Lipid nanoparticle compositions and methods of using same

Abstract

Lipid nanoparticle compositions and methods of using the same are provided for treating or preventing diseases or disorders.

Description

Attorney Docket No.133279-5010-PC LIPID NANOPARTICLE COMPOSITIONS AND METHODS OF USING SAME FIELD

[0001] The disclosure relates generally to ionizable lipids and lipid nanoparticle compositions and methods of making and using the same, including for nucleic acid delivery to cells and tissues. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 578,598, filed on August 24, 2023, the entire content of which are hereby incorporated herein by reference in its entirety. BACKGROUND

[0003] The delivery of a therapeutic compound, such as a nucleic acid, to cells and tissues of a subject can be impeded by limited ability of the compound to reach and be internalized by targeted cells and tissues. Delivery of such compounds can be conducted using lipid nanoparticles (LNPs) comprising an ionizable lipid. When ionizable lipids are used to deliver therapeutic compounds such as nucleic acid by inclusion in LNPs, these compounds are protonated or ionized at acidic endosomal pH to become amphiphilic and destabilize the endosomal membrane for endosome escape. Ionizable lipids that produce stable LNPs and that are effective for delivery of compounds such as nucleic acids to various tissues are needed. SUMMARY

[0004] In one aspect, the disclosure provides lipid nanoparticle compositions and methods of using the same. In some embodiments, the disclosure provides an ionizable lipid comprising a moiety of Formula (1): Formula (1) wherein in Formula (1): Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10 alkylene;Attorney Docket No.133279-5010-PC Het is substituted or unsubstituted heterocycle; Cyc is a substituted or unsubstituted cyclic moiety; L1and L2are linking moieties; and R is substituted or unsubstituted C1-20alkyl or substituted or unsubstituted C1-20alkenyl. In some embodiments, the disclosure provides an ionizable lipid of Formula (2):wherein in Formula (2): Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-10alkylene, L1and L2are linking moieties; R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-20alkyl, and substituted or unsubstituted C1-20alkenyl; and a and b are each independently an integer from 1 to 3. In some embodiments, the disclosure provides a lipid nanoparticle composition, comprising an ionizable lipid disclosed herein, and at least one of a phospholipid, a PEGylated lipid, and a sterol. In some embodiments, the disclosure provides a pharmaceutical composition for treating a disorder in a subject in need thereof comprising a lipid nanoparticle composition described herein. In some embodiments, the disclosure provides a method for treating a disorder in a subject in need thereof comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disclosure provides a method of preparing a lipid nanoparticle composition described herein comprising mixing a lipid mixture and an aqueous solution to provide the lipid nanoparticle composition, wherein the lipid mixture comprises an organic solvent, an ionizable lipid, and at least one of a phospholipid, a PEGylated lipid, and a sterol, and the aqueous solution comprises a nucleic acid.Attorney Docket No.133279-5010-PC BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Fig.1 shows eGFP tissue quantitation following single dose of LNP containing MAPC- 100010.

[0006] Fig.2 shows liver eGFP quantitation following a single dose of lipid nanoparticle formulations consisting of varying ionizable lipid compositions. DETAILED DESCRIPTION

[0007] The disclosure relates to ionizable lipids, lipid nanoparticle (LNP) compositions, pharmaceutical compositions, and methods of preparing and using the same. The ionizable lipids as components of LNPs support delivery of compounds such as nucleic acids to cells and tissues. The LNPs of this disclosure are useful for delivery of nucleic acids such as mRNA, oligonucleotides, and siRNA, to various organs and tissues, and in connection with methods for treating disease, including but not limited to proliferative, inflammatory, and genetic diseases. The ionizable lipids disclosed herein further provide enhanced encapsulation efficiency of the compound (e.g., nucleic acids).

[0008] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0009] As used herein, the term “a”, “an”, or “the” generally is construed to cover both the singular and the plural forms.

[0010] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that compositions, amounts, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like,Attorney Docket No.133279-5010-PC and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate,” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0011] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., (C1-10)alkyl or C1-10alkyl). Whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range, e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition is also intended to cover the occurrence of the term “alkyl” where no numerical range is specifically designated. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl. The alkyl moiety may be attached to the rest of the molecule by a single bond, such as for example, methyl (Me), ethyl (Et), n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t- butyl) and 3-methylhexyl. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of substituents which are independently heteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -S(O)tRa- (where t is 1 or 2), -OC(O)-Ra, -N(Ra)2, - C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0012] “Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.Attorney Docket No.133279-5010-PC

[0013] “Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.

[0014] “Alkylheterocycloalkyl” refers to an -(alkyl) heterocyclyl radical where alkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heterocycloalkyl and alkyl respectively.

[0015] An “alkene” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.

[0016] “Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., (C2-10)alkenyl or C2-10 alkenyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkenyl moiety may be attached to the rest of the molecule by a single bond, such as for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl and penta-1,4-dienyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, - N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0017] “Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical where alkenyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkenyl and cycloalkyl respectively.Attorney Docket No.133279-5010-PC

[0018] “Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (i.e., (C2-10)alkynyl or C2-10 alkynyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkynyl may be attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl and hexynyl. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, - N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0019] “Alkynyl-cycloalkyl” refers to an -(alkynyl)cycloalkyl radical where alkynyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkynyl and cycloalkyl respectively.

[0020] “Carboxaldehyde” refers to a -(C=O)H radical.

[0021] “Carboxyl” refers to a -(C=O)OH radical.

[0022] “Cyano” refers to a -CN radical.

[0023] “Cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e. (C3-10)cycloalkyl or C3-10 cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range - e.g., “3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are notAttorney Docket No.133279-5010-PC limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, - N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0024] “Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical where cycloalkyl and alkenyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and alkenyl, respectively.

[0025] “Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl)heterocycloalkyl radical where cycloalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heterocycloalkyl, respectively.

[0026] “Cycloalkyl-heteroaryl” refers to a -(cycloalkyl)heteroaryl radical where cycloalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heteroaryl, respectively.

[0027] The term “alkoxy” refers to the group -O-alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers to alkoxy groups containing one to six carbons.

[0028] The term “substituted alkoxy” refers to alkoxy wherein the alkyl constituent is substituted (i.e., -O-(substituted alkyl)). Unless stated otherwise specifically in the specification, the alkylAttorney Docket No.133279-5010-PC moiety of an alkoxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, - C(O)N(Ra)2, -N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, - N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0029] The term “alkoxycarbonyl” refers to a group of the formula (alkoxy)(C=O)- attached through the carbonyl carbon wherein the alkoxy group has the indicated number of carbon atoms. Thus a (C1-6)alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbon atoms attached through its oxygen to a carbonyl linker. “Lower alkoxycarbonyl” refers to an alkoxycarbonyl group wherein the alkoxy group is a lower alkoxy group.

[0030] The term “substituted alkoxycarbonyl” refers to the group (substituted alkyl)-O-C(O)- wherein the group is attached to the parent structure through the carbonyl functionality. Unless stated otherwise specifically in the specification, the alkyl moiety of an alkoxycarbonyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, - OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0031] “Acyl” refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-, (heteroalkyl)- C(O)- and (heterocycloalkyl)-C(O)-, wherein the group is attached to the parent structure through the carbonyl functionality. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless statedAttorney Docket No.133279-5010-PC otherwise specifically in the specification, the alkyl, aryl or heteroaryl moiety of the acyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, - OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0032] “Acyloxy” refers to a R(C=O)O- radical wherein R is alkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl, which are as described herein. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless stated otherwise specifically in the specification, the R of an acyloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, - OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0033] “Acylsulfonamide” refers a -S(O)2-N(Ra)-C(=O)- radical, where Rais hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. Unless stated otherwise specifically in the specification, an acylsulfonamide group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -Attorney Docket No.133279-5010-PC N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl

[0034] “Amino” or “amine” refers to a -N(Ra)2 radical group, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification. When a -N(Ra)2 group has two Rasubstituents other than hydrogen, they can be combined with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -N(Ra)2is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless stated otherwise specifically in the specification, an amino group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, - N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0035] The term “substituted amino” also refers to N-oxides of the groups -NHRa, and NRaRaeach as described above. N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid.

[0036] “Amide” or “amido” refers to a chemical moiety with formula -C(O)N(R)2 or -NHC(O)R, where R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted. The R2 of -N(R)2 of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7- membered ring. Unless stated otherwise specifically in the specification, an amido group isAttorney Docket No.133279-5010-PC optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. An amide may be an amino acid or a peptide molecule attached to a compound disclosed herein, thereby forming a prodrug. The procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety.

[0037] “Aromatic” or “aryl” or “Ar” refers to an aromatic radical with six to ten ring atoms (e.g., C6-C10 aromatic or C6-C10 aryl) which has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in “-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Whenever it appears herein, a numerical range such as “6 to 10” refers to each integer in the given range; e.g., “6 to 10 ring atoms” means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Unless stated otherwise specifically in the specification, an aryl moiety is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, - OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. It is understood that a substituent RAttorney Docket No.133279-5010-PC attached to an aromatic ring at an unspecified position (e.g., ) includes one or more, and up to the maximum number of possible substituents.

[0038] The term “aryloxy” refers to the group -O-aryl.

[0039] The term “substituted aryloxy” refers to aryloxy wherein the aryl substituent is substituted (i.e., -O-(substituted aryl)). Unless stated otherwise specifically in the specification, the aryl moiety of an aryloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, - C(O)N(Ra)2, -N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, - N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0040] “Aralkyl” or “arylalkyl” refers to an (aryl)alkyl-radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.

[0041] “Ester” refers to a chemical radical of formula -COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The procedures and specific groups to make esters are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety. Unless stated otherwise specifically in the specification, an ester group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(O)- Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -Attorney Docket No.133279-5010-PC N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0042] “Fluoroalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, 2,2,2- trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of the fluoroalkyl radical may be optionally substituted as defined above for an alkyl group.

[0043] “Halo,” “halide,” or, alternatively, “halogen” is intended to mean fluoro, chloro, bromo or iodo. The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl,” and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.

[0044] “Heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” refer to optionally substituted alkyl, alkenyl and alkynyl radicals and which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, or combinations thereof. A numerical range may be given, e.g., C1-C4 heteroalkyl which refers to the chain length in total, which in this example is 4 atoms long. A heteroalkyl group may be substituted with one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, - C(O)N(Ra)2, -N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, - N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0045] “Heteroalkylaryl” refers to an -(heteroalkyl)aryl radical where heteroalkyl and aryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and aryl, respectively.Attorney Docket No.133279-5010-PC

[0046] “Heteroalkylheteroaryl” refers to an -(heteroalkyl)heteroaryl radical where heteroalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and heteroaryl, respectively.

[0047] “Heteroalkylheterocycloalkyl” refers to an -(heteroalkyl)heterocycloalkyl radical where heteroalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and heterocycloalkyl, respectively.

[0048] “Heteroalkylcycloalkyl” refers to an -(heteroalkyl)cycloalkyl radical where heteroalkyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and cycloalkyl, respectively.

[0049] “Heteroaryl” or “heteroaromatic” or “HetAr” or “Het” refers to a 5- to 18-membered aromatic radical (e.g., C5-C13 heteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system. Whenever it appears herein, a numerical range such as “5 to 18” refers to each integer in the given range - e.g., “5 to 18 ring atoms” means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. Bivalent radicals derived from univalent heteroaryl radicals whose names end in “-yl” by removal of one hydrogen atom from the atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical - e.g., a pyridyl group with two points of attachment is a pyridylidene. A N-containing “heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non-fused. The heteroatom(s) in the heteroaryl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl may be attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl,Attorney Docket No.133279-5010-PC cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H- benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10- hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6- naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a- octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4- d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8- tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -ORa, -SRa, -OC(O)- Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, - N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0050] Substituted heteroaryl also includes ring systems substituted with one or more oxide (-O-) substituents, such as, for example, pyridinyl N-oxides.Attorney Docket No.133279-5010-PC

[0051] “Heteroarylalkyl” refers to a moiety having an aryl moiety, as described herein, connected to an alkylene moiety, as described herein, wherein the connection to the remainder of the molecule is through the alkylene group.

[0052] “Heterocycloalkyl” or “heterocyclyl” refer to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Whenever it appears herein, a numerical range such as “3 to 18” refers to each integer in the given range - e.g., “3 to 18 ring atoms” means that the heterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc., up to and including 18 ring atoms. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocycloalkyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. The heterocycloalkyl may be attached to the rest of the molecule through any atom of the ring(s). Examples of such heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo- thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocycloalkyl moiety is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, - ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, - N(Ra)C(O)ORa, -N(Ra)C(O)Ra, -N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tN(Ra)2 (where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.Attorney Docket No.133279-5010-PC

[0053] “Heterocycloalkyl” also includes bicyclic ring systems wherein one non-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2 carbon atoms in addition to 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, as well as combinations comprising at least one of the foregoing heteroatoms; and the other ring, usually with 3 to 7 ring atoms, optionally contains 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen and is not aromatic.

[0054] “Nitro” refers to the -NO2radical.

[0055] “Oxa” refers to the -O- radical.

[0056] “Oxo” refers to the =O radical.

[0057] “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space - i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon can be specified by either (R) or (S). Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S). The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.Attorney Docket No.133279-5010-PC

[0058] “Moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

[0059] “Substituted” means that the referenced group may have attached one or more additional groups, radicals or moieties individually and independently selected from, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivatives thereof. The substituents themselves may be substituted, for example, a cycloalkyl substituent may itself have a halide substituent at one or more of its ring carbons. The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.

[0060] “Sulfanyl” refers to groups that include -S-(optionally substituted alkyl), -S-(optionally substituted aryl), -S-(optionally substituted heteroaryl) and -S-(optionally substituted heterocycloalkyl).

[0061] “Sulfinyl” refers to groups that include -S(O)-H, -S(O)-(optionally substituted alkyl), -S(O)-(optionally substituted amino), -S(O)-(optionally substituted aryl), -S(O)- (optionally substituted heteroaryl) and -S(O)-(optionally substituted heterocycloalkyl).

[0062] “Sulfonyl” refers to groups that include -S(O2)-H, -S(O2)-(optionally substituted alkyl), -S(O2)-(optionally substituted amino), -S(O2)-(optionally substituted aryl), -S(O2)- (optionally substituted heteroaryl), and -S(O2)-(optionally substituted heterocycloalkyl).

[0063] “Sulfonamidyl” or “sulfonamido” refers to a -S(=O)2-NRR radical, where each R is selected independently from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The R groups in -NRR of the -S(=O)2-NRR radical may be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7-membered ring. A sulfonamido group is optionally substituted by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl, respectively.Attorney Docket No.133279-5010-PC

[0064] “Sulfoxyl” refers to a -S(=O)2OH radical. “Sulfonate” refers to a -S(=O)2-OR radical, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). A sulfonate group is optionally substituted on R by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl, respectively.

[0065] The term “hydrophobic lipid” refers to compounds having a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N—N- dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0066] As used herein, “lipid conjugate” or “polymer-conjugated lipid” means a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEGylated lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEGylated lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In some embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used. As used herein, a “PEGylated lipid,” “PEG lipid conjugate,” “PEG- modified lipids,” or “PEG lipid” refers to a lipid comprising a polyethylene glycol component.

[0067] As used herein, “neutral lipid” means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0068] “Amphipathic lipid” means the material in which the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, andAttorney Docket No.133279-5010-PC other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0069] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

[0070] “Anionic lipid” means a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0071] The terms “cationic lipid” and “amino lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). The cationicAttorney Docket No.133279-5010-PC lipid is typically protonated (i.e., positively charged) at a pH below the pKaof the cationic lipid and is substantially neutral at a pH above the pKa. The cationic lipids of the invention may also be termed titratable cationic lipids.

[0072] As used herein, “lipid nanoparticle” means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA or oligonucleotide) to a target site of interest (e.g., cell, tissue, organ, and the like). In preferred embodiments, the lipid nanoparticle is a nucleic acid- lipid nanoparticle, which is typically formed from an ionizable lipid, a neutral lipid (e.g., a phospholipid), a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA or oligonucleotide) may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0073] “Lipid encapsulated” can mean a lipid nanoparticle composition that provides a therapeutic nucleic acid such as an mRNA or oligonucleotide with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle.

[0074] The term “treatment” refers to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. For example, a composition, method, or system of the present disclosure may be administered as a prophylactic treatment to a subject that has a predisposition for a given condition (e.g., arthritis). “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, canine, feline, or equine, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms.

[0075] “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine. It is understood thatAttorney Docket No.133279-5010-PC compositions and methods of the present disclosure are applicable to treat all mammals, including, but not limited to human, canine, feline, equine, and bovine subjects.

[0076] By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Nucleotides generally comprise a base, sugar, and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides. By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine, and uracil.

[0077] As used herein complementary nucleotide bases are a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or with uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acid that hybridize (i.e., join by hydrogen bonding) with each other. By “complementary” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by traditional Watson-Crick or by other non-traditional modes of binding.

[0078] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid includes single stranded nucleic acid as well as double stranded nucleic acids. Nucleic acids include mRNA (e.g., encoding for a protein of interest), DNA expression vectors encoding for an mRNA, cDNA, oligonucleotides (e.g., antisense oligonucleotides and splice switching oligonucleotides), morpholino oligonucleotides, siRNA, and non-coding RNAs such as but not limited to miRNAs, lncRNA, shRNA, gRNA, aptamers, and toll-like receptor agonists and antagonists. Such nucleic acids may include chemical modifications as known in the art. In exemplary embodiments, the nucleic acid is an oligonucleotide, which may be a gapmer comprising a central block of DNA nucleotides for recruitment of RNase H. Such oligonucleotide compounds may be in the range of 10 to 24 nucleotides in length, such as 10 to 18 nucleotides in length. Oligonucleotides may comprise one or more 2´ modifications such as a 2´ to 4´ bridge (e.g., locked nucleic acid or cEt), 2´-OMe, 2´-MOE, and 2´-F. Oligonucleotides may comprise one or more backbone modifications such as but not limited to phosphorothioate and phosphorodithioate. Exemplary oligonucleotides andAttorney Docket No.133279-5010-PC chemical modifications to oligonucleotides are described in WO 2022 / 226377 and WO 2023 / 034537, which are hereby incorporated by reference in their entireties.

[0079] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0080] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0081] As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a nanoparticle composition including the therapeutic and / or prophylactic agent to the subject (e.g., by an intravenous, intramuscular, intradermal, subcutaneous, intraarticular, or intradiscal route). Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0082] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific doseAttorney Docket No.133279-5010-PC will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0083] Any of the compositions disclosed herein can be administered to a non-human subject, such as a laboratory or farm animal. Non-limiting examples of a non-human subject include laboratory or research animals, pets, wild or domestic animals, farm animals, etc., e.g., a dog, a goat, a guinea pig, a hamster, a mouse, a pig, a non-human primate (e.g., a gorilla, an ape, an orangutan, a lemur, a baboon, etc.), a rat, a sheep, a horse, a cow, or the like. As used herein, a “lipid component” is that component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more cationic / ionizable, PEGylated, structural, or other lipids, such as phospholipids.

[0084] In one aspect, the disclosure provides an ionizable lipid.

[0085] In some embodiments, the ionizable lipid comprises a moiety of Formula (1): Formula (1) wherein in Formula (1): Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10alkylene; Het is substituted or unsubstituted heteroalkylene or substituted or unsubstituted heterocycle; Cyc is a substituted or unsubstituted cyclic moiety; L1and L2are linking moieties; and R is substituted or unsubstituted C1-20 alkyl or substituted or unsubstituted C1-20 alkenyl.

[0086] In some embodiments, the ionizable lipid is of Formula (10): Formula (10)Attorney Docket No.133279-5010-PC wherein in Formula (10): Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10 alkylene; Het is independently selected at each occurrence from substituted or unsubstituted heteroalkylene; Cyc is independently selected at each occurrence from a substituted or unsubstituted cyclic moiety; L1and L2are independently selected at each occurrence from a linking moiety; and R is independently selected at each occurrence from substituted or unsubstituted C10-20 alkyl or substituted or unsubstituted C10-20alkenyl.

[0087] In some embodiments, the ionizable lipid comprises one or more moieties of Formula (1) (e.g., 1, 2, 3, 4, 5, 6 moieties of Formula (1)). In some embodiments, the ionizable lipid comprises 1 moiety of Formula (1). In some embodiments, the ionizable lipid comprises 2 moieties of Formula (1).

[0088] In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10 alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-9alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-8 alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-7alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-6 alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-5 alkylene. In some embodiments, Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-4 alkylene.

[0089] In some embodiments, Alk1is independently selected at each occurrence from C1-10 alkylene. In In some embodiments, Alk1is independently selected at each occurrence from C1-4alkylene. In some embodiments, Alk1is C1 alkylene at one occurrence. In some embodiments, Alk1is C1 alkylene at each occurrence. In some embodiments, Alk1is C2 alkylene at one occurrence. In some embodiments, Alk1is C2alkylene at each occurrence. In someAttorney Docket No.133279-5010-PC embodiments, Alk1is C3alkylene at one occurrence. In some embodiments, Alk1is C3alkylene at each occurrence. In some embodiments, Alk1is C4 alkylene at one occurrence. In some embodiments, Alk1is C4 alkylene at each occurrence.

[0090] In some embodiments, Alk2is independently selected at each occurrence from C1-10alkylene. In some embodiments, Alk2is independently selected at each occurrence from C1-4 alkylene. In some embodiments, Alk2is C1 alkylene at one occurrence. In some embodiments, Alk2is C1alkylene at one occurrence. In some embodiments, Alk2is C2alkylene at one occurrence. In some embodiments, Alk2is C2alkylene at one occurrence. In some embodiments, Alk2is C3 alkylene at one occurrence. In some embodiments, Alk2is C3 alkylene at one occurrence. In some embodiments, Alk2is C4alkylene at one occurrence. In some embodiments, Alk2is C4alkylene at one occurrence.

[0091] In some embodiments, Alk1and Alk2are C1 alkylene. In some embodiments, Alk1and Alk2are C2 alkylene. In some embodiments, Alk1and Alk2are C3 alkylene. In some embodiments, Alk1and Alk2are C4alkylene.

[0092] In some embodiments, Het is independently selected at each occurrence from substituted or unsubstituted heteroalkylene. In some embodiments, Het is independently selected at each occurrence from an aromatic or saturated, optionally substituted heterocycle. In some embodiments, Het is a saturated cyclic moiety.

[0093] In some embodiments, Het comprises a 3- to 7-membered ring, for example, a 3- membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, or a 7-membered ring. In some embodiments, Het comprises a 6-membered ring. In some embodiments, the 6- membered ring is 1,2-substituted, 1,3-substituted, 1,4-substituted, 2,3-substituted, 2,4- substituted, 2,5-substituted, or 2,6-substituted.

[0094] In some embodiments, Het comprises one or more of oxygen, sulfur, and nitrogen. In some embodiments, Het comprises one or more nitrogen (e.g., 1 or 2 nitrogen). In some embodiments, Het is selected at each occurrence from a saturated nitrogen-containing heterocycle. Examples of a suitable saturated nitrogen-containing heterocycle include, but are not limited to, ethylenimine, azetidine, hexahydropyrimidine, 1,3-diazetidine, piperidine, piperazine, pyrrolidine, and morpholine. In some embodiments, Het is independently selected atAttorney Docket No.133279-5010-PC each occurrence from 1,2-substituted piperidine, 1,3-substituted piperidine, 1,4-substituted piperidine, 2,3-substituted piperidine, 2,4-substituted piperidine, 2,5-substituted piperidine, 2,6- substituted piperidine, 1,2-substituted piperazine, 1,3-substituted piperazine, 1,4-substituted piperazine, 2,3-substituted piperazine, 2,5-substituted piperazine, 2,6-substituted piperazine, 1,2- substituted pyrrolidine, 1,3-substituted pyrrolidine, 2,3-substituted pyrrolidine, 2,4-substituted pyrrolidine, and 2,5-substituted pyrrolidine. In some embodiments, Het is 1,4-substituted piperidine at one occurrence. In some embodiments, Het is 1,4-substituted piperidine.

[0095] In some embodiments, the ionizable lipid comprises a moiety of Formula (100):wherein in Formula 100: Cyc is a substituted or unsubstituted cyclic moiety; L2is a linking moiety; R is substituted or unsubstituted C10-20 alkyl or substituted or unsubstituted C10-20 alkenyl; and a, b, and c are integers from 1 to 5.

[0096] In some embodiments, the ionizable lipid comprises one or more moieties of Formula (100) (e.g., 1, 2, 3, 4, 5, 6 moieties of Formula (100)). In some embodiments, the ionizable lipid comprises 1 moiety of Formula (100). In some embodiments, the ionizable lipid comprises 2 moieties of Formula (100).

[0097] In some embodiments, the ionizable lipid is of Formula (101):wherein in Formula 101:Attorney Docket No.133279-5010-PC Cyc is independent selected at each occurrence from a substituted or unsubstituted cyclic moiety; L2is independently selected at each occurrence from a linking moiety; R is independently selected at each occurrence from substituted or unsubstituted C10-20alkyl or a substituted or unsubstituted C10-20 alkenyl; and a, b, and c are integers independently selected at each occurrence from 1 to 5.

[0098] In some embodiments, Cyc is independently selected at each occurrence from a substituted or unsubstituted cyclic moiety. In some embodiments, Cyc comprises a 3- to 7- membered ring, for example, a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6- membered ring, or a 7-membered ring. In some embodiments, Cyc comprises a 5-membered ring. In some embodiments, the 5-membered ring is 1,2-substituted, 1,3-substituted, 2,3- substituted, 2,4-substituted, or 2,5-substituted. In some embodiments, Cyc comprises a 6- membered ring. In some embodiments, the 6-membered ring is 1,2-substituted, 1,3-substituted, 1,4-substituted, 2,3-substituted, 2,4-substituted, 2,5-substituted, or 2,6-substituted.

[0099] In some embodiments, Cyc comprises substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted bicycloalkylene, substituted or unsubstituted bicycloheteroalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0100] In some embodiments, Cyc is selected from, but not limited to, 1,2-substituted cyclopropane, 1,2-substituted cyclobutane, 1,3-substituted cyclobutane, 1,2-substituted cyclopentane, 1,3-substituted cyclopentane, 1,2-substituted cyclohexane, 1,3-substituted cyclohexane, 1,4-substituted cyclohexane, 1,2-substiuted heptane, 1,3-substituted heptane, and 1,4-substituted heptane.

[0101] In some embodiments, Cyc is bicycloalkylene. Examples of suitable bicycloalkylene include, but is not limited to, 1,2-substituted bicyclo[1.1.1]pentane, 1,3-substituted bicyclo[1.1.1]pentane, 1,2-substituted bicyclo[2.1.1]hexane, 1,3-substituted bicyclo[2.1.1]hexane, 1,4-substituted bicyclo[2.1.1]hexane, 1,5-substituted bicyclo[2.1.1]hexane, 1,2-substituted bicyclo[2.2.1]heptane, 1,3-substituted bicyclo[2.2.1]heptane, 1,4-substituted bicyclo[2.2.1]heptane, 1,7-substituted bicyclo[2.2.1]heptane, 2,3-substituted bicyclo[2.2.1]heptane, 2,5-substituted bicyclo[2.2.1]heptane, 2.6-substitutedAttorney Docket No.133279-5010-PC bicyclo[2.2.1]heptane, and 2,7-substituted bicyclo[2.2.1]heptane. In some embodiments, Cyc is 1,3-substituted bicyclo[1.1.1]pentane at one occurrence. In some embodiments, Cyc is 1,3- substituted bicyclo[1.1.1]pentane at each occurrence.

[0102] In some embodiments, Cyc is arylene. Examples of suitable arylene include, but are not limited to, 1,2-substituted benzene, 1,3-substituted benzene, and 1,4-substituted benzene. In some embodiments, Cyc is 1,4-substituted benzene at one occurrence. In some embodiments, Cyc is 1,4-substituted benzene at each occurrence.

[0103] In some embodiments, L1and L2are independently selected at each occurrence from a linking moiety. In some embodiments, L1and L2independently at each occurrence comprise one or more moieties selected from C1-5alkylene, –O–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, – OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–.

[0104] In some embodiments, L1independently at each occurrence comprises one or more moieties selected from C1-5 alkylene, –O–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, – OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–. In some embodiments, L1comprises a moiety of C1-5 alkylene. In some embodiments, L1comprises a moiety of –C(O)O–. In some embodiments, L1comprises a moiety of –OC(O)–. In some embodiments, L1comprises a moiety of –OC(O)O–. In some embodiments, L1comprises a moiety of –OC(O)NH–. In some embodiments, L1comprises a moiety of –NHC(O)O–. In some embodiments, L1comprises a moiety of –C(O)NH–. In some embodiments, L1comprises a moiety of –NHC(O)–.

[0105] In some embodiments, L1is independently at each occurrence selected from –CH2– C(O)O–, –CH2–OC(O)–, –CH2–OC(O)O–, –CH2–OC(O)NH–, –CH2–NHC(O)O–, –CH2– C(O)NH–, –CH2–NHC(O)–, –C(O)O–CH2–, –OC(O)–CH2–, –OC(O)O–CH2–, –OC(O)NH– CH2–, –NHC(O)O–CH2–, –C(O)NH–CH2–, and –NHC(O)–CH2–. In some embodiments, L1is – CH2–C(O)O–. In some embodiments, L1is –CH2–OC(O)–. In some embodiments, L1is –CH2– OC(O)O–. In some embodiments, L1is –CH2–OC(O)NH–. In some embodiments, L1is –CH2– NHC(O)O–. In some embodiments, L1is –CH2–C(O)NH–. In some embodiments, L1is –CH2– NHC(O)–. In some embodiments, L1is –C(O)O–CH2–. In some embodiments, L1is –OC(O)– CH2–. In some embodiments, L1is –OC(O)O–CH2–. In some embodiments, L1is –OC(O)NH– CH2–. In some embodiments, L1is –NHC(O)O–CH2–. In some embodiments, L1is –C(O)NH– CH2–. In some embodiments, L1is –NHC(O)–CH2–. In some embodiments, L1is –CH2–Attorney Docket No.133279-5010-PC C(O)O–, –CH2–OC(O)–, –OC(O)–CH2–, or –C(O)O–CH2– at one occurrence. In some embodiments, L1is –CH2–C(O)O–, –CH2–OC(O)–, –OC(O)–CH2–, or –C(O)O–CH2– at each occurrence.

[0106] In some embodiments, L2is independently at each occurrence selected from –C(O)O–, – OC(O)–, –OC(O)O–, –OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–. In some embodiments, L2is –CH2–C(O)O–. In some embodiments, L2is –CH2–OC(O)–. In some embodiments, L2is –CH2–OC(O)O–. In some embodiments, L2is –CH2–OC(O)NH–. In some embodiments, L2is –CH2–NHC(O)O–. In some embodiments, L2is –CH2–C(O)NH–. In some embodiments, L2is –CH2–NHC(O)–. In some embodiments, L2is –C(O)O–CH2–. In some embodiments, L2is –OC(O)–CH2–. In some embodiments, L2is –OC(O)O–CH2–. In some embodiments, L2is –OC(O)NH–CH2–. In some embodiments, L2is –NHC(O)O–CH2–. In some embodiments, L2is –C(O)NH–CH2–. In some embodiments, L2is –NHC(O)–CH2–. In some embodiments, L2is –OC(O)O– at one occurrence. In some embodiments, L2is –OC(O)O– at each occurrence. In some embodiments, L2is –OC(O)NH– or –NHC(O)O– at one occurrence. In some embodiments, L2is –OC(O)NH– or –NHC(O)O– at each occurrence.

[0107] In some embodiments, R is independently selected at each occurrence from substituted or unsubstituted C10-20alkyl or substituted or unsubstituted C10-20alkenyl.

[0108] In some embodiments, R is independently selected at each occurrence from substituted or unsubstituted C10-20 alkyl. In some embodiments, R is independently selected at each occurrence from C14-20alkyl, for example, C14alkyl, C15alkyl, C16alkyl, C17alkyl, C18alkyl, C19alkyl, or C20alkyl.

[0109] In some embodiments, R is independently selected at each occurrence from substituted or unsubstituted C10-20alkenyl. In some embodiments, R is independently selected at each occurrence from C14-20alkenyl, for example, C14alkenyl, C15alkenyl, C16alkenyl, C17alkenyl, C18 alkenyl, C19 alkenyl, or C20 alkenyl. In some embodiments, R is independently selected at each occurrence from C14-20 polyunsaturated alkenyl (e.g., an alkenyl containing two or more double bonds, for example, 2, 3, 4, or 5 carbon-carbon double bonds). In some embodiments, R is independently selected at each occurrence from a C14-20monounsaturated alkenyl (e.g., an alkenyl containing one carbon-carbon double bond). In some embodiments, a double bond of the C14-20polyunsaturated alkenyl and / or C14-20monounsaturated alkenyl is in the trans-Attorney Docket No.133279-5010-PC configuration. In some embodiments, a double bond of the C14-20polyunsaturated alkenyl and / or C14-20 monounsaturated alkenyl is in the cis-configuration. In some embodiments, a double bond of the C14-20 polyunsaturated alkenyl and / or C14-20 monounsaturated alkenyl is between the C1and C2carbons, between the C2and C3carbons, between the C3and C4carbons, between the C4 and C5 carbons, between the C5 and C6 carbons, between the C6 and C7 carbons, between the C7 and C8 carbons, between the C8 and C9 carbons, between the C9 and C10 carbons, between the C10and C11carbons, between the C11and C12carbons, between the C12and C13carbons, between the C13 and C14 carbons, between the C14 and C15 carbons, between the C15 and C16 carbons, between the C16 and C17 carbons, between the C17 and C18 carbons, between the C18 and C19carbons, and / or between the C19and C20carbons. In some embodiments, R is C16alkenyl at one occurrence. In some embodiments, R is C16alkenyl at each occurrence. In some embodiments, R is C17 alkenyl at one occurrence. In some embodiments, R is C17 alkenyl at each occurrence. In some embodiments, R is C18 alkenyl at one occurrence. In some embodiments, R is C18alkenyl at each occurrence. In some embodiments, R is , ,from 1 to 5, for example, from 1 to 4, from 1 to 3, from 2 to 5, from 2 to 4, or from 3 to 5.

[0111] In some embodiments, a is independently selected at each occurrence from an integer from 1 to 5, for example, 1, 2, 3, 4, or 5. In some embodiments, a is 2 at one occurrence. In some embodiments, a is 2 at each occurrence.

[0112] In some embodiments, b is independently selected at each occurrence from an integer from 1 to 5, for example, 1, 2, 3, 4, or 5. In some embodiments, b is 2 at one occurrence. In some embodiments, b is 2 at each occurrence.

[0113] In some embodiments, c is independently selected at each occurrence from an integer from 1 to 5, for example, 1, 2, 3, 4, or 5. In some embodiments, c is 1 at one occurrence. In some embodiments, c is 1 at each occurrence.Attorney Docket No.133279-5010-PC

[0114] In some embodiments, the ionizable lipid is of Formula (1001), Formula (1002), Formula (1003), Formula (1004), or Formula (1005):Attorney Docket No.133279-5010-PC

[0115] In some embodiments, the ionizable lipid is of Formula (1001). In some embodiments, the ionizable lipid is of Formula (1002). In some embodiments, the ionizable lipid is of Formula (1003). In some embodiments, the ionizable lipid is of Formula (1004). In some embodiments, the ionizable lipid is of Formula (1005).

[0116] In some embodiments, a compound of Formula (I), a compound of Formula (10), a compound of Formula (100), and / or a compound of Formula (101), excludes a moiety of Alk1and / or Alk2of any of C1 alkylene, C3 alkylene, C4 alkylene, and C5 alkylene.

[0117] In some embodiments, a compound of Formula (I), a compound of Formula (10), a compound of Formula (100), and / or a compound of Formula (101), excludes a moiety of Het of any of formulas: .

[0118] In some, (10), a compound of Formula (100), and / or a compound of Formula (101), excludes a moiety of Cyc of formula: .

[0119] In some embodiments, a compound of Formula (I), a compound of Formula (10), a compound of Formula (100), and / or a compound of Formula (101), excludes a moiety of L1and / or L2of any of formulas:Attorney Docket No.133279-5010-PCcompound of Formula (100), and / or a compound of Formula (101), excludes a moiety of R of any of formulas: , ,wherein in Formula (2): Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1- 10 alkylene, L1and L2are linking moieties; R1and R2are at each independent occurrence selected from substituted or unsubstituted C1-20 alkyl, and substituted or unsubstituted C1-20 alkenyl; and a and b are each independently an integer from 1 to 3.

[0122] In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-10 alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-9 alkylene. In someAttorney Docket No.133279-5010-PC embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-8 alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-7 alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-6alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-5 alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently selected from C1-4alkylene. In some embodiments, Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-3 alkylene.

[0123] In some embodiments, Alk1is C1-10 alkylene. In some embodiments, Alk1is C1-4 alkylene. In some embodiments, Alk1is C1alkylene. In some embodiments, Alk1is C2alkylene. In some embodiments, Alk1is C3alkylene. In some embodiments, Alk1is C4alkylene.

[0124] In some embodiments, Alk2is C1-10 alkylene. In some embodiments, Alk2is C1-4 alkylene. In some embodiments, Alk2is C1alkylene. In some embodiments, Alk2is C2alkylene. In some embodiments, Alk2is C3 alkylene. In some embodiments, Alk2is C4 alkylene.

[0125] In some embodiments, Alk3is C1-10alkylene. In some embodiments, Alk3is C1-4alkylene. In some embodiments, Alk3is C1 alkylene. In some embodiments, Alk3is C2 alkylene. In some embodiments, Alk3is C3 alkylene. In some embodiments, Alk3is C4 alkylene.

[0126] In some embodiments, L1and L2are linking moieties. In some embodiments, L1and / or L2comprise one or more moieties selected from –O–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–.

[0127] In some embodiments, L1is selected from –O–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O– , –OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–. In some embodiments, L1is – C(O)O–. In some embodiments, L1is –OC(O)–. In some embodiments, L1is –OC(O)O–. In some embodiments, L1is –OC(O)NH–. In some embodiments, L1is –NHC(O)O–. In some embodiments, L1is –C(O)NH–. In some embodiments, L1is –NHC(O)–.Attorney Docket No.133279-5010-PC

[0128] In some embodiments, L2is selected from –O–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O– , –OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–. In some embodiments, L2is – C(O)O–. In some embodiments, L2is –OC(O)–. In some embodiments, L2is –OC(O)O–. In some embodiments, L2is –OC(O)NH–. In some embodiments, L2is –NHC(O)O–. In some embodiments, L2is –C(O)NH–. In some embodiments, L2is –NHC(O)–.

[0129] In some embodiments, L1and L2are independently selected from –OC(O)O–, –C(O)O– and –OC(O)–.

[0130] In some embodiments, a and b are each independently an integer from 1 to 3. In some embodiments, a is an integer from 1 to 3, for example 1, 2 or 3. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, b is an integer from 1 to 3, for example 1, 2 or 3. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.

[0131] In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-20alkyl, and substituted or unsubstituted C1-20alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-18 alkyl, and substituted or unsubstituted C1-18 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-16alkyl, and substituted or unsubstituted C1-16 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-14 alkyl, and substituted or unsubstituted C1-14alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-12alkyl, and substituted or unsubstituted C1-12 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-10 alkyl, and substituted or unsubstituted C1-10alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-8 alkyl, and substituted or unsubstituted C1-8 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-20alkyl, and substituted or unsubstituted C4-20alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-18 alkyl, and substituted or unsubstituted C4-18 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-16alkyl, andAttorney Docket No.133279-5010-PC substituted or unsubstituted C4-16alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-14 alkyl, and substituted or unsubstituted C4-14 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-12alkyl, and substituted or unsubstituted C4-12 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-10 alkyl, and substituted or unsubstituted C4-10 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C4-8 alkyl, and substituted or unsubstituted C4-8 alkenyl. In some embodiments, R1and R2are independently at each occurrence selected from substituted or unsubstituted C6-8 alkyl, and substituted or unsubstituted C6-8alkenyl.

[0132] In some embodiments, R1and / or R2orone occurrence, wherein m, n, and o are each independently an integer fromm is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In some embodiments, R1and / or R2are at one occurrence. In some embodiments, R1and / or R2are at one occurrence. In some embodiments, R1and / or R2areat one occurrence. In some embodiments, R1and / or R2are at one occurrence.Attorney Docket No.133279-5010-PC In some embodiments, R1at one occurrence. In some embodiments, R2one occurrence.R1and / or R2are ,embodiments, m1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, m2is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, n2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, o1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, o2is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, R1and / or R2are atone occurrence. In some embodiments, R1and / or R2are at one occurrence. In some embodiments, R1and / or R2are at one occurrence. In some embodiments, R1and / or R2at oneoccurrence. In some embodiments, R1and / or R2at oneoccurrence. In some embodiments, R2at one occurrence.Attorney Docket No.133279-5010-PC

[0134] In some embodiments, a double bond of R1and / or R2is in the trans-configuration. In some embodiments, a double bond of R1and / or R2is in the cis-configuration.

[0135] In some embodiments, the ionizable lipid is of Formula (1006) or Formula (1007):

[0136] In somesome embodiments, the ionizable lipid is of Formula (1007).

[0137] In some embodiments, a compound of Formula (2) excludes a moiety of Alk1, Alk2, and / or Alk3of any of C1alkylene, C4alkylene, C5alkylene, C6alkylene, C7alkylene, and .some embodiments, a compound of Formula (2) excludes a moiety of L1and / or L2of any of formulas: O .

[0139] In some embodiments, a compound of Formula (2) excludes a moiety of R1and / or R2of any of formulas:Attorney Docket No.133279-5010-PC , , , , ,

[0141] Lipid nanoparticle compositions may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic agent. The elements of a lipid nanoparticle composition may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a lipid nanoparticle composition may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.

[0142] The lipid component of a lipid nanoparticle composition may include, for example, an ionizable lipid (such as a cationic lipid) described herein, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a PEGylated lipid, and a structural lipid (such as a sterol, e.g., cholesterol). In some embodiments, the lipid component comprises an ionizable lipid describedAttorney Docket No.133279-5010-PC herein, and at least one of a phospholipid, a PEGylated lipid, and a sterol. The elements of the lipid component may be provided in specific fractions.

[0143] In some embodiments, the lipid component comprises an ionizable lipid (as described herein), a phospholipid, a PEGylated lipid, and a sterol. In some embodiments, lipid component comprises about 40 mol% to about 60 mol% of the ionizable lipid, about 1 mol% to 15 mol% of the phospholipid, about 30 mol% to about 50 mol% of the sterol, and from about 0.1 mol% to about 5 mol% of the PEGylated lipid. In some embodiments, lipid component comprises about 45 mol% to about 55 mol% of the ionizable lipid as described herein, about 5 mol% to 15 mol% of the phospholipid, about 35 mol% to about 45 mol% of the sterol, and from about 0.5 mol% to about 2.5 mol% of the PEGylated lipid.

[0144] In some embodiments, lipid component comprises about 52.5 mol% of the ionizable lipid, about 7.5 mol% of the phospholipid, about 40 mol% of the sterol, and from about 1.5 mol% of the PEGylated lipid. In some embodiments, lipid component comprises from about 52 mol% to about 53 mol% of the ionizable lipid, from about 7 mol% to about 8 mol% of the phospholipid, about 40 mol% of the sterol, and from about 1 mol% to about 2 mol% of the PEGylated lipid.

[0145] In some embodiments, the ionizable lipid comprises from about 30 mol% to about 70 mol% of the lipid component, for example, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 50.5 mol%, about 51 mol%, about 51.5 mol%, about 52 mol%, about 52.5 mol%, about 53 mol%, about 53.5 mol%, about 54 mol%, about 54.5 mol%, about 55 mol%, about 55.5 mol%, about 56 mol%, about 56.5 mol%, about 57 mol%, about 57.5 mol%, about 58 mol%, about 58.5 mol%, about 59 mol%, about 59.5 mol%, about 60 mol%, about 65 mol%, or about 70 mol% of the lipid component. In some embodiments, the ionizable lipid comprises about 52 mol% of the lipid component. In some embodiments, the ionizable lipid comprises about 52.5 mol% of the lipid component. In some embodiments, the ionizable lipid comprises about 53 mol% of the lipid component. In some embodiments, the ionizable lipid comprises from about 30 mol% to about 70 mol%, from about 40 mol% to about 60 mol%, from about 40 mol% to about 55 mol%, from about 40 mol% to about 50 mol%, from about 45 mol% to about 60 mol%, from about 50 mol% to about 60 mol%, about 50 mol% to about 55 mol%, or from about 45 mol% to about 55 mol% of the lipid component.Attorney Docket No.133279-5010-PC

[0146] In some embodiments, the phospholipid comprises from about 1 mol% to 15 mol% of the lipid component, for example, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 8 mol%, about 8.5 mol%, about 9 mol%, about 9.5 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol% of the lipid component. In some embodiments, the phospholipid comprises about 7 mol% of the lipid component. In some embodiments, the phospholipid comprises about 7.5 mol% of the lipid component. In some embodiments, the phospholipid comprises about 8 mol% of the lipid component. In some embodiments, the phospholipid comprises from about 1 mol% to 15 mol%, from about 5 mol% to 15 mol%, from about 5 mol% to about 10 mol%, from about 6 mol% to about 10 mol%, from about 7 mol% to about 10 mol%, from about 8 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, from about 5 mol% to about 8 mol%, or from about 7 mol% to about 8 mol% of the lipid component.

[0147] In some embodiments, the PEGylated lipid comprises from about 0.1 mol% to about 5 mol% of the lipid component, for example, about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol% of the lipid component. In some embodiments, the PEGylated lipid comprises about 1 mol% of the lipid component. In some embodiments, the PEGylated lipid comprises about 1.5 mol% of the lipid component. In some embodiments, the PEGylated lipid comprises about 2 mol% of the lipid component. In some embodiments, the PEGylated lipid comprises from about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 5 mol%, about 2 mol% to about 5 mol%, about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, or about 1 mol% to about 2 mol% of the lipid component.Attorney Docket No.133279-5010-PC

[0148] In some embodiments, the sterol comprises from about 30 mol% to about 50 mol% of the lipid component, for example, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol% of the lipid component. In some embodiments, the sterol comprises about 40 mol% of the lipid component. In some embodiments, the sterol comprises from about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 35 mol% to about 45 mol% of the lipid component.

[0149] Lipid nanoparticle compositions may be designed for one or more specific applications or targets. For example, a lipid nanoparticle composition may be designed to deliver an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of lipid nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. Lipid nanoparticles can encapsulate a nucleic acid or polynucleotide as described herein, including, for example, an mRNA or oligonucleotide agent.

[0150] The amount of a therapeutic and / or prophylactic agent in a lipid nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the lipid nanoparticle composition as well as on the properties of the therapeutic and / or prophylactic agent. For example, the amount of an RNA or an oligonucleotide useful in a lipid nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA or the oligonucleotide. The relative amounts of a therapeutic and / or prophylactic agent and other elements (e.g., lipids) in a lipid nanoparticle composition may also vary. In some embodiments, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic agent in a lipid nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. In some embodiments, the lipid nanoparticle composition may comprise from about 0.1 wt% to about 50 wt%, from about 0.1 wt% to about 20 wt%, from about 1 wt% to about 20Attorney Docket No.133279-5010-PC wt%, from about 1 wt% to about 10 wt%, or from about 1 wt% to about 5 wt% of the therapeutic and / or prophylactic agent.

[0151] For example, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic agent may be from about 10:1 to about 40:1. In certain embodiments, the wt / wt ratio is about 20:1. The amount of a therapeutic and / or prophylactic agent in a lipid nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0152] In some embodiments, the therapeutic and / or prophylactic agent comprises a nucleic acid component. In some embodiments, the nucleic acid component comprises, but is not limited to, messenger RNA (mRNA), CRISPR RNA (crRNA), tracrRNA, single-guide RNA (sgRNA), short interfering RNA (siRNA), miRNA, TLR agonist or antagonist, antisense oligonucleotides (ASO), and splice switching oligonucleotides, and / or mixtures thereof. In other embodiments, the nucleic acid component comprises DNA including, but not limited to, DNA selected from linear DNA, plasmid DNA, circular DNA, and mixtures thereof.

[0153] In some embodiments, the lipid nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. The one or more RNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1. In certain embodiments, the N:P ratio may be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1.

[0154] The characteristics of a plurality of lipid nanoparticles of the lipid nanoparticle composition may depend on the components thereof. For example, a plurality of lipid nanoparticles including cholesterol as a structural lipid may have different characteristics than a plurality of lipid nanoparticles that includes a different structural lipid. Similarly, the characteristics of a plurality of lipid nanoparticles may depend on the absolute or relative amounts of its components. For instance, a plurality of lipid nanoparticles including a higher molar fraction of a phospholipid may have different characteristics than a plurality of lipid nanoparticles including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the plurality of lipid nanoparticles.Attorney Docket No.133279-5010-PC

[0155] The plurality of lipid nanoparticles of the lipid nanoparticle composition may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a plurality of lipid nanoparticles. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a plurality of lipid nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0156] The average diameter of a plurality of lipid nanoparticles of the lipid nanoparticle composition may be between about 10 nm and about 1 micrometer, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm. In some embodiments, a plurality of lipid nanoparticles of the lipid nanoparticle composition has an average diameter of from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, from about 90 nm to about 100 nm, from about 50 nm to about 150 nm, about 50 nm to about 140 nm, about 50 nm to about 130 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, or about 70 nm to about 130 nm.

[0157] A polydispersity index may be used to indicate the homogeneity of a plurality of lipid nanoparticles of the lipid nanoparticle composition, e.g., the particle size distribution of the plurality of lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. In some embodiments, a particle size distribution of the plurality of lipid nanoparticles of the lipid nanoparticle composition is relativelyAttorney Docket No.133279-5010-PC homogenous. In some embodiments, a plurality of lipid nanoparticles of the lipid nanoparticle composition has a polydispersity index of about 0 to about 0.25, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, or about 0.25. In some embodiments, a plurality of lipid nanoparticles of the lipid nanoparticle composition has a polydispersity index of from about 0.01 to about 0.5, about 0.01 to about 0.4, about 0.01 to about 0.3, about 0.01 to about 0.2, or from about 0.01 to about 0.1.

[0158] The zeta potential of a plurality of lipid nanoparticles of the lipid nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a plurality of lipid nanoparticles. A plurality of lipid nanoparticles with relatively low charges, positive or negative, is generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a plurality of lipid nanoparticles may be from about -10 mV to about +20 mV, from about -10 mV to about +10 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, or from about +5 mV to about +10 mV at physiological pH.

[0159] In some embodiments, the lipid nanoparticle composition further comprises neutral lipids including, but not limited to, a phospholipid selected from the group consisting of 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-distearoyl-sn-glycero-3-phosphocholine (DSPC), 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-Attorney Docket No.133279-5010-PC phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), and sphingomyelin (SM).

[0160] In some embodiments, the phospholipid comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

[0161] In some embodiments, the lipid nanoparticle composition further comprises polymer- conjugated lipids, including, but not limited to, a PEGylated lipid selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof. For example, a PEGylated lipid may be PEG-c- DOMG, PEG-DMG, PEG2000-c-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA or a PEG-DSPE lipid.

[0162] In some embodiments, the PEGylated lipid is selected from PEGylated lipid is selected from R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000), PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (PEG-DMPE), PEGylated 1,2-Dilauroyl-sn-glycero-3-phosphorylethanolamine (PEG-DLPE), PEGylated 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine (PEG-DPPE), PEGylated 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (PEG-DOPE), PEGylated 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (PEG-DSPE), PEGylated 1,2-palmitoyl-sn-glycero-3-phosphatidylcholine (PEG-DPPC), and N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl- 3-amine (PEG-C-DMA).

[0163] Non-exhaustive and non-limiting examples of PEGylated lipids include:Attorney Docket No.133279-5010-PCAttorney Docket No.133279-5010-PC .

[0164] In some embodiments, the PEGylated lipid comprises 1,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG 2000).

[0165] In some embodiments, the lipid nanoparticle composition further comprises a structural component. See generally Patel, S., et al. (2020). Nature Communications, 11(1), 1-13. In some embodiments, the lipid nanoparticle composition further comprises a sterol including, but notAttorney Docket No.133279-5010-PC limited to, a sterol selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11-dehydroergosterol, tomatidine, tomatine, α- tocopherol, and mixtures thereof. In other embodiments, the structural lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0166] Non-exhaustive and non-limiting examples of structural lipids include:Attorney Docket No.133279-5010-PCsterol comprises dexamethasone. Without wishing to be bound by any particular theory, it is believed that the use of dexamethasone in LNPs may reduce inflammation.

[0168] In some embodiments, the lipid nanoparticle composition further comprises a nucleic acid as already described, and which can include single stranded nucleic acid as well as double stranded nucleic acids. Nucleic acids include mRNA (e.g., encoding for a protein of interest), DNA expression vectors encoding for an mRNA, cDNA, oligonucleotides (e.g., antisense oligonucleotides and splice switching oligonucleotides), morpholino oligonucleotides, siRNA, and non-coding RNAs such as but not limited to miRNAs, lncRNA, shRNA, gRNA, aptamers, and toll-like receptor agonists and antagonists. Such nucleic acids may include chemical modifications as known in the art. In exemplary embodiments, the nucleic acid is an oligonucleotide, which may be a gapmer comprising a central block of DNA nucleotides for recruitment of RNaseH. Such oligonucleotide compounds may be in the range of 10 to 24 nucleotides in length, such as 10 to 18 nucleotides in length. Oligonucleotides may comprise one or more 2´ modifications such as a 2´ to 4´ bridge (e.g., locked nucleic acid or cEt), 2´-OMe, 2´-MOE, and 2´-F. Oligonucleotides may comprise one or more backbone modifications such as but not limited to phosphorothioate and phosphorodithioate. Exemplary oligonucleotides and chemical modifications to oligonucleotides are described in WO 2022 / 226377 and WO 2023 / 034537, which are hereby incorporated by reference in their entireties.Attorney Docket No.133279-5010-PC

[0169] The pharmaceutical compositions and methods of treatment described herein include a lipid nanoparticle composition described herein comprising an ionizable lipid (e.g., an ionizable lipid of Formula (1) and / or Formula (2)), and at least one of a phospholipid, a PEGylated lipid, and a sterol.

[0170] In one aspect, the present disclosure provides a pharmaceutical composition for treating a disorder in a subject in need thereof comprising a lipid nanoparticle composition described herein. In some embodiments, the disclosure provides a method for treating a disorder in a subject in need thereof comprising administering to the subject a pharmaceutical composition described herein.

[0171] Depending on the method / route of administration, pharmaceutical dosage forms come in several types. These include many kinds of liquid, solid, and semisolid dosage forms. Common pharmaceutical dosage forms include pill, tablet, or capsule, drink or syrup, and natural or herbal form such as plant or food of sorts, among many others. Notably, the route of administration (ROA) for drug delivery is dependent on the dosage form of the substance in question. A liquid pharmaceutical dosage form is the liquid form of a dose of a chemical compound used as a drug or medication intended for administration or consumption.

[0172] In some embodiments, a pharmaceutical formulation of the present disclosure can be administered orally or parenterally. In some embodiments, a pharmaceutical composition of the present disclosure can be delivered to a subject orally, intravenously, subcutaneously (e.g., intradiscal injection), dermally (e.g., transdermally via patch), and / or via implant.

[0173] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acidAttorney Docket No.133279-5010-PC or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0174] In an aspect the disclosure provides a method for treating a disorder in a subject in need thereof, the method comprising administering to the subject an LNP or pharmaceutical composition disclosed herein. In various embodiments, the disorder is a proliferative disorder such as cancer, or is an inflammatory disorder, or is a genetic disorder. The disorder can impact one or more organs or tissues, such as the liver, kidneys, heart, GI, pancreas, spleen, lungs, skeletal muscle, skin, or CNS, for example.

[0175] In one aspect, the disclosure provides a method of preparing a lipid nanoparticle composition described herein comprising an ionizable lipid (e.g., an ionizable lipid of Formula (1) and / or Formula (2)), and at least one of a phospholipid, a PEGylated lipid, and a sterol.

[0176] In some embodiments, the method comprising mixing a lipid mixture and an aqueous solution to provide the lipid nanoparticle composition.

[0177] Mixing can be performed by any method known to one of ordinary skill in the art, including, but not limited to, microfluidic devices, T- or Y-junction mixers, ethanol injection, and hand-mixing. In some embodiments, the mixing is rapid mixing. In some embodiments, the mixing is performed by a microfluidic device. The microfluidic device may be an automated microfluidic device or a microfluidic chip, in accordance with some embodiments. In some embodiments, the microfluidic device has a flow rate of the aqueous solution to lipid mixture of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In some embodiments, the microfluidic device has a flow rate of the aqueous solution to the lipid mixture of about 3:1. In some embodiments, the microfluidic device has a total flow rate of from about 1 mL / min to about 50 mL / min, from about 5 mL / min to about 50 mL / min, from about 1 mL / min to about 40 mL / min, from about 1 mL / min to about 30 mL / min, from about 1 mL / min to about 20 mL / min, from about 10 mL / min to about 40 mL / min, from about 10 mL / min to about 30 mL / min, or from about 10 mL / min to about 20 mL / min. In some embodiments, the microfluidic device has a total flow rate of about 18 mL / min.Attorney Docket No.133279-5010-PC

[0178] In some embodiments, the lipid mixture comprises an organic solvent, an ionizable lipid (e.g., an ionizable lipid of Formula (1) and / or Formula (2)), and at least one of a phospholipid, a PEGylated lipid, and a sterol. Suitable ionizable lipids, phospholipids, PEGylated lipids, and sterols are described elsewhere herein.

[0179] The organic solvent for use in the method is not particularly limited. Examples of suitable organic solvents include, but are not limited to, dimethyl sulfoxide, acetone, methanol, ethanol, isopropanol, n-propanol, diethyl ether, glycerin, ethyl acetate, diethylene glycol, acetonitrile, and tetrahydrofuran. In some embodiments, the organic solvent is an alcohol. In some embodiments, the organic solvent comprises ethanol.

[0180] In some embodiments, the aqueous solution comprises a nucleic acid. Suitable nucleic acids are described elsewhere herein, which include DNA (e.g., ssDNA and dsDNA) and RNA (e.g., RNA, an antisense oligonucleotide, siRNA, and mRNA).

[0181] In some embodiments, the aqueous solution comprises a buffer. The buffer for use in the method is not particularly limited. Examples of suitable buffers include, but are not limited to, HEPES, MOPS, Tris, MES, PIPES, phosphate buffer, PBS, bis-Tris, ADA, ACES, MOPSO, bis- Tris propane, BES, TES, citrate buffer, SSC, TAE, TBE, and Tris-EDTA. In some embodiments, the aqueous solution comprises a citrate buffer.

[0182] In some embodiments, the buffer is acidic (i.e., pH < 7). In some embodiments, the buffer is basic (i.e., pH > 7). In some embodiments, the buffer is neutral (i.e., pH = 7). In some embodiments, the pH is about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7. In some embodiments, the pH is from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, from about 3 to about 6, from about 3 to about 5, or from about 3 to about 4.

[0183] In some embodiments, the buffer has a concentration of from about 1 µM to about 1 M, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. In some embodiments, the buffer has a concentration of from about 1 mM to about 1 M, from about 1 mM to about 20 mM, or from about 1 mM to about 10 mM.Attorney Docket No.133279-5010-PC

[0184] In some embodiments, the buffer is a citrate buffer. In some embodiments, the citrate buffer has a pH of from about 3 to about 4. In some embodiments, the citrate buffer has a concentration of about 1 mM to about 10 mM.

[0185] In some embodiments, the method further comprises filtering the lipid nanoparticle composition. Filtering can be performed by any method known to one of ordinary skill in the art including, but not limited to, gravity filtration, vacuum filtration, mechanical filtration, and centrifugal filtration. In some embodiments, the filtering comprises mechanical filtration. In some embodiments, the filtering comprises syringe filtration. In some embodiments, the syringe filtration comprises a syringe filter with a pore size of from about 0.01 to about 10 µm, for example, about 0.01 µm, about 0.05 µm, about 0.1 µm, about 0.2 µm, about 0.22 µm, about 0.3 µm, about 0.4 µm, about 0.45 µm, about 0.5 µm, about 0.6 µm, about 0.7 µm, about 0.8 µm, about 0.9 µm, about 1 µm, about 1.1 µm, about 1.2 µm, about 1.3 µm, about 1.4 µm, about 1.5 µm, about 1.6 µm, about 1.7 µm, about 1.8 µm, about 1.9 µm, about 2 µm, about 2.5 µm, about 2.7 µm, about 3 µm, about 3.1 µm, about 3.5 µm, about 4 µm, about 4.5 µm, about 6 µm, about 6.5 µm, about 7 µm, about 7.5 µm, about 8 µm, about 8.5 µm, about 9 µm, about 9.5 µm, or about 10 µm.

[0186] In some embodiments, the method further comprises diluting the lipid nanoparticle composition. The term “diluting” as used herein refers to adding a solvent (e.g., a buffer, an organic solvent, etc.) to a liquid composition (e.g., a mixture, a solution, a colloid, a dispersion, a suspension, etc.). In some embodiments, the method further comprises diluting the lipid nanoparticle composition in a buffer solution. Examples of suitable buffer solutions include, but are not limited to, HEPES, MOPS, Tris, MES, PIPES, phosphate buffer, PBS, bis-Tris, ADA, ACES, MOPSO, bis-Tris propane, BES, TES, citrate buffer, SSC, TAE, TBE, and Tris-EDTA.

[0187] In some embodiments, the buffer solution is acidic (i.e., pH < 7). In some embodiments, the buffer solution is basic (i.e., pH > 7). In some embodiments, the buffer solution is neutral (i.e., pH = 7). In some embodiments, the buffer solution is at physiological pH. In some embodiments, the buffer solution has a pH of about 7.4.

[0188] In some embodiments, the buffer solution has a concentration of from about 1 µM to about 1 M, for example, about 1 µM, about 10 µM, about 100 µM, about 1 µM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM,Attorney Docket No.133279-5010-PC about 9 mM, about 10 mM, about 50 mM, about 100 mM, or about 1 M. In some embodiments, the buffer solution has a concentration of about 5 mM. In some embodiments, the buffer solution has a concentration of from about 1 µM to about 1 M, from about 10 µM to about 1 M, from about 100 µM to about 1 M, from about 1 mM to about 1 M, from about 1 mM to about 100 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, or from about 1 mM to about 10 mM. In some embodiments, the buffer solution has a concentration of from about 1 mM to about 50 mM.

[0189] In some embodiments, the buffer solution comprises HEPES. In some embodiments, the buffer solution comprises HEPES at a pH of about 7.4. In some embodiments, the buffer solution comprises HEPES at a concentration of from about 1 mM to about 50 mM.

[0190] In some embodiments, the method further comprises concentrating the lipid nanoparticle composition. The term “concentrating” as used herein refers to removing a solvent (e.g., a buffer, an organic solvent, etc.) from a liquid composition (e.g., a mixture, a solution, a colloid, a dispersion, a suspension, etc.). The concentrating can be performed by any method known to one of ordinary skill in the art, including, but not limited to, evaporation (e.g., by heating) and ultrafiltration. In some embodiments, the concentrating is performed by ultrafiltration. In some embodiments, the concentrating is performed by a Tangential Flow Filter.

[0191] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with a lipid nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the lipid nanoparticle composition before and after breaking up the lipid nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA or oligonucleotide) in a solution. For the lipid nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent may be at least about 50%, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%,Attorney Docket No.133279-5010-PC at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency is from about 50% to about 100%, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 85% to about 100%, about 50% to about 99%, from about 55% to about 99%, from about 60% to about 99%, from about 65% to about 99%, from about 70% to about 99%, from about 75% to about 99%, from about 80% to about 99%, from about 85% to about 99%, from about 90% to about 99%, or from about 85% to about 99%. In some embodiments, the encapsulation efficiency is from about 80% to about 99%. EXAMPLES Example 1: Assembly of lipid nanoparticles containing MAPC-100010.

[0192] A lipid nanoparticle was encapsulated with mRNA by mixing an aqueous solution of RNA with ethanolic solution of lipids. An ionizable lipids of Table 1, DSPC (Avanti), Cholesterol (MP), and PEG-DMG 2000 (Avanti) were diluted in Ethanol in a molar ratio of 52.5:7.5:40:1.5%. eGFP mRNA (Trilink) was diluted in 5mM citrate buffer at pH 3.5. An aqueous solution of eGFP mRNA and mixture of lipid in ethanol were mixed in microfluidic device using Ignite (Precision Nanosystems) at a flow rate of 3:1. The LNPs were diluted in HEPES at pH 7.4 and concentrated using Tangential Flow filter (TFF). LNPs were sterile filtered by syringe filtration before being stored at −80 °C.

[0193] Particle size and polydispersity were measured by using dynamic light scattering on Zetasizer Nano (Malvern). The LNPs were diluted hundred-fold in PBS to measure.

[0194] A RiboGreen assay was performed to measure RNA content and encapsulation efficiency using Quant-IT™ RiboGreen RNA assay kit according to manufacturer instructions. The florescence was read on Tecan Infinity.Attorney Docket No.133279-5010-PC

[0195] Table 1 depicts the structure of exemplary ionizable lipids being used in the formation of lipid nanoparticles to encapsulate nucleic acid and Table 2 describes the physical properties of the resulting lipid nanoparticle formulations.

[0196] Table 1: Chemical structure of exemplary ionizable lipids. Lipid Structure MAPC 100010Attorney Docket No.133279-5010-PC

[0197] Table 2: Characterization of exemplary lipid nanoparticle formulations. P Encapsulation LipidParticle sizeolydispersity (d. nm)Index Efficiency (%)Example 2: Functional delivery of eGFP mRNA using lipid nanoparticle comprising of MAPC-100010.

[0198] Lipid nanoparticles (LNP) containing MAPC-100010 were injected via lateral tail vain in adult CD-1 mice. 48 hours post-dose, animals were sacrificed, and necropsies were performed. All harvested organs were snap frozen prior to quantitation of eGFP protein.

[0199] To evaluate the function of lipid nanoparticle formulations, organ concentrations of eGFP protein were performed by enzyme-linked immunosorbent assay (ELISA) in homogenates. Snap-frozen organs were homogenized in 1 mL of RIPA buffer (supplemented with cOmpleteTMprotease inhibitor, Roche) on a Bead Mill Homogenizer (Fisher) for 30 seconds on setting 6, followed by centrifugation at 300xg for 1 min.

[0200] eGFP protein concentrations in homogenates were then measured by GFP ELISA kit (Abcam, ab17181) according to manufacturer instructions.

[0201] Fig.1 demonstrates the functional delivery of a nucleic acid payload via a lipid nanoparticle formulation containing MAPC-100010 by the measurement of translated eGFP protein in various tissues following a single intravenous dose. Translated GFP levels are shown for liver, kidney, heart, gastrocnemius muscle, and spleen.Attorney Docket No.133279-5010-PC Example 3: Functional delivery of eGFP mRNA using lipid nanoparticle formulations consisting of various ionizable lipids

[0202] Lipid nanoparticles (LNP) containing either Coatsome™ SS-OP (NOF Corporation), MAPC-100010, MAPC-100018, MAPC-100020, MAPC-100021, MAPC-100101, MAPC- 100104, or MAPC-100105 were injected via lateral tail vain in adult CD-1 mice. A dose of 1 mg / kg mRNA was used for all cohorts to evaluate relative potency of each formulation, with 3 animals injected per formulation. 48 hours post-dose, animals were sacrificed, and liver tissue necropsies were performed. All harvested organs were snap frozen prior to quantitation of eGFP protein.

[0203] To evaluate the function of lipid nanoparticle formulations, organ concentrations eGFP protein are performed by enzyme-linked immunosorbent assay (ELISA) in homogenates. Snap- frozen organs were homogenized in 1 mL of RIPA buffer (supplemented with cOmpleteTMprotease inhibitor, Roche) on a Bead Mill Homogenizer (Fisher) for 30 seconds on setting 6, followed by centrifugation at 300xg for 1 min.

[0204] eGFP protein concentrations in homogenates were then measured by GFP ELISA kit (Abcam, ab17181) according to manufacturer instructions.

[0205] Fig.2 demonstrates the functional delivery of a nucleic acid payload via lipid nanoparticle formulations containing varying ionizable lipids by the measurement of translated eGFP protein in liver following a single intravenous dose. As shown, MAPC-100010 resulted in high levels of translated GFP in liver.

Claims

Attorney Docket No.133279-5010-PC CLAIMS 1. An ionizable lipid comprising a moiety of Formula (1): Formula (1) wherein in Formula (1): Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10alkylene; Het is substituted or unsubstituted heterocycle; Cyc is a substituted or unsubstituted cyclic moiety; L1and L2are linking moieties; and R is substituted or unsubstituted C1-20 alkyl or substituted or unsubstituted C1-20 alkenyl.

2. The ionizable lipid of claim 1, wherein the ionizable lipid is of Formula (10): Formula (10) wherein in Formula (10): Alk1and Alk2are independently selected at each occurrence from substituted or unsubstituted C1-10 alkylene; Het is independently selected at each occurrence from substituted or unsubstituted heteroalkylene; Cyc is independently selected at each occurrence from a substituted or unsubstituted cyclic moiety; L1and L2are independently selected at each occurrence from a linking moiety; and R is independently selected at each occurrence from substituted or unsubstituted C10-20alkyl or substituted or unsubstituted C10-20 alkenyl.

3. The ionizable lipid of claim 1 or 2, wherein Alk1and Alk2are independently selected at each occurrence from C1-4 alkylene.Attorney Docket No.133279-5010-PC 4. The ionizable lipid of any one of claims 1 to 3, wherein Het is a saturated cyclic moiety.

5. The ionizable lipid of claim 4, wherein Het is selected at each occurrence from a saturated nitrogen-containing heterocycle.

6. The ionizable lipid of any one of claims 1 to 5, wherein Het is independently selected at each occurrence from 1,2-substituted piperidine, 1,3-substituted piperidine, 1,4-substituted piperidine, 2,3-substituted piperidine, 2,4-substituted piperidine, 2,5-substituted piperidine, 2,6- substituted piperidine, 1,2-substituted piperazine, 1,3-substituted piperazine, 1,4-substituted piperazine, 2,3-substituted piperazine, 2,5-substituted piperazine, 2,6-substituted piperazine, 1,2- substituted pyrrolidine, 1,3-substituted pyrrolidine, 2,3-substituted pyrrolidine, 2,4-substituted pyrrolidine, and 2,5-substituted pyrrolidine.

7. The ionizable lipid of claim 6, wherein Het is 1,4-substituted piperidine.

8. The ionizable lipid of any one of claims 1 to 7, wherein the ionizable lipid comprises a moiety of Formula (100):wherein in Formula 100: Cyc is a substituted or unsubstituted cyclic moiety; L2is a linking moiety; R is substituted or unsubstituted C10-20 alkyl or substituted or unsubstituted C10-20 alkenyl; and a, b, and c are integers from 1 to 5.Attorney Docket No.133279-5010-PC 9. The ionizable lipid of any one of claims 1 to 8, wherein the ionizable lipid is of Formula (101):wherein in Formula 101: Cyc is independent selected at each occurrence from a substituted or unsubstituted cyclic moiety; L2is independently selected at each occurrence from a linking moiety; R is independently selected at each occurrence from substituted or unsubstituted C10-20alkyl or a substituted or unsubstituted C10-20 alkenyl; and a, b, and c are integers independently selected at each occurrence from 1 to 5.

10. The ionizable lipid of any one of claims 1 to 9, wherein Cyc is bicyclo.

11. The ionizable lipid of any one of claims 1 to 9, wherein Cyc is arylene.

12. The ionizable lipid of any one of claims 1 to 11, wherein Cyc is selected at each occurrence from 1,2-substituted bicyclo[1.1.1]pentane, 1,3-substituted bicyclo[1.1.1]pentane, 1,2-substituted benzene, 1,3-substituted benzene, and 1,4-substituted benzene.

13. The ionizable lipid of any one of claims 1 to 12, wherein L1, independently at each occurrence comprises one or more moieties selected from C1-5alkylene, –C(O)O–, –OC(O)–, – OC(O)O–, –OC(O)NH–, –NHC(O)O–, –C(O)NH–, and –NHC(O)–.

14. The ionizable lipid of any one of claims 1 to 13, wherein L1is independently at each occurrence selected from –CH2–C(O)O–, –CH2–OC(O)–, –CH2–OC(O)O–, –CH2–OC(O)NH–, – CH2–NHC(O)O–, –CH2–C(O)NH–, –CH2–NHC(O)–, –C(O)O–CH2–, –OC(O)–CH2–, – OC(O)O–CH2–, –OC(O)NH–CH2–, –NHC(O)O–CH2–, –C(O)NH–CH2–, and –NHC(O)–CH2–.Attorney Docket No.133279-5010-PC 15. The ionizable lipid of any one of claims 1 to 14, wherein L2is independently at each occurrence selected from –C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)NH–, –NHC(O)O–, – C(O)NH–, and –NHC(O)–.

16. The ionizable lipid of any one of claims 1 to 15, wherein R is independently selected at each occurrence from C14-20 monounsaturated alkenyl.

17. The ionizable lipid of any one of claims 1 to 16, wherein the ionizable lipid is of Formula (1001), Formula (1002), Formula (1003), Formula (1004), or Formula (1005):Attorney Docket No.133279-5010-PC18. An ionizable lipid of Formula (2):wherein in Formula (2): Alk1, Alk2, and Alk3are independently at each occurrence substituted or unsubstituted C1-10 alkylene, L1and L2are linking moieties; R1and R2are independently at each occurrence selected from substituted or unsubstituted C1-20 alkyl, and substituted or unsubstituted C1-20 alkenyl; and a and b are each independently an integer from 1 to 3.

19. The ionizable lipid of claim 18, wherein Alk1, Alk2, and Alk3are independently selected from C1-4 alkylene.Attorney Docket No.133279-5010-PC 20. The ionizable lipid of claim 18 or 19, wherein L1and L2are independently selected from –OC(O)O–, –C(O)O– and –OC(O)–.

21. The ionizable lipid of any one of claims 18 to 20, wherein R1and R2are independently at each occurrence selected from C6-8 alkyl and C6-8 monounsaturated alkenyl.

22. The ionizable lipid of any one of claims 18 to 21, wherein R1is one occurrence. lipid of any one of claims 18 to 22, wherein R2is one occurrence.

24. The ionizable lipid of any one of claims 18 to 23, wherein R2at one occurrence.

25. The ionizable lipid of any one of claims 1 to 24, wherein the ionizable lipid is of Formula (1006) or Formula (1007):Attorney Docket No.133279-5010-PC26. A lipid nanoparticle composition, comprising an ionizable lipid of any one of claims 1 to 24, and at least one of a phospholipid, a PEGylated lipid, and a sterol.

27. The lipid nanoparticle composition of claim 26, wherein the ionizable lipid comprises from about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 45 mol% to about 55 mol% of the lipid nanoparticle composition.

28. The lipid nanoparticle composition of claim 26 or 27, wherein the phospholipid is selected from 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-distearoyl-sn-glycero-3-phosphocholine (DSPC), 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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), and sphingomyelin (SM).Attorney Docket No.133279-5010-PC 29. The lipid nanoparticle composition of any one of claims 26 to 28, wherein the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

30. The lipid nanoparticle composition of any one of claims 26 to 29, wherein the phospholipid comprises from about 5 mol% to about 10 mol%, about 6 mol% to about 10 mol%, about 7 mol% to about 10 mol%, about 8 mol% to about 10 mol%, about 5 mol% to about 9 mol%, about 5 mol% to about 8 mol%, or about 7 mol% to about 8 mol% of the lipid nanoparticle composition.

31. The lipid nanoparticle composition of any one of claims 26 to 30, wherein the PEGylated lipid is selected from R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2- dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 1,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG 2000), PEGylated 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (PEG-DMPE), PEGylated 1,2-Dilauroyl-sn-glycero-3- phosphorylethanolamine (PEG-DLPE), PEGylated 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (PEG-DPPE), PEGylated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PEG-DOPE), PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), PEGylated 1,2-palmitoyl-sn-glycero-3-phosphatidylcholine (PEG-DPPC), and N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DMA).

32. The lipid nanoparticle composition of any one of claims 26 to 31, wherein the PEGylated lipid comprises 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000).

33. The lipid nanoparticle composition of any one of claims 26 to 32, wherein the PEGylated lipid comprises from about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 5 mol%, about 2 mol% to about 5 mol%, about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, or about 1 mol% to about 2 mol% of the lipid nanoparticle composition.Attorney Docket No.133279-5010-PC 34. The lipid nanoparticle composition of any one of claims 26 to 33, wherein the sterol is selected from cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11- dehydroergosterol, tomatidine, tomatine, dexamethasone, and α-tocopherol.

35. The lipid nanoparticle composition of any one of claims 26 to 34, wherein the sterol comprises cholesterol.

36. The lipid nanoparticle composition of any one of claims 26 to 35, wherein the sterol comprises from about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 35 mol% to about 45 mol% of the lipid nanoparticle composition.

37. The lipid nanoparticle composition of any one of claims 26 to 36, further comprising a nucleic acid.

38. The lipid nanoparticle composition of claim 37, wherein the nucleic acid is selected from an RNA, an antisense oligonucleotide, and siRNA.

39. The lipid nanoparticle composition of claim 38, wherein the RNA comprises an mRNA.

40. The lipid nanoparticle of claim 37, wherein the nucleic acid is selected from a dsDNA and a ssDNA.

41. The lipid nanoparticle composition of any one of claims 26 to 40, wherein a plurality of lipid nanoparticles of the lipid nanoparticle composition has an average diameter of from about 50 nm to about 150 nm, about 50 nm to about 140 nm, about 50 nm to about 130 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, or about 70 nm to about 130 nm.Attorney Docket No.133279-5010-PC 42. The lipid nanoparticle composition of any one of claims 26 to 41, wherein a plurality of lipid nanoparticles of the lipid nanoparticle composition has a polydispersity index of from about 0.01 to about 0.5, about 0.01 to about 0.4, about 0.01 to about 0.3, about 0.01 to about 0.2, or from about 0.01 to about 0.

1.

43. A pharmaceutical composition for treating a disorder in a subject in need thereof, the composition comprising the lipid nanoparticle composition of any one of claims 26 to 42.

44. A method for treating a disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of claim 43.

45. A method of preparing a lipid nanoparticle composition of any one of claims 26 to 42, the method comprising mixing a lipid mixture and an aqueous solution to provide the lipid nanoparticle composition, wherein the lipid mixture comprises an organic solvent, an ionizable lipid, and at least one of a phospholipid, a PEGylated lipid, and a sterol, and the aqueous solution comprises a nucleic acid.

46. The method of claim 45, wherein the ionizable lipid comprises the ionizable lipid of any one of claims 1 to 25.

47. The method of claim 45 or 46, further comprising filtering the lipid nanoparticle composition.

48. The method of any one of claims 45 to 47, further comprising diluting the lipid nanoparticle composition in a buffer solution.

49. The method of claim 48, wherein the buffer solution has a pH of about 7.

4.

50. The method of any one of claims 45 to 49, further comprising concentrating the lipid nanoparticle composition.Attorney Docket No.133279-5010-PC 51. The method of claim 50, wherein the concentrating is performed by a Tangential Flow Filter.

52. The method of any one of claims 45 to 51, wherein the organic solvent comprises ethanol.

53. The method of any one of claims 45 to 52, wherein the aqueous solution comprises a citrate buffer.

54. The method of claim 53, wherein the citrate buffer has a pH of from about 3 to about 4.

55. The method of claim 53 or 54, wherein the citrate buffer has a concentration of about 1 mM to about 10 mM.

56. The method of any one of claims 45 to 55, wherein the mixing is performed by a microfluidic device.

57. The method of claim 56, wherein the microfluidic device has a flow rate of the aqueous solution to the lipid mixture of about 3:

1.

58. The method of any one of claims 45 to 57, wherein the filtering comprises syringe filtration.

59. The method of any one of claims 45 to 58, wherein the method has an encapsulation efficiency of from about 80% to about 99%.

Images