embodiments, a heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 6-membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heteroaryl"). In some embodiments, the 5- to 6-membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5- to 14-membered heteroaryl.

[0377] Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0378] The term "unsaturated bond" refers to a double or triple bond. The terms "unsaturated" or "partially unsaturated" refer to a moiety that contains at least one double or triple bond. The term "saturated" refers to a moiety that contains no double or triple bonds, i.e., only single bonds.

[0379] The addition of the suffix "ene" to a group indicates that the group is a divalent moiety, for example, alkylene is a divalent moiety of alkyl, alkenylene is a divalent moiety of alkenyl, alkynylene is a divalent moiety of alkynyl, heteroalkylene is a divalent moiety of heteroalkyl, heteroalkenylene is a divalent moiety of heteroalkenyl, heteroalkynylene is a divalent moiety of heteroalkynyl, carbocyclylene is a divalent moiety of carbocyclyl, heterocyclylene is a divalent moiety of heterocyclyl, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0380] A group is optionally substituted unless expressly specified otherwise. The phrase "optionally substituted" refers to substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a group that can be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl, a "substituted" or "unsubstituted" alkenyl, a "substituted" or "unsubstituted" alkynyl, a "substituted" or "unsubstituted" heteroalkyl, a "substituted" or "unsubstituted" heteroalkenyl, a "substituted" or "unsubstituted" heteroalkynyl, a "substituted" or "unsubstituted" carbocyclyl, a "substituted" or "unsubstituted" heterocyclyl, a "substituted" or "unsubstituted" aryl, or a "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means that at least one hydrogen atom present in a group is replaced with an acceptable substituent, e.g., a substituent that, upon substitution, results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group; when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, including any of the substituents described herein that result in the formation of stable compounds. The present invention contemplates any and all combinations that result in stable compounds. For purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety. The present invention is not intended to be limited in any way by the exemplary substituents described herein.

[0381] Exemplary carbon atom substituents include halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OR aa , -ON(R bb )2, -N(R bb)2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SRaa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)(R aa )2, -P(=O)(OR cc )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)(N(R bb )2)2, -OP(=O)(N(R bb )2)2, -NR bb P(=O)(R aa )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(N(R bb )2)2, -P(R cc )2, -P(OR cc )2, -P(R cc )3 + X - , -P(OR cc )3 + X - , -P(R cc )4, -P(OR cc )4, -OP(R cc )2, -OP(R cc )3 + X - , -OP(OR cc )2, -OP(OR cc )3 + X - , -OP(R cc )4, -OP(OR cc )4, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C 3-10Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with X groups - is the counterion, Or, the two geminal hydrogens on the carbon atom can be =O, =S, or =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb , or =NOR cc are exchanged on the basis of R aa Independently, for each case, C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C 3-10 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R aa and the groups taken together form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd is substituted with a group, R bb In each case, independently, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa, -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)(R aa )2, -P(=O)(OR cc )2, -P(=O)(N(R cc )2)2, C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C 3-10 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R bb and the groups taken together form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd substituted with X groups - is the counterion, R cc In each case, independently, hydrogen, C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C3-10 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R cc and the groups taken together form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd is substituted with a group, R dd In each case, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff ) OR ee , -OC(=NR ff )R ee , -OC(=NR ff ) OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff)2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)(OR ee )2, -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hetero C 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, 3-10 membered heterocyclyl, C 6-10 aryl, and 5- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg or substituted with two geminal R dd The substituents can be taken together to form =O or =S, and X - is the counterion, R ee Independently, for each case, C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hetero C 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C3-10 Carbocyclyl, C 6-10 aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg is substituted with a group, R ff In each case, independently, hydrogen, C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hetero C 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, 3-10 membered heterocyclyl, C 6-10 aryl and 5- to 10-membered heteroaryl, or two R ff and the groups taken together form a 3- to 10-membered heterocyclyl or a 5- to 10-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R gg is substituted with a group, R gg In each case, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 Alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6Alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)(OC 1-6 alkyl)2, -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, ...

Claims

1. A compound of formula (I): 【Chemistry 1】 or a salt thereof, wherein r is independently an integer from 42 to 48, inclusive, or a salt thereof; The compounds of formula (I) or salts thereof, wherein the compounds of formula (I) have a purity of 87 mol % or more with respect to the compound of formula (I).

2. r is independently an integer from 44 to 46, inclusive; or 2. The compounds of claim 1, wherein r is 45.

3. the purity is greater than 87 mol %, or the purity being greater than 90 mol %, or the purity being greater than 95 mol %, or the purity being greater than 98 mol %, or the purity being greater than 99 mol %, or the purity being between 90 and 100 mol %, inclusive; or the purity being between 90 and 95 mol%, inclusive; or 3. The plurality of compounds according to claim 1 or 2, wherein the purity is between 95 and 100 mole %, inclusive.

4. A plurality of compounds according to any one of claims 1 to 3, comprising an impurity, the impurity being 【Chemistry 2】 or one or more of salts thereof, where r is independently an integer from 42 to 48, inclusive; the compounds contain less than 13 mol % of impurities in total; or the compounds contain less than 10 mol % of impurities in total; or the compounds contain less than 5 mol % of impurities in total; or the compounds contain less than 2 mol % of impurities in total; or A plurality of compounds, the plurality of compounds comprising less than 1 mole percent impurities in total.

5. the plurality of compounds are free of impurities; or The plurality of compounds is free of the one or more impurities of claim 4; or the compounds are PEG-free; or The plurality of compounds does not include any lipid other than the compound of formula (I); or 4. The method of claim 1, wherein the compound is free of fatty acids.

6. A compound of formula (I): 【Chemistry 3】 or a salt thereof, wherein r is independently an integer from 35 to 55, inclusive; A lipid nanoparticle (LNP) comprising a plurality of compounds or salts thereof, wherein the plurality of compounds of formula (I) have a purity of 87 mol % or more with respect to the compound of formula (I).

7. r is independently an integer from 40 to 50, inclusive; or r is independently an integer from 42 to 48, inclusive; or r is independently an integer from 44 to 46, inclusive; or The LNP of claim 6, wherein r is 45.

8. the purity is greater than 87 mol %, or the purity being greater than 90 mol %, or the purity being greater than 95 mol %, or the purity being greater than 98 mol %, or the purity being greater than 99 mol %, or the purity being between 90 and 100 mol %, inclusive; or the purity being between 90 and 95 mol%, inclusive; or 8. The LNP of claim 6 or 7, wherein the purity is 95 to 100 mol%, inclusive.

9. The plurality of compounds includes an impurity, and the impurity is 【Chemistry 4】 or one or more of salts thereof, where r is independently an integer from 35 to 55, inclusive; the compounds contain less than 13 mol % of impurities in total; or the compounds contain less than 10 mol % of impurities in total; or the compounds contain less than 5 mol % of impurities in total; or the compounds contain less than 2 mol % of impurities in total; or The LNP of any one of claims 6 to 8, wherein the plurality of compounds contain less than 1 mol% impurities in total.

10. the plurality of compounds are free of impurities; or The plurality of compounds is free of the one or more impurities of claim 9; or the compounds are PEG-free; or The plurality of compounds does not include any lipid other than the compound of formula (I); or The LNP of any one of claims 6 to 8, wherein the plurality of compounds does not include a fatty acid.

11. comprising 0.15 to 15 mol % of said compounds of formula (I); or comprising 1-2 mol % of said compounds of formula (I); or The LNP of any one of claims 6 to 10, comprising 1.5 mol% of the compounds of formula (I).

12. The LNP according to any one of claims 6 to 11, further comprising an ionic amino lipid, optionally The ionic amino lipid is 【Chemistry 5】 【Chemistry 6】 and pharma- ceutically acceptable salts thereof, or The ionic amino lipid is 【Chemistry 7】 and pharma- ceutically acceptable salts thereof, or The ionic amino lipid is 【Chemistry 8】 and pharma- ceutically acceptable salts thereof, or The ionic amino lipid has the structure: 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof; and / or The ionic amino lipid is present in a molar ratio of 25-65% relative to other lipids; or The ionic amino lipid is present in a molar ratio of 40% or less relative to other lipids; or LNPs, wherein the ionic amino lipid is present at a molar ratio of 25-40% relative to other lipids.

13. The ionic amino lipid is 【Chemistry 10】 or a pharma- ceutically acceptable salt thereof.

14. The LNP according to any one of claims 6 to 13, further comprising a helper lipid, optionally the helper lipid is DSPC or an analogue thereof; or The helper lipid is DSPC, or The helper lipid has the structure: 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof; or the helper lipid is DOPE, or the helper lipid is oleic acid or an analogue thereof; and / or The helper lipid is present in a molar ratio of 10-40% relative to other lipids, or The helper lipid is present in a molar ratio of 30% relative to other lipids; or LNPs in which the helper lipid is present at a molar ratio of 20% relative to other lipids.

15. The LNP according to any one of claims 6 to 14, further comprising a structured lipid, the structured lipid is a sterol, or the structural lipid is cholesterol; and / or The structured lipids are present in a molar ratio of 30-50% relative to other lipids; or LNPs in which the structured lipids are present at a molar ratio of 38.5% relative to other lipids.

16. the LNPs comprise a plurality of compounds of formula (I), an ionic amino lipid, a helper lipid, and a structural lipid; or The LNP according to any one of claims 6 to 15, comprising a molar ratio of 25 to 65% ionic amino lipid, 0.15 to 15% of a plurality of compounds of formula (I), 30 to 50% structural lipid, and 10 to 40% helper lipid.

17. Compound (A): 【Chemistry 12】 A lipid nanoparticle (LNP) comprising a plurality of compounds of formula (I) according to any one of claims 1 to 5, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol, optionally Compound (A) is present in a molar ratio of 40% or less relative to other lipids; or Compound (A) is present in a molar ratio of 25-35%, including 25% and 35%, relative to other lipids; or Compound (A) is present in a molar ratio of 30% relative to other lipids, and / or The compound of formula (I) is present in a molar ratio of 0.15 to 15% relative to other lipids, or The compound of formula (I) is present in a molar ratio of 1-2% relative to other lipids, or the compound of formula (I) is present in a molar ratio of 1.5% relative to the other lipids; and / or DOPE is present in a molar ratio of 20-40% relative to other lipids, or DOPE is present in a molar ratio of 30% relative to other lipids; and / or Cholesterol is present in a molar ratio of 30-50% relative to other lipids, or LNPs in which cholesterol is present at a molar ratio of 38.5% relative to other lipids.

18. the LNPs comprise up to 40% molar ratio of compound (A), 0.15-15% of a compound of formula (I), 20-40% DOPE, and 30-50% cholesterol; or the LNPs comprise a molar ratio of 25-35% compound (A), 1-2% compound of formula (I), 25-35% DOPE, and 35-42% cholesterol; or the LNPs comprise compound (A): compound of formula (I): DOPE: cholesterol in a molar ratio of 30:30:38.5:1.5; or 18. The LNP of claim 17, wherein the LNP comprises compound (A): compound of formula (I): DOPE: cholesterol in a molar ratio of 40:20:38.5:1.

5.

19. Compound (B): 【Chemistry 13】 A lipid nanoparticle (LNP) comprising a plurality of compounds of formula (I) according to any one of claims 1 to 5, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, optionally Compound (B) is present in a molar ratio of 30-45% relative to other lipids, or Compound (B) is present in a molar ratio of 40% or less relative to other lipids, or Compound (B) is present in a molar ratio of 25-35%, inclusive, relative to other lipids; or Compound (B) is present in a molar ratio of 30% relative to other lipids, and / or The compound of formula (I) is present in a molar ratio of 0.15 to 15% relative to other lipids, or The compound of formula (I) is present in a molar ratio of 1-2% relative to other lipids, or the compound of formula (I) is present in a molar ratio of 1.5% relative to the other lipids; and / or DSPC is present in a molar ratio of 20-40% relative to other lipids, or DSPC is present in a molar ratio of 30% relative to other lipids; and / or Cholesterol is present in a molar ratio of 30-50% relative to other lipids, or LNPs in which cholesterol is present at a molar ratio of 38.5% relative to other lipids.

20. the LNPs comprise up to 40% molar ratio of compound (B), 0.15-15% of a compound of formula (I), 20-40% DSPC, and 30-50% cholesterol; or the LNPs comprise a molar ratio of 25-35% compound (B), 1-2% compound of formula (I), 25-35% DSPC, and 35-42% cholesterol; or Compound (B): comprising the compound of formula (I):DSPC:cholesterol in a molar ratio of 30:30:38.5:1.5; or The LNP of claim 19, comprising compound (B): compound of formula (I): DSPC: cholesterol in a molar ratio of 40:20:38.5:1.

5.

21. Compound (C): 【Chemistry 14】 A lipid nanoparticle (LNP) comprising a plurality of compounds of formula (I) according to any one of claims 1 to 5, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, optionally Compound (C) is present in a molar ratio of 30-45% relative to other lipids, or Compound (C) is present in a molar ratio of 40% or less relative to other lipids; or Compound (C) is present in a molar ratio of 25-35%, including 25% and 35%, relative to other lipids; or Compound (C) is present in a molar ratio of 30% relative to other lipids, and / or The compound of formula (I) is present in a molar ratio of 0.15 to 15% relative to other lipids, or The compound of formula (I) is present in a molar ratio of 1-2% relative to other lipids, or the compound of formula (I) is present in a molar ratio of 1.5% relative to the other lipids; and / or DSPC is present in a molar ratio of 20-40% relative to other lipids, or DSPC is present in a molar ratio of 30% relative to other lipids; and / or Cholesterol is present in a molar ratio of 30-50% relative to other lipids, or LNPs in which cholesterol is present at a molar ratio of 38.5% relative to other lipids.

22. the LNPs comprise up to 40% molar ratio of compound (C), 0.15-15% of a compound of formula (I), 20-40% DSPC, and 30-50% cholesterol; or the LNPs comprise a molar ratio of 25-35% compound (C), 1-2% compound of formula (I), 25-35% DSPC, and 35-42% cholesterol; or the LNPs comprise compound (C): compound of formula (I): DSPC: cholesterol in a molar ratio of 30:30:38.5:1.5; or The LNP of claim 21, wherein the LNP comprises compound (C): compound of formula (I): DSPC: cholesterol in a molar ratio of 40:20:38.5:1.

5.

23. The LNP of any one of claims 6 to 22, further comprising a therapeutic agent, the therapeutic agent is a nucleic acid; or the therapeutic agent is RNA; or the therapeutic agent is an mRNA; and / or an N / P ratio between 2.0 and 5.0, inclusive; or The N / P ratio is between 2.5 and 3.5, or LNP with an N / P ratio of 3.

0.

24. 24. The LNP of claim 23, further comprising an agent that inhibits an immune response in a subject, optionally the LNPs have reduced IgM binding compared to LNPs comprising a plurality of compounds of formula (I) having a purity of less than 87 mole %; or the LNPs have increased ApoE binding compared to LNPs comprising a plurality of compounds of formula (I) having a purity of less than 87 mole %; or The LNPs are less susceptible to accelerated blood clearance (ABC) compared to LNPs comprising a plurality of compounds of formula (I) having a purity of less than 87 mol%.

25. A pharmaceutical composition comprising a plurality of compounds according to any one of claims 1 to 5, or an LNP according to any one of claims 6 to 24.

26. A method for delivering a therapeutic agent to a subject, the method comprising administering to the subject an LNP according to any one of claims 6 to 24, wherein the LNP encapsulates the therapeutic agent.

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