WO2024044728A1

WO2024044728A1 - Pegylated lipid compounds and methods of use thereof

Info

Publication number: WO2024044728A1
Application number: PCT/US2023/072878
Authority: WO
Inventors: Muthusamy Jayaraman; Ganapathy Subramanian SANKARAN
Original assignee: Renagade Therapeutics Management Inc.
Priority date: 2022-08-26
Filing date: 2023-08-25
Publication date: 2024-02-29

Abstract

The present disclosure relates to PEGylated lipid compounds and pharmaceutically acceptable salts thereof. Such compounds are useful, for example, as constituent parts of lipid nanoparticle (LNP) formulations for delivery of various active agents. The present disclosure further provides LNPs comprising a disclosed compound. Also provided herein are methods of preparing such PEGylated lipid compounds, as well as pharmaceutical compositions comprising an LNP and an active agent; and methods of use thereof.

Description

PEGYLATED LIPID COMPOUNDS AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to United States Provisional Patent Application serial number 63/373,649, filed August 26, 2022 and United States Provisional Patent Application serial number 63/511,515, filed June 30, 2023; the contents of each of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to PEGylated lipid compounds and pharmaceutically acceptable salts thereof. Such compounds are useful, for example, as constituent parts of lipid nanoparticle (LNP) formulations for delivery of various active agents. The present disclosure further provides LNPs comprising a disclosed compound. Also provided herein are methods of preparing such PEGylated lipid compounds, as well as pharmaceutical compositions comprising an LNP and an active agent; and methods of use thereof.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 23, 2023, is named 203250_seqlist.XML and is 9,675 bytes in size.

BACKGROUND

[0004] Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds, such as therapeutic nucleic acids, proteins, and peptides, which are otherwise cell impermeable. Drugs based on nucleic acids, which include large nucleic acid molecules such as, e.g., in vitro transcribed messenger RNA (mRNA) as well as smaller polynucleotides that interact with a messenger RNA or a gene, have to be delivered to the proper cellular compartment in order to be effective. For example, double-stranded nucleic acids such as double-stranded RNA molecules (dsRNA), including, e.g., siRNAs, suffer from poor physico-chemical properties that render them impermeable to cells. If successful delivery into the proper cellular compartment is achieved, siRNAs block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). Typically, siRNAs are large in size with a molecular weight ranging from 12-17 kDa and are highly anionic due to their phosphate backbone with up to 50 negative charges. In addition, the two complementary RNA strands result in a rigid helix. When administered intravenously, siRNA is rapidly excreted from the body with a typical half-life in the range of only 10 minutes. Additionally, siRNAs are rapidly degraded by nucleases present in blood and other fluids or in tissues and have been shown to stimulate strong immune responses in vitro and in vivo. These features contribute to siRNAs’ poor drug-like properties. See, e.g., Robbins et al., Oligonucleotides 19:89-102, 2009. mRNA molecules suffer from similar issues. [0005] Lipid nanoparticle (LNP) formulations have improved nucleic acid delivery in vivo. For example, such formulations can significantly reduce the siRNA doses necessary to achieve target knockdown in vivo. See Zimmermann et al., Nature 441 : 111-114, 2006. Typically, such lipid nanoparticle drug delivery systems are multi-component formulations comprising cationic (or ionizable) lipids, helper lipids, and lipids containing polyethylene glycol (PEG lipids). It should be noted that the terms “cationic” and “ionizable” as they relate to lipids herein, unless otherwise described, are used interchangeably. The positively charged cationic lipids bind to the anionic nucleic acid, while the other components support a stable self-assembly of the lipid nanoparticles. Efforts have been directed toward improving delivery efficacy of lipid nanoparticle formulations. Many such efforts have been aimed toward developing more appropriate cationic lipids. See, e.g., Akinc etal., Nature Biotechnology 26:561-569, 2008; Love et al., Proc. Natl. Acad. Sci. USA 107: 1864-1869, 2010; Baigude et al., Journal of Controlled Release 107:276-287, 2005; Semple et al., Nature Biotechnology 28: 172-176, 2010.

[0006] Further, the PEG lipid PEG2000-C-DMA has been used in LNP formulations that have entered human clinical trials in applications as diverse as oncology, vaccines, antivirals and metabolic diseases. Lipid-containing nanoparticles or lipid nanoparticles, liposomes, and lipoplexes have proven effective as transport vehicles into cells and/or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids. Though a variety of such lipid-containing nanoparticles have been demonstrated, the desired safety, efficacy, and/or specificity are still lacking. For example, there is a need for additional PEG lipids having, e.g., unique or improved properties for use in lipid nanoparticle formulations.

[0007] The present disclosure meets this need and provides additional, related advantages. SUMMARY

[0008] In an aspect of the present disclosure, provided herein is a compound of formula PL-

or a pharmaceutically acceptable salt thereof, wherein each variable is as defined herein.

[0009] In another aspect of the present disclosure, provided herein is a compound of Formula

PL-II:

[0010] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising a PEGylated lipid compound described herein, such as a compound of formula PL-I or PL-II, or a pharmaceutically acceptable salt thereof. The present disclosure also provides pharmaceutical compositions comprising such LNPs.

[0011] In another aspect, provided herein is a method of treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the subject is suffering from a disease or disorder such as those described herein.

DETAILED DESCRIPTION

[0012] In one aspect, the present disclosure provides a compound of formula PL-I:

or a pharmaceutically acceptable salt thereof, wherein:

A¹ is a saturated 5-6 membered carbocyclic ring or a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the carbocyclic ring and heterocyclic ring are substituted with t occurrences of R⁴;

X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-; L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH₂-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-;

L² and L³ are independently a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O- , -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)- , -C(R⁵)=N-, or -C(R⁵)=N-O-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R² and R³ are independently a straight or branched C6-30 alkyl, straight or branched C6-30 alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x;

R⁴ is Ci-4 alkyl;

R⁵ is C1-6 alkyl or C2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R^x is independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or -OC(O)OR; n is an integer from 10-75, inclusive; m is 0, 1, 2, 3, or 4; and t is 0, 1, or 2.

[0013] In another aspect, the present disclosure provides a compound of Formula PL-II:

or a pharmaceutically acceptable salt thereof, wherein: X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-;

L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-;

L² and L³ are a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, - OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁴)=N- , or -C(R⁴)=N-O-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R² and R³ are independently straight or branched C6-30 alkyl, straight or branched C6-30 alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x;

R⁴ is C1-6 alkyl or C2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R^x is independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or OC(O)OR; n is an integer from 10-75, inclusive; and m is 0, 1, 2, 3, or 4.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof; a pharmaceutical agent; and a carrier, excipient, or adjuvant.

[0015] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a PEGylated lipid, and a phospholipid. A. Definitions

[0016] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[0017] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0018] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value. This disclosure encompasses embodiments where the value is within ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, or ±9% of the stated value.

[0019] As used herein, the term “biologically active” refers to a characteristic of an agent (e.g., DNA, RNA, or protein) that has activity in a biological system (including in vitro and in vivo biological system), and particularly in a living organism, such as in a mammal, including human and non-human mammals. For instance, an agent when administered to an organism has a biological effect on that organism, is considered to be biologically active.

[0020] As used herein, the term “bulge” refers to a small region of unpaired base(s) that interrupts a “stem” of base-paired nucleotides. The bulge may comprise one or two singlestranded or unbase-paired nucleotides joined at both ends by base-paired nucleotides of the stem. The bulge can be symmetrical (viz., the two unbase-paired single-stranded regions have the same number of nucleotides), or asymmetrical (viz., the unbase-paired single stranded region(s) have different or unequal numbers of nucleotides), or there is only one unbase-paired nucleotide on one strand. A bulge can be described as A/B (such as a “2/2 bulge,” or a “1/0 bulge”) wherein A represents the number of unpaired nucleotides on the upstream strand of the stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem. An upstream strand of a bulge is more 5’ to a downstream strand of the bulge in the primary nucleotide sequence.

[0021] The term “recombinant nucleic acid” or “recombinant nucleotide,” as used herein, refers to a molecule that is constructed by joining nucleic acid molecules, which optionally may self-replicate in a live cell.

[0022] The term “synthetic or artificial nucleic acid,” as used herein, refers to nucleic acids that are non-naturally occuring sequences. Such sequences do not originate from, or are not known to be present in any living organism (e.g., based on sequence search in existing sequence databases).

[0023] Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.

[0024] Engineered nucleic acid constructs of the present disclosure, such as the engineered retron described herein, may be encoded by a single molecule (e.g., encoded by or present on the same plasmid or other suitable vector) or by multiple different molecules (e.g., multiple independently-replicating vectors).

[0025] As used herein, the term “exosomes” refer to small membrane bound vesicles with an endocytic origin. Without wishing to be bound by theory, exosomes are generally released into an extracellular environment from host/progenitor cells post fusion of multivesicular bodies the cellular plasma membrane. As such, exosomes can include components of the progenitor membrane in addition to designed components (e.g. engineered retron). Exosome membranes are generally lamellar, composed of a bilayer of lipids, with an aqueous inter-nanoparticle space.

[0026] As used herein, the term “heterologous nucleic acid” refers to a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (e.g., DNA or RNA) and, if expressed, can encode a heterologous polypeptide. Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0027] As used herein, the term “liposomes” refers to small vesicles that contain at least one lipid bilayer membrane surrounding an aqueous inner-nanoparticle space that is generally not derived from a progenitor/host cell.

[0028] As used herein, the term “nanoparticle” refers to any particle ranging in size from 10- 1,000 nm. [0029] As used herein, the terms “nucleic acid” or “nucleic acid molecule” or “nucleic acid sequence” or “polynucleotide” generally refer to deoxyribonucleic or ribonucleic oligonucleotides in either single- or double-stranded form. The terms may (or may not) encompass oligonucleotides containing known analogues of natural nucleotides. The terms also may (or may not) encompass nucleic acid-like structures with synthetic backbones, see, e.g, Eckstein, 1991; Baserga et ah, 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. The terms encompass both ribonucleic acid (RNA) and DNA, including cDNA, genomic DNA, synthetic, synthesized (e.g., chemically synthesized) DNA, and/or DNA (or RNA) containing nucleic acid analogs. The nucleotides Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) also may (or may not) encompass nucleotide modifications, e.g., methylated and/or hydroxylated nucleotides, e.g., Cytosine (C) encompasses 5-methylcytosine and 5- hydroxymethylcytosine.

[0030] As used herein, the term “sequence identity” refers to the overall relatedness between polymeric molecules, e.g. , between polynucleotide molecules (e.g. , DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). For example, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna. CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).

[0031] As used herein, the term “identical” refers to two or more sequences or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the “percent identity” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence.

[0032] As used herein, the term “stem” refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, formed by inverted repeat sequences connected at a “tip,” where the more 5’ or “upstream” strand of the stem bends to allows the more 3’ or “downstream” strand to base-pair with the upstream strand. The number of base pairs in a stem is the “length” of the stem. The tip of the stem is typically at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. Larger tips with more than 5 nucleotides are also referred to as a “loop.” An otherwise continuous stem may be interrupted by one or more bulges as defined herein. The number of unpaired nucleotides in the bulge(s) are not included in the length of the stem. The position of a bulge closest to the tip can be described by the number of base pairs between the bulge and the tip (e.g., the bulge is 4 bps from the tip). The position of the other bulges (if any) further away from the tip can be described by the number of base pairs in the stem between the bulge in question and the tip, excluding any unpaired bases of other bulges in between.

[0033] As used herein, the term “loop” in the polynucleotide refers to a single stranded stretch of one or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, wherein the most 5’ nucleotide and the most 3’ nucleotide of the loop are each linked to a base-paired nucleotide in a stem.

[0034] As used herein, the term “operably linked” or “under transcriptional control,” when used in conjunction with the description of a promoter, refers to the correct location and orientation in relation to a polynucleotide (e.g., a coding sequence) to control the initiation of transcription by RNA polymerase and expression of the coding sequence, such as one for the msr gene, msd gene, and/or the ret gene.

[0035] As used herein, the term “vector” permits or facilitates the transfer of a polynucleotide from one environment to another. It is a replicon such as a plasmid, phage, or cosmid into which another DNA segment may be inserted so as to bring about the replication of the inserted segment (e.g. , the subj ect engineered retron). Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” may include cloning and expression vectors, as well as viral vectors and integrating vectors.

[0036] As used herein, the term “expression vector” or “expression construct” refers to a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available, such as from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). The present invention comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof.

[0037] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from one to thirty or more carbon atoms (e.g., C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1 -methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, propyl enyl, but-l-enyl, pent-l-enyl, penta-1, 4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Alkyl groups that include one or more units of unsaturation (one or more double and/or triple bond) can be C2-C24, C2-C12, C2-C8 or C2-C6 groups, for example. Unless specifically stated otherwise, an alkyl group is optionally substituted. The term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-6 means one to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups.

[0038] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to thirty or more carbon atoms (e.g., C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. Alkylene groups that include one or more units of unsaturation (one or more double and/or triple bond) can be C2-C24, C2-C12, C2-C8 or C2-C6 groups, for example. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0039] “Cycloalkyl” or “carbocyclic ring” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0040] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted. [0041] As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two or more heteroatoms typically selected from the group consisting of O, N, Si, P, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be a primary, secondary, tertiary or quaternary nitrogen. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples of heteroalkyl groups include: -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, - CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0042] As used herein, the term “heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms typically selected from the group consisting of N, O, Si, P, and S. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,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 -di oxo- thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted.

[0043] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized p (pi) electrons, where n is an integer.

[0044] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl. [0045] As used herein, the term “heteroaryl” or “heteroaromatic” refers to aryl groups which contain at least one heteroatom typically selected from N, O, Si, P, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally teriatry or quatemized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline, 2,3- dihydrobenzofuryl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2 -furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3 -dihydrofuran, 2, 5 -dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran,

2.3 -dihydropyran, tetrahydropyran, 1,4-di oxane, 1,3 -di oxane, homopiperazine, homopiperidine,

1.3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethyleneoxide. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl. Examples of polycyclic heterocycles include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1, 2,3,4- tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3- dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7- benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5- benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl. The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.

[0046] As used herein, the term “amino aryl” refers to an aryl moiety which contains an amino moiety. Such amino moieties may include, but are not limited to primary amines, secondary amines, tertiary amines, quaternary amines, masked amines, or protected amines. Such tertiary amines, masked amines, or protected amines may be converted to primary amine or secondary amine moieties. Additionally, the amine moiety may include an amine-like moiety which has similar chemical characteristics as amine moieties, including but not limited to chemical reactivity.

[0047] As used herein, the terms “alkoxy,” “alkylamino” and “alkylthio” are used in their conventional sense, and refer to alkyl groups linked to molecules via an oxygen atom, an amino group, a sulfur atom, respectively.

[0048] For example, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (C1-C3) alkoxy, particularly ethoxy and methoxy.

[0049] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

[0050] As described herein, compounds of the present disclosure may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0051] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; — (CH₂)0-4R°; — (CH₂)0-40R°; — 0(CH2)0-4R°, — O— (CH₂)0-4C(0)OR°; — (CH₂)0-4CH(OR°)2; — (CH₂)o.₄SR°; — (CH₂)_0.4Ph, which may be substituted with R°; — (CH₂)0-40(CH₂)0-iPh which may be substituted with R°; — CH=CHPh, which may be substituted with R°; — (CH₂)0-40(CH₂)0-i-pyridyl which may be substituted with R°; — NO₂; — CN; — N₃; — (CH₂)0-4N(R°)₂; — (CH₂)0-4N(R°)C(0)R°; — N(R°)C(S)R°; — (CH₂)0- ₄N(R°)C(O)NR° 2; — N(R°)C(S)NR° 2; — (CH₂)0-4N(R°)C(0)OR°; — N(R°)N(R°)C(O)R°; — N(R°)N(R°)C(O)NR° 2; — N(R°)N(R°)C(O)OR°; — (CH₂)0-4C(0)R°; — C(S)R°; — (CH₂)0- ₄C(O)OR°; — (CH₂)0-4C(0)SR°; — (CH₂)0-4C(0)OSiR° 3; — (CH₂)0-4OC(0)R°; — OC(0)(CH₂)0- ₄SR°, SC(S)SR°; — (CH₂)0-4SC(0)R°; — (CH₂)0-4C(0)NR° 2; — C(S)NR° 2; — C(S)SR°; — SC(S)SR°, — (CH₂)0-4OC(0)NR° 2; — C(O)N(OR°)R°; — C(O)C(O)R°; — C(O)CH₂C(O)R°; — C(NOR°)R°; — (CH₂)0-4SSR°; — (CH₂)0-4S(0)2R°; — (CH₂)0-4S(0)20R°; — (CH₂)0-4OS(0)2R°; — S(O)₂NR° 2; — (CH₂)0-4S(0)R°; — N(R°)S(O)₂NR° 2; — N(R°)S(O)₂R°; — N(OR°)R°; — C(NH)NR° 2; — P(O)₂R°; — P(O)R° 2; — OP(O)R° 2; — 0P(0)(0R°)2; SiR° 3; — (C1-4 straight or branched alkylene)O — N(R°)2; or — (C1-4 straight or branched alkylene)C(O)O — N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, C1- 6 aliphatic, — CH₂Ph, — 0(CH2)0-iPh, — CH2-(5-6 membered heteroaryl ring), or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12- membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0052] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, — (CH₂)0- ₂R●, -(haloR●), — (CH₂)0-2OH, — (CH₂)0-20R●, — (CH₂)0-2CH(OR●)2; — O(haloR●), — CN, — N₃, — (CH₂)0-2C(0)R●, — (CH₂)0-2C(0)OH, — (CH₂)0-2C(0)OR●, — (CH₂)0-2SR●, — (CH₂)0-2SH, — (CH₂)0-2NH₂, — (CH₂)0-2NHR●, — (CH₂)0-2NR● 2, — NO₂, — SiR● 3, — OSiR● 3, — C(O)SR●, — (Ci-4 straight or branched alkylene)C(O)OR●, or — SSR● wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, — CH2Ph, — 0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0053] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)₂R*, =NR*, =N0R*, — O(C(R*2))2-3O— , or — S(C(R*₂))₂-3S— , wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: — O(CR*2)2-3O — , wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0054] Suitable substituents on the aliphatic group of R* include halogen, — R●, -(haloR●), —OH, —OR●, — O(haloR●), — CN, — C(O)OH, — C(O)OR●, — NH₂, — NHR●, —NR● 2, or — NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, — CH2Ph, — 0(CH2)0-iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0055] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include — R^†, — NR^† ₂, — C(O)R^†, — C(O)OR^†, — C(O)C(O)R^†, — C(O)CH₂C(O)R^†, — S(O)₂R^†, — S(O)₂NRb, — CISJNR^†N, — C(NH)NR^† 2, or — N(R^†)S(O)₂R^†; wherein each R^† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted — OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0056] Suitable substituents on the aliphatic group of R^† are independently halogen, — R●, - (haloR●), —OH, —OR●, — O(haloR●), — CN, — C(O)OH, — C(O)OR●, — NH₂, —NHR●, — NR● 2, or — NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, — CH2Ph, — 0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0057] Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, or elimination.

[0058] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. The heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.

[0059] In various embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.

[0060] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carb oxami doaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0061] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0062] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0063] As used herein, the term “antibody” is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”). Antibodies are primarily amino-acid based molecules but may also comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). Non-limiting examples of antibodies or fragments thereof include VH and VL domains, scFvs, Fab, Fab', F(ab')2, Fv fragment, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intrabodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, codon- optimized antibodies, tandem scFv antibodies, bispecific T-cell engagers, mAb2 antibodies, chimeric antigen receptors (CAR), tetravalent bispecific antibodies, biosynthetic antibodies, native antibodies, miniaturized antibodies, unibodies, maxibodies, antibodies to senescent cells, antibodies to conformers, antibodies to disease specific epitopes, or antibodies to innate defense molecules.

[0064] As used herein, a “lipid nanoparticle” or “LNP” is a composition comprising one or more lipids. LNPs are typically sized on the order of micrometers or smaller and may include a lipid bilayer, and preferably have an average size of less than 1 micrometer.

[0065] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0066] As used herein, the interchangeable terms “ionizable lipid” and “cationic lipid” refer to a lipid capable of being either positively charged or uncharged (neutral), depending on pH. Exemplary ionizable lipids comprise one or more fatty acid or fatty aliphatic chains and one or more moieties capable of bearing a positive charge. In preferred embodiments, the moiety capable of bearing the positive charge is a protonatable amine group. Preferred ionizable or cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH of the surrounding environment. Preferable ionizable lipids are protonated to form a cation at acidic physiological pH (about pH 4) and are neutral at neutral pH (pH 7).

[0067] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0068] An “isolated nucleic acid” refers to a nucleic acid segment or fragment, which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment, which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components, which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA or RNA, which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA or RNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA or RNA, which is part of a hybrid gene encoding additional polypeptide sequence.

[0069] The term “DNA” is a well-known term of art that refers to deoxyribonucleic acid.

[0070] The term “RNA” is a well-known term of art that refers to ribonucleic acid. [0071] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0072] As used herein, the term “homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0073] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0074] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0075] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit under the conditions of administration.

[0076] The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder state.

[0077] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0078] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0079] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0080] As used herein, “encapsulation efficiency” (abbreviated as “ee”) refers to the amount of a therapeutic and/or prophylactic that becomes part of a nanoparticle composition, relative to theinitial total amount of therapeutic and/or prophylactic used in the preparation of a nanoparticle composition. For example, if 97 mg of a polynucleotide are encapsulated in a nanoparticle composition out of a total 100 mg of therapeutic and/or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0081] Throughout the disclosure, chemical substituents described in Markush structures are represented by variables. Where a variable is given multiple definitions as applied to different Markush formulas in different sections of the disclosure, it is to be understood that each definition should only apply to the applicable formula in the appropriate section of the disclosure.

[0082] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control.

[0083] As used herein, the following abbreviations and initialisms have the indicated meanings:

[0084] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

B. Exemplary PEGylated Lipid Compounds

[0085] In one aspect, the present disclosure provides a compound of formula PL-I’:

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is -N(H)-, -N(C1-6 alkyl)-, -C1-6 aliphatic-N(H)-, -C1-6 aliphatic-N(C1-6 alkyl)-, -O- or -C1-6 aliphatic-O-;

L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH₂-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-; L² and L³ are independently a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O- , -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)- , -C(R⁵)=N-, or -C(R⁵)=N-O-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R⁴ is Ci-4 alkyl;

[0086] In another aspect, the present disclosure provides a compound of formula PL-I” :

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is -C1-8 aliphatic-N(H)-, -C1-8 aliphatic-N(C1-6 alkyl)-, or -C1-8 aliphatic-O-; L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH₂-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R⁴ is Ci-4 alkyl;

[0087] In one aspect, the present disclosure provides a compound of formula PL-I:

or a pharmaceutically acceptable salt thereof, wherein: A¹ is a saturated 5-6 membered carbocyclic ring or a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the carbocyclic ring and heterocyclic ring are substituted with t occurrences of R⁴;

X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-;

L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH₂-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-;

L² and L³ are independently a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O- , -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)- , -C(R⁵)=N-, or -C(R⁵)=N-O-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N₂, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R⁴ is Ci-4 alkyl;

R¹

[0088] As defined generally above, R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl. In some embodiments, R¹ is H. In some embodiments, R¹ is C1-6 alkyl. In some embodiments, R¹ is -(C1-6 alkyl)-N3. In some embodiments, R¹ is -(C1-6 alkyl)-SH. In some embodiments, R¹ is C3-8 alkynyl. In some embodiments, R¹ is a C1-3 alkyl. In some embodiments, R¹ is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ is propyl. In some embodiments, R¹ is -(C1-3 alkyl)-N3. In some embodiments, R¹ is -CH2N3. In some embodiments, R¹ is -(C1.3 alkyl)-SH. In some embodiments, R¹ is -CH2SH. In some embodiments, R¹ is C3-5 alkynyl. In some embodiments, R¹ is C3 alkynyl. In some embodiments, R¹ is C4 alkynyl. In some embodiments, R¹ is C5 alkynyl. In some embodiments, R¹ is C5-8 alkynyl. In some embodiments, R¹ is C5 alkynyl. In some embodiments, R¹ is C6 alkynyl. In some embodiments, R¹ is C7 alkynyl. In some embodiments, R¹ is C8 alkynyl. In some embodiments, R¹ is not methyl. In some embodiments, R¹ is selected from those depicted in Table 1, below.

R² and R³

[0089] As defined generally above, R² and R³ are independently a straight or branched C6-30 alkyl, straight or branched C6-30 alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x.

[0090] In some embodiments, R² is a straight or branched C6-30 alkyl. In some embodiments, R² is -(CH2) C6-25. In some embodiments, R² is -(CH2)10-25. In some embodiments, R² is -(CH2)10- 14. In some embodiemtns, R² is -(CH2)14-16. In some embodiments, R² is -(CH2)18-20.

[0091] In some embodiments, R² is a straight or branched C6-30 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkyl substituted with m instances of R^x.

[0092] In some embodiments, R² is a straight or branched C6-25 alkyl. In some embodiments, R² is a straight or branched C6-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkyl substituted with m instances of R^x. [0093] In some embodiments, R² is a straight or branched C10-25 alkyl. In some embodiments, R² is a straight or branched C10-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkyl substituted with m instances of R^x. [0094] In some embodiments, R² is a straight or branched C10-14 alkyl. In some embodiments, R² is a straight or branched C10-14 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-14 alkyl substituted with m instances of R^x. [0095] In some embodiments, R² is a straight or branched C14-16 alkyl. In some embodiments, R² is a straight or branched C14-16 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkyl substituted with m instances of R^x. [0096] In some embodiments, R² is a straight or branched C18-20 alkyl. In some embodiments, R² is a straight or branched C18-20 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkyl substituted with m instances of R^x.

[0097] In some embodiments, R² is a straight or branched C6-30 alkenyl. In some embodiments, R² is a straight or branched C6-25 alkenyl. In some embodiments, R² is C10-25 alkenyl. In some embodiments, R² is C10-14 alkenyl. In some embodiments, R² is C14-16 alkenyl. In some embodiments, R² is C18-20 alkenyl.

[0098] In some embodiments, R² includes one, two, three, or four carbon-carbon double bonds.

[0099] In some embodiments, R² is a straight or branched C6-30 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkenyl substituted with m instances of R^x.

[0100] In some embodiments, R² is a straight or branched C6-25 alkenyl. In some embodiments, R² is a straight or branched C6-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkenyl substituted with m instances of R^x.

[0101] In some embodiments, R² is a straight or branched C10-25 alkenyl. In some embodiments, R² is a straight or branched C10-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkenyl substituted with m instances of R^x.

[0102] In some embodiments, R² is a straight or branched C10-14 alkenyl. In some embodiments, R² is a straight or branched C10-14 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C 10-14 alkenyl substituted with m instances of R^x.

[0103] In some embodiments, R² is a straight or branched C14-16 alkenyl. In some embodiments, R² is a straight or branched C14-16 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkenyl substituted with m instances of R^x.

[0104] In some embodiments, R² is a straight or branched C18-20 alkenyl. In some embodiments, R² is a straight or branched C18-20 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkenyl substituted with m instances of R^x.

[0105] In some embodiments, R² is a straight or branched C6-30 alkynyl. In some embodiments, R² is -(CH2)C6-2.5 In some embodiments R² is -(CH2)10-25. In some embodiments R² is -(CH2)10-14. In some embodiemtns R² is -(CH2)14-16. In some embodiments R² is -(CH2)18-20.

[0106] In some embodiments, R² has one, two, three, four, or more carbon-carbon triple bonds.

[0107] In some embodiments, R² is a straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkynyl substituted with m instances of R^x.

[0108] In some embodiments, R² is a straight or branched C6-25 alkynyl. In some embodiments, R² is a straight or branched C6-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkynyl substituted with m instances of R^x.

[0109] In some embodiments, R² is a straight or branched C10-25 alkynyl. In some embodiments, R² is a straight or branched C10-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkynyl substituted with m instances of R^x.

[0110] In some embodiments, R² is a straight or branched C10-14 alkynyl. In some embodiments, R² is a straight or branched C10-14 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-14 alkynyl substituted with m instances of R^x.

[OlH] In some embodiments, R² is a straight or branched C14-16 alkynyl. In some embodiments, R² is a straight or branched C14-16 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkynyl substituted with m instances of R^x.

[0112] In some embodiments, R² is a straight or branched C18-20 alkynyl. In some embodiments, R² is a straight or branched C18-20 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkynyl substituted with m instances of R^x.

[0001] In some embodiments, R² is selected from those depicted in Table 1, below.

[0113] In some embodiments, R³ is a straight or branched C6-30 alkyl. In some embodiments, R³ is -(CH2)C6-2.5 In some embodiments R³ is -(CH2)10-25. In some embodiments R³ is -(CH2)i0-i4. In some embodiemtns R³ is -(CH2)14-16. In some embodiments R³ is -(CH2)18-20.

[0114] In some embodiments, R³ is a straight or branched C6-30 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkyl substituted with m instances of R^x.

[0115] In some embodiments, R³ is a straight or branched C6-25 alkyl. In some embodiments, R³ is a straight or branched C6-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkyl substituted with m instances of R^x.

[0116] In some embodiments, R³ is a straight or branched C10-25 alkyl. In some embodiments, R³ is a straight or branched C10-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkyl substituted with m instances of R^x.

[0117] In some embodiments, R³ is a straight or branched C10-14 alkyl. In some embodiments, R³ is a straight or branched C10-14 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-14 alkyl substituted with m instances of R^x.

[0118] In some embodiments, R³ is a straight or branched C14-16 alkyl. In some embodiments, R³ is a straight or branched C14-16 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkyl substituted with m instances of R^x.

[0119] In some embodiments, R³ is a straight or branched C18-20 alkyl. In some embodiments, R³ is a straight or branched C18-20 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkyl substituted with m instances of R^x.

[0120] In some embodiments, R³ is a straight or branched C6-30 alkenyl. In some embodiments, R³ is C6-25 alkenyl. In some embodiments, R³ is C10-25 alkenyl. In some embodiments, R³ is C10-14 alkenyl. In some embodiments, R³ is C14-16 alkenyl. In some embodiments, R³ is C18-20 alkenyl.

[0121] In some embodiments, R³ is a straight or branched C6-30 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkenyl substituted with m instances of R^x.

[0122] In some embodiments, R³ is a straight or branched C6-25 alkenyl. In some embodiments, R³ is a straight or branched C6-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkenyl substituted with m instances of R^x.

[0123] In some embodiments, R³ is a straight or branched C10-25 alkenyl. In some embodiments, R³ is a straight or branched C10-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkenyl substituted with m instances of R^x.

[0124] In some embodiments, R³ is a straight or branched C10-14 alkenyl. In some embodiments, R³ is a straight or branched C10-14 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C 10-14 alkenyl substituted with m instances of R^x.

[0125] In some embodiments, R³ is a straight or branched C14-16 alkenyl. In some embodiments, R³ is a straight or branched C14-16 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkenyl substituted with m instances of R^x.

[0126] In some embodiments, R³ is a straight or branched C18-20 alkenyl. In some embodiments, R³ is a straight or branched C18-20 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkenyl substituted with m instances of R^x. [0127] In some embodiments, R³ is a straight or branched C6-30 alkynyl. In some embodiments, R³ is C6-25 alkynyl. In some embodiments R³ is C10-25 alkynyl. In some embodiments R³ is C10-14 alkynyl. In some embodiemtns R³ is C14-16 alkynyl. In some embodiments R³ is C18-20 alkynyl.

[0128] In some embodiments, R³ has one, two, three, four, or more carbon-carbon triple bonds.

[0129] In some embodiments, R³ is a straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkynyl substituted with m instances of R^x.

[0130] In some embodiments, R³ is a straight or branched C6-25 alkynyl. In some embodiments, R³ is a straight or branched C6-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkynyl substituted with m instances of R^x.

[0131] In some embodiments, R³ is a straight or branched C10-25 alkynyl. In some embodiments, R³ is a straight or branched C10-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkynyl substituted with m instances of R^x.

[0132] In some embodiments, R³ is a straight or branched C10-14 alkynyl. In some embodiments, R³ is a straight or branched C10-14 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-14 alkynyl substituted with m instances of R^x.

[0133] In some embodiments, R³ is a straight or branched C14-16 alkynyl. In some embodiments, R³ is a straight or branched C14-16 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkynyl substituted with m instances of R^x.

[0134] In some embodiments, R³ is a straight or branched C18-20 alkynyl. In some embodiments, R³ is a straight or branched C18-20 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkynyl substituted with m instances of R^x.

[0135] In some embodiments, R³ is selected from those depicted in Table 1, below.

[0136] In some embodiments, R² and R³ are the same. In some embodiments R² and R³ are different.

R⁴

[0137] As generally defined above, R⁴ is C1-4 alkyl. In some embodiments, R⁴ is C1.3 alkyl. In some embodiments, R⁴ is C1.2 alkyl. In some embodiments, R⁴ is C2-4 alkyl.

[0138] In some embodiments, R⁴ is methyl. In some embodiments R⁴ is ethyl. In some embodiments R⁴ is propyl. In some embodiments R⁴ is isopropyl. In some embodiments R⁴ is butyl. In some embodiments R⁴ is isobutyl.

[0139] In some embodiments, R⁴ is selected from those depicted in Table 1, below.

R⁵

[0140] As generally defined above, R⁵ is C1-6 alkyl. In some embodiments, R⁵ is C1.5 alkyl. In some embodiments, R⁵ is C1.4 alkyl. In some embodiments, R⁵ is C1.3 alkyl. In some embodiments, R⁵ is C1.2 alkyl. In some embodiments, R⁵ is C2-6 alkyl.

[0141] In some embodiments, R⁵ is methyl. In some embodiments, R⁵ is ethyl. In some embodiments, R⁵ is propyl. In some embodiments, R⁵ is isopropyl. In some embodiments, R⁵ is butyl. In some embodiments, R⁵ is isobutyl. In some embodiments, R⁵ is pentyl. In some embodiments, R⁵ is hexyl.

[0142] In some embodiments, R⁵ is selected from those depicted in Table 1, below.

X¹

[0143] As generally defined above, X¹ is -N(H)-, -N(C1-6 alkyl)-, -C1-6 aliphatic-N(H)-, -C1-6 aliphatic-N(C1-6 alkyl)-, -O- or -C1-6 aliphatic-O-. Alternatively, as generally defined above, X¹ is -C1-8 aliphatic-N(H)-, -C1-8 aliphatic-N(C1-6 alkyl)-, or -C1-8 aliphatic-O-.

[0144] Alternatively, as generally defined above, X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-. In some embodiments, X¹ is -N(H)-. In some embodiments, X¹ is -N(C1-6 alkyl)-. In some embodiments, X¹ is -O-. [0145] In some embodiments, X¹ is -C1-6 aliphatic-N(H)-. In some embodiments, X¹ is -C1-6 alkyl-N(H)-. In some embodiments, X¹ is -C1-8 aliphatic-N(H)-. In some embodiments, X¹ is - C1-8 alkyl-N(H)-. In some embodiments, X¹ is -Ci alkyl-N(H)-. In some embodiments, X¹ is - C2 alkyl-N(H)-. In some embodiments, X¹ is -C3 alkyl-N(H)-. In some embodiments, X¹ is -C₄ alkyl-N(H)-. In some embodiments, X¹ is -C5 alkyl-N(H)-. In some embodiments, X¹ is -C6 alkyl-N(H)-. In some embodiments, X¹ is -CH2N(H)-. In some embodiments, X¹ is -

(CH₂)₂N(H)-. In some embodiments, X¹ is -(CH₂)3N(H)-. In some embodiments, X¹ is -

(CH₂)₄N(H)-. In some embodiments, X¹ is -(CH₂)5N(H)-. In some embodiments, X¹ is -

(CH₂)₆N(H)-.

[0146] In some embodiments, X¹ is -C1-6 aliphatic-N(C1-6 alkyl)-. In some embodiments, X¹ is -C1-6 alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C1-8 aliphatic-N(C1-6 alkyl)-. In some embodiments, X¹ is -C1-8 alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -Ci alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C₂ alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C3 alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C₄ alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C5 alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -C6 alkyl-N(C1-6 alkyl)-. In some embodiments, X¹ is -CH2N(C1-6 alkyl)-. In some embodiments, X¹ is - (CH₂)₂N(C1-6 alkyl)-. In some embodiments, X¹ is -(CH₂)3N(C1-6 alkyl)-. In some embodiments, X¹ is -(CH₂)₄N(C1-6 alkyl)-. In some embodiments, X¹ is -(CH2)5N(C1-6 alkyl)-. In some embodiments, X¹ is -(CH₂)6N(C1-6 alkyl)-.

[0147] In some embodiments, X¹ is -C1-6 aliphatic-O-. In some embodiments, X¹ is -C1-6 alkyl-O-. In some embodiments, X¹ is -C1-8 aliphatic-O-. In some embodiments, X¹ is -C1-8 alkyl- O-. In some embodiments, X¹ is -Ci alkyl-O-. In some embodiments, X¹ is -C₂ alkyl-O-. In some embodiments, X¹ is -C3 alkyl-O-. In some embodiments, X¹ is -C₄ alkyl-O-. In some embodiments, X¹ is -C5 alkyl-O-. In some embodiments, X¹ is - C6 alkyl-O-. In some embodiments, X¹ is -CH2N(H)-. In some embodiments, X¹ is -(CH₂)₂O-. In some embodiments, X¹ is — (CH₂)3O-. In some embodiments, X¹ is -(CH2)₄O-. In some embodiments, X¹ is - (CH2)SO-. In some embodiments, X¹ is -(CH2)6O-.

[0148] In some embodiments, X¹ is -N(CI-5 alkyl)-. In some embodiments, X¹ is -N(CI-₄ alkyl)-. In some embodiments, X¹ is -N(CI-3 alkyl)-. In some embodiments, X¹ is -N(CI-₂ alkyl)- . In some embodiments, X¹ is -N(C₂-6 alkyl)-.

[0149] In some embodiments, X¹ is -N(CH3)-. In some embodiments, X¹ is -N(CH2CH3)-. In some embodiments, X¹ is -N((CH2)2CH3)-. In some embodiments, X¹ is -N((CH2)3CH3)-. In some embodiments, X¹ is -N((CH2)₄CH3)-. In some embodiments, X¹ is -N((CH2)5CH3)-.

[0150] In some embodiments, X¹ is selected from those depicted in Table 1, below. L¹

[0151] As defined generally above, L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH2-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-.

[0152] In some embodiments, L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)- N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)₂-, or -C(O)(C1-6 aliphatic)C(O)-.

[0153] In some embodiments, L¹ is -C(O)(C1-6 aliphatic)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(C1-6 aliphatic)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(C1-6 aliphatic)C(O)O- . In some embodiments, L¹ is -C(O)(C1-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(Ci- 6 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(C1-6 aliphatic)-. In some embodiments, L¹ is -C(O)(C1-6 aliphatic)-N(R)-. In some embodiments, L¹ is-C(O)-.

[0154] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)C(O)- N(R)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)C(O)-N(R)-.

[0155] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)C(O)- N(R)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)C(O)-N(R)-.

[0156] In some embodiments, L¹ is -C(O)(CH2)C(O)-N(R)-. In some embodiments, L¹ is - C(O)(CH2CH2)C(O)-N(R)-. In some embodiments, L¹ is -C(O)(CH2)3C(O)-N(R)-. In some embodiments, L¹ is -C(O)(CH2)4C(O)-N(R)-. In some embodiments, L¹ is -C(O)(CH2)5C(O)- N(R)-. In some embodiments, L¹ is -C(O)(CH2)6C(O)-N(R)-.

[0157] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)- N(R)C(O)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)-N(R)C(O)-.

[0158] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)- N(R)C(O)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)-N(R)C(O)-.

[0159] In some embodiments, L¹ is -C(O)(CH2)-N(R)C(O)-. In some embodiments, L¹ is - C(O)(CH2CH2)-N(R)C(O)-. In some embodiments, L¹ is -C(O)(CH2)3-N(R)C(O)-. In some embodiments, L¹ is -C(O)(CH2)4-N(R)C(O)-. In some embodiments, L¹ is -C(O)(CH2)5- N(R)C(O)-. In some embodiments, L¹ is -C(O)(CH2)6-N(R)C(O)-.

[0160] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)C(O)O-. [0161] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)C(O)O-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)C(O)O-.

[0162] In some embodiments, L¹ is -C(O)(CH2)C(O)O-. In some embodiments, L¹ is - C(O)(CH2CH2)C(O)O-. In some embodiments, L¹ is -C(O)(CH2)3C(O)O-. In some embodiments, L¹ is -C(O)(CH2)4C(O)O-. In some embodiments, L¹ is -C(O)(CH2)5C(O)O-. In some embodiments, L¹ is -C(O)(CH2)6C(O)O-.

[0163] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)C(O)-.

[0164] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)C(O)-.

[0165] In some embodiments, L¹ is -C(O)(CH2)C(O)-. In some embodiments, L¹ is - C(O)(CH2CH2)C(O)-. In some embodiments, L¹ is -C(O)(CH2)3C(O)-. In some embodiments, L¹ is -C(O)(CH2)4C(O)-. In some embodiments, L¹ is -C(O)(CH2)5C(O)-. In some embodiments, L¹ is -C(O)(CH₂)₆C(O)-.

[0166] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)C(O)OCH2-.

[0167] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)C(O)OCH2-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)C(O)OCH2-.

[0168] In some embodiments, L¹ is -C(O)(CH2)C(O)OCH2-. In some embodiments, L¹ is - C(O)(CH2CH2)C(O)OCH2-. In some embodiments, L¹ is -C(O)(CH2)3C(O)OCH2-. In some embodiments, L¹ is -C(O)(CH2)4C(O)OCH2-. In some embodiments, L¹ is C(O)(CH2)5C(O)OCH2-. In some embodiments, L¹ is -C(O)(CH2)6C(O)OCH2-.

[0169] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)-. In some embodiments, L¹ is - C(O)(Ci-4 aliphatic)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)-.

[0170] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)- . In some embodiments, L¹ is - C(O)(C3-6 aliphatic)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)- . [0171] In some embodiments, L¹ is -C(O)(CH2)-. In some embodiments, L¹ is - C(O)(CH2CH2)-. In some embodiments, L¹ is -C(O)(CH2)3-. In some embodiments, L¹ is - C(O)(CH₂)₄-. In some embodiments, L¹ is -C(O)(CH2)5-. In some embodiments, L¹ is - C(O)(CH₂)₆-.

[0172] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)-N(R)-.

[0173] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)-N(R)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)-N(R)-.

[0174] In some embodiments, L¹ is -C(O)(CH2)-N(R)-. In some embodiments, L¹ is - C(O)(CH2CH2)-N(R)-. In some embodiments, L¹ is -C(O)(CH2)3-N(R)-. In some embodiments, L¹ is -C(O)(CH2)4-N(R)-. In some embodiments, L¹ is -C(O)(CH2)5-N(R)-. In some embodiments, L¹ is -C(O)(CH2)6-N(R)-.

[0175] In some embodiments, L¹ is selected from those depicted in Table 1, below. L² and L³

[0176] As defined generally above, L² and L³ are independently a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁵)=N-, or -C(R⁵)=N-O-.

[0177] In some embodiments L² is a covalent bond.

[0178] In some embodiments, L² is C1-6 alkylene. In some embodiments, L² is C1.5 alkylene. In some embodiments, L² is C1.4 alkylene. In some embodiments, L² is C1.3 alkylene. In some embodiments, L² is C1.2 alkylene.

[0179] In some embodiments, L² is C2-6 alkylene. In some embodiments, L² is C3-6 alkylene. In some embodiments, L² is C4-6 alkylene. In some embodiments, L² is C5-6 alkylene.

[0180] In some embodiments L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, - OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, - C(R⁵)=N-, or -C(R⁵)=N-O-.

[0181] In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-. [0182] In some embodiments, L² is a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or -OC(O)N(R)-.

[0183] In some embodiments, L² is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0184] In some embodiments, L² is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or - OC(O)N(R)-.

[0185] In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -NR-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-S-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)2-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(R⁵)=N-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the Ci- 6 alkylene is replaced with -C(R⁵)=N-O-.

[0186] In some embodiments L² is selected from -O-, -OC(O)-, -C(O)O-, -OC(O)O-, -CH2O- , -CH₂OC(O)-, and -CH₂OC(O)O-. [0187] In some embodiments L² is -O-. In some embodiments L² is -OC(O)-. In some embodiments L² is -C(O)O-. In some embodiments L² is -OC(O)O-. In some embodiments L² is -CH2O-. In some embodiments L² is -CH2OC(O)-. In some embodiments L² is -CH2OC(O)O-. [0188] In some embodiments L³ is a covalent bond.

[0189] In some embodiments, L³ is C1-6 alkylene. In some embodiments, L³ is C1.5 alkylene. In some embodiments, L³ is C1.4 alkylene. In some embodiments, L³ is C1.3 alkylene. In some embodiments, L³ is C1.2 alkylene.

[0190] In some embodiments, L³ is C2-6 alkylene. In some embodiments, L³ is C3-6 alkylene. In some embodiments, L³ is C4-6 alkylene. In some embodiments, L³ is C5-6 alkylene.

[0191] In some embodiments L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, - OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, - C(R⁵)=N-, or -C(R⁵)=N-O-.

[0192] In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0193] In some embodiments, L³ is a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or -OC(O)N(R)-.

[0194] In some embodiments, L³ is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0195] In some embodiments, L³ is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or - OC(O)N(R)-.

[0196] In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -NR-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-S-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)2-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(R⁵)=N-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the Ci- 6 alkylene is replaced with -C(R⁵)=N-O-.

[0197] In some embodiments L³ is selected from -O-, -OC(O)-, -C(O)O-, -OC(O)O-, -CH₂O- , -CH₂OC(O)-, and -CH₂OC(O)O-.

[0198] In some embodiments L³ is -O-. In some embodiments L³ is -OC(O)-. In some embodiments L³ is -C(O)O-. In some embodiments L³ is -OC(O)O-. In some embodiments L³ is -CH₂O-. In some embodiments L³ is -CH₂OC(O)-. In some embodiments L³ is -CH₂OC(O)O-. [0199] In some embodiments, L² and L³ are a covalent bond.

[0200] In some embodiments, L² and L³ are independently a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, - S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, - N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁵)=N-, or -C(R⁵)=N-O-.

[0201] In some embodiments, L² and L³ are independently C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, - S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, - N(R)C(O)-, or -N(R)C(O)N(R)-.

[0202] In some embodiments, L² and L³ are independently selected from -O-, -OC(O)-, - C(O)O-, -OC(O)O-, -CH₂O-, -CH₂OC(O)-, and -CH₂OC(O)O-.

[0203] In some embodiments, L² and L³ are -O-. In some embodiments, L² and L³ are -OC(O)- . In some embodiments, L² and L³ are -C(O)O-. In some embodiments, L² and L³ are -OC(O)O- . In some embodiments, L² and L³ are -CH₂O-. In some embodiments, L² and L³ are -CH₂OC(O)- . In some embodiments, L² and L³ are -CH₂OC(O)O-.

[0204] In some embodiments, L² and L³ are independently selected from -O-, -OC(O)-, - C(O)O-, -OC(O)O-, -CH₂O-, -CH₂OC(O)-, and -CH₂OC(O)O-. [0205] In some embodiments, L² is -OC(O)- and L³ is -CH2OC(O)-.

[0206] In some embodiments, L² and L³ are the same. In some embodiments, L² and L³ are not the same.

[0207] In some embodiments, L² is selected from those depicted in Table 1, below. In some embodiments, L³ is selected from those depicted in Table 1, below.

A¹

[0208] As defined generally above, A¹ is a saturated 5-6 membered carbocyclic ring or a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the carbocyclic ring and heterocyclic ring are substituted with t occurrences of R⁴.

[0209] In some embodiments, A¹ is a saturated 5-6 membered carbocyclic ring substituted with t occurrences of R⁴. In some embodiments, a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur substituted with t occurrences of R⁴.

[0210] In some embodiments, A¹ is a saturated 5 membered carbocyclic ring substituted with t occurrences of R⁴. In some embodiments, A¹ is a saturated 6 membered carbocyclic ring substituted with t occurrences of R⁴.

[0211] In some embodiments, A¹ is cyclopentyl. In some embodiments, A¹ is cyclohexyl.

[0212] In some embodiments, A¹ is selected from pyrrolidinylene, tetrahydrofuranylene, tetrahydrothiophenylene, imidazolidinylene, thiazolidinylene, oxazolidinylene, piperidinylene, tetrahydro-2H-pyranylene, tetrahydro-2H-thiopyranylene, piperazinylene, morpholinylene, and hexahydropyrimidinylene.

[0213] In some embodiments, A¹ is selected from those depicted in Table 1, below.

R^x

[0214] As generally defined above, each R^x is independently halogen, -CN, -OR, -SR, - C(O)R, -C(O)OR, or OC(O)OR.

[0215] In some embodiments, R^x is halogen. In some embodiments, R^x is -CN. In some embodiments, R^x is -OR. In some embodiments, R^x is -SR. In some embodiments, R^x is -C(O)R. In some embodiments, R^x is -C(O)OR. In some embodiments, R^x is OC(O)OR.

[0216] In some embodiments, R^x is selected from those depicted in Table 1, below. n

[0217] As defined generally above, n is an integer from 10-75.

[0218] In some embodiments, n is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-75. In some embodiments, n is 10-30, 20-40, 30-50, 40-60, or 50-75. In some embodiments, n is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75. In some embodiments, n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.

[0219] In some embodiments, n is an integer from 30-55, inclusive. In some embodiments, n is an integer from 40-50, inclusive. In some embodiments, n is 44, 45, or 46. In some embodiments, n is 44. In some embodiments, n is 45. In some embodiments, n is 46. m

[0220] As defined generally above, m is 0, 1, 2, 3, or 4. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 0, 1, or 2. In some embodiments, m is 1, 2, or 3. t

[0221] As defined generally above, t is 0, 1, or 2. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 1 or 2.

[0222] In some embodiments, the present invention provides a compound of formula PL-Ia,

PL-Ib, or PL-Ic:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, X¹, L¹, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-.

[0223] In some embodiments, the present invention provides a compound of formula PL-Iaa, PL-Iab, PL-Iac, PL-Iad, PL-Iae, PL-Iaf, PL-Iag, or PL-Iah:

[0224] In some embodiments, the present invention provides a compound of formula PL-Iba,

PL-Ibb, PL-Ibc, PL-Ibd, PL-Ibe, PL-Ibf, PL-Ibg, or PL-Ibh:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R , R⁴, R⁵, X¹, L¹, L², L , R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-.

[0225] In some embodiments, the present invention provides a compound of formula PL-Ica,

PL-Icb, PL-Icc, or PL-Icd:

[0226] In some embodiments, the present invention provides a compound of formula PL-Id or PL-Ie:

[0227] In some embodiments, the present invention provides a compound of formula PL-If:

[0228] In some embodiments, the present invention provides a compound of formula PL-Ig:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, X¹, L¹, L², L³, A¹, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-.

[0229] In some embodiments, the present invention provides a compound of formula PL-Ih, PL-Ii, PL-Iha, PL-Ihb, PL-Ihc, PL-Ihd, PL-Iia, PL-lib, PL-Iic, or PL-Iid:

[0230] In some embodiments, the present invention provides a compound of formula PL-Ij or PL-Ik:

[0231] In some embodiments, the present invention provides a compound of formula PL-11, PL-Im, or PL-In:

[0232] In some embodiments, the present invention provides a compound of formula PL-Io,

PL-Ip, or PL-Iq:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, L¹, L², L³, R,

R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination.

[0233] In some embodiments, the present invention provides a compound of formula PL-Ioa,

PL-Iob, PL-Ioc, PL-Iod, PL-Ioe, PL-Iof, PL-Iog, or PL-Ioh:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, L¹, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination.

[0234] In some embodiments, the present invention provides a compound of formula PL-Ipa,

PL-Ipb, PL-Ipc, PL-Ipd, PL-Ipe, PL-Ipf, PL-Ipg, or PL-Iph:

[0235] In some embodiments, the present invention provides a compound of formula PL-Iqa,

PL-Iqb, PL-Iqc, or PL-Iqd:

[0236] In some embodiments, the present invention provides a compound of formula PL-Ir or PL-Is:

[0237] In some embodiments, the present invention provides a compound of formula PL-It:

[0238] In some embodiments, the present invention provides a compound of formula PL-Iu:

[0239] In some embodiments, the present invention provides a compound of formula PL-Iv, PL-Iw, PL-Iva, PL-Ivb, PL-Ivc, PL-Ivd, PL-Iwa, PL-Iwb, PL-Iwc, or PL-Iwd:

[0240] In some embodiments, the present invention provides a compound of formula PL-Ix or PL-Ixx:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination.

[0241] In some embodiments, the present invention provides a compound of formula PL-Iy, PL-Iyy, or PL-Iyyy:

[0242] In some embodiments, the present invention provides a compound of formula PL-Iz, PL-Izz, or PL-Izzz:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination.

[0243] In one aspect, the present disclosure provides a compound of formula PL-II’ :

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-;

L² and L³ are a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, - OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁶)=N- , or -C(R⁶)=N-O-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl; R² and R³ are independently straight or branched C6-30 alkyl, straight or branched C6-30 alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x;

R⁶ is C1-6 alkyl or C2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R^x is independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or OC(O)OR; n is an integer from 10-75, inclusive; and m is 0, 1, 2, 3, or 4.

[0244] In one aspect, the present disclosure provides a compound of formula PL-II”:

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is -C1-8 aliphatic-N(H)-, -C1-8 aliphatic-N(C1-6 alkyl)-, or -C1-8 aliphatic-O-;

L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

[0245] In one aspect, the present disclosure provides a compound of formula PL-II:

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-;

L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

X¹

[0246] As generally defined above, X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-. In some embodiments, X¹ is -N(H)-. In some embodiments, X¹ is -N(C1-6 alkyl)-. In some embodiments, X¹ is -O-.

[0247] In some embodiments, X¹ is -N(CI-5 alkyl)-. In some embodiments, X¹ is -N(CI-4 alkyl)-. In some embodiments, X¹ is -N(C1-3 alkyl)-. In some embodiments, X¹ is -N(CI-2 alkyl)- . In some embodiments, X¹ is -N(C2-6 alkyl)-.

[0248] In some embodiments, X¹ is -NCH3-. In some embodiments, X¹ is -NCH2CH3- . In some embodiments, X¹ is -N(CH2)2CH3-. In some embodiments, X¹ is -N(CH2)3CH4-. In some embodiments, X¹ is -N(CH2)4CH5-. In some embodiments, X¹ is -N(CH2)5CH6-.

[0249] In some embodiments, X¹ is selected from those depicted in Table 1, below.

L¹

[0250] As defined generally above, L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-.

[0251] In some embodiments, L¹ is -C(O)(C1-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C1-6 aliphatic)-. In some embodiments, L¹ is -C(O)-.

[0252] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(Ci-4 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)C(O)-.

[0253] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C3-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)C(O)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)C(O)-. [0254] In some embodiments, L¹ is -C(O)(CH2)C(O)-. In some embodiments, L¹ is - C(O)(CH2CH2)C(O)-. In some embodiments, L¹ is -C(O)(CH2)3C(O)-. In some embodiments, L¹ is -C(O)(CH2)4C(O)-. In some embodiments, L¹ is -C(O)(CH2)5C(O)-. In some embodiments, L¹ is -C(O)(CH₂)₆C(O)-.

[0255] In some embodiments, L¹ is -C(O)(C1-5 aliphatic)-. In some embodiments, L¹ is - C(O)(Ci-4 aliphatic)-. In some embodiments, L¹ is -C(O)(C1-3 aliphatic)-. In some embodiments, L¹ is -C(O)(Ci-2 aliphatic)-.

[0256] In some embodiments, L¹ is -C(O)(C2-6 aliphatic)- . In some embodiments, L¹ is - C(O)(C3-6 aliphatic)-. In some embodiments, L¹ is -C(O)(C4-6 aliphatic)-. In some embodiments, L¹ is -C(O)(C5-6 aliphatic)- .

[0257] In some embodiments, L¹ is -C(O)(CH2)-. In some embodiments, L¹ is - C(O)(CH2CH2)-. In some embodiments, L¹ is -C(O)(CH2)3-. In some embodiments, L¹ is - C(O)(CH₂)₄-. In some embodiments, L¹ is -C(O)(CH2)5-. In some embodiments, L¹ is - C(O)(CH₂)₆-.

[0258] In some embodiments, L¹ is selected from those depicted in Table 1, below. L² and L³

[0259] As defined generally above, L² and L³ are independently a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁶)=N-, or -C(R⁶)=N-O-.

[0260] In some embodiments L² is a covalent bond.

[0261] In some embodiments, L² is C1-6 alkylene. In some embodiments, L² is C1.5 alkylene. In some embodiments, L² is C1.4 alkylene. In some embodiments, L² is C1.3 alkylene. In some embodiments, L² is C1.2 alkylene.

[0262] In some embodiments, L² is C2-6 alkylene. In some embodiments, L² is C3-6 alkylene. In some embodiments, L² is C4-6 alkylene. In some embodiments, L² is C5-6 alkylene.

[0263] In some embodiments L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, - OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, - C(R⁶)=N-, or -C(R⁶)=N-O-.

[0264] In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-. [0265] In some embodiments, L² is a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or -OC(O)N(R)-.

[0266] In some embodiments, L² is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0267] In some embodiments, L² is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or - OC(O)N(R)-.

[0268] In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -NR-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-S-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)2-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)O-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)N(R)-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(R⁶)=N-. In some embodiments, L² is C1-6 alkylene wherein one methylene unit of the Ci- 6 alkylene is replaced with -C(R⁶)=N-O-.

[0269] In some embodiments L² is selected from -O-, -OC(O)-, -C(O)O-, -OC(O)O-, -CH2O- , -CH₂OC(O)-, and -CH₂OC(O)O-. [0270] In some embodiments L² is -O-. In some embodiments L² is -OC(O)-. In some embodiments L² is -C(O)O-. In some embodiments L² is -OC(O)O-. In some embodiments L² is -CH2O-. In some embodiments L² is -CH2OC(O)-. In some embodiments L² is -CH2OC(O)O-. [0271] In some embodiments L³ is a covalent bond.

[0272] In some embodiments, L³ is C1-6 alkylene. In some embodiments, L³ is C1.5 alkylene. In some embodiments, L³ is C1.4 alkylene. In some embodiments, L³ is C1.3 alkylene. In some embodiments, L³ is C1.2 alkylene.

[0273] In some embodiments, L³ is C2-6 alkylene. In some embodiments, L³ is C3-6 alkylene. In some embodiments, L³ is C4-6 alkylene. In some embodiments, L³ is C5-6 alkylene.

[0274] In some embodiments L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, - OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, - C(R⁶)=N-, or -C(R⁶)=N-O-.

[0275] In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0276] In some embodiments, L³ is a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or -OC(O)N(R)-.

[0277] In some embodiments, L³ is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, or -N(R)C(O)-.

[0278] In some embodiments, L³ is a covalent bond or C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or - OC(O)N(R)-.

[0279] In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -NR-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S-S-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -S(O)2-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -OC(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)O-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -N(R)C(O)N(R)-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with -C(R⁶)=N-. In some embodiments, L³ is C1-6 alkylene wherein one methylene unit of the Ci- 6 alkylene is replaced with -C(R⁶)=N-O-.

[0280] In some embodiments L³ is selected from -O-, -OC(O)-, -C(O)O-, -OC(O)O-, -CH₂O- , -CH₂OC(O)-, and -CH₂OC(O)O-.

[0281] In some embodiments L³ is -O-. In some embodiments L³ is -OC(O)-. In some embodiments L³ is -C(O)O-. In some embodiments L³ is -OC(O)O-. In some embodiments L³ is -CH₂O-. In some embodiments L³ is -CH₂OC(O)-. In some embodiments L³ is -CH₂OC(O)O-. [0282] In some embodiments, L² and L³ are a covalent bond.

[0283] In some embodiments, L² and L³ are independently a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, - S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, - N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁶)=N-, or -C(R⁶)=N-O-.

[0284] In some embodiments, L² and L³ are independently selected from -O-, -OC(O)-, - C(O)O-, -OC(O)O-, -CH₂O-, -CH₂OC(O)-, and -CH₂OC(O)O-.

[0285] In some embodiments, L² and L³ are -O-. In some embodiments, L² and L³ are -OC(O)- . In some embodiments, L² and L³ are -C(O)O-. In some embodiments, L² and L³ are -OC(O)O- . In some embodiments, L² and L³ are -CH₂O-. In some embodiments, L² and L³ are -CH₂OC(O)- . In some embodiments, L² and L³ are -CH₂OC(O)O-.

[0286] In some embodiments, L² and L³ are independently selected from -O-, -OC(O)-, - C(O)O-, -OC(O)O-, -CH₂O-, -CH₂OC(O)-, and -CH₂OC(O)O-.

[0287] In some embodiments, L² is -OC(O)- and L³ is -CH₂OC(O)-.

[0288] In some embodiments, L² and L³ are the same. In some embodiments, L² and L³ are not the same. [0289] In some embodiments, L² is selected from those depicted in Table 1, below. In some embodiments, L³ is selected from those depicted in Table 1, below.

R¹

[0290] As defined generally above, R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl. In some embodiments, R¹ is H. In some embodiments, R¹ is C1-6 alkyl. In some embodiments, R¹ is -(C1-6 alkyl)-N3. In some embodiments, R¹ is -(C1-6 alkyl)-SH. In some embodiments, R¹ is C3-8 alkynyl. In some embodiments, R¹ is a C1-3 alkyl. In some embodiments, R¹ is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ is propyl. In some embodiments, R¹ is -(C1.3 alkyl)-N3. In some embodiments, R¹ is -CH2N3. In some embodiments, R¹ is -(C1.3 alkyl)-SH. In some embodiments, R¹ is -CH2SH. In some embodiments, R¹ is C3-5 alkynyl. In some embodiments, R¹ is C3 alkynyl. In some embodiments, R¹ is C4 alkynyl. In some embodiments, R¹ is C5 alkynyl. In some embodiments, R¹ is C5-8 alkynyl. In some embodiments, R¹ is C5 alkynyl. In some embodiments, R¹ is C6 alkynyl. In some embodiments, R¹ is C7 alkynyl. In some embodiments, R¹ is C8 alkynyl. In some embodiments, R¹ is not methyl. In some embodiments, R¹ is selected from those depicted in Table 1, below.

R² and R³

[0291] As defined generally above, R² and R³ are independently a straight or branched C6-30 alkyl, straight or branched C6-30 alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x.

[0292] In some embodiments, R² is a straight or branched C6-30 alkyl. In some embodiments, R² is -(CH2)6-25. In some embodiments, R² is -(CH2)10-25. In some embodiments, R² is -(CH2)10- 14. In some embodiments, R² is -(CH2)14-16. In some embodiments, R² is -(CH2)18-20.

[0293] In some embodiments, R² is a straight or branched C6-30 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkyl substituted with m instances of R^x.

[0294] In some embodiments, R² is a straight or branched C6-25 alkyl. In some embodiments, R² is a straight or branched C6-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkyl substituted with m instances of R^x. [0295] In some embodiments, R² is a straight or branched C10-25 alkyl. In some embodiments, R² is a straight or branched C10-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkyl substituted with m instances of R^x. [0296] In some embodiments, R² is a straight or branched C10-14 alkyl. In some embodiments, R² is a straight or branched C10-14 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-14 alkyl substituted with m instances of R^x. [0297] In some embodiments, R² is a straight or branched C14-16 alkyl. In some embodiments, R² is a straight or branched C14-16 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkyl substituted with m instances of R^x. [0298] In some embodiments, R² is a straight or branched C18-20 alkyl. In some embodiments, R² is a straight or branched C18-20 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkyl substituted with m instances of R^x.

[0299] In some embodiments, R² is a straight or branched C6-30 alkenyl. In some embodiments, R² is a straight or branched C6-25 alkenyl. In some embodiments, R² is C10-25 alkenyl. In some embodiments, R² is C10-14 alkenyl. In some embodiments, R² is C14-16 alkenyl. In some embodiments, R² is C18-20 alkenyl.

[0300] In some embodiments, R² includes one, two, three, or four carbon-carbon double bonds.

[0301] In some embodiments, R² is a straight or branched C6-30 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkenyl substituted with m instances of R^x.

[0302] In some embodiments, R² is a straight or branched C6-25 alkenyl. In some embodiments, R² is a straight or branched C6-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkenyl substituted with m instances of R^x.

[0303] In some embodiments, R² is a straight or branched C10-25 alkenyl. In some embodiments, R² is a straight or branched C10-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkenyl substituted with m instances of R^x.

[0304] In some embodiments, R² is a straight or branched C10-14 alkenyl. In some embodiments, R² is a straight or branched C10-14 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C 10-14 alkenyl substituted with m instances of R^x.

[0305] In some embodiments, R² is a straight or branched C14-16 alkenyl. In some embodiments, R² is a straight or branched C14-16 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkenyl substituted with m instances of R^x.

[0306] In some embodiments, R² is a straight or branched C18-20 alkenyl. In some embodiments, R² is a straight or branched C18-20 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkenyl substituted with m instances of R^x.

[0307] In some embodiments, R² is a straight or branched C6-30 alkynyl. In some embodiments, R² is -(CH2)C6-2.5 In some embodiments, R² is -(CH2)10-25. In some embodiments, R² is -(CH2)10-14. In some embodiments, R² is -(CH2)14-16. In some embodiments, R² is -(CH2)18- 20.

[0308] In some embodiments, R² has one, two, three, four, or more carbon-carbon triple bonds. [0309] In some embodiments, R² is a straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-30 alkynyl substituted with m instances of R^x.

[0310] In some embodiments, R² is a straight or branched C6-25 alkynyl. In some embodiments, R² is a straight or branched C6-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C6-25 alkynyl substituted with m instances of R^x.

[0311] In some embodiments, R² is a straight or branched C10-25 alkynyl. In some embodiments, R² is a straight or branched C10-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-25 alkynyl substituted with m instances of R^x.

[0312] In some embodiments, R² is a straight or branched C10-14 alkynyl. In some embodiments, R² is a straight or branched C10-14 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C10-14 alkynyl substituted with m instances of R^x.

[0313] In some embodiments, R² is a straight or branched C14-16 alkynyl. In some embodiments, R² is a straight or branched C14-16 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C14-16 alkynyl substituted with m instances of R^x.

[0314] In some embodiments, R² is a straight or branched C18-20 alkynyl. In some embodiments, R² is a straight or branched C18-20 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R² is a straight or branched C18-20 alkynyl substituted with m instances of R^x.

[0002] In some embodiments, R² is selected from those depicted in Table 1, below.

[0315] In some embodiments, R³ is a straight or branched C6-30 alkyl. In some embodiments, R³ is -(CH2)6-25. In some embodiments, R³ is -(CH2)10-25. In some embodiments, R³ is -(CH2)10- 14. In some embodiments, R³ is -(CH2)14-16. In some embodiments, R³ is -(CH2)18-20.

[0316] In some embodiments, R³ is a straight or branched C6-30 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkyl substituted with m instances of R^x.

[0317] In some embodiments, R³ is a straight or branched C6-25 alkyl. In some embodiments, R³ is a straight or branched C6-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkyl substituted with m instances of R^x.

[0318] In some embodiments, R³ is a straight or branched C10-25 alkyl. In some embodiments, R³ is a straight or branched C10-25 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkyl substituted with m instances of R^x.

[0319] In some embodiments, R³ is a straight or branched C10-14 alkyl. In some embodiments, R³ is a straight or branched C10-14 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-14 alkyl substituted with m instances of R^x.

[0320] In some embodiments, R³ is a straight or branched C14-16 alkyl. In some embodiments, R³ is a straight or branched C14-16 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkyl substituted with m instances of R^x.

[0321] In some embodiments, R³ is a straight or branched C18-20 alkyl. In some embodiments, R³ is a straight or branched C18-20 alkyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkyl substituted with m instances of R^x.

[0322] In some embodiments, R³ is a straight or branched C6-30 alkenyl. In some embodiments, R³ is C6-25 alkenyl. In some embodiments, R³ is C10-25 alkenyl. In some embodiments, R³ is C10-14 alkenyl. In some embodiments, R³ is C14-16 alkenyl. In some embodiments, R³ is C18-20 alkenyl.

[0323] In some embodiments, R³ is a straight or branched C6-30 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkenyl substituted with m instances of R^x.

[0324] In some embodiments, R³ is a straight or branched C6-25 alkenyl. In some embodiments, R³ is a straight or branched C6-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkenyl substituted with m instances of R^x.

[0325] In some embodiments, R³ is a straight or branched C10-25 alkenyl. In some embodiments, R³ is a straight or branched C10-25 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkenyl substituted with m instances of R^x.

[0326] In some embodiments, R³ is a straight or branched C10-14 alkenyl. In some embodiments, R³ is a straight or branched C10-14 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C 10-14 alkenyl substituted with m instances of R^x.

[0327] In some embodiments, R³ is a straight or branched C14-16 alkenyl. In some embodiments, R³ is a straight or branched C14-16 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkenyl substituted with m instances of R^x.

[0328] In some embodiments, R³ is a straight or branched C18-20 alkenyl. In some embodiments, R³ is a straight or branched C18-20 alkenyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkenyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkenyl substituted with m instances of R^x.

[0329] In some embodiments, R³ is a straight or branched C6-30 alkynyl. In some embodiments, R³ is C6-25 alkynyl. In some embodiments, R³ is C10-25 alkynyl. In some embodiments, R³ is C10-14 alkynyl. In some embodiments, R³ is C14-16 alkynyl. In some embodiments, R³ is C18-20 alkynyl.

[0330] In some embodiments, R³ has one, two, three, four, or more carbon-carbon triple bonds.

[0331] In some embodiments, R³ is a straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-30 alkynyl substituted with m instances of R^x.

[0332] In some embodiments, R³ is a straight or branched C6-25 alkynyl. In some embodiments, R³ is a straight or branched C6-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C6-25 alkynyl substituted with m instances of R^x.

[0333] In some embodiments, R³ is a straight or branched C10-25 alkynyl. In some embodiments, R³ is a straight or branched C10-25 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-25 alkynyl substituted with m instances of R^x.

[0334] In some embodiments, R³ is a straight or branched C10-14 alkynyl. In some embodiments, R³ is a straight or branched C10-14 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C10-14 alkynyl substituted with m instances of R^x.

[0335] In some embodiments, R³ is a straight or branched C14-16 alkynyl. In some embodiments, R³ is a straight or branched C14-16 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C14-16 alkynyl substituted with m instances of R^x.

[0336] In some embodiments, R³ is a straight or branched C18-20 alkynyl. In some embodiments, R³ is a straight or branched C18-20 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x. In some embodiments, R³ is a straight or branched C18-20 alkynyl substituted with m instances of R^x.

[0337] In some embodiments, R³ is selected from those depicted in Table 1, below.

[0338] In some embodiments, R² and R³ are the same. In some embodiments R² and R³ are different.

R⁶

[0339] As generally defined above, R⁶ is C1-6 alkyl or C2-14 alkenyl. In some embodiments, R⁶ is C1-6 alkyl. In some embodiments, R⁶ is C2-14 alkenyl. In some embodiments, R⁶ is C1.5 alkyl. In some embodiments, R⁶ is C1.4 alkyl. In some embodiments, R⁶ is C1.3 alkyl. In some embodiments, R⁶ is C1.2 alkyl. In some embodiments, R⁶ is C2-6 alkyl. In some embodiments, R⁶ is methyl. In some embodiments, R⁶ is ethyl. In some embodiments, R⁶ is propyl. In some embodiments, R⁶ is isopropyl. In some embodiments, R⁶ is butyl. In some embodiments, R⁶ is isobutyl. In some embodiments, R⁶ is pentyl. In some embodiments, R⁶ is hexyl. In some embodiments, R⁶ is C2-14 alkenyl. In some embodiments, R⁶ is C2-10 alkenyl. In some embodiments, R⁶ is C2-8 alkenyl. In some embodiments, R⁶ is C2-6 alkenyl. In some embodiments, R⁶ is C2-4 alkenyl. In some embodiments, R⁶ is selected from those depicted in Table 1, below. R^x

[0340] As generally defined above, each R^x is independently halogen, -CN, -OR, -SR, - C(O)R, -C(O)OR, or OC(O)OR. In some embodiments, R^x is halogen. In some embodiments, R^x is -CN. In some embodiments, R^x is -OR. In some embodiments, R^x is -SR. In some embodiments, R^x is -C(O)R. In some embodiments, R^x is -C(O)OR. In some embodiments, R^x is OC(O)OR. In some embodiments, R^x is selected from those depicted in Table 1, below. n

[0341] As defined generally above, n is an integer from 10-75.

[0342] In some embodiments, n is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-75. In some embodiments, n is 10-30, 20-40, 30-50, 40-60, or 50-75. In some embodiments, n is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75. In some embodiments, n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.

[0343] In some embodiments, n is an integer from 30-55, inclusive. In some embodiments, n is an integer from 40-50, inclusive. In some embodiments, n is 44, 45, or 46. In some embodiments, n is 44. In some embodiments, n is 45. In some embodiments, n is 46. m

[0344] As defined generally above, m is 0, 1, 2, 3, or 4. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 0, 1, or 2. In some embodiments, m is 1, 2, or 3.

[0345] In some embodiments, the present invention provides a compound of formula PL-IIc or PL-IId:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, X¹, R, R^x, m, and n, is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-.

[0346] In some embodiments, the present invention provides a compound of formula PL-IIe:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, X¹, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-.

[0347] In some embodiments, the present invention provides a compound of formula PL-IIf:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. [0348] In some embodiments, the present invention provides a compound of formula PL-IIg or PL-IIh:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, X¹, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. In some embodiments, X¹ is -N(H)-. [0349] In some embodiments, the present invention provides a compound of formula PL-IIa or PL-IIb:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, R, R^x, m, and n, is as defined above and described in embodiments herein, both singly and in combination.

[0350] In some embodiments, the present invention provides a compound of formula PL-IIk:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, L², L³, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination. [0351] In some embodiments, the present invention provides a compound of formula PL-IIm or PL-IIn:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁶, R, R^x, m, and n is as defined above and described in embodiments herein, both singly and in combination.

[0352] In some embodiments, the PEGylated lipid compound is one of those shown in Table

1. In some embodiments, the PEGylated lipid compound is selected from Compounds PL-1, PL-

2, PL-3, PL-4, PL-5, PL-6, PL-7, PL-8, PL-9, PL-10, PL-11, PL-12, PL-13, PL-14, PL-15, and PL-16; or a pharmaceutically acceptable salt thereof.

Table 1. Exemplary Compounds

c. Lipid Nanoparticle Compositions

[0353] In one aspect, the present disclosure further provides delivery systems for delivery of a therapeutic payload disclosed herein. In some embodiments, a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.

[0354] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid. In some embodiments, an LNP further comprises a 5^th lipid, besides any of the aforementioned lipid components. In some embodiments, the LNP encapsulates one or more elements of the active agent of the present disclosure. In some embodiments, an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system.

[0355] In some embodiments, an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about lOOnm, HOnm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, an LNP has a diameter of less than about lOOnm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60-100nm. In some embodiments, an LNP has a diameter of about 75-80nm.

[0356] In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%.

[0357] In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol- % of the phospholipid may be from about 5 mol-% to about 20 mol-%.

[0358] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%.

[0359] In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol- % to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. i. Ionizable lipids

[0360] In some embodiments, an LNP disclosed herein comprises an ionizable lipid. In some embodiments, an LNP comprises two or more ionizable lipids.

[0361] In some embodiments, an ionizable lipid has a dimethylamine or an ethanolamine head. In some embodiments, an ionizable lipid has an alkyl tail. In some embodiments, a tail has one or more ester linkages, which may enhance biodegradability. In some embodiments, a tail is branched, such as with 3 or more branches. In some embodiments, a branched tail may enhance endosomal escape. In some embodiments, an ionizable lipid has a pKa between 6 and 7, which may be measured, for example, by TNS assay.

[0362] In some embodiments, an ionizable lipid has a structure of any of the formulas disclosed below, and all formulas disclosed in a reference publication and patent application publication cited below. In some embodiments, an ionizable lipid comprises a head group of any structure or formula disclosed below. In some embodiments, an ionizable lipid comprises a bridging moiety of any structure or formula disclosed below. In some embodiments, an ionizable lipid comprises any tail group, or combination of tail groups disclosed below. The present disclosure contemplates all permutations and combinations of head group, bridging moiety and tail group, or tail groups, disclosed herein.

[0363] In some embodiments, a head, tail, or structure of an ionizable lipid is described in US patent application US20170210697A1.

[0364] In some embodiments, a compound has a structure according to formula 1 :

wherein:

R¹ is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, - R*YR", YR", and -R"M'R^†; R² and R³ are independently selected from the group consisting ofH, Cl-14 alkyl, C2-14 alkenyl, — R*YR", — YR", and — R*OR", or R² and R³, together with the atom to which they are attached, form a heterocycle or carbocycle;

R⁴ is selected from the group consisting of a C3-6 carbocycle, — (CH2)nQ, — (CH2)nCHQR, — CHQR, CO(R)2, and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, — O(CH₂)nN(R)₂, — C(O)OR, — OC(O)R, — CX₃, — CX₂H, -CXH₂, -CN, N(R)₂, - C(O)N(R)₂, - N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)₂, -N(R)R⁸, - O(CH₂)_nOR, -N(R)C(=NR⁹)N(R)₂ -N(R)C(=CHR⁹)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR, -N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂ - N(OR)C(— NR)N(R) - N(OR)C(=CHR⁹)N(R)₂, -C(=NR⁹)N(R)₂, — C(=NR⁹)R, -C(O)N(R)OR, and — C(R)N(R)2, C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5 or ahead group disclosed in Table 2A; each R⁵ is independently selected from the group consisting of Cl -3 alkyl, C2-3 alkenyl, and H; each R⁶ is independently selected from the group consisting of Cl -3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from — C(O)O-, -OC(O) — , -C(O)N(R’)-, -N(R')C(O)-, - C(O)—, — C(S)— , — C(S)S-, — SC(S)— , — CH(OH)— , — P(O)(OR’)O-, — S(O)— , — S-S-, an aryl group, and a heteroaryl group;

R⁷ is selected from the group consisting of C 1 -3alkyl, C2-3 alkenyl, and H;

R⁸ is selected from the group consisting of C3-6 carbocycle and heterocycle;

R⁹ is selected from the group consisting of H. CN, NCh, Cl -6 alkyl, -OR, — S(O)2R, — S(O)₂N(R)₂, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of Cl -3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, — R*YR", —YR", and H; each R” is independently selected from the group consisting of C3-14 alkyl, C3-14 alkenyl, and H; each R* is independently selected from the group consisting of Cl-12 alkyl and C2-12 alkenyl: each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; each Q is is -OH, -NHC(S)N(R)₂, -NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R⁸, - NHC(=NR⁹)N(R)₂, -NHC(=CHR⁹)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13: and wherein when R⁴ is — (CH₂)_nQ, — (CH₂)_nCHQR, — CHQR, or — CQ(R)₂, then (i) Q is not — N(R), when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

[0365] In some embodiments, R⁴ is in Table 2A.

[0366] In some embodiments, R⁴ in formula 1 is selected from head groups 1-47.

Table 2A - Ionizable lipid head groups

[0367] In some embodiments, a subset of the compounds of formula 1 are also described by formula lb:

[0368] Wherein 1 is selected from 1, 2, 3, 4, and 5; M¹ is a bond or M¹; R⁴is unsubstituted C1- 3 alkyl, or -(CH2)_nQ, in which n is 2, 3, or 4, and Q is -OH, -NHC(S)N(R)2, -NHC(O)N(R)2, - N(R)C(O)R, -N(R)S(O)₂R, -N(R)R⁸, -NHC(=NR⁹)N(R)₂, -NHC(=CHR⁹)N(R)₂, -OC(O)N(R)₂, - N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M' are independently selected from - C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -S-S-, an ary l group, and a heteroaryl group; and R² and R3 are independently selected from the group consisting of H, Ci-i4 alkyl, and C2-14 alkenyl.

[0369] In some embodiments, a head, tail, or structure of an ionizable lipid is described in international patent application PCT/US2018/058555.

[0370] In some embodiments, an ionizable lipid has a structure according to formula 2:

wherein: one of L¹ or L² is -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0)_x-, -S-S-, -C(=O)S-, -SC(=O)-, - NR^aC(=0)-, -C(=O)NR^a-, -NR^aC(=0)NR^a-, -0C(=0)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0)_x-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^aC(=0)-, - C(=O)NR^a-, -NR^aC(=0)NR^a-, -0C(=0)NR^a- or -NR^aC(=O)O- or a direct bond;

R^a is H or Ci -C 12 alkyl;

R^la and R^lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond; R^2a and R^2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R^3a and R^3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R^4a and R^4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently methyl or cycloalkyl;

R⁷ is, at each occurrence, independently H or C1-C12 alkyl;

R⁸ and R⁹ are each independently unsubstituted C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; and x is 0, 1 or 2.

[0371] In some embodiments, an ionizable lipid has a structure according to formula 3 :

wherein: one of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, -SC(=O)-, - NR^aC(=O)-, -C(=O)NR^a-, -NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^aC(=O)-, - C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O- or a direct bond;

G¹ is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR^aC(=O)- or a direct bond:

G² is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR^a- or a direct bond;

G³ is C1-C6 alkylene;

R^a is H or C1-C12 alkyl; R^la and R^lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond; R^2a and R^2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R^3a and R^3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R^4a and R^4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently H or methyl;

R⁷ is C4-C20 alkyl;

R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0372] In some embodiments, an ionizable lipid has a structure according to formula 4:

[0373] wherein: one of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, -SC(=O)-, - NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^aC(=O)-, - C(=O)NR^a-, -NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O- or a direct bond;

G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G³ is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

R^a is H or C1-C12 alkyl;

R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl; R⁵ is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0374] In some embodiments, an ionizable lipid has a structure according to formula 5:

wherein: one of G¹ or G² is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)y, -S-S-, - C(=O)S-, SC(=O)-, -N(R^a)C(=O)-, -C(=O)N(R^a)-, -N(R^a)C(=O)N(R^a)-, -OC(=O)N(R^a)- or - N(R^a)C(=O)O-, and the other of G¹ or G² is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_y, -S-S-, -C(=O)S-, -SC(=O)-, -N(R^a)C(=O)-, -C(=O)N(R^a)-, -N(R^a)C(=O)N(R^a)-, - OC(=O)N(R^a)- or -N(R^a)C(=O)O- or a direct bond;

L is, at each occurrence, ~O(C=O)-, wherein ~ represents a covalent bond to X;

X is CR^a;

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;

R^a is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;

R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;

R¹ and R² have, at each occurrence, the following structure, respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to 12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c² are, at each occurrence, independently an integer from 5 to 10; d¹ and d² are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.

[0375] In some embodiments, an ionizable lipid has a structure according to formula 6:

wherein: one of G¹ or G² is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)y, -S-S-, - C(=O)S-, SC(=O)-, -N(R^a)C(=O)-, -C(=O)N(R^a)-, -N(R^a)C(=O)N(R^a)-, -OC(=O)N(R^a)- or - N(R^a)C(=O)O-, and the other of G¹ or G² is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_y-, -S-S-, -C(=O)S-, -SC(=O)-, -N(R^a)C(=O)-, -C(=O)N(R^a)-, -N(R^a)C(=O)N(R^a)-, - OC(=O)N(R^a)- or -N(R^a)C(=O)O- or a direct bond;

L is, at each occurrence, ~O(C=O)-, wherein ~ represents a covalent bond to X;

X is CR^a; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;

R¹ and R² have, at each occurrence, the following structure, respectively:

R' is, at each occurrence, independently H or C1-C12 alkyl; a¹ and a² are, at each occurrence, independently an integer from 3 to 12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c² are, at each occurrence, independently an integer from 2 to 12; d¹ and d² are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a¹, a², c¹, c², d¹ and d² are selected such that the sum of a¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d² is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent. [0376] In certain embodiments of Formula (V), G¹ and G² are each independently -O(C=O)- or -(C=O)O-.

[0377] In some embodiments, an ionizable lipid has a disulfide tail.

[0378] In some embodiments, an ionizable lipid includes short peptides of 12-15 mer length as head groups. [0379] In some embodiments, the head of an ionizable lipid comprises the structure of Vitamin A, D, E, or K as described in the published Patent Application WO2019232095A1, which is incorporated by herein by reference in its entirety.

[0380] In some embodiments, a lipid is described in international patent applications W02021077067, or WO2019152557, each of which is incorporated herein by reference in its entirety.

[0381] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2019/0240354, which is incorporated herein by reference in its entirety.

[0382] In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula I:

or salts thereof, wherein:

R¹ and R² are either the same or different and are independently hydrogen (H) or an optionally substituted C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, or R³ and R² may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof;

R³ is either absent or is hydrogen (H) or a C1-C6 alkyl to provide a quaternary amine; R⁴ and R⁵ are either the same or different and are independently an optionally substituted C10-C24 alkyl, C10-C24 alkenyl, C10-C24 alkynyl, or C10-C24 acyl, wherein at least one of R⁴ and R⁵ comprises at least two sites of unsaturation; and n is 0, 1, 2, 3, or 4.

[0383] In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula II:

wherein R¹ and R² are either the same or different and are independently an optionally substituted C12-C24 alkyl, C12-C24 alkenyl, C12-C24 alkynyl, or C12-C24 acyl; R³ and R⁴ are either the same or different and are independently an optionally substituted C1-C6 alkyl, C2-C6 alkenyl, or C2- C6, alkynyl, or R³ and R⁴ may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or is hydrogen (H) or a Ci-C6 alkyl to provide a quaternary amine; m, n, and p are either the same or different and are independently either 0, 1, or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and are independently O, S, or NH. In some embodiments, q is 2.

[0384] In some embodiments, the cationic lipid of Formula II is 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane, 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]- dioxolane, 2,2-dihnoleyl-4-(4-dimethylaminobutyl)-[l,3]-dioxolane, 2,2-dilinoleyl-5- dimethylaminomethyl-[l,3]-dioxane, 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane, 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane, 2,2-dioleoyl-4-dimethylaminomethyl-[l,3]- dioxolane, 2,2-distearoyl-4-dimethylaminomethyl-[l,3]-dioxolane, 2,2-dilinoleyl-4-N- morpholino-[l,3]-dioxolane, 2,2-Dilinoleyl-4-trimethylamino-[l,3]-dioxolane chloride, 2,2- dilinoleyl-4,5-bis(dimethylaminomethyl)-[l,3]-di oxolane, 2,2-dilinoleyl-4-methylpiperzine- [l,3]-dioxolane, or mixtures thereof In some embodiments, the cationic lipid of Formula II is 2, 2-dilinol ey 1-4 -(2 -dimethylaminoethyl)- [1,3] -di oxolane.

[0385] In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula III:

[0386] or salts thereof, wherein: RJ and R² are either the same or different and are independently an optionally substituted Ci-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, or R¹ and R² may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (0), and mixtures thereof; R³ is either absent or is hydrogen (H) or a C1-C6 alkyl to provide a quaternary amine; R⁴ and R⁵ are either absent or present and when present are either the same or different and are independently an optionally substituted C1-C10 alkyl or C2-C10 alkenyl; and n is 0, 1, 2, 3, or 4.

[0387] In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula C:

X-A-Y — Z¹; (Formula C) or salts thereof, wherein:

X is — N(H)R or — NR₂;

A is absent, Ci to C6 alkyl, C2 to C6 alkenyl, or C2 to C6 alkynyl, which Cito C6 alkyl, C2to C6 alkenyl, and C2 to C6 alkynyl is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, — CN, — OR^X, — NR^xR^y, — NR^xC(=O)R^y, — NR^xSO₂R^y, — C(=O)R^X, — C(=O)OR^X, — C(=O)NR^xR^y, — SO_nR^x, and — SOnNR^xR^y, wherein n is 0, 1, or 2, and R^xand R^y are each independently hydrogen, alkyl, or heterocycle, wherein each alkyl and heterocycle of R^x and R^y may be further substituted with one or more groups independently selected from oxo, halogen, — OH, — CN, alkyl, — OR^X, heterocycle, — NR^xR^y , — NR^x'C(=O)R^y', — NR^X SO₂R^y, — C(=O)R^X, — C(=O)OR^X', — C(=O)NR^xR^y', — SO_nR^x', and — SO_n'NR^xR^y , wherein n' is 0, 1, or 2, and R^x and R^y are each independently hydrogen, alkyl, or heterocycle;

Y is selected from the group consisting of absent, — C(=O) — , — O — , — OC(=O) — , — C(=O)O— , — N(R^b)C(=O)— , — C(=O)N(R^b)— , — N(R^b)C(=O)O— , and — OC(=O)N(R^b)—

Z¹ is a Ci to C6 alkyl that is substituted with three or four R^x groups, wherein each R^xis independently selected from C6to C11 alkyl, C6 to C11 alkenyl, and C6to C11 alkynyl, which C6 to C11 alkyl, C6 to C11 alkenyl, and C6to C11 alkynyl is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, — CN, — OR^X, — NR^XR^y, — NR^xC(=O)R^y, — NR^xSO₂R^y, — C(=O)R^X, — C(=O)OR^X, — C(=O)NR^xR^y, — SO_nR^x, and — SO_nNR^xR^y, wherein n is 0, 1, or 2, and R^xand R^y are each independently hydrogen, alkyl, or heterocycle, wherein any alkyl and heterocycle of R^xand R^y may be further substituted with one or more groups independently selected from oxo, halogen, — OH, — CN, alkyl, — OR^X, heterocycle, — NR^xR^y , — NR^xC(=O)R^y', — NR^x SO₂R^y', — C(=O)R^X', — C(=O)OR^X', — C(=O)NR^xR^y , — SO_nR^x, and — SO_nNR^xR^y , wherein n' is 0, 1, or 2, and R^x and R^y are each independently hydrogen, alkyl, or heterocycle; each R is independently alkyl, alkenyl, or alkynyl, that is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, — CN, — OR^X, — NR^XR^y, — NR^xC(=O)R^y, — NR^xSO₂R^y, — C(=O)R^X, — C(=O)OR^X, — C(=O)NR^xR^y, — SO_nR^x, and — SO_nNR^xR^y, wherein n is 0, 1, or 2, and R^xand R^y are each independently hydrogen, alkyl, or heterocycle, wherein any alkyl and heterocycle of R^xand R^y may be further substituted with one or more groups independently selected from oxo, halogen, — OH, — CN, alkyl, — OR^X, heterocycle, — NR^xR^y , — NR^xC(=O)R^y', — NR^x SO₂R^y', — C(=O)R^X', — C(=O)OR^X', — C(=O)NR^xR^y , — SO_nR^x, and — SO_nNR^xR^y , wherein n' is 0, 1, or 2, and R^x and R^y are each independently hydrogen, alkyl, or heterocycle; and each R^b is H or Ci to C6alkyl.

[0388] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2010/0130588, which is incorporated herein by reference in its entirety.

[0389] In some embodiments, the lipids disclosed in US 2010/0130588 are of Formula I:

wherein R¹ and R² are independently selected and are H or C1-C3 alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation. In some embodiments, R³ and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C18), etc. In some embodiments, R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C14) and R⁴ is linoleyl (Cis).

[0390] In some embodiments, the lipid of Formula I is l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA) or l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0391] In some embodiments, the lipids disclosed in US 2010/0130588 are of Formula II:

wherein R¹ and R² are independently selected and are H or C1-C3 alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation.

[0392] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2021/0087135, which is incorporated herein by reference in its entirety.

[0393] In some embodiments, the lipids disclosed in US 2021/0087135 are of Formula (A):

or its N-oxide, or a salt or isomer thereof, wherein R'^a is R'^branched or R'^cyclic; wherein R 'branched is :

wherein:

denotes a point of attachment; wherein R^aα is H, and R^aβ . R^{a γ}. and R^aδ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl, wherein at least one of R^aβ , R^{a γ}, and R^aδ is selected from the group consisting of C2-12 alkyl and C2-12 alkenyl;

R² and R³ are each C1-14 alkyl;

R⁴ is selected from the group consisting of — (CH2)2OH, — (CH2)3OH, — (CH2)4OH, — (CH₂)₅OH and wherein:

denotes a point of attachment;

R¹⁰ is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C2- 3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R⁵ is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R⁶ is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; R⁷ is H;

M and M' are each independently selected from the group consisting of — C(O)O — and — OC(O)— ;

R' is a Ci-i2 alkyl or C2-12 alkenyl;

Y^a is a C3-6 carbocycle;

R* ^ais selected from the group consisting of C145 alkyl and C2-15 alkenyl;

1 is selected from the group consisting of 1, 2, 3, 4, and 5; s is 2 or 3; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0394] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2021/0128488, which is incorporated herein by reference in its entirety

[0395] In some embodiments, the lipids disclosed in US 2021/0128488 are of structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

L¹ is — O(C=O)R', — (C=O)OR¹, — C(=O)R¹, —OR¹, — S(O)_XR¹, — S— SR¹, — C('O)SR', — SC(=O)R', — NR^aC(=O)R¹, — C(=O)NR^bR^c, — NR^aC(=O)NR^bR^c, — OC(=O)NR^bR^c or — NR^aC(=O)OR¹;

L² is — O(C=O)R², — (C=O)OR², — C(=O)R², —OR², — S(O)_XR², — S— SR², — C(=O)SR², — SC(=O)R², — NR^dC(=O)R², — C(=O)NR^eR^f, — NR^dC(=O)NR^eR^f, — OC(=O)NR^eR^f; — NR^dC(=O)OR² or a direct bond to R²;

G¹ and G² are each independently C2-C12 alkylene or C2-C12 alkenylene;

G³ is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

R^a, R^b, R^d and R^e are each independently H or C1-C12 alkyl or C1-C12 alkenyl;

R^c and R^f are each independently C1-C12 alkyl or C2-C12 alkenyl;

R¹ and R² are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl;

R³ is — N(R⁴)R⁵;

R⁴ is Ci-C 12 alkyl;

R⁵ is substituted C1-C12 alkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted unless otherwise specified. [0396] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2020/0121809, which is incorporated herein by reference in its entirety.

[0397] In some embodiments the lipids disclosed in US 2020/0121809 have a structure of Formula II:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)_X— , — S— S— , — C(=O)S— , SC(=O)— , — NR^aC(=O)— , — C(=O)NR^a— , NR^aC(=O)NR^a— , — OC(=O)NR^a— or — NR^aC(=O)O— , and the other of L¹ or L² is — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)_X— , — S— S— , — C(=O)S— , SC(=O)— , — NR^aC(=O)— , — C(=O)NR^a— , NR^aC(=O)NR^a— , — OC(=O)NR^a— or — NR^aC(=O)O— or a direct bond;

G¹ is C1-C2 alkylene, — (C=O)— , — O(C=O)— , — SC(=O)— , — NR^aC(=O)— or a direct bond;

G² is — C(=O)— , — (C=O)O— , — C(=O)S— , — C(=O)NR^a— or a direct bond;

G³ is C1-C6 alkylene;

R^ais H or C1-C12 alkyl;

R^laand R^lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^lais H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^2aand R^2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^2ais H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^3aand R^3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R^3ais H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^4aand R^4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^4ais H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R⁵ and R⁶ are each independently H or methyl; R⁷ is C4-C20 alkyl;

[0398] In some embodiments, the lipids disclosed in US 2020/0121809 have a structure of Formula III:

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)_X— , — S— S— , — C(=O)S— , SC(=O)— , — NR^aC(=O)— , — C(=O)NR^a— , NR^aC(=O)NR^a— , — OC(=O)NR^a— or — NR^aC(=O)O— , and the other of L¹ or L² is — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)_X— , — S— S— , — C(=O)S— , SC(=O)— , — NR^aC(=O)— , — C(=O)NR^a— , NR^aC(=O)NR^a— , — OC(=O)NR^a— or — NR^aC(=O)O— or a direct bond;

R^ais H or C1-C12 alkyl;

R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR^S, CN, — C(=O)OR⁴, — OC(=O)R⁴ or — NR⁵C(=O)R⁴;

R⁴ is Ci-C 12 alkyl;

R⁵ is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0399] In some embodiments, the lipids disclosed in US 2020/0121809 have a structure of Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G¹ or G² is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)_y— , — S— S— , — C(=O)S— , SC(=O)— , — N(R^a)C(=O)— , — C(=O)N(R^a)— , — N(R^a)C(=O)N(R^a)— , — OC(=O)N(R^a)— or — N(R^a)C(=O)O— , and the other of G¹ or G² is, at each occurrence, — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)_y— , — S— S— , — C(=O)S— , — SC(=O)— , — N(R^a)C(=O)— , — C(=O)N(R^a)— , — N(R^a)C(=O)N(R^a)— , — OC(=O)N(R^a) — or — N(R^a)C(=O)O — or a direct bond;

L is, at each occurrence, — O(C=O) — , wherein - represents a covalent bond to X;

X is CR^a;

R^ais, at each occurrence, independently H, C1-C12 alkyl, Ci-C 12 hydroxylalkyl, Ci- C 12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, Ci-C 12 alkoxy carbonyl, Ci- C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;

R¹ and R² have, at each occurrence, the following structure, respectively:

[0400] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0108685, which is incorporated herein by reference in its entirety. [0401] In some embodiments, the lipids disclosed in US 2013/0108685 are represented by the following formula (I):

wherein:

R¹ and R² are, the same or different, each linear or branched alkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, or R¹ and R² are combined together to form dialkylmethylene, dialkenylmethylene, dialkynylmethylene or alkylalkenylmethylene,

X¹ and X³ are hydrogen atoms, or are combined together to form a single bond or alkylene, X³ is absent or represents alkyl having 1 to 6 carbon atoms, or alkenyl having 3 to 6 carbon atoms, when X³ is absent,

Y is absent, a and b are 0, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-3-yl, piperi din-3 -yl, piperidin-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L² are — O — ,

Y is absent, a and b are, the same or different, 0 to 3, and are not 0 at the same time, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-3- yl, piperi din-3 -yl, piperi din-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and L² are, the same or different, — O — , — CO — O — or — O — CO — ,

Y is absent, a and b are, the same or different, 0 to 3, L³ is a single bond, R³ is a hydrogen atom, and L¹ and L² are, the same or different, — O — , — CO — O — or — O — CO — , or

Y is absent, a and b are, the same or different, 0 to 3, L³ is — CO — or — CO — O — , R³ is pyrrolidin-2-yl, pyrrolidin-3-yl, piperi din-2 -yl, piperi din-3 -yl, piperi din-4-yl, morpholin-2-yl, morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, wherein at least one of the substituents is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L² are, the same or different, — O — , — CO — O — or — O — CO — , and when X³ is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms,

Y is a pharmaceutically acceptable anion, a and b are, the same or different, 0 to 3, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-2- yl, pyrrolidin-3-yl, piperidin-2-yl, piperi din-3 -yl, piperidin-4-yl, morpholin-2-yl, morpholin-3- yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and L² are, the same or different, — O — , — CO — O — or — O— CO— ).

[0402] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0195920, which is incorporated herein by reference in its entirety. [0403] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (I), which has a branched alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group and the teriary carbon):

Formula (I)

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); with respect to R¹ and R²,

(i) R¹ and R² are each, independently, optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R¹⁰;

(ii) R¹ and R², together with the nitrogen atom to which they are attached, form an optionally substituted heterocylic ring; or

(iii) one ofR¹ and R² is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 member heterocyclic ring or heteroaryl (e.g., a 6- member ring) with (a) the adjacent nitrogen atom and (b) the (R)_a group adjacent to the nitrogen atom; each occurrence of R is, independently, — (CR³R⁴) — ; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH2, R¹⁰, alkylamino, or dialkylamino (In some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl); each occurrence of R¹⁰ is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein the compound of formula has at most two R¹⁰ groups (preferably at most one R¹⁰ group); the dashed line to Q is absent or a bond; when the dashed line to Q is absent then Q is absent or is — O — , — NH — , — S — , — C(O) — , — C(O)O — , — OC(O)— , — C(O)N(R⁴)— , — N(R⁵)C(O)— , — S— S— , — OC(O)O— , — O— N=C(R⁵)— , — C(R⁵)=N— O— , — OC(O)N(R⁵)— , — N(R⁵)C(O)N(R⁵)— , — N(R⁵)C(O)O— , — C(O)S— , — C(S)O— or — C(R⁵)=N— O— C(O)— ; or when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R⁵ is, independently, H or alkyl (e.g. C1-C4 alkyl);

X and Y are each, independently, alkylene or alkenylene (e.g., C4to C20 alkylene or C4to C20 alkenylene);

M¹ and M² are each, independently, a biodegradable group (e.g., — OC(O) — , — C(O)O — , — SC(O)— , — C(O)S— , — OC(S)— , — C(S)O, — S— S— , C(R⁵)=N— , — N=C(R⁵)— , — C(R⁵)=N— O— , — O— N=C(R⁵)— , — C(O)(NR⁵)— , — N(R⁵)C(O)— , — C(S)(NR⁵)— , — N(R⁵)C(O)— , — N(R⁵)C(O)N(R⁵)— , — OC(O)O— , — OSi(R⁵)₂O— , — C(O)(CR³R⁴)C(O)O— , — OC(O)(CR³R⁴)C(O)— , or

wherein R¹¹ is a C2-C8 alkyl or alkenyl; each occurrence of R^zis, independently, Ci-C8 alkyl (e.g., methyl, ethyl, isopropyl, n-butyl, n- pentyl, or n-hexyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3; and

Z¹ and Z² are each, independently, C8-C11 alkyl or C8-C11 alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

[0404] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (II), which has a branched alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

Formula (It)

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

(iii) one ofR¹ and R² is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 membered heterocyclic ring or heteroaryl (e.g., a 6- member ring) with (a) the adjacent nitrogen atom and (b) the (R)_a group adjacent to the nitrogen atom; each occurrence of R is, independently, — (CR³R⁴) — ; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH2, R¹⁰, alkylamino, or dialkylamino (In some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl); each occurrence of R¹⁰ is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein the compound of formula has at most two R¹⁰ groups (preferably at most one R¹⁰ group); the dashed line to Q is absent or a bond; when the dashed line to Q is absent then Q is absent or is — O — , — NH — , — S — , — C(O) — , — C(O)O — , — OC(O)— , — C(O)N(R⁴)— , — N(R⁵)C(O)— , — S— S— , — OC(O)O— , — O— N=C(R⁵)— , — C(R⁵)=N— O— , — OC(O)N(R⁵)— , — N(R⁵)C(O)N(R⁵)— , — N(R⁵)C(O)O— , — C(O)S— , — C(S)O— or — C(R⁵)=N— O— C(O)— ; or when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R⁵ is, independently, H or alkyl;

X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group

wherein R¹¹ is a C₂-C8 alkyl or alkenyl; each occurrence of R^zis, independently, Ci-C8 alkyl (e.g., methyl, ethyl, isopropyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3; and

Z¹ and Z² are each, independently, C8-C11 alkyl or C8-C11 alkenyl, wherein (i) the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z²; and (ii) the terminus of at least one of Z¹ and Z² is separated from the group M¹ or M² by at least 8 carbon atoms.

[0405] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (III), which has a branching point at a position that is 2-6 carbon atoms (i.e., at the beta (P), gamma (γ), delta (δ), epsilon (ε) or zeta position (ζ) adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

Formula (III)

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R', R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², R^z, a, and b are defined as in formula (I);

L¹ and L² are each, independently, C1-C5 alkylene or C2-C5 alkenylene;

X and Y are each, independently, alkylene (e.g., CUo C20 alkylene or C6-C8 alkylene) or alkenylene (e.g., C4 to C20 alkenylene); and

Z¹ and Z² are each, independently, C8-C11 alkyl or C8-C11 alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z². and with the proviso that the terminus of at least one of Z¹ and Z² is separated from the group M¹ or M² by at least 8 carbon atoms.

[0406] In some embodiments, the cationic lipid disclosed in US 2013/0195920 is a compound of formula (IV), which has a branching point at a position that is 2-6 carbon atoms (i.e., at beta (P), gamma (y), delta (5), epsilon (a) or zeta position (Q adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

Formula (IV)

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R', R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M², R^z, a, and b are defined as in formula (I);

L¹ and L² and are each, independently, C1-C5 alkylene or C2-C5 alkenylene; X and Y are each, independently, alkylene or alkenylene (e.g., C12-C 20 alkylene or C12- C20 alkenylene); and each occurrence of Z is independently C1-C4 alkyl (preferably, methyl).

[0407] For example, in some embodiments, -L¹-C(Z)3 is — CH2C(CH3)3. In some embodiments, -L¹-C(Z)3 is — CH2CH2C(CH3)3.

[0408] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (V), which has an alkoxy or thioalkoxy (i.e., — S-alkyl) group substitution on at least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R', R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as in formula (I);

X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group;

Z¹ and Z² are each, independently, C8-C11 alkyl or C8-C11 alkenyl, wherein (i) the C8-C11 alkyl or Cx-C 14 alkenyl of at least one of Z¹ and Z² is substituted by one or more alkoxy (e.g., a Ci- C4 alkoxy such as — OCH3) or thioalkoxy (e.g., a C1-C4 thioalkoxy such as — SCH3) groups, and (ii) the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

[0409] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VIA), which has one or more fluoro substituents on at least one tail at a position that is either alpha to a double bond or alpha to a biodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R¹, R², R, a, and b are as defined with respect to formula (I);

Q is absent or is — O— , — NH— , — S— , — C(O)— , — C(O)O— , — OC(O)— , — C(O)N(R⁴)— , — N(R⁵)C(O)— , — S— S— , — OC(O)O— , — O— N=C(R⁵)— , — C(R⁵)=N— O— , — OC(O)N(R⁵)— , — N(R⁵)C(O)N(R⁵)— , — N(R⁵)C(O)O — , — C(O)S— , — C(S)O— or — C(R⁵)=N— O— C(O)— ; R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); and each of R⁹ and R¹⁰ are independently C12-C24 alkyl (e.g., C12-C20 alkyl), C12-C24 alkenyl (e.g., C12- C20 alkenyl), or C12-C24 alkoxy (e.g., C12-C20 alkoxy) (a) having one or more biodegradable groups and (b) optionally substituted with one or more fluorine atoms at a position which is (i) alpha to a biodegradable group and between the biodegradable group and the tertiary carbon atom marked with an asterisk (*), or (ii) alpha to a carbon-carbon double bond and between the double bond and the terminus of the R⁹ or R¹⁰ group; each biodegradable group independently interrupts the C 12-C24 alkyl, alkenyl, or alkoxy group or is substituted at the terminus of the C12-C24 alkyl, alkenyl, or alkoxy group, wherein

(i) at least one of R⁹ and R¹⁰ contains a fluoro group;

(ii) the compound does not contain the following moiety:

wherein - is an optional bond; and

(iii) the terminus of R⁹ and R¹⁰ is separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms).

[0410] In some embodiments, the terminus of R⁹ and R¹⁰ is separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 18-22 carbon atoms (e.g., 18-20 carbon atoms).

[0411] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VIB), which has one or more fluoro substituents on at least one tail at a position that is either alpha to a double bond or alpha to a biodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R', R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as in formula (I); X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group; and

Z¹ and Z² are each, independently, C8-C11 alkyl or C8-C11 alkenyl, wherein said C8-C11 alkenyl is optionally substituted by one or more fluorine atoms at a position that is alpha to a double bond, wherein at least one of X, Y, Z¹, and Z² contains a fluorine atom.

[0412] In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VII), which has an acetal group as a biodegradable group in at least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

R', R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, a, and b are defined as in formula (I);

wherein R¹¹ is a C4-C10 alkyl or C4-C10 alkenyl; with the proviso that at least one of M¹ and M² is

, and

Z¹ and Z² are each, independently, C4-C14 alkyl or C4-C14 alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

[0413] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2015/0005363, which is incorporated herein by reference in its entirety.

[0414] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2014/0308304, which is incorporated herein by reference in its entirety.

[0415] In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of formula (I):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

Xaa is a D- or L-amino acid residue having the formula — NR^N — CR ¹ R² — (C=O) — , or a peptide of amino acid residues having the formula — {NR^N — CR'R² — (C=O)}_n — , wherein n is 2 to 20; R¹ is independently, for each occurrence, a non-hydrogen, substituted or un substituted side chain of an amino acid;

R² and R^Nare independently, for each occurrence, hydrogen, an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, or any combination of the foregoing, and having from 1 to 20 carbon atoms, C(1-5)alkyl, cycloalkyl, cycloalkylalkyl, C(3-5)alkenyl, C(3- 5)alkynyl, C(1-5)alkanoyl, C(1-5)alkanoyloxy, C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl, C(1-5)alkoxy- C(1-5)alkoxy, C(1-5)alkyl-amino-C(1-5)alkyl-, C(1-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl, cyano-C(1-5)alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl, carboxyl, or hydroxyl;

Z is NH, O, S, — CH2S — , — CH2S(O) — , or an organic linker consisting of 1-40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms (preferably, Z is NH or O);

R^x and R^y are, independently, (i) a lipophilic tail derived from a lipid (which can be naturally- occurring or synthetic), phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail optionally includes a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and an organic protecting group; or (iii) a substituted or unsubstituted C(3-22)alkyl, C(6- i2)Cycloalkyl, C(6-i2)Cycloalkyl-C(3-22)alkyl, C(3-22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or C(6-i2)- alkoxy-C(3-22)alkyl; one of R^x and R^y is a lipophilic tail as defined above and the other is an amino acid terminal group, or both R^x and R^y are lipophilic tails; at least one of R^x and R^y is interrupted by one or more biodegradable groups (e.g., — OC(O) — , — C(O)O— , — SC(O)— , — C(O)S— , — OC(S)—, — C(S)O— , — S— S— — C(R⁵)=N— , — N=C(R⁵)— , — C(R⁵)=N— O— , — O— N=C(R⁵)— , — C(O)(NR⁵)— , — N(R⁵)C(O)— , — C(S)(NR⁵)— , — N(R⁵)C(O)—, — N(R⁵)C(O)N(R⁵)— , — OC(O)O— , — OSi(R⁵)₂O— , — C(O)(CR³R⁴)C(O)O— , — OC(O)(CR³R⁴)C(O)— or

(wherein R¹¹ is a C₂-C8 alkyl or alkenyl), in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH₂, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl)); and

R^xand R^y each, independently, optionally have one or more carbon-carbon double bonds.

[0416] In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of formula (IA):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

Z and Xaa are as defined with respect to formula (I) (the variables which are used in the definition of Xaa, namely R^N, R¹ and R², are also as defined in formula (I)); each occurrence of R is, independently, — (CR³R⁴) — ; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH₂, alkylamino, or dialkylamino (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl); or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group, wherein no more than three R groups in each chain between the — Z-Xaa-C(O) — and Z² moieties are cycloalkyl (e.g., cyclopropyl);

Q¹ and Q² are each, independently, absent, — O — , — S — , — OC(O) — , — C(O)O — , — SC(O) — , — C(O)S— , — OC(S)— , — C(S)O— , — S— S— , — C(O)(NR⁵)— , — N(R⁵)C(O)— , — C(S)(NR⁵)— , — N(R⁵)C(O)— , — N(R⁵)C(O)N(R⁵)— , or — OC(O)O— ;

Q³ and Q⁴ are each, independently, H, — (CR³R⁴) — , cycloalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, or a cholesterol moiety; each occurrence of A¹, A², A³ and A⁴ is, independently, — (CR⁵R⁵ — CR⁵=CR⁵) — ;

M¹ and M² are each, independently, a biodegradable group (e.g., — OC(O) — , — C(O)O — , — SC(O)— , — C(O)S— , — OC(S)— , — C(S)O— , — S— S— , — C(R⁵)=N— , — N=C(R⁵)— , — C(R⁵)=N— O— , — O— N=C(R⁵)— , — C(O)(NR⁵)— , — N(R⁵)C(O)— , — C(S)(NR⁵)— , — N(R⁵)C(O)— , — N(R⁵)C(O)N(R⁵)— , — OC(O)O— , — OSi(R⁵)₂O— , — C(O)(CR³R⁴)C(O)O— , — OC(O)(CR³R⁴)C(O)— , or

(wherein R¹¹ is a C₂-C8 alkyl or alkenyl)); each occurrence of R⁵ is, independently, H or alkyl (e.g., C1-C4 alkyl);

Z² is absent, alkylene or — O — P(O)(OH) — O — ; each - attached to Z² is an optional bond, such that when Z² is absent, Q³ and Q⁴ are not directly covalently bound together; c, d, e, f, i, j, m, n, q and r are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; g and h are each, independently, 0, 1 or 2; k and 1 are each, independently, 0 or 1, wherein at least one of k and 1 is 1; o and p are each, independently, 0, 1 or 2; and

Q³ and Q⁴ are each, independently, separated from the — Z-Xaa-C(O) — moiety by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms). [0417] In some embodiments the lipids disclosed in US 2014/0308304 are of the formula (IC):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein

Z and Xaa are as defined with respect to formula (I) (the variables which are used in the definition of Xaa, namely R^N, R¹ and R², are also as defined in formula (I)); each of R⁹ and R¹⁰ are, independently, alkylene or alkenylene; each of Rⁿ and R¹² are, independently, alkyl or alkenyl, optionally terminated by COOR¹³ wherein each R¹³ is independently unsubstituted alkyl (e.g., C1-C4 alkyl such as methyl or ethyl), substituted alkyl (such as benzyl), or cycloalkyl;

wherein Rⁿ is a C₂-C8 alkyl or alkenyl, in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH₂, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl));

R⁹, M¹, and Rⁿ are together at least 8 carbon atoms in length (e.g., 12 or 14 carbon atoms or longer); and

R¹⁰, M², and R¹² are together at least 8 carbon atoms in length (e.g., 12 or 14 carbon atoms or longer).

[0418] In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula II:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: s is 1, 2, 3 or 4; and

R⁷ is selected from lysyl, ornithyl, 2,3-diaminobutyryl, histidyl and an acyl moiety of the formula:

t is 1, 2 or 3; the NH3 ⁺ moiety in the acyl moiety in R⁷ is optionally absent; each occurrence of Y“ is independently a pharmaceutically acceptable anion (e.g., halide, such as chloride);

R⁵ and R⁶ are each, independently a lipophilic tail derived from a naturally-occurring or synthetic lipid, phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail may contain a steroid; or a substituted or unsubstituted C(3-22)alkyl, C(6-i2)Cycloalkyl, C(6-i2)Cycloalkyl-C(3-22)alkyl, C(3- 22)alkenyl, C(3-22)alkynyl, C(3-22)alkoxy, or C(6-i2)alkoxy-C(3-22)alkyl; at least one of R⁵ and R⁶ is interrupted by one or more biodegradable groups (e.g., — SC(O) — , — C(O)S— , — OC(S)— , — C(S)O— , — S— S— , — C(O)(NR^a)— , — N(R^a)C(O)— , — C(S)(NR^a)— , — N(R^a)C(O)— , — N(R^a)C(O)N(R^a)— , or — OC(O)O— ); each occurrence of R^ais, independently, H or alkyl; and

R⁵ and R⁶ each, independently, optionally contain one or more carbon-carbon double bonds. [0419] In some embodiments, the lipids disclosed in US 2014/0308304 are of the formula (IIA):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein:

R⁷ and s are as defined with respect to formula (II); each occurrence of R is, independently, — (CR³R⁴) — ; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH₂, alkylamino, or dialkylamino (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C1-C4 alkyl); or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group, wherein no more than three R groups in each chain attached to the nitrogen N* are cycloalkyl (e.g., cyclopropyl);

Q³ and Q⁴ are each, independently, H, — (CR³R⁴) — , aryl, cycloalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or a cholesterol moiety; each occurrence of A¹, A², A³ and A⁴ is, independently, — (CR⁵R⁵ — CR⁵=CR⁵) — ;

wherein R¹¹ is a C₂-C8 alkyl or alkenyl; each occurrence of R⁵ is, independently, H or alkyl;

Z is absent, alkylene or — O — P(O)(OH) — O — ; each - attached to Z is an optional bond, such that when Z is absent, Q³ and Q⁴ are not directly covalently bound together; c, d, e, f, i, j, m, n, q and r are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; g and h are each, independently, 0, 1 or 2; k and 1 are each, independently, 0 or 1, where at least one of k and 1 is 1; and o and p are each, independently, 0, 1 or 2.

[0420] In some embodiments the lipid disclosed in US 2014/0308304 are of the formula (IIC):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein:

R⁷ and s are as defined with respect to formula (II); each of R⁹ and R¹⁰ are independently alkyl (e.g., C12-C24 alkyl) or alkenyl (e.g., C12-C24 alkenyl); each of R¹¹ and R¹² are independently alkyl or alkenyl, optionally terminated by COOR¹³ where each R¹³ is independently alkyl (e.g., C1-C4 alkyl such as methyl or ethyl);

wherein R¹¹ is a C2-C8 alkyl or alkenyl; in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, — NH2, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently, H or Ci- C4 alkyl));

R⁹, M¹, and R¹¹ are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer); and

R¹⁰, M², and R¹² are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer).

[0421] In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula (4):

wherein:

X is N or P;

R¹, R², R, a, b, M¹, and M² are as defined with respect to formula (I);

Q is absent or is — O— , — NH— , — S— , — C(O)O— , — OC(O)— , — C(O)N(R⁴)— , — N(R⁵)C(O)— , — S— S— , — OC(O)O— , — O— N=C(R⁵)— , — C(R⁵)=N— O— , — OC(O)N(R⁵)— , — N(R⁵)C(O)N(R⁵)— , — N(R⁵)C(O)O— , — C(O)S— , — C(S)O— or — C(R⁵)=N— O— C(O)— ;

R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); each of R⁹ and R¹⁰ are independently alkylene, or alkenylene; and each of R¹¹ and R¹² are independently alkyl or alkenyl, optionally terminated by COOR¹³ where each R¹³ is independently alkyl (e.g., C1-C4 alkyl such as methyl or ethyl);

[0422] In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula (5)

wherein:

X is N or P;

R¹, R², R, a, and b are as defined with respect to formula (I);

Q is absent or is — O— , — NH— , — S— , — C(O)O— , — OC(O)— , — C(O)N(R⁴)— , — N(R⁵)C(O)— , — S— S— , — OC(O)O— , — O— N=C(R⁵)— , — C(R⁵)=N— O— , — OC(O)N(R⁵)— , — N(R⁵)C(O)N(R⁵)— , — N(R⁵)C(O)O— , — C(O)S— , — C(S)O— or — C(R⁵)=N — O — C(O) — ;R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); each of R⁹ and R¹⁰ are independently C12-C24 alkyl or alkenyl substituted at its terminus with a biodegradable group, such as — COOR¹³ where each R¹³ is independently alkyl (preferably Ci- C4 alkyl such as methyl or ethyl).

[0423] In some embodiments the lipids disclosed in US 2014/0308304 are of Formula A:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0-6 (e.g., n is 0, 1 or 2);

R¹ and R² are independently selected from H, (C1-C6)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more sub stituents selected from R', or R¹ and R² can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 3-7 (e.g., 4-7) members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';

R³ is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R³ can be taken together with R¹ to form a monocyclic heterocycle with 3-7 (e.g., 4-7) members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'; each occurrence of R⁴, R³ and R⁴ is independently selected from H, (C1-C6)alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R'; or R³ and R⁴ when directly bound to the same carbon atom form an oxo (=0) group, cyclopropyl or cyclobutyl; or R³ and R⁴ form an oxo (=0) group;

R⁵ is selected from H and (C1-C6)alkyl; or R⁵ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; each occurrence of R" is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;

L¹ is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl is optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with one or more sub stituents selected from R'; and

L² is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl is optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with one or more sub stituents selected from R'; with the proviso that the CR³R⁴ group when present adjacent to the nitrogen atom in formula A is not a ketone ( — C(O) — ).

[0424] In some embodiments the lipids disclosed in US 2014/0308304 are of formula B:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0, 1, 2, 3, 4, or 5;

R⁶ and R⁷ are each independently (i) C1-C4 linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R', or (ii) C3-C8 cycloalkyl (e.g., C3-C6 cycloalkyl); or R⁶ and R⁷ together with the nitrogen atom adjacent to them form a 3-6 membered ring;

L¹ is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 sub stituents selected from R'; and

L² is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 sub stituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; and each occurrence of R" is independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

[0425] In some embodiments, lipids disclosed in US 2014/0308304 are of formula C:

L¹ is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally has one or more biodegradable groups; each biodegradable group independently interrupts the alkyl or alkenyl group or is substituted at the terminus of the alkyl or alkenyl group, and said alkyl or alkenyl is optionally substituted with 1-5 sub stituents selected from R'; and

[0426] In some embodiments, the lipid disclosed in US 2014/0308304 are of formula D:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is 0, 1, 2, or 3; n is 0, 1, 2, 3, 4, or 5;

L¹ is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R'; and L² is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; and each occurrence of R" is independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

[0427] In some embodiments lipid disclosed in US 2014/0308304 are of formula E:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is 0, 1, 2, 3, 4, or 5; the group “amino acid” is an amino acid residue;

L¹ is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 sub stituents selected from R'; and

L² is a C4-C22 alkyl or C4-C22 alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 sub stituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; and each occurrence of R" is independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

[0428] The amino acid residue in formula E may have the formula — C(O) — C(R⁹)(NH2), where R⁹ is an amino acid side chain.

[0429] In some embodiments, the lipid disclosed in US 2014/0308304 are of formula F:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R⁶ and R⁷ are independently (i) C1-C4 linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R', or (ii) C3-C8 cycloalkyl (e.g., C3-C6 cycloalkyl); or R⁶ and R⁷ together with the nitrogen atom adjacent to them form a 3-6 membered ring;

L¹ is a C4-C22 alkyl or C4-C22 alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R'; and

L² is a C4-C22 alkyl or C4-C22 alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; each occurrence of R" is independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

[0430] In some embodiments, the lipid disclosed in US 2014/0308304 are of formula G:

Formula G

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0, 1, 2, 3, 4, or 5; q is 1, 2, 3, or 4

R⁶ and R⁷ are independently (i) C1-C4 linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R', or (ii) C3-C8 cycloalkyl (e.g., C3-C6 cycloalkyl);

L² is a C4-C22 alkyl or C4-C22 alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R'; each occurrence of R' is independently selected from halogen, R", OR", SR", CN, CO2R" and CON(R")₂; each occurrence of R" is independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH. [0431] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0053572, which is incorporated herein by reference in its entirety.

[0432] In some embodiments, the lipids disclosed in US 2013/0053572 are of Formula A:

wherein: n is 0, 1 or 2;

R¹ and R² are independently selected from H, (C1-C6)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more substituents selected from R', or R¹, and R² can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'; R³ is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R³ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';

R⁴ is selected from H, (C1-C6)alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R';

R⁵ is selected from H and (C1-C6)alkyl; or R⁵ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';

R' is independently selected from halogen, R", OR", CN, CO2R" and CON(R")2;

R" is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;

Li is a C4-C22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R'; and L2 is a C4-C22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R'; or any pharmaceutically acceptable salt or stereoisomer thereof.

[0433] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US Application publication US2017/0119904, which is incorporated by reference herein, in its entirety.

[0434] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application publication WO2021/204179, which is incorporated by reference herein, in its entirety.

[0435] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application WO2022251665A1, which is incorporated by reference herein, in its entirety.

[0436] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, selected from one of those in Table 2B below, or a pharmaceutically acceptable salt thereof.

Table 2B. Exemplary Ionizable Lipids

[0437] In some embodiments, the ionizable lipid is MC3.

[0438] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044343A1, which is incorporated by reference herein, in its entirety. Formula (VII-A)

[0439] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-A):

or a pharmaceutically acceptable salt thereof, wherein:

A is -N(-X'R')-, -C(R')(-L1-N(R")R6)-, -C(R')(-OR^7a)-, -C(R')(-N(R")R^8a)- , -C(R')(-C(=O)OR^9a)-, -C(R')(-C(=O)N(R")R^10a)-, or -C(=N-R^lla)-;

T is -X^2a-Y^la-Q^la or -X³-C(=O)OR⁴;

X¹ is optionally substituted C2-C6 alkylenyl;

R¹ is -OH, -R^la,

Z¹ is optionally substituted C1-C6 alkyl;

Z^la is hydrogen or optionally substituted C1-C6 alkyl;

X² and X^2a are independently optionally substituted C2-C14 alkylenyl or optionally substituted C2-C14 alkenyl enyl;

X³ is optionally substituted C2-C14 alkylenyl or optionally substituted C2-C14 alkenylenyl;

(i) Y¹ is

wherein the bond marked with an is attached to X^2a; each Z² is independently H or optionally substituted Ci-C8 alkyl; each Z³ is indpendently optionally substituted C1-C6 alkylenyl;

Q¹ is -NR²R³, -CH(OR²)(OR³), -CR²=C(R³)(R¹²), or -C(R²)(R³)(R¹²);

Q^la is -NR²'R³', -CH(OR²')(OR³'), -CR²=C(R³)(R¹²), or -C(R²')(R³')(R¹²'); or

wherein the bond marked with an "*" is attached to X²;

wherein the bond marked with an is attached to X^2a; each Z² is independently H or optionally substituted Ci-C8 alkyl; each Z³ is independently optionally substituted C1-C6 alkylenyl;

Q¹ is -NR²R³;

Q^la is -NR²R³ ;

R², R³, and R¹² are independently hydrogen, optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenylenyl, or -(CH2)m-G-(CH2)_nH;

R², R³ , and R¹²' are independently hydrogen, optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenylenyl, or -(CH2)m-G-(CH2)_nH;

G is a C3-C8 cycloalkylenyl; each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

X³ is optionally substituted C2-C14 alkylenyl;

R⁴ is optionally substituted C4-C14 alkyl;

L¹ is Ci-C8 alkylenyl;

R⁶ is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl

R^7a is -C(=O)N(R"')R^7b, -C(=S)N(R"')R^7b, -N=C(R^7b)(R^7c), or

R^7b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^7C is hydrogen or C1-C6 alkyl;

R^8a is -C(=O)N(R"')R^8b, -C(=S)N(R"')R^8b, -N=C(R^8b)(R^8c), or

R^8b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^8C is hydrogen or C1-C6 alkyl; R^9a is -N=C(R^9b)(R^9c);

R^9b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)Ci-C6 alkyl;

R^9C is hydrogen or C1-C6 alkyl;

Ri°^a i_s -N=C(R^10b)(R^10c);

R^10b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^10c is hydrogen or C1-C6 alkyl;

R^lla is -OR^llb, -N(R")R^llb, -OC(=O)R^llb, or -N(R")C(=O)R^llb;

R^llb is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R' is hydrogen or C1-C6 alkyl;

R" is hydrogen or C1-C6 alkyl; and

R'" is hydrogen or C1-C6 alkyl.

Formula (VIII- A)

[0440] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-A), wherein the Lipids of the Disclosure have a structure of Formula (VIILA):

or a pharmaceutically acceptable salt thereof.

Formula (VII-B)

[0441] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B):

or a pharmaceutically acceptable salt thereof, wherein:

A is -C(R')(-L1-N(R")R6)-, -C(R')(-OR^7a)-, -C(R')(-N(R")R^8a)-

, -C(R')(-C(=O)OR^9a)-, -C(R')(-C(=O)N(R")R^10a)-, or -C(=N-R^lla)-;

T is -X^2a-Y^la-Q^la or -X³-C(=O)OR⁴;

X² and X^2a are independently optionally substituted C2-C14 alkylenyl or optionally subsituted C2-C14 alkenylenyl;

X³ is optionally substituted C1-C14 alkylenyl or optionally substituted C2-C14 alkenylenyl;

Y¹ is

wherein the bond marked with an "*" is attached to X²;

Y^la is

wherein the bond marked with an is attached to X^2a; each Z³ is independently optionally substituted C1-C6 alkylenyl or optionally substituted C2-C14 alkenyl enyl;

Q¹ is -NR²R³, -CH(OR²)(OR³), -CR²=C(R³)(R¹²), or -C(R²)(R³)(R¹²);

Q^la is -NR²R³', -CH(OR²')(OR³'), -CR²=C(R³)(R¹²), or -C(R²')(R³')(R¹²');

X³ is optionally substituted C2-C14 alkylenyl;

R⁴ is optionally substituted C4-C14 alkyl;

L¹ is Ci-C8 alkylenyl;

R⁶ is (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl.

R^7a is -C(=O)N(R"')R^7b, -C(=S)N(R"')R^7b, -N=C(R^7b)(R^7c),

Z¹ is optionally substituted C1-C6 alkyl;

R¹⁰ is C1-C6 alkylenyl;

R^7b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^7C is hydrogen or C1-C6 alkyl;

R^8a is -C(=O)N(R"')R^8b, -C(=S)N(R"')R^8b, -N=C(R^8b)(R^8c),

R^8b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)Ci-C6 alkyl;

R^8C is hydrogen or C1-C6 alkyl;

R^9a is -N=C(R^9b)(R^9c);

R^9b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^9C is hydrogen or C1-C6 alkyl;

Ri°^a i_s -N=C(R^10b)(R^10c);

R^10b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R^10c is hydrogen or C1-C6 alkyl;

R^lla is -OR^llb, -N(R")R^llb, -OC(=O)R^llb, or -N(R")C(=O)R^llb;

R^llb is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;

R' is hydrogen or C1-C6 alkyl;

R" is hydrogen or C1-C6 alkyl; and R'" is hydrogen or C1-C6 alkyl.

[0442] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(R)(-L¹-N(R")R⁶)-.

[0443] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(R')(-OR^7a)-.

[0444] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(R')(-N(R")R^8a).

[0445] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(R')(-C(=O)OR^9a).

[0446] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(R')(-C(=O)N(R")R^10a)-.

[0447] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein A is -C(=N-R^lla)-.

[0448] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein T is -X^2a-Y^la-Q^la.

[0449] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein T is -X³-C(=O)OR⁴.

[0450] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein X² and/or X^2a are/is optionally substituted C2-C14 alkylenyl (e.g., C2-C10 alkylenyl, C2- C8 alkylenyl, C2, C3, C4, C5, C6, C7, or C8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein X² is C2-C14 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein X^2a is C2- C14 alkylenyl.

[0451] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y¹ and/or Y^la are/is

[0452] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y¹ is

[0453] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y^la is

[0454] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y¹ and/or Y^la are/is

[0455] In some embodiments, Lipids of the Disclosure have structure of Formula (VII-B), wherein Y¹ is

[0456] In some embodiments, Lipids of the Disclosure have structure of Formula (VII-B), wherein Y^la is

[0457] In some embodiments, Lipids of the Disclosure have structure of Formula (VII-B), wherein Y¹ and/or Y^la are/is

[0458] In some embodiments, Lipids of the Disclosure have structure of Formula (VII-B), wherein Y¹ is

[0459] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y^la is

[0460] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y¹ and/or Y^la are/is

[0461] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y¹ is

[0462] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Y^la is

[0463] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Q¹ and/or Q^la are/is -C(R²)(R³)(R¹²). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Q¹ is -C(R²)(R³ )(R¹²). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein Q^la is -C(R²)(R³)(R¹²).

[0464] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein X³ is optionally substituted C1-C14 alkylenyl (e.g., C1-C6, C1-C4 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein X³ is Ci- C14 alkylenyl.

[0465] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R², R³, R¹², R², R³ , and/or R¹² are hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R² is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R³ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R¹² is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R² is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R³ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R¹² is hydrogen.

[0466] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R², R³, R¹², R², R³ , and/or R¹²' are optionally substituted C1-C14 alkyl (e.g., C4-C10 alkyl, C5, C6. C7. C8, C9 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R² is C4-C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R³ is C4-C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R¹² is C4-C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R² is C4- C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R³ is C4-C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R¹² is C4-C10 alkyl.

[0467] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R⁴ is optionally substituted C4-C14 alkyl (e.g., C8-C11 alkyl, linear C8-C11 alkyl, C8, C9, C10, C11, C12, C13, or C14 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R⁴ is linear C8-C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R⁴ is linear C11 alkyl.

[0468] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein L¹ is C1-C3 alkylenyl.

[0469] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R⁶ is (hydroxy)C1-C6 alkyl.

[0470] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^7a is

In some embodiments, Lipids of the Disclosure have a

structure of Formula (VII-B), wherein R^7a is

In some embodiments,

Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^7a is

[0471] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^7a is selected from the group consisting of -C(=O)N(R"')R^7b, -C(=S)N(R"')R^7b, and - N=C(R^7b)(R^7c). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII- B), wherein R^7a is -C(=O)N(R"')R^7b. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^7a is -C(=S)N(R"')R^7b. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^7a is -N=C(R^7b)(R^7c).

[0472] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^8a is selected from the group consisting of -C(=O)N(R"')R^8b, -C(=S)N(R"')R^8b, and - N=C(R^8b)(R^8c). In some embodiments, Lipids of the Disclosure have a structure of Formula (VII- B), wherein R^8a is -C(=O)N(R"')R^8b. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^8a is -C(=S)N(R"')R^8b. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^8a is -N=C(R^8b)(R^8c).

[0473] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^8a is

[0474] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^9b is (hydroxy)C1-C6 alkyl.

[0475] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^10b is (amino)C1-C6 alkyl.

[0476] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -OR^llb or -OC(=O)R^llb. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -OR^llb. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -OC(=O)R^llb.

[0477] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -N(R")R^llb or -N(R")C(=O)R^llb. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -N(R")R^llb. In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^lla is -N(R")C(=O)R^llb.

[0478] In some embodiments, Lipids of the Disclosure have a structure of Formula (VII-B), wherein R^llb is (amino)C1-C6 alkyl.

Formula (III-C)

[0479] In some embodiments, Lipids of the Disclosure have a structure of Formula (IILC):

or a pharmaceutically acceptable salt thereof, wherein

R²⁰ is Ci-C₆ alkylenyl-NR²⁰ C(O)OR²⁰ ;

R²⁰' is hydrogen or optionally substituted C1-C6 alkyl;

R²⁰" is optionally substituted C1-C6 alkyl, phenyl, or benzyl;

Z¹ is optionally substituted C1-C6 alkyl;

X² and X^2a are independently optionally substituted C2-C14 alkylenyl;

Y¹ and Y^la are independently

wherein the bond marked with an is attached to X² or X^2a;

Z³ is independently optionally substituted C2-C6 alkylenyl;

R² and R³ are independently optionally substituted C4-C14 alkyl; and R²' and R³' are independently optionally substituted C4-C14 alkyl.

[0480] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R²⁰ is -CH2CH2CH2NHC(O)O-t-butyl or -CH2CH2CH2NH(O)O-benzyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R²⁰ is - CH2CH2CH2NHC(O)O-t-butyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R²⁰ is -CH2CH2CH2NHC(O)O-benzyl. [0481] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein X² and X^2a are independently C4-C8 alkylenyl (e.g., C5, C6, C7 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein X² is C6 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein X^2a is C6 alkyl.

[0482] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein Y¹ and Y^la are

wherein Z³ is C2-C4alkylenyl (e.g., C2 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein Y is

wherein Z is C2-C4alkylenyl (e.g., C2 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein Y^la is wherein Z

³ is C2-C4alkylenyl (e.g., C2 alkylenyl).

[0483] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R², R³, R²' and R³' are independently optionally substituted C4-C10 alkyl (e.g., C6-Cgalkyl, C6, C7, C8, C9 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R² is C6-Cgalkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R³ is C6-Cgalkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R² is C6-Cgalkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-C), wherein R³ is C6-Cgalkyl.

Formula (III-D)

[0484] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D):

or a pharmaceutically acceptable salt thereof, wherein R¹ is -OH; is optionally substituted C4 alkylenyl; and X^2a are independently optionally substituted C2-C14 alkylenyl; and Y^la are independently

Z³ is independently optionally substituted C2-C6 alkylenyl;

R² and R³ are independently optionally substituted C4-C14 alkyl or C1-C2 alkyl substituted with optionally substituted cyclopropyl; or

R²' and R³' are independently optionally substituted C4-C14 alkyl or C1-C2 alkyl substituted with optionally substituted cyclopropyl.

[0485] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein X¹ is C4 alkylenyl.

[0486] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein X² and X^2a are independently optionally substituted C4-C10 alkylenyl (e.g., C5, C6, C7, C8, C9, or C10 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein X² is C4-C10 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein X^2a is C4-C10 alkylenyl.

[0487] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein Y¹ and Y^la are independently

, wherein Z is independently C2-C4 alkylenyl (e.g., C2, C4 alkylenyl).

[0488] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R², R³, R²' and R³' are independently C6-C11 alkyl (e.g., C6, C7, C8, C9, C10, C11, C12, C13, or C14 alkyl) or C1-C2 alkyl substituted with optionally substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R², R³, R²' and R³' are independently C6-C11 alkyl (e.g., C6, C7, C8, C9, C10, C11, C12, C13, or C14 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R² is C6-Ci4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III- D), wherein R³ is C6-C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R² is C6-C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R³ is C6-C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R² is C1-C2 alkyl substituted with substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R³ is C1-C2 alkyl substituted with substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R²' is C1-C2 alkyl substituted with substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R³' is C1-C2 alkyl substituted with substituted cyclopropyl.

[0489] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R², R³, R²' and R³' are independently C1-C2 alkyl substituted with cyclopropylene-(Ci- C⁶alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R² is C1-C2 alkyl substituted with cyclopropylene-(Ci-C6alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R³ is C1-C2 alkyl substituted with cyclopropylene- (Ci-C⁶alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R²' is C1-C2 alkyl substituted with cyclopropylene-(Ci-C⁶alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-D), wherein R³' is C1-C2 alkyl substituted with cyclopropylene- (Ci-C⁶alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl).

Formula (III-E)

[0490] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is -OH;

X¹ is branched C2-C8 alkylenyl

X² and X^2a are independently optionally substituted C2-C14 alkylenyl;

Y¹ and Y^la are independently

Z³ is independently optionally substituted C2-C6 alkylenyl;

R² and R³ are independently optionally substituted C4-C14 alkyl;

R²' and R³' are independently optionally substituted C4-C14 alkyl.

[0491] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X¹ is branched C6 alkylenyl. [0492] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X² and X^2a are independently C4-C10 alkylenyl (e.g., C6, C7, C8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X² is C4-C10 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X^2a is C4-C 10 alkylenyl.

[0493] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein Y¹ and Y^la are

, wherein Z³ is independently optionally substituted C2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein Y¹ is

, wherein Z³ is independently optionally substituted C2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein Y^la is

, wherein Z³ is independently optionally substituted C2 alkylenyl.

[0494] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R², R³, R²' and R³' are independently C6-C12 alkyl (e.g., C9 alkyl) or C4-C10 alkyl (e.g.,

C4, C6 alkyl) optionally substituted with C2-C8alkenylene (e.g., C4, C6 alkenylene). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C4-C10 alkyl optionally substituted with C2-C8alkenylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C4-C10 alkyl optionally substituted with C2-C8alkenylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C4-C10 alkyl optionally substituted with C2-C8alkenylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C4-C10 alkyl optionally substituted with C2-C8alkenylene.

Formula (IILF)

[0495] In some embodiments, Lipids of the Disclosure have a structure of Formula (IILF):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is -OH;

X¹ is optionally substituted C2-C6 alkylenyl;

X² and X^2a are independently optionally substituted C2-C14 alkylenyl; each of Y¹ and Y^la is a bond;

[0496] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X¹ is C4 alkylenyl.

[0497] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X² and X^2a are independently C4-C10 alkylenyl (e.g., C6-C8 alkylenyl, C6, C7, C8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X² is C4-C10 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein X^2a is C4-C10 alkylenyl.

[0498] In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R², R³, R²' and R³' are independently C6-Cio alkyl (e.g., C7. C8 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C6-Cio alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C6-Cio alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R² is C6-Cio alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (III-E), wherein R³ is C6-Cio alkyl.

Formula (VIII-B)

[0499] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B):

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is a bond, R¹ is C1-C6 alkyl,

X² is is C2-C6 alkylenyl,

X^2a is C2-C14 alkylenyl, wherein X² or X^2a is substituted with OH or Ci.4alkylenyl-OH,

Y¹ is

wherein the bond marked with an is attached to X²;

Y^la is

Q¹ is -C(R²)(R³)(R¹²);

Q^la is -C(R²)(R³ )(R¹²);

R², R³, and R¹² are independently hydrogen, optionally substituted C1-C14 alkyl, or optionally substituted C2-C14 alkenylenyl, and

R², R³ , and R¹²' are independently hydrogen, optionally substituted C1-C14 alkyl, or optionally substituted C2-C14 alkenylenyl.

[0500] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R¹ is methyl.

[0501] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein X² is C4, C5, or C6 alkylenyl.

[0502] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein X^2a is C4-C8 alkylenyl (e.g., C5, C6, or C7 alkylenyl).

[0503] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein Y¹ is

or

in some embodiments, Lipids of the Disclosure have a structure of

Formula (VIII-B), wherein Y¹ is

In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein Y¹ is

In some embodiments, Lipids of T the Disclosure have a structure of Formula (VIII-B), wherein Y^la is

In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein Y^la is

[0504] In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R², R³, R¹², R², R³ , and R¹²' are independently hydrogen or C5-C12 alkyl (e.g., C6, C7, C8, C9, C10, C11 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R² is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R³ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R² is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R³ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R² is C5- C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R³ is C5-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R² is C5-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (VIII-B), wherein R³ is C5-C12 alkyl.

Formula (X)

[0505] In some embodiments, Lipids of the Disclosure have a structure of Formula (X):

or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 9;

R™ is selected from hydrogen and optionally substituted C1-C6 alkyl; and (i) ee is 1, each dd is independently selected from 1 to 4; and each R^ww is independently selected from the group consisting of C4-C14 alkyl, branched C4-C12 alkenyl, C4-C12 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any -(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl;

(ii) ee is 0, each dd is 1; and each R^ww is linear C4-C12 alkyl.

[0506] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R™ is H. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is optionally substituted C1-C6 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is Ci alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is C2 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R™ is C3 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is C4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is C5 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein R^xx is C₆ alkyl.

[0507] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently selected from the group consisting of C4-C14 alkyl, branched C4-C12 alkenyl, C4-C12 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any -(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C4- C14 alkyl, wherein any -(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C4-C14 alkyl, wherein any -(CH2)2- of the C4-C14 alkyl can be optionally replaced with cyclopropylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C4-C12 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C4-C12 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9-C12 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C4-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently selected from the group consisting of C6-C11 alkyl, branched C8-Ci2 alkenyl, C8-Ci2 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any -(CH2)2- of the C6-C11 alkyl can be optionally replaced with cyclopropylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C6-C11 alkyl, wherein any - (CH2)2- of the C6-C14 alkyl can be optionally replaced with cyclopropylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C8-C11 alkenyl, e.g., (linear or branched C3-C5 alkylenyl)-(branched C5-C?alkenyl), e.g., (branched C5 alkylenyl)-(branched C8alkenyl), e.g.,

[0508] . In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C8-C11 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9-C12 alkenyl.

[0509] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently selected from the group consisting of C6-C11 alkyl (e.g., C6, C8, C9, C10, C11, C13 alkyl), wherein any -(CH2)2- of the C6-C11 alkyl can be optionally replaced with cyclopropylene.

[0510] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently branched C8-C11 alkenyl (e.g., branched C10 alkenyl).

[0511] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently C8-C11 alkenyl comprising at least two double bonds (e.g., C9 or C10 alkenyl comprising two double bonds).

[0512] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently (Ci alkylenyl)-(cyclopropylene-C6 alkyl) or (C2 alkylenyl)- (cyclopropylene-C2 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently (Ci alkylenyl)-(cyclopropylene-C6 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is independently (C2 alkylenyl)-(cyclopropylene-C2 alkyl).

[0513] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C5 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C6 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C7 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C8 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C13 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C14 alkyl.

[0514] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C10 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C11 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C12 alkenyl.

[0515] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C8 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C10 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C11 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C12 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C13 alkenyl comprising at least two double bonds. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C14 alkenyl comprising at least two double bonds.

[0516] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkyl, wherein one -(CH2)2- of the C9 alkyl is replaced with C2-C6 cycloalkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R'™ is C9 alkyl, wherein one -(CH2)2- of the C9 alkyl is replaced with cyclopropylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkyl, wherein two -(CH2)2- of the C9 alkyl are replaced with C2-C6 cycloalkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is C9 alkyl, wherein two -(CH2)2- of the C9 alkyl are replaced with cyclopropylene. [0517] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C5 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C6 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C7 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C8 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C9 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C13 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is linear C14 alkyl.

[0518] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C8 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C9 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C10 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C11 alkenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each R^ww is branched C12 alkenyl.

[0519] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is independently selected from 3 to 7. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 4. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 5. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 6. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 7. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 8. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each cc is 9.

[0520] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each dd is independently selected from 1 to 4. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each dd is 1. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each dd is 2. In some embodiments,

Lipids of the Disclosure have a structure of Formula (X), wherein each dd is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein each dd is 4.

[0521] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein ee is 1.

[0522] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein ee is 0.

Formula (X-A)

[0523] In some embodiments, Lipids of the Disclosure have a structure of Formula (X), wherein the Lipids of the Disclosure have a structure of Formula (X-A):

or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 7; each dd is independently selected from 1 to 4;

R™ is selected from hydrogen and optionally substituted C1-C6 alkyl; and each R^ww is independently selected from the group consisting of C4-C14 alkyl or (linear or branched C3-C5 alkylenyl)-(branched C8-Cvalkenyl).

[0524] In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is Ci alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is C2 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is C3 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R™ is C4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is C5 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein R^xx is C₆ alkyl.

[0525] In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 4, 5, 6, or 7. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 4. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 5. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 6. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each cc is 7.

[0526] In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each dd is 1 or 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each dd is 1. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each dd is 2. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each dd is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each dd is 4.

[0527] In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C4-C14 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C4 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C5 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C6 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C7 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C8 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C9 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C11 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R'™ is C13 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is C14 alkyl.

[0528] In some embodiments, Lipids of the Disclosure have a structure of Formula (X-A), wherein each R^ww is (linear or branched C3-C5 alkylenyl)-(branched C8-Cvalkenyl), e.g., (branched C5 alkylenyl)-(branched C8alkenyl), e.g.,

[0529] In some embodiments, Lipids of the Disclosure comprise an acyclic core. In some embodiments, Lipids of the Disclosure are selected from any lipid in Table (I) below or a pharmaceutically acceptable salt thereof: Table (I). Non-Limiting Examples of Ionizable Lipids with an Acyclic Core

[0530] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044333A1, which is incorporated by reference herein, in its entirety.

Formula (CY)

[0531] In some embodiments, an LNP disclosed herein comprises an ionizable lipid of Formula (CY)

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from the group consisting of -OH, -OAc, R^la,

Z¹ is optionally substituted C1-C6 alkyl;

X¹ is optionally substituted C2-C6 alkylenyl;

X² is selected from the group consisting of a bond, -CH2- and -CH2CH2-;

X²’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-;

X'^r is selected from the group consisting of a bond, -CH2- and -CH2CH2

X¹ is selected from the group consisting of a bond, -CH2- and -CH2CH2-;

X⁴ and X⁵ are independently optionally substituted C2-C14 alkylenyl or optionally substituted

C2-C14 alkenylenyl;

Y¹ and Y² are independently selected from the group consisting of

wherein the bond marked with an is attached to X⁴ or X⁵; each Z² is independently H or optionally substituted Ci-C8 alkyl; each Z³ is indpendently optionally substituted Ci-C8 alkylenyl;

R² is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, and -(CH2)pCH(OR⁶)(OR⁷);

R³ is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)_qCH(OR⁸)(OR⁹);

R^ia is:

R^2a, R^2b, and R^2c are independently hydrogen and C1-C6 alkyl;

R^3a, R^3b, and R^3c are independently hydrogen and C1-C6 alkyl;

R^4a, R^4b, and R^4C are independently hydrogen and C1-C6 alkyl;

R³³, R^5b, and R^5c are independently hydrogen and C1-C6 alkyl;

R⁶, R^z, R⁸, and R⁹ are independently optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)m-A-(CH2)_nH; each A is independently a C3-C8 cycloalkylenyl; each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12; each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; p is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, and 7; and q is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, and 7.

Formulas (CY-I), (CY-II), (CY-III), (CY-IV), and (CY-V)

[0532] In some embodiments, the present disclosure includes a compound of Formula (CY-

I), (CY-II), (CY-III), (CY-IV), or (CY-V):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X² , X³, X³ , X⁴, X⁵, Y¹, Y², R¹, R², and R³ are defined herein.

Formulas (CY-VI) and (CY-VII)

[0533] In some embodiments, the present disclosure includes a compound of Formula (CY- VI) or (CY-VII):

(CY-VI) (CY-VII) or a pharmaceutically acceptable salt thereof, wherein X¹, X⁴, X⁵, R¹, R², and R³ are defined herein.

Formulas (CY-VIII) and (CY-IX)

[0534] In some embodiments, the present disclosure includes a compound of Formula (CY- VIII) or (CY-IX):

(CY- VIII) (CY- IX), or pharmaceutically acceptable salt thereof. wherein X¹, X⁴, X⁵, R¹, R², and R³ are defined herein.

Formulas (CY-IV-a), (CY-IV-b), and (CY-IV-c)

[0535] In some embodiments, the present disclosure includes a compound of Formula (CY-

IV-a), (CY-IV-b), or (CY-IV-c)

(CY-IV-a) (CY-IV-b) (CY-IV-c), or pharmaceutically acceptable salt thereof. wherein X¹, X⁴, X⁵, R², and R³ are defined herein.

Formulas (CY-IV-d), (CY-IV-e), and (CY-IV-f)

[0536] In some embodiments, the present disclosure includes a compound of Formula (CY- IV-d), (CY-IV-e), or (CY-IV-f)

or pharmaceutically acceptable salt thereof. wherein X¹, X⁴, X⁵, R², and R³ are defined herein.

Formula (CY-IV)

[0537] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R⁵, X¹, X², X’, X⁴, X⁵, Y¹, and

Y² are as defined in connection with Formula (CY-F).

[0538] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’), wherein:

R¹ is -OH, R^la,

wherein Z¹ is optionally substituted Ci-C8 alkyl;

X¹ is optionally substituted C2-C6 alkylenyl;

X² and X³ are independently a bond, -CH2-, or -CH2CH2-;

X⁴ and X⁵ are independently optionally substituted C2-C14 alkylenyl;

Y¹ and Y² are independently

R² and R3 are independently optionally substituted C4-C20 alkyl;

R^la is:

R^2a, R^2b, and R^2c are independently hydrogen and C1-C6 alkyl;

R^3a, R^3b, and R^3c are independently hydrogen and C1-C6 alkyl;

R^4a, R^4b, and R^4c are independently hydrogen and C1-C6 alkyl; and

R^5a, R^5b, and R^5c are independently hydrogen and C1-C6 alkyl

[0539] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’), wherein R¹ is -OH,

[0540] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’), wherein Y¹ and Y² are independently:

[0541] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’), wherein R² is -CH(OR⁶)(OR⁷).

[0542] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-IV’), wherein R³ is -CH(OR⁸)(OR⁹).

[0543] Non-limiting examples of lipids having a structure of Formula (CY-IV’) include compounds CY7, CY8, CY19, CY20, CY21, CY28, CY29, CY40, CY41, CY42, CY48, CY49, CY58, CY59, and CY60.

Formula (CY-VF)

[0544] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF):

or a pharmaceutically acceptable salt thereof, wherein R¹, R⁶, R⁷, R⁸, R⁹, X¹, X², X³, X⁴, X⁵, Y¹, and Y² are as defined in connection with Formula (CY-F). [0545] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R¹ is -OH.

[0546] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein X¹ is C2-C6 alkylenyl.

[0547] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein X² is -CH2CH2-.

[0548] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein X⁴ is C2-C6 alkylenyl.

[0549] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein X⁵ is C2-C6 alkylenyl.

[0550] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein Y¹ is:

[0551] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein Y² is:

[0552] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein each Z³ is independently optionally substituted C1-C6 alkylenyl.

[0553] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein each Z³ is -CH2CH2-.

[0554] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁶ is C5-C14 alkyl.

[0555] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁷ is C5-C14 alkyl.

[0556] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁶ is C6-C11 alkenyl.

[0557] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁷ is C6-C11 alkenyl.

[0558] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁸ is C5-C16 alkyl. [0559] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁹ is C5-C14 alkyl.

[0560] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁸ is C6-C11 alkenyl.

[0561] In some embodiments, Lipids of the Disclosure have a structure of Formula (CY-VF), or a pharmaceutically acceptable salt thereof, wherein R⁹ is C6-C11 alkenyl.

[0562] In some embodiments, Lipids of the Disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. In some embodiments, Lipids of the Disclosure are selected from any lipid in Table (II) below or a pharmaceutically acceptable salt thereof:

R¹

[0563] In some embodiments, R¹ is selected from the group consisting of -OH, -OAc, R^la,

and In some embodiments, R¹ is -OH or -OAc. In some

embodiments, R¹ is OH. In some emobodiments, R¹ is -OAc. In some embodiments, R¹ is R^la.

In some embodiments, R¹ is imidazolyl. In some embodiments, R¹ is

.

R²

[0564] In some embodiments, R² is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, and -(CH2)_PCH(OR⁶)(OR⁷).

[0565] In some embodiments, R² is optionally substituted C4-C20 alkyl. In some embodiments, R² is optionally substituted C8-C11 alkyl. In some embodiments, R² is optionally substituted C9-C16 alkyl. In some embodiments, R² is optionally substituted C8-Cio alkyl. In some embodiments, R² is optionally substituted C11-C13 alkyl. In some embodiments, R² is optionally substituted C14-C16 alkyl. In some embodiments, R² is optionally substituted C9 alkyl. In some embodiments, R² is optionally substituted C10 alkyl. In some embodiments, R² is optionally substituted C11 alkyl. In some embodiments, R² is optionally substituted C12 alkyl. In some embodiments, R² is optionally substituted C13 alkyl. In some embodiments, R² is optionally substituted C14 alkyl. In some embodiments, R² is optionally substituted C15 alkyl. In some embodiments, R² is optionally substituted Ci6 alkyl.

[0566] In some embodiments, R² is optionally substituted C2-C14 alkenyl. In some embodiments, R² is optionally substituted C5-C14 alkenyl. In some embodiments, R² is optionally substituted C7-C14 alkenyl. In some embodiments, R² is optionally substituted C9-C14 alkenyl. In some embodiments, R² is optionally substituted C10-C14 alkenyl. In some embodiments, R² is optionally substituted C12-C14 alkenyl.

[0567] In some embodiments, R² is -(CH2)_PCH(OR⁶)(OR⁷). In some embodiments, R² is - CH(OR⁶)(OR⁷). In some embodiments, R² is -CH2CH(OR⁶)(OR⁷). In some embodiments, R² is -(CH2)2CH(OR⁶)(OR⁷). In some embodiments, R² is -(CH2)3CH(OR⁶)(OR⁷). In some embodiments, R² is -(CH2)4CH(OR⁶)(OR⁷).

[0568] In some embodiments, R² is selected from the group consisting of

R³

[0569] In some embodiments, R³ is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, and -(CH2)_qCH(OR⁶)(OR⁷).

[0570] In some embodiments, R³ is optionally substituted C4-C20 alkyl. In some embodiments, R³ is optionally substituted C8-C11 alkyl. In some embodiments, R³ is optionally substituted C9-C16 alkyl. In some embodiments, R³ is optionally substituted C8-Cio alkyl. In some embodiments, R³ is optionally substituted C11-C13 alkyl. In some embodiments, R³ is optionally substituted C14-C16 alkyl. In some embodiments, R³ is optionally substituted C9 alkyl. In some embodiments, R³ is optionally substituted C10 alkyl. In some embodiments, R³ is optionally substituted C11 alkyl. In some embodiments, R³ is optionally substituted C12 alkyl. In some embodiments, R³ is optionally substituted C13 alkyl. In some embodiments, R³ is optionally substituted C14 alkyl. In some embodiments, R³ is optionally substituted C15 alkyl. In some embodiments, R³ is optionally substituted Ci6 alkyl.

[0571] In some embodiments, R³ is optionally substituted C2-C14 alkenyl. In some embodiments, R³ is optionally substituted C5-C14 alkenyl. In some embodiments, R³ is optionally substituted C7-C14 alkenyl. In some embodiments, R³ is optionally substituted C9-C14 alkenyl. In some embodiments, R³ is optionally substituted C10-C14 alkenyl. In some embodiments, R³ is optionally substituted C12-C14 alkenyl.

[0572] In some embodiments, R³ is -(CH2)_qCH(OR⁸)(OR⁹). In some embodiments, R³ is -CH(OR⁸)(OR⁹). In some embodiments, R³ is -CH2CH(OR⁸)(OR⁹). In some embodiments, R³ is -(CH2)2CH(OR⁸)(OR⁹). In some embodiments, R³ is -(CH2)3CH(OR⁸)(OR⁹). In some embodiments, R³ is -(CH2)4CH(OR⁸)(OR⁹).

[0573] In some embodiments, R³ is selected from the group consisting of

R⁶, R⁷, R⁸, R⁹

[0574] In some embodiments, R⁶, R⁷, R⁸, and R⁹ are independently optionally substituted Ci- C14 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)m-A-(CH2)_nH. In some embodiments, R⁶, R⁷, R⁸, and R⁹ are independently optionally substituted C1-C14 alkyl. In some embodiments, R⁶, R⁷, R⁸, and R⁹ are independently optionally substituted C2-C14 alkenyl. In some embodiments, R⁶, R⁷, R⁸, and R⁹ are independently -(CH2)m-A-(CH2)_nH.

[0575] In some embodiments, R⁶ is optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)m-A-(CH2)_nH. In some embodiments, R⁶ is optionally substituted C3- C10 alkyl. In some embodiments, R⁶ is optionally substituted C4-C10 alkyl. In some embodiments, R⁶ is independently optionally substituted C5-C10 alkyl. In some embodiments, R⁶ is optionally substituted C9-C10 alkyl. In some embodiments, R⁶ is optionally substituted C1-C14 alkyl. In some embodiments, R⁶ is optionally substituted C2-C14 alkenyl. In some embodiments, R⁶ is -(CH2)m- A-(CH₂)_nH.

[0576] In some embodiments, R⁷ is optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)m-A-(CH2)_nH. In some embodiments, R⁷ is optionally substituted C3- C10 alkyl. In some embodiments, R⁷ is optionally substituted C4-C10 alkyl. In some embodiments, R⁷ is optionally substituted C5-C10 alkyl. In some embodiments, R⁷ is optionally substituted C9- Cio alkyl. In some embodiments, R⁷ is optionally substituted C1-C14 alkyl. In some embodiments, R⁷ is optionally substituted optionally substituted C2-C14 alkenyl. In some embodiments, R⁷ is - (CH₂)m-A-(CH₂)nH.

[0577] In some embodiments, R⁸ is optionally substituted C1-C14 alkyl, optionally substituted C₂-Ci4 alkenyl, or -(CH₂)m-A-(CH₂)_nH. In some embodiments, R⁸ is optionally substituted C3- C10 alkyl. In some embodiments, R⁸ is optionally substituted C4-C10 alkyl. In some embodiments, R⁸ is optionally substituted C5-C10 alkyl. In some embodiments, R⁸ is optionally substituted C9- C10 alkyl. In some embodiments, R⁸ is optionally substituted C1-C14 alkyl. In some embodiments, R⁸ is optionally substituted C₂-Ci4 alkenyl. In some embodiments, R⁸ is -(CH₂)m-A-(CH₂)_nH.

[0578] In some embodiments, R⁹ is optionally substituted C1-C14 alkyl, optionally substituted C₂-Ci4 alkenyl, or -(CH₂)m-A-(CH₂)_nH. In some embodiments, R⁹ is optionally substituted C3- C10 alkyl. In some embodiments, R⁹is optionally substituted C4-C10 alkyl. In some embodiments, R⁹ is optionally substituted C5-C10 alkyl. In some embodiments, R⁹ is optionally substituted C9- C10 alkyl. In some embodiments, R⁹is optionally substituted C1-C14 alkyl. In some embodiments, R⁹ is optionally substituted C₂-Ci4 alkenyl. In some embodiments, R⁹ is -(CH₂)m-A-(CH₂)_nH.

[0579] In some embodiments, each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, each m is 0. In some embodiments, each m is 1. In some embodiments, each m is 2. In some embodiments, each m is 3. In some embodiments, each m is 4. In some embodiments, each m is 5. In some embodiments, each m is 6. In some embodiments, each m is 7. In some embodiments, each m is 8. In some embodiments, each m is 9. In some embodiments, each m is 10. In some embodiments, each m is 11. In some embodiments, each m is 12.

[0580] In some embodiments, each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, each n is 0. In some embodiments, each n is 1. In some embodiments, each n is 2. In some embodiments, each n is 3. In some embodiments, each n is 4. In some embodiments, each n is 5. In some embodiments, each n is 6. In some embodiments, each n is 7. In some embodiments, each n is 8. In some embodiments, each n is 9. In some embodiments, each n is 10. In some embodiments, each n is 11. In some embodiments, each n is 12.

[0581] In some embodiments, each A is independently a C3-C8 cycloalkylenyl. In some embodiments, each A is cyclopropylenyl.

X¹

[0582] In some embodiments, X¹ is optionally substituted C₂-C6 alkylenyl. In some embodiments, X¹ is optionally substituted C₂-C8 alkylenyl. In some embodiments, X¹ is optionally substituted C₂-C4 alkylenyl. In some embodiments, X¹ is optionally substituted C₂-C3 alkylenyl. In some embodiments, X¹ is optionally substituted C₂ alkylenyl. In some embodiments, X¹ is optionally substituted C3 alkylenyl. In some embodiments, X¹ is optionally substituted C4 alkylenyl. In some embodiments, X¹ is optionally substituted C5 alkylenyl. In some embodiments, X¹ is optionally substituted C6 alkylenyl. In some embodiments, X¹ is optionally substituted -(CH2)2-. In some embodiments, X¹ is optionally substituted -(CH2)3-. In some embodiments, X¹ is optionally substituted -(CH2)4-. In some embodiments, X¹ is optionally substituted -(CH2)5-. In some embodiments, X¹ is optionally substituted -(CH2)6-.

X²

[0583] In some embodiments, X² is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In some embodiments, X² is a bond. In some embodiments, X² is -CH2-. In some embodiments, X² is -CH2CH2-.

X²’

[0584] In some embodiments, X² is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In some embodiments, X² is a bond. In some embodiments, X² is -CH2-. In some embodiments, X² is -CH2CH2-.

[0585] In some embodiments, X³ is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In some embodiments, X³ is a bond. In some embodiments, X³ is -CH2-. In some embodiments, X³ is -CH2CH2-.

[0586] In some embodiments, X³ is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In some embodiments, X³ is a bond. In some embodiments, X³ is -CH2-. In some embodiments, X³ is -CH2CH2-.

[0587] In some embodiments, X⁴ is selected from the group consting of optionally substituted C2-C14 alkylenyl and optionally substituted C2-C14 alkenylenyl. In some embodiments, X⁴ is optionally substituted C2-C14 alkylenyl. In some embodiments, X⁴ is optionally substituted C2- C10 alkylenyl. In some embodiments, X⁴ is optionally substituted C2-C8 alkylenyl. In some embodiments, X⁴ is optionally substituted C2-C6 alkylenyl. In some embodiments, X⁴ is optionally substituted C3-C6 alkylenyl. In some embodiments, X⁴ is optionally substituted C3 alkylenyl. In some embodiments, X⁴ is optionally substituted C4 alkylenyl. In some embodiments, X⁴ is optionally substituted C5 alkylenyl. In some embodiments, X⁴ is optionally substituted C6 alkylenyl. In some embodiments, X⁴ is optionally substituted -(CH2)2-. In some embodiments, X⁴ is optionally substituted -(CH2)3-. In some embodiments, X⁴ is optionally substituted -(CH2)4-. In some embodiments, X⁴ is optionally substituted -(CH2)s-. In some embodiments, X⁴ is optionally substituted -(CH2)6-.

[0588] In some embodiments, X⁵ is selected from the group consting of optionally substituted C2-C14 alkylenyl and optionally substituted C2-C14 alkenylenyl. In some embodiments, X⁵ is optionally substituted C2-C14 alkylenyl. In some embodiments, X⁵ is optionally substituted C2- C10 alkylenyl. In some embodiments, X⁵ is optionally substituted C2-C8 alkylenyl. In some embodiments, X⁵ is optionally substituted C2-C6 alkylenyl. In some embodiments, X⁵ is optionally substituted C3-C6 alkylenyl. In some embodiments, X⁵ is optionally substituted C3 alkylenyl. In some embodiments, X⁵ is optionally substituted C4 alkylenyl. In some embodiments, X⁵ is optionally substituted C5 alkylenyl. In some embodiments, X⁵ is optionally substituted C6> alkylenyl. In some embodiments, X⁵ is optionally substituted -(CH2)2-. In some embodiments, X⁵ is optionally substituted -(CH2)3-. In some embodiments, X⁵ is optionally substituted -(CH2)4-. In some embodiments, X⁵ is optionally substituted -(CH2)5-. In some embodiments, X⁵ is optionally substituted -(CH2)6-.

Y¹

[0589] In some embodiments, Y¹ is selected from the group consisting of

Y²

[0591] In some embodiments, Y² is selected from the group consisting of

[0592] In some embodiments, Y² is

Table (II). Non-Limiting Examples of Ionizable Lipids with a Cyclic Core

[0593] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application PCT/US2022/082276, published as PCT Publication WO2023122752, which is incorporated by reference herein, in its entirety.

[0594] In one embodiment, the disclosure provides a compound of Formula IA:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^la)- and -C(R')-OC(=O)(R^8a)-;

R^la is -L^R¹;

L¹ is C₂-C₆ alkylenyl or -(CH₂)₂-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^2a, R^2b, and R^2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;

R^3a, R^3b, and R^3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R^4a, R^4b, and R^4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;

R^5a, R^5b, and R^5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;

R^6a, R^6b, and R^6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or

R^6a and R^6b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo; and R^6c is selected from the group consisting of hydrogen and Ci-C₆ alkyl;

R^7a R7b and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or

R^7a and R^7b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo; and R^7c is selected from the group consisting of hydrogen and Ci-C₆ alkyl;

R' is selected from the group consisting of hydrogen and C1-C6 alkyl;

R^8a is - L²-R⁸;

L² is C2-C6 alkylenyl;

R⁸ is selected from the group consisting of -NR^9aR^9b,

R^9a and R^9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or

R^9a and R^9b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo;

Q¹ is C1-C20 alkylenyl; W¹ is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R^12a)-

, -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a is selected from the group consisting of hydrogen and C1-C6 alkyl;

X¹ is optionally substituted C1-C15 alkylenyl; or

X¹ is a bond;

Y¹ is selected from the group consisting of -(CH2)_m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6;

Z¹ is selected from the group consisting of optionally substituted C4-C12 cycloalkylenyl,

R¹⁰ is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl;

Q² is C1-C20 alkylenyl;

W² is selected from the group consisting of -C(=O)O-, -C(=O)N(R^12b)-, -OC(=O)N(R^12b)-, and -OC(=O)O-;

R^12b is selected from the group consisting of hydrogen and C1-C6 alkyl;

X² is optionally substituted C1-C15 alkylenyl; or

X² is a bond;

Y² is selected from the group consisting of -(CH2)_n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6;

Z² is selected from the group consisting of -(CH2)_P-, optionally substituted C4-C12

p is 0 or 1; and

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C 10 alkenyl; wherein one or more methylene linkages of X¹, X², Y¹, Y², Z¹, Z², R¹⁰, and R¹¹, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. [0595] In one embodiment, the disclosure provides a compound of Formula IB:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^la)- and -C(R')-OC(=O)(R^8a)-;

R^la is -L¹-R¹;

L¹ is C₂-C₆ alkylenyl or -(CH₂)₂-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^3a, R^3b, and R^3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;

R^4a, R^4b, and R^4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;

R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R^7a and R^7b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo; and R^7c is selected from the group consisting of hydrogen and Ci-C₆ alkyl;

R' is selected from the group consisting of hydrogen and C1-C6 alkyl;

R^8a is - L²-R⁸;

L² is C2-C6 alkylenyl;

Q¹ is C1-C20 alkylenyl;

W¹ is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R^12a)-

, -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a is selected from the group consisting of hydrogen and C1-C6 alkyl;

X¹ is optionally substituted C1-C15 alkylenyl; or

X¹ is a bond;

Y¹ is selected from the group consisting of-(CH2)_m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6;

Z¹ is selected from the group consisting of optionally substituted C5-C12 bridged cycloalkylenyl,

Q² is C1-C20 alkylenyl;

R^12b is selected from the group consisting of hydrogen and C1-C6 alkyl;

X² is optionally substituted C1-C15 alkylenyl; or

X² is a bond;

Y² is selected from the group consisting of-(CH2)_n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6;

Z² is selected from the group consisting of-(CH2)_p-, optionally substituted C4-C12

p is 0 or 1; and

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C 10 alkenyl; wherein one or more methylene linkages of X¹, X², Y¹, Y², Z¹, Z², R¹⁰, and R¹¹, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl.

[0596] In one embodiment, the disclosure provides a compound of Formula IC:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^la)- and -C(R’)-OC(=O)(R^8a)-;

R^la is -L1-R¹; L¹ is C2-C6 alkylenyl or -(CH₂)₂-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or

R’ is selected from the group consisting of hydrogen and C1-C6 alkyl;

R^8a is - L²-R⁸;

L² is C2-C6 alkylenyl; R⁸ is selected from the group consisting of -NR^9aR^9b,

Q¹ is C1-C20 alkylenyl;

, -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a is selected from the group consisting of hydrogen and C1-C6 alkyl;

X¹ is optionally substituted branched C1-C15 alkylenyl; or

X¹ is a bond;

Q² is C1-C20 alkylenyl;

R^12b is selected from the group consisting of hydrogen and C1-C6 alkyl;

X² is optionally substituted C1-C15 alkylenyl; or Y² is selected from the group consisting of -(CH2)_n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6;

Z² is of -(CH₂)_P-; p is 0 or 1; and

R¹¹ is C1-C20 branched alkyl; wherein one or more methylene linkages of X¹, X², Y¹, Y², Z¹, Z², R¹⁰, and R¹¹, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl.

[0597] In some embodiments, the disclosure provides a compound of any one of Formulae IA, IB, IC, or a pharmaceutically acceptable salt or solvate thereof, wherein Z¹ is optionally substituted C5-C12 bridged cycloalkylenyl.

[0598] In some embodiments, the disclosure provides a compound of any one of Formulae IA, IB, IC, or a pharmaceutically acceptable salt or solvate thereof, wherein Z¹ is not adamantyl.

[0599] In one embodiment, the disclosure provides a compound of Formula ID:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^la)- and -C(R')-OC(=O)(R^8a)-;

R^la is -LkR¹;

L¹ is C2-C6 alkylenyl or -(CH₂)₂-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^6a and R^6b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo; and R^6c is selected from the group consisting of hydrogen and C1-C6 alkyl;

R' is selected from the group consisting of hydrogen and C1-C6 alkyl;

R^8a is - L²-R⁸;

L² is C2-C6 alkylenyl;

R⁸ is selected from the group consisting of -NR^9aR^9b,

R^9a and R^9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R^9a and R^9b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo;

Q¹ is C1-C20 alkylenyl;

, -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a is selected from the group consisting of hydrogen and C1-C6 alkyl;

X¹ is optionally substituted branched C1-C15 alkylenyl; or

X¹ is a bond;

Z¹ is optionally substituted C5-C12 bridged cycloalkylenyl;

Q² is C1-C20 alkylenyl;

W² is selected from the group consisting of -C(=O)O-, -C(=O)N(R^12b)-, -OC(=O)N(R^12b)-, and

-OC(=O)O-;

R^12b is selected from the group consisting of hydrogen and C1-C6 alkyl;

X² is optionally substituted C1-C15 alkylenyl; or

Y² is -(CH₂)n-; n is 0, 1, 2, 3, 4, 5, or 6;

Z² is of -(CH₂)_P-; p is 0 or 1; and

R¹¹ is C1-C20 branched alkyl.

[0600] In some embodiments, the disclosure provides a compound of Formula ID or a pharmaceutically acceptable salt or solvate thereof, wherein Z¹ is not adamantyl.

[0601] In one embodiment, the disclosure provides a compound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^la)- and -C(R')-OC(=O)(R^8a)-;

R^la is -LkR¹;

L¹ is C2-C6 alkylenyl; R¹ is selected from the group consisting of -OH,

R' is selected from the group consisting of hydrogen and C1-C6 alkyl;

R^8a is - L²-R⁸;

L² is C2-C6 alkylenyl;

R⁸ is -NR^9aR^9b;

R^9a and R^9b taken together with the nitrogen atom to which they are attached form a 4-to 8- membered heterocyclo; Q¹ is C1-C20 alkylenyl;

W¹ is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R^12a)- , -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a is selected from the group consisting of hydrogen and C1-C6 alkyl;

X¹ is C1-C15 alkylenyl; or

X¹ is a bond;

Z¹ is selected from the group consisting of C4-C12 cycloalkylenyl,

Q² is C1-C20 alkylenyl;

R^12b is selected from the group consisting of hydrogen and C1-C6 alkyl;

X² is C1-C15 alkylenyl; or

X² is a bond;

Z² is selected from the group consisting of -(CH2)_P-, C4-C12 cycloalkylenyl,

p is 0 or 1; and

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C 10 alkenyl. [0602] In another embodiment, the disclosure provides a compound of Formula II:

or a pharmaceutically acceptable salt or solvate thereof, wherein R¹, R¹⁰, R¹¹, Q¹, Q², W¹, W², X¹, X², Y¹, Y², Z¹, and Z² are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0603] In another embodiment, the disclosure provides a compound of Formula III:

or a pharmaceutically acceptable salt or solvate thereof, wherein R¹, R^9a, R^9b, R¹⁰, R¹¹, L², Q¹, Q², W¹, W², X¹, X², Y¹, Y², Z¹, and Z² are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0604] In another embodiment, the disclosure provides a compound of Formula IV:

or a pharmaceutically acceptable salt or solvate thereof, wherein R^9a, R^9b, L², Q¹, Q², X¹, Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0605] In another embodiment, the disclosure provides a compound of Formula VI’ :

[0606] In another embodiment, the disclosure provides a compound of Formula VI” :

or a pharmaceutically acceptable salt or solvate thereof, wherein R^9a, R^9b, L², Q¹, Q², X¹, i Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0607] In another embodiment, the disclosure provides a compound of Formula VI’”:

[0608] In another embodiment, the disclosure provides a compound of Formula VII:

or a pharmaceutically acceptable salt or solvate thereof, wherein R¹, L¹, Q¹, Q², X¹, X², Y¹, Y² Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below. [0609] In another embodiment, the disclosure provides a compound of Formula VII’:

or a pharmaceutically acceptable salt or solvate thereof, wherein R¹, L¹, Q¹, Q², X¹, X², Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0610] In another embodiment, the disclosure provides a compound of Formula VII”:

[0611] In another embodiment, the disclosure provides a compound of Formula VII’”:

[0612] Formula IA, Formula IB, Formula IC, Formula I, In another embodiment, the disclosure provides a compound of Formula VIII:

or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

A, X¹, X², Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0613] In certain embodiments, the compound is a compound of Formula VIII, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0614] In another embodiment, the disclosure provides a compound of Formula VIII’ :

VIII’ or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

[0615] In certain embodiments, the compound is a compound of Formula VIII’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0616] In another embodiment, the disclosure provides a compound of Formula VIII”:

VIII or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

A, X¹, X², Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below. [0617] In certain embodiments, the compound is a compound of Formula VIII”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0618] In another embodiment, the disclosure provides a compound of Formula VIII’”:

[0619] In certain embodiments, the compound is a compound of Formula VIII’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0620] In another embodiment, the disclosure provides a compound of Formula IX:

IX or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

L¹, X¹, X², Y¹, Y², Z¹, Z², R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0621] In certain embodiments, the compound is a compound of Formula IX, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. [0622] In another embodiment, the disclosure provides a compound of Formula IX’:

[0623] In certain embodiments, the compound is a compound of Formula IX’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0624] In another embodiment, the disclosure provides a compound of Formula IX” :

[0625] In certain embodiments, the compound is a compound of Formula IX”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. [0626] In another embodiment, the disclosure provides a compound of Formula IX’” :

IX’” or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

[0627] In certain embodiments, the compound is a compound of Formula IX’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0628] In another embodiment, the disclosure provides a compound of Formula X:

L¹, X¹, X², Y¹, Y², Z¹, Z², R^9a, R^9b, R¹⁰, an R¹¹ are as defined herein in Formula IA, Formula

IB, Formula IC, Formula ID, Formula I or below.

[0629] In certain embodiments, the compound is a compound of Formula X, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. [0630] In another embodiment, the disclosure provides a compound of Formula X’:

IB, Formula IC, Formula ID, Formula I or below.

[0631] In certain embodiments, the compound is a compound of Formula X’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0632] In another embodiment, the disclosure provides a compound of Formula X”:

IB, Formula IC, Formula ID, Formula I or below.

[0633] In certain embodiments, the compound is a compound of Formula X”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. [0634] In another embodiment, the disclosure provides a compound of Formula X’”:

IB, Formula IC, Formula ID, Formula I or below.

[0635] In certain embodiments, the compound is a compound of Formula X’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0636] In another embodiment, the disclosure provides a compound of Formula XI:

or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and

A, X¹, Y¹, Z¹, R¹⁰, and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0637] In certain embodiments, the compound is a compound of Formula XI, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. [0638] In another embodiment, the disclosure provides a compound of Formula XI’ :

[0639] In certain embodiments, the compound is a compound of Formula XI’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0640] In another embodiment, the disclosure provides a compound of Formula XI” :

A, X¹, Y¹, Z¹, R¹⁰, and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below. [0641] In certain embodiments, the compound is a compound of Formula XI”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0642] In another embodiment, the disclosure provides a compound of Formula XI’”:

[0643] In certain embodiments, the compound is a compound of Formula XI’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0644] In another embodiment, the disclosure provides a compound of Formula XII:

or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and L¹, X¹, Y¹, Z¹, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0645] In certain embodiments, the compound is a compound of Formula XII, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0646] In another embodiment, the disclosure provides a compound of Formula XII’:

L¹, X¹, Y¹, Z¹, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0647] In certain embodiments, the compound is a compound of Formula XII’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0648] In another embodiment, the disclosure provides a compound of Formula XII”:

L¹, X¹, Y¹, Z¹, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

[0649] In certain embodiments, the compound is a compound of Formula XII”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0650] In another embodiment, the disclosure provides a compound of Formula XII’” :

[0651] In certain embodiments, the compound is a compound of Formula XII’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0652] In another embodiment, the disclosure provides a compound of Formula XIII:

XIII or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and

L¹, X¹, Y¹, Z¹, R^9a, R^9b, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula

IC, Formula I or below.

[0653] In certain embodiments, the compound is a compound of Formula XIII, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0654] In another embodiment, the disclosure provides a compound of Formula XIII’ :

XIII’ or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and

L¹, X¹, Y¹, Z¹, R^9a, R^9b, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0655] In certain embodiments, the compound is a compound of Formula XIII’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0656] In another embodiment, the disclosure provides a compound of Formula XIII”:

[0657] In certain embodiments, the compound is a compound of Formula XIII”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0658] In another embodiment, the disclosure provides a compound of Formula XIII’”:

L¹, X¹, Y¹, Z¹, R^9a, R^9b, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

[0659] In certain embodiments, the compound is a compound of Formula XIII’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl.

[0660] In another embodiment, the disclosure provides a compound of Formula XIV:

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C 10 alkenyl; q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and

[0661] In certain embodiments, the compound is a compound of Formula XIV, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. In certain embodiments, Z¹ is not adamantyl.

[0662] In another embodiment, the disclosure provides a compound of Formula XIV’:

or a pharmaceutically acceptable salt or solvate thereof, wherein

[0663] In certain embodiments, the compound is a compound of Formula XIV’, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. In certain embodiments, Z¹ is not adamantyl.

[0664] In another embodiment, the disclosure provides a compound of Formula XIV” :

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C10 alkenyl; q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and

A, X¹, Y¹, Z¹, R¹⁰, and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I or below.

[0665] In certain embodiments, the compound is a compound of Formula XIV”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. In certain embodiments, Z¹ is not adamantyl.

[0666] In another embodiment, the disclosure provides a compound of Formula XIV’” :

or a pharmaceutically acceptable salt or solvate thereof, wherein

[0667] In certain embodiments, the compound is a compound of Formula XIV’”, wherein Z¹ is an optionally substituted C5-C12 bridged cycloalkylenyl. In certain embodiments, Z¹ is not adamantyl. [0668] In another embodiment, the disclosure provides a compound of Formula XV:

XV or a pharmaceutically acceptable salt or solvate thereof, wherein

L¹, X¹, Y¹, Z¹, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula, IC, Formula I or below; wherein Z¹ is not adamantyl.

[0669] In another embodiment, the disclosure provides a compound of Formula XV’ :

or a pharmaceutically acceptable salt or solvate thereof, wherein

[0670] In another embodiment, the disclosure provides a compound of Formula XV” :

XV or a pharmaceutically acceptable salt or solvate thereof, wherein

[0671] In another embodiment, the disclosure provides a compound of Formula XV’”:

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C10 alkenyl; q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and L¹, X¹, Y¹, Z¹, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula, IC, Formula I or below; wherein Z¹ is not adamantyl.

[0672] In another embodiment, the disclosure provides a compound of Formula XVI:

XVI or a pharmaceutically acceptable salt or solvate thereof, wherein

[0673] In another embodiment, the disclosure provides a compound of Formula XVI’ :

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹¹ is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C 10 alkenyl; q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3; r² is 0, 1, or 2; s² is 0, 1, 2, 3, 4, 5, 6; and L¹, X¹, Y¹, Z¹, R^9a, R^9b, R¹⁰ and R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0674] In another embodiment, the disclosure provides a compound of Formula XVI” :

or a pharmaceutically acceptable salt or solvate thereof, wherein

[0675] In another embodiment, the disclosure provides a compound of Formula XVI’”:

or a pharmaceutically acceptable salt or solvate thereof, wherein

[0676] In another embodiment, the disclosure provides a compound of Formula XVII:

XVII or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

[0677] In certain embodiments, the compound is a compound of Formula XVII, wherein one or more methylene linkages of X², Y², Z², and R¹¹, are not replaced with a group selected from - O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl.

[0678] In another embodiment, the disclosure provides a compound of Formula XVIII:

L¹, X¹, X², Y¹, Y², Z², an R¹¹ are as defined herein in Formula IA, Formula IB, Formula IC, Formula ID, Formula I, or below.

[0679] In certain embodiments, the compound is a compound of Formula XVIII, wherein one or more methylene linkages of X², Y², Z², and R¹¹, are not replaced with a group selected from - O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. [0680] In another embodiment, the disclosure provides a compound of Formula XVIII’ :

XVIII’ or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

[0681] In another embodiment, the disclosure provides a compound of Formula XIX:

[0682] In another embodiment, the disclosure provides a compound of Formula XX:

[0683] In another embodiment, the disclosure provides a compound of Formula XXI:

XXI or a pharmaceutically acceptable salt or solvate thereof, wherein q¹ is 0, 1, 2, or 3; q² is 0, 1, 2, or 3;

L¹

[0684] In another embodiment, L¹ is selected from the group consisting of -CH2CH2-, - CH2CH2CH2-, and -CH2CH2CH2CH2-. In another embodiment, L¹ is -CH2CH2-. In another embodiment, L¹ is - CH2CH2CH2-. In another embodiment, L¹ is -CH2CH2CH2CH2-. In certain embodiments, L¹ is -(CH2)2-6-OC(=O)-. In some embodiments, L¹ is -(CH2)2-OC(=O)-.

R¹

[0685] In some embodiments, R¹ is

In another embodiment, R¹ is -OH. In some embodiments, R¹ is -N(R^9a)(R^9b). In some embodiments, R¹ is -NMe2. In some embodiments, R¹ is -NEt2. In another embodiment, R¹ is

. In another embodiment, R¹ is

L²

[0686] In another embodiment, L² is selected from the group consisting of -CH2CH2-, - CH2CH2CH2-, and -CH2CH2CH2CH2-. In another embodiment, L² is - CH2CH2-. In another embodiment, L² is -CH2CH2CH2-. In another embodiment, L² is -CH2CH2CH2CH2-. R⁸

[0687] In some embodiments, R⁸ is In another embodiment, R⁸ is -NR^9aR^9b. In

some embodiments, R⁸ is -NMe2. In some embodiments, R⁸ is -NEt2. In another embodiment, R⁸ is -OH.

R9a, R9b

[0688] In another embodiment, R^9a and R^9b are independently selected from the group consisting of hydrogen and C1-C4 alkyl. In another embodiment, R^9a and R^9b are each methyl. In another embodiment, R^9a and R^9b are each ethyl.

R ’

[0689] In another embodiment, R' is hydrogen. In some embodiments, R’ is C1-C6 alkyl.

Q¹

[0690] In another embodiment, Q¹ is straight chain C1-C20 alkylenyl. In another embodiment, Q¹ is straight chain C1-C10 alkylenyl. In another embodiment, Q¹ is C1-C10 alkylenyl. In another embodiment, Q¹ is C2-C5 alkylenyl. Q¹ is C6-C9 alkylenyl. In another embodiment, Q¹ is selected from the group consisting of -CH2CH2-, -CH2CH2CH2-, -CH₂(CH₂)2CH₂-, -CH₂(CH₂)3CH₂- , -CH₂(CH₂)4CH₂-, -CH₂(CH₂)5CH2-, -CH₂(CH₂)6CH₂-, -CH₂(CH₂)7CH₂-, and -CH₂(CH₂)₈CH2- . In another embodiment, Q¹ is -CH2CH2-. In another embodiment, Q¹ is -CH2CH2CH2-. In another embodiment, Q¹ is -CH2(CH2)2CH2-. In another embodiment, Q¹ is -CH2(CH2)3CH2-. In another embodiment, Q¹ is -CH2CH2-. In another embodiment, Q¹ is -CH2(CH2)4CH2-. In another embodiment, Q¹ is -CH2(CH2)5CH2-. In another embodiment, Q¹ is -CH2(CH2)6CH2-. In another embodiment, Q¹ is -CH2(CH2)?CH2-. In another embodiment, Q¹ is -CH2(CH2)8.CH2-.

W¹

[0691] In another embodiment, W¹ is selected from the group consisting of -C(=O)O-, - OC(=O)-, -C(=O)N(R^12a)-, -N(R^12a)C(=O)-, -OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and - OC(=O)O-. In another embodiment, W¹ is -C(=O)O-. In another embodiment, W¹ is -OC(=O)-. In another embodiment, W¹ is -C(=O)N(R^12a)-. In another embodiment, W¹ is -N(R^12a)C(=O)-. In another embodiment, W¹ is -OC(=O)N(R^12a)-. In another embodiment, W¹ is - N(R^12a)C(=O)O-. In another embodiment, W¹ is -OC(=O)O-.

X¹

[0692] In another embodiment, X² is optionally substituted C1-C15 alkylenyl. In another embodiment, X² is branched C1-C15 alkylenyl. In another embodiment, X¹ is a bond or C1-C15 alkylenyl. In another embodiment, X¹ is a bond. In another embodiment, X¹ is C2-C5 alkylenyl. In another embodiment, X¹ is C6-C9 alkylenyl. In another embodiment, X¹ is -CH2-. In another embodiment, X² is -CH2CH2-. In another embodiment, X² is -CH2CH2CH2-. In another embodiment, X² is -CH2CH2CH2CH2-. In another embodiment, X² is -CH2CH2CH2CH2CH2-.

Y¹

[0693] In another embodiment, Y¹ is selected from the group consisting of -(CH2)_m-, -O-, -S- , and -S-S-. In another embodiment, Y¹ is -(CH2)_m-. In some embodiments, Y¹ is -O-. In some embodiments, Y¹ is -S-. In another embodiment, Y¹ is -CH2-. In another embodiment, Y² is - CH2CH2-. m

[0694] In another embodiment, m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4. In another embodiment, m is 5. In another embodiment, m is 6. n

[0695] In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6.

P

[0696] In another embodiment, p is 0. In another embodiment, p is 1.

Z¹

[0697] In another embodiment, Z¹ is selected from the group consisting of C4-C12

is optionally subtituted.

[0698] In another embodiment, Z¹ is

[0699] In another embodiment, Z¹ is C4-C12 cycloalkylenyl. In another embodiment, Z¹ is a monocyclic C4-C8 cycloalkylenyl. In another embodiment, Z¹ is a monocyclic C4-C6 cycloalkylenyl. In another embodiment, Z¹ is a monocyclic C4 cycloalkylenyl. In another embodiment, Z¹ is a monocyclic C5 cycloalkylenyl. In another embodiment, Z¹ is a monocyclic C6 cycloalkylenyl.

[0700] In another embodiment, Z¹ is an optionally substituted bridged bicyclic or multicyclic cycloalkylenyl. In some embodiments, Z¹ is optionally substituted C5-C12 bridged cycloalkylenyl. In some embodiments, Z¹ is optionally substituted C6-Cio bridged cycloalkylenyl. In some embodiments, Z¹ is a optionally substituted C5-C10 bridged cycloalkylenyl, selected from the group consisting of adamantyl, cubanyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[l.l. l]pentyl, bicyclo[3.2.1]octyl, and bicyclo[3.1.1]heptyl.

[0701] In another embodiment, Z¹ is selected from the group consisting of:

[0702] In another embodiment, Z¹ is selected from the group consisting of:

[0703] In another embodiment, R¹⁰ is hydrogen.

[0704] In another embodiment, R¹⁰ is C1-C10 alkyl. In another embodiment, R¹⁰ is C3-C7 alkyl. In another embodiment, R¹⁰ is C4-C6 alkyl. In another embodiment, R¹⁰ is C4. In another embodiment, R¹⁰ is C5. In another embodiment, R¹⁰ is C6.

[0705] In another embodiment, R¹⁰ is C2-C12 alkenyl. In another embodiment, R¹⁰ is C6-C12 alkenyl. In another embodiment, R¹⁰ is C2-C8 alkenyl.

R¹¹

[0706] In another embodiment, R¹¹ is C1-C10 alkyl. In another embodiment, R¹¹ is optionally substituted C1-C20 alkyl. In another embodiment, R¹¹ is optionally substituted branched C1-C20 alkyl. In another embodiment, R¹¹ is optionally substituted C1-C15 alkyl. In another embodiment, R¹¹ is optionally substituted C1-C15 branched alkyl. In another embodiment, R¹¹ is optionally substituted C10-C15 alkyl. In another embodiment, R¹¹ is optionally substituted C10-C15 branched alkyl. In another embodiment, R¹¹ is selected from the group consisting of -CH3, -CH2CH3, and -CH2CH2CH3. In another embodiment, R¹¹ is selected from the group consisting of -CH₂(CH₂)2CH3, -CH₂(CH₂)3CH3, -CH₂(CH₂)4CH3, -CH₂(CH₂)₅CH3,

CH₂(CH₂)6CH3, -CH₂(CH₂)7CH3, and -CH₂(CH₂)₈CH3. In another embodiment, R¹¹ is -CH₃. In another embodiment, R¹¹ is -CH2CH3. In another embodiment, R¹¹ is -CH2CH2CH3. In another embodiment, R¹¹ is -CH2(CH2)2CH3. In another embodiment, R¹¹ is -CH2(CH2)3CH3. In another embodiment, R¹¹ is -CH2(CH2)4CH3. In another embodiment, R¹¹ is -CH2(CH2)5CH3. In another embodiment, R¹¹ is CH2(CH2)6CH3. In another embodiment, R¹¹ is -CH2(CH2)7CH3. In another embodiment, R¹¹ is -CH2(CH2)sCH3.

[0707] In another embodiment, R¹¹ is C2-C10 alkenyl. In another embodiment, R¹¹ is C2-C12 alkenyl. In another embodiment, R¹¹ is C6-C12 alkenyl. In another embodiment, R¹¹ is C2-C8 alkenyl.

[0708] In another embodiment, the disclosure provides a compound of any one of Formulae IA, IB, IC, or I-XXI or a pharmaceutically acceptable salt or solvate thereof, wherein R¹¹ is hydrogen.

Q²

[0709] In another embodiment, Q² is straight chain C1-C20 alkylenyl. In another embodiment, Q² is straight chain C1-C10 alkylenyl. In another embodiment, Q² is C2-C10 alkylenyl. In another embodiment, Q² is selected from the group consisting of -CH2CH2-, -CH2CH2CH2- , -CH₂(CH₂)2CH₂-, -CH₂(CH₂)3CH₂-, -CH₂(CH₂)4CH₂-, -CH₂(CH₂)5CH2-, -CH₂(CH₂)6CH₂- , -CH₂(CH₂)7CH₂-, and -CH2(CH2)₈ CH2-. In another embodiment, Q² is -CH2CH2-. In another embodiment, Q² is -CH2CH2CH2-. In another embodiment, Q² is -CH2(CH2)3CH2-. In another embodiment, Q² is -CH2(CH2)4CH2-. In another embodiment, Q² is -CH2(CH2)5CH2-. In another embodiment, Q² is -CH2(CH2)6CH2-. In another embodiment, Q² is -CH2(CH2)7CH2-. In another embodiment, Q² is -CH2(CH2)₈ CH2-.

W²

[0710] In another embodiment, W² is selected from the group consisting of -C(=O)O- and - OC(=O)-. In another embodiment, W² is -C(=O)O-. In another embodiment, W² is -OC(=O)-. X²

[0711] In another embodiment, X² is optionally substituted C1-C15 alkylenyl. In another embodiment, X² is C1-C15 branched alkylenyl. In another embodiment, X² is C1-C6 alkylenyl or a bond. In another embodiment, X² is C2-C4 alkylenyl. In another embodiment, X² is C3-C5 alkylenyl. In another embodiment, X² is selected from the group consisting of -CH2CH2- , -CH2CH2CH2-, -CH₂(CH₂)2CH₂-, -CH2(CH₂)₃CH2-, and -CH₂(CH₂)4CH₂-. In another embodiment, X² is -CH2-. In another embodiment, X² is a bond. In another embodiment, X² is branched C1-C15 alkylenyl, wherein one or more methylene linkages of X² are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. Y²

[0712] In another embodiment, Y² is selected from the group consisting of -(CH2)_m- and -S-. In another embodiment, Y² is -(CH2)_m-. In another embodiment, Y² is -S-.

Z²

[0713] In another embodiment, Z² is -(CH2)_P-. In another embodiment, Z² is -CH2-. In another embodiment, Z² is -CH2CH2-. In another embodiment, Z² is C4-C12 cycloalkylenyl. In another embodiment, Z² is a monocyclic C4-C8 cycloalkylenyl. In certain embodiments, Z² is optionally subtituted.

[0714] In another embodiment, Z² is an optionally substituted bridged bicyclic or multicyclic cycloalkylenyl. In some embodiments, Z² is optionally substituted C5-C12 bridged cycloalkylenyl. In some embodiments, Z² is optionally substituted Ce-Cio bridged cycloalkylenyl. In some embodiments, Z² is an optionally substituted C5-C10 bridged cycloalkylenyl, selected from the group consisting of adamantyl, cubanyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[l.l.l]pentyl, bicyclo[3.2.1]octyl, and bicyclo[3.1.1]heptyl.

[0715] In another embodiment, Z² is selected from the group consisting of:

[0716] In another embodiment, Z² is selected from the group consisting of:

[0717] In another embodiment, the disclosure provides a compound selected from any one of more of the compounds of Table (III), or a pharmaceutically acceptable salt or solvate thereof. Table (III). Non-Limiting Examples of Ionizable Lipids with a Constrained Arm

[0718] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application PCT/US2023/065477, which is incorporated by reference herein, in its entirety.

[0719] In some embodiments, lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. [0720] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I):

or a pharmaceutically acceptable salt thereof, wherein

each Y is independently selected from the group consisting of

R¹ is -(CH₂)1-6N(R^a)2 or -(CH₂)1-6OH;

R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-;

R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted Ci- C6> alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2.

[0721] In some embodiments, a compound of the present disclosure is represented by Formula (CX-i):

(CX-i) or a pharmaceutically acceptable salt thereof, wherein

O

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of

R¹ is -(CH₂)i-6N(R^a)₂;

R² is optionally substituted C1-C36 alkyl or optionally substituted C₂-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2. [0722] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-F), (CX-I”), (CX-I’”),

(CX-F) (CX-I”) (CX-I’”) or a pharmaceutically acceptable salt thereof.

[0723] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-a), (CX-I-b), (CX-I-c), or (CX-I-d):

or a pharmaceutically acceptable salt thereof.

[0724] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-a’), (CX-I-b’), (CX-I-c’), or (CX-I-d’):

(CX-I-c’) (CX-I-d’) or a pharmaceutically acceptable salt thereof.

[0725] In some embodiments, a compound of the present disclosure is represented by Formula (CX-I-a”), (CX-I-b”), (CX-I-c”), or (CX-I-d”):

(CX-I-c”) (CX-I-d”) or a pharmaceutically acceptable salt thereof.

[0726] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-a’”), (CX-I-b’”), (CX-I-c’”), or (CX-I-d’”):

or a pharmaceutically acceptable salt thereof. [0727] In some embodiments, a compound of the present disclosure is represented by Formula (CX-I-e) or (CX-I-f):

(CX-I-e) (CX-I-f) or a pharmaceutically acceptable salt thereof.

[0728] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-e’) or (CX-I-f):

(CX-I-e’) (CX-I-f) or a pharmaceutically acceptable salt thereof.

[0729] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-e”) or (CX-I-f ’):

(CX-I-e”) (CX-I-f ’) or a pharmaceutically acceptable salt thereof. [0730] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-I-e’”) or (CX-I-f”):

(CX-I-e’”) (CX-I-f ”) or a pharmaceutically acceptable salt thereof.

[0731] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II):

(CX-II) or a pharmaceutically acceptable salt thereof, wherein

each Y is independently selected from the group consisting of

R¹ is -(CH₂)1-6N(R^a)2 or -(CH₂)1-6OH;

R² is optionally substituted C5-C36 alkyl or optionally substituted C5-C36 alkenyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R², and wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-;

R² is optionally substituted C1-C36 alkyl or optionally substituted C5-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2.

[0732] In some embodiments, a compound of the present disclosure is represented by Formula (CX-ii):

(CX-ii) or a pharmaceutically acceptable salt thereof, wherein

O

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of ^H , H

R¹ is -(CH₂)i-6N(R^a)₂; R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-;

R² is optionally substituted C1-C36 alkyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and - C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2.

[0733] In some embodiments, a compound of the present disclosure is represented by Formula

(cx-ir), (cx-ii”), (cx-ir”),

or a pharmaceutically acceptable salt thereof.

[0734] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-a)

(CX-II-a) or a pharmaceutically acceptable salt thereof. [0735] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-a’), (CX-II-a”), or (CX-II-a’”),

(CX-II-a’”) or a pharmaceutically acceptable salt thereof.

[0736] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-b), (CX-II-c), or (CX-II-d)

(CX-II-b) (CX-II-c) (CX-II-d) or a pharmaceutically acceptable salt thereof.

[0737] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-b’), (CX-II-c’), or (CX-II-d’)

(CX-II-b’) (CX-II-c’) (CX-II-d’) or a pharmaceutically acceptable salt thereof. [0738] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-b”), (CX-II-c”), or (CX-II-d”)

or a pharmaceutically acceptable salt thereof.

[0739] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-II-b’”), (CX-II-c’”), or (CX-II-d’”)

or a pharmaceutically acceptable salt thereof.

[0740] In some embodiments, a compound of the present disclosure is represented by is represented by formula (CX-II-e):

or a pharmaceutically acceptable salt thereof. [0741] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-III)

or a pharmaceutically acceptable salt thereof, wherein

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of

R¹ is -(CH₂)1-6N(R^a)2 or -(CH₂)1-6OH;

R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; and n is 1 or 2.

[0742] In some embodiments, a compound of the present disclosure is represented by Formula (CX-iii)

or a pharmaceutically acceptable salt thereof, wherein

each Y is independently selected from the group consisting of

R¹ is -(CH₂)i-6N(R^a)₂; each R² is independently optionally substituted C1-C36 alkyl or optionally substituted C₂-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; and n is 1 or 2. [0743] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-III-a), (CX-III-b), or (CX-III-c):

or a pharmaceutically acceptable salt thereof.

[0744] In some embodiments, a compound of the present disclosure is represented by Formula (CX-III-d) or (CX-III-e)

or a pharmaceutically acceptable salt thereof. [0745] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-IV)

or a pharmaceutically acceptable salt thereof, wherein

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of

R¹ is -(CH₂)1-6N(R^a)2 or -(CH₂)1-6OH;

R² is C3-C36 branched alkyl or optionally substituted C3-C36 branched alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene and -O-;

R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; and n is 1 or 2. [0746] In some embodiments, a compound is represented by formula (CX-IV-a), (CX-IV-b), or (CX-IV-c):

or a pharmaceutically acceptable salt thereof.

[0747] In some embodiments, a compound of the present disclosure compound is represented by formula (CX-IV-d) or (CX-IV-e):

or a pharmaceutically acceptable salt thereof.

[0748] In some embodiments, Z is selected from the group consisting of a bond,

[0749] In some embodiments, Z is selected from the group consisting of

O

[0750] In some embodiments, Z is selected from the group consisting of a bond,

wherein R¹ is attached at the position denoted by *.

O

[0751] In some embodiments, Z is selected from the group consisting of

wherein R¹ is attached at the position denoted by *.

O O

[0752] In some embodiments, Z is

In some embodiments, Z is

0 wherein R¹ is attached at the position denoted by *. In some embodiments, Z is

O wherein R¹ is attached at the position denoted by *. In some embodiments, Z is

wherein R¹ is attached at the position denoted by *.

[0753] In some embodiments, each Y is independently selected from the group consisting of

[0754] In some embodiments, Y is selected from the group consisting of ⁰

[0755] In some embodiments, Y is selected from the group consisting

,

wherein R² is attached at the position denoted by *

[0756] In some embodiments, Y is

, wherein R² is attached at the position denoted

by *. In some embodiments, Y is wherein R² is attached at the position denoted by

0

*. In some embodiments, Y is wherein R² is attached at the position denoted by

In some embodiments, Y is

wherein R² is attached at the position denoted by

[0757] In some embodiments,

[0758] In some embodiments,

[0759] In some embodiments,

R¹

[0760] In some embodiments, R¹ is -(CH2)1-6N(R^a)2 or -(CH2)1-6OH. In some embodiments, R¹ is -(CH₂)I.₆OH. In some embodiments, R¹ is -(CH2)1-6N(R^a)2. In some embodiments, R¹ is - (CH₂)₂N(R^a)₂. In some embodiments, R¹ is -(CH2)3N(R^a)2. In some embodiments, R¹ is - (CH₂)₄N(R^a)2. In some embodiments, R¹ is -(CH2)1-6N(Me)2. In some embodiments, R¹ is - (CH₂)i-6N(Et)₂. In some embodiments, R¹ is -(CH₂)i-6N(n-Pr)₂. In some embodiments, R¹ is - (CH2)1-6N(i-Pr)2. In some embodiments, R¹ is -(CH2)2N(Me)2. In some embodiments, R¹ is - (CH2)3N(Me)2. In some embodiments, R¹ is -(CH2)4N(Me)2. In some embodiments, R¹ is - (CH₂)₂N(Et)₂. In some embodiments, R¹ is -(CH2)3N(Et)2. In some embodiments, R¹ is - (CH₂)₄N(Et)₂.

[0761] In some embodiments, R¹ is selected from the group consisting of

[0762] In some embodiments, R¹ is selected from the group consisting of

[0763] In some embodiments, R¹ is selected from the group consisting of

R² and R²

[0764] In some embodiments, R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C32 alkyl or optionally substituted C2-C32 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C30 alkyl or optionally substituted C2-C30 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)- , and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl, wherein 1-6 methylene units of R² are replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl. In some embodiments, R² is optionally substituted C10-C24 alkyl or optionally substituted C10-C24 alkenyl, wherein 1-6 methylene units of R² are replaced with -O-.

[0765] In some embodiments, R² is optionally substituted C5-C36 alkyl or optionally substituted C5-C36 alkenyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R², and wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; and R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-.

[0766] In some embodiments, R² is optionally substituted C10-C24 alkyl or optionally substituted C10-C24 alkenyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R² and wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; and R² is optionally substituted C10-C36 branched alkyl or optionally substituted C10-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-.

[0767] In some embodiments, R² is C3-C36 branched alkyl or optionally substituted C3-C36 branched alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene and -O-; and R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and - C(O)O-. In some embodiments, R² is optionally substituted C10-C24 branched alkyl or optionally substituted C10-C24 branched alkenyl, wherein 1-3 methylene units of R² are optionally replaced with -O-; and R² is optionally substituted C10-C36 alkyl or optionally substituted C10-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-.

[0768] In some embodiments, R² and/or R² is

wherein each q is independently selected from 0-12 and each R° is independently selected, and is as described and defined herein. [0769] In some embodiments, R² and/or R² is

wherein each q is independently selected from 0-12.

[0770] In some embodiments, R² is optionally substituted C5-C36 alkyl or optionally substituted C5-C36 alkenyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R², and wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; R² is optionally substituted C1-C36 alkyl or optionally substituted C5-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-.

[0771] In some embodiments, R² is optionally substituted C10-C24 alkyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R²; and R² is optionally substituted C10-C24 alkyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R² .

[0772] In some embodiments, each q is independently selected from 0-6. In some embodiments, each q is independently selected from 0-8. In some embodiments, each q is independently selected from 0-10. In some embodiments, each q is independently selected from 0-12.

[0773] In some embodiments, R² is optionally substituted C10-C24 alkyl or optionally substituted C10-C24 alkenyl, wherein 2 methylene units of R² are replaced with -O- to form an acetal within R² and wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; and R² is optionally substituted C10-C24 alkenyl, wherein 1-3 methylene units of R² are optionally replaced with -O-

[0774] In some embodiments, R² is selected from the group consisting of

[0776] In some embodiments, R² is selected from the group consisting of

[0777] In some embodiments, R² is optionally substituted C1-C36 alkyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C32 alkyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C30 alkyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and - C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl, wherein 1-6 methylene units of R² are replaced with a group each independently selected from -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl. In some embodiments, R² is optionally substituted C10-C24 alkyl, wherein 1-6 methylene units of R² are replaced with -O-.

[0778] In some embodiments, R² is selected from the group consisting of

[0779] In some embodiments, R² and R² are each independently selected from the group consisting of

[0780] In some embodiments, R² is selected from the group consisting of

[0781] In some embodiments, R² is

In some embodiments, R² is

[0782] In some embodiments, R² is selected from the group consisting of

[0783] In some embodiments, R² is selected from the group consisting of

[0785] In some embodiments, the present disclosure includes a compound selected from any lipid in Table (IV) below or a pharmaceutically acceptable salt thereof: Table (IV). Non-Limiting Examples of Ionizable Lipids

[0786] In some embodiments, lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof.

[0787] In some embodiments, a compound of the present disclosure is represented by Formula

(CZ-I)

or a pharmaceutically acceptable salt thereof, wherein

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of , and

R¹ is -(CH₂)i-6N(R^a)₂; each R² is independently optionally substituted C1-C36 alkyl or optionally substituted C₂-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2.

[0788] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-I-a), (CZ-I-b), (CZ-I-c), or (CZ-I-d)

or a pharmaceutically acceptable salt thereof. [0789] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-I-e) or (CZ-I-f)

or a pharmaceutically acceptable salt thereof.

[0790] In some embodiments, a compound of the present disclosure is represented by Formula

(CZ-I-g)

or a pharmaceutically acceptable salt thereof.

[0791] In some embodiments, a compound of the present disclosure is represented by Formula

(CZ-II)

or a pharmaceutically acceptable salt thereof, wherein

Z is selected from the group consisting of a bond,

each Y is independently selected from the group consisting of ,

R¹ is -(CH₂)i-6N(R^a)₂; each R² is independently optionally substituted C1-C36 alkyl or optionally substituted C₂-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each R^a is independently optionally substituted C1-C6 alkyl; or two R^a are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2. or a pharmaceutically acceptable salt thereof.

[0792] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-II-a), (CZ-II-b), (CZ-II-c) or (CZ-II-d):

or a pharmaceutically acceptable salt thereof. [0793] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-II-e)

or a pharmaceutically acceptable salt thereof.

[0794] In some embodiments, Z is selected from the group consisting of a bond,

[0795] In some embodiments, Z is selected from the group consisting of

[0796] In some embodiments, Z is selected from the group consisting of a bond,

wherein R¹ is attached at the position denoted by *

[0797] In some embodiments, Z is selected from the group consisting of

, wherein R¹ is attached at the position denoted by *. o

[0798] In some embodiments, Z is

In some embodiments, Z is

wherein R¹ is attached at the position denoted by * In some embodiments, Z is

wherein R¹ is attached at the position denoted by *. In some embodiments,

wherein R¹ is attached at the position denoted by *

[0799] In some embodiments, Y is selected from the group consisting of ,

In some embodiments, Y is or .

[0800] In some embodiments, Y is selected from the group consisting of ,

, wherein R² is attached at the position denoted by *

[0801] In some embodiments, Y is , wherein R² is attached at the position denoted by *. In some embodiments, Y is , wherein R² is attached at the position denoted by *. In some

embodiments, Y is ^H , wherein R² is attached at the position denoted by *. In some embodiments, Y is

wherein R² is attached at the position denoted by *. In some embodiments,

[0802] In some embodiments,

[0803] In some embodiments,

[0804] In some embodiments,

R¹

[0805] In some embodiments, R¹ is -(CH2)1-6N(R^a)2. In some embodiments, R¹ is - (CH₂)₂N(R^a)₂. In some embodiments, R¹ is -(CH2)3N(R^a)2. In some embodiments, R¹ is - (CH₂)₄N(R^a)₂. In some embodiments, R¹ is -(CH2)1-6N(Me)2. In some embodiments, R¹ is - (CH₂)i-6N(Et)₂. In some embodiments, R¹ is -(CH₂)i-6N(n-Pr)₂. In some embodiments, R¹ is - (CH2)1-6N(CZ-I-Pr)2. In some embodiments, R¹ is -(CH2)2N(Me)2. In some embodiments, R¹ is -(CH2)3N(Me)2. In some embodiments, R¹ is -(CH2)4N(Me)2. In some embodiments, R¹ is - (CH₂)₂N(Et)₂. In some embodiments, R¹ is -(CH2)3N(Et)2. In some embodiments, R¹ is - (CH₂)₄N(Et)₂.

[0806] In some embodiments, R¹ is selected from the group consisting of

[0807] In some embodiments, R¹ is selected from the group consisting of

[0808] In some embodiments, R¹ is selected from the group consisting of

R²

[0809] In some embodiments, R² is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C32 alkyl or optionally substituted C2-C32 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C30 alkyl or optionally substituted C2-C30 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl, wherein 1-6 methylene units of R² are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)- , and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl, wherein 1-6 methylene units of R² are replaced with a group each independently selected from -O-, -OC(O)-, and -C(O)O-. In some embodiments, R² is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl. In some embodiments, R² is optionally substituted C10-C24 alkyl or optionally substituted C10-C24 alkenyl, wherein 1-6 methylene units of R² are replaced with -O-.

[0810] In some embodiments, R² is

wherein each q is independently selected from 0-12 and each R° is independently selected and defined herein.

[0811] In some embodiments, R² is

wherein each q is independently selected from 0-12.

[0812] In some embodiments, each q is independently selected from 0-6. In some embodiments, each q is independently selected from 0-8. In some embodiments, each q is independently selected from 0-10. In some embodiments, each q is independently selected from 0-12.

[0813] In some embodiments, R² is selected from the group consisting of

[0814] In some embodiments, R² is selected from the group consisting of

[0815] In some embodiments, the present disclosure includes a compound selected from any lipid in Table (V) below or a pharmaceutically acceptable salt thereof:

Table (V). Non-Limiting Examples of Ionizable Lipids

ii. Structural lipids

[0816] In some embodiments, an LNP comprises a structural lipid. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof. In some embodiments, a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety. [0817] In some embodiments, a structural lipid is a cholesterol analog. Using a cholesterol analog may enhance endosomal escape as described in Patel et al., Naturally-occuring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications (2020), which is incorporated herein by reference.

[0818] In some embodiments, a structural lipid is a phytosterol. Using a phytosterol may enhance endosomal escape as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference.

[0819] In some embodiments, a structural lipid contains plant sterol mimetics for enhanced endosomal release. iii. PEGylated lipids

[0820] A PEGylated lipid is a lipid modified with polyethylene glycol. In some embodiments, the LNP comprises a compound of Formula I, Formula I’, Formula I” or a pharmaceutically acceptable salt thereof, as described herein above. In some embodiments, the LNP comprises a compound of Formula II, Formula II’, Formula II” or a pharmaceutically acceptable salt thereof, as described herein above. In some embodiments, the LNP comprises a compound set forth in Table 1, as described herein above.

[0821] In some embodiments, an LNP comprises an additional PEGylated lipid or PEG- modified lipid. A PEGylated lipid may be selected from the non-limiting 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 PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0822] In some embodiments, an LNP comprises an additional PEGylated lipid selected from (R)-2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG- S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG-DSG, PEG- DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG Cl 4, PEG- DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-Cl l, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C- DMA, PEG2000, PEG200, PEG(2k)-DMG, PEGDSPE C18, PEGDMPE C14, PEGDLPE C12, PEG Click DMG C14, PEG Click Cl 2, PEG Click CIO, N(Carbonyl-m ethoxypoly ethylengly col- 2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000- DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE- PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG- PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3-bis(octadecyloxy)propyl-l- (methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[0823] In some embodiments, the LNP comprises a PEGylated lipid disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095 Al; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828 Al, each of which is incorporated by reference herein in their entirety.

[0824] In some embodiments, the LNP comprises a PEGylated lipid substitute in place of the PEGylated lipid. All embodiments disclosed herein that contemplate a PEGylated lipid should be understood to also apply to PEGylated lipid substitutes. In some embodiments, the LNP comprises a polysarcosine-lipid conjugate, such as those disclosed in US 2022/0001025 Al, which is incorporated by reference herein in its entirety. v. Phospholipids

[0825] In some embodiments, an LNP of the present disclosure comprises a phospholipid. Phospholipids useful in the compositions and methods may be selected from the non-limiting group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine

(DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 - phosphocho line (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Di ether PC),

1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoylsn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3-

(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-a-phosphatidyl serine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), di oleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2- dioleoyl-sn-glycero-3 -phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3- (oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), l,2-dioleoyl-sn-glycero-3-phospho-(l’-myo- inositol) (DOPI; 18: 1 PI), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2- dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-

2-hydroxy-sn-glycero-3-phospho-L-serine (18: 1 Lyso PS), l-stearoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (18:0 Lyso PS), and sphingomyelin. In some embodiments, an LNP includes DSPC. In certain embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE.

[0826] In some embodiments, an LNP comprises a phospholipid selected from 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine, l-myristoyl-2-palmitoyl-sn-glycero-3- phosphocholine, l-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-myristoyl- sn-glycero-3 -phosphocholine, 1 -palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 - palmitoyl-2-oleoyl-glycero-3 -phosphocholine, l-palmitoyl-2-linoleoyl-sn-glycero-3- phosphocholine, 1 -palmitoyl-2-arachidonoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl-2- docosahexaenoyl-sn-glycero-3-phosphocholine, l-stearoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine, l-stearoyl-2-oleoyl-sn- glycero-3 -phosphocholine, 1 -stearoyl-2-linoleoyl-sn-glycero-3 -phosphocholine, 1 -stearoyl-2- arachidonoyl-sn-glycero-3-phosphocholine, l-stearoyl-2-docosahexaenoyl-sn-glycero-3- phosphocholine, l-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine, l-oleoyl-2-palmitoyl-sn- glycero-3 -phosphocholine, 1 -oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl-2- acetyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn-glycero-3 -phospho-( 1 ’ -myo-inositol-3 ’ ,4’ - bisphosphate), 1 ,2-dioleoyl-sn-glycero-3 -phospho-( 1 ’ -myo-inositol-3 ’ ,5 ’ -bisphosphate), 1 ,2- dioleoyl-sn-glycero-3 -phospho-( 1 ’ -myo-inositol -4 ’ , 5 ’ -bisphosphate), 1 ,2-dioleoyl-sn-glycero- 3 -phospho-(l'-myo-inositol-3 ',4', 5'-trisphosphate), 1 ,2-dioleoyl-sn-glycero-3 -phospho-( 1 ’ -myo- inositol-3 ’-phosphate), l,2-dioleoyl-sn-glycero-3-phospho-(r-myo-inositol-4’-phosphate), 1,2- dioleoyl-sn-glycero-3-phospho-(l'-myo-inositol-5'-phosphate), l,2-dioleoyl-sn-glycero-3- phospho-(l’ -myo-inositol), l,2-dioleoyl-sn-glycero-3-phospho-L-serine, and 1-(8Z- octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine.

[0827] In some embodiments, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S. 2021/0121411, which is incorporated herein by reference. [0828] In some embodiments, the LNP comprises a phospholipid disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095 Al; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety.

[0829] In some embodiments, phospholipids disclosed in US 2020/0121809 have the following structure:

wherein Ri and R2 are each independently a branched or straight, saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl). vi. Targeting moi eties

[0830] In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen.

[0831] In some embodiments, the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed more in the target organ than it is in the liver.

[0832] In some embodiments, the targeting moiety is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeted particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows specific delivery of the siRNAs encapsulated within the particles at a greater percentage to B-cell lymphocytes malignancies (such as MCL) than to other subtypes of leukocytes.

[0833] In some embodiments, the lipid nanoparticles may be targeted when conjugated/attached/associated with a targeting moiety such as an antibody. vii. Zwitterionic amino lipids

[0834] In some embodiments, an LNP comprises a zwitterionic lipid. In some embodiments, an LNP comprising a zwitterionic lipid does not comprise a phospholipid.

[0835] Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs without phospholipids to load, stabilize, and release mRNAs intracellular as described in U.S. Patent Application 20210121411, which is incorporated herein by reference in its entirety. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs as described in Liu et al., Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety.

[0836] The zwitterionic lipids may have head groups containing a cationic amine and an anionic carboxylate as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine a-amine may reduce immunogenicity as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013). viii. Additional Lipid Components

[0001] In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of influencing the tropism of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and Cl 2-200 (see Cheng, et al. Nat Nanotechnol. 2020 April; 15(4): 313-320.; Dillard, et al. PNAS 2021 Vol. 118 No. 52.).

[0002] In some embodiments, the LNP compositions of the present disclosure comprise, or further comprise one or more lipids selected from l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine (18:3 PC), Acylcamosine (AC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), N-oleoyl- sphingomyelin (SPM) (Cl 8:1), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8- 9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), l,2-Dipalmitoylglycerol-3- hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), di oleyloxybenzyl alcohol (DOB A), 1,2- dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy- propoxy)-ethoxy ] -ethoxy } -ethoxy)-ethyl] -3 -(3 ,4, 5 - 1 rihy droxy-6-hy droxymethyl- 1 etrahy dro- pyran-2-ylsulfanyl)-propionamide (DOGP4aMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), di oleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedi stearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Na- Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-a- histidinyl-Na-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2-Dioleoyl-sn-Glycero-3- Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), l-myristoyl-2-hydroxy-sn- glycero-phosphocholine (MHPC), a thiol -reactive maleimide headgroup lipid e.g.l,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3- [phosphoethanolamine-N-dodecanoyl (NC12-DOPE), l-oleoyl-2-cholesteryl hemisuccinoyl-sn- glycero-3 -phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycoldistearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamine B-phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans- PE,l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha, alpha-trehalose-6,6'-dibehenate (TDB), 1,2-dielaidoyl-sn-glycero- 3-phophoethanolamine (transDOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl- 2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)tetrahydrofuran-2-yl)methylmethylphosphate, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphocholine, l,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinoleoyl-sn- glycero-3 -phosphoethanolamine, 1 ,2-dioleyl-sn-glycero-3 -phosphoethanolamine, 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine, 16-0-monom ethyl PE, 16-O-dimethyl PE, and di ol ey Iphosphati dy 1 ethanol amine . IX. Polynucleotides

[0837] In some embodiments, an LNP of the present disclosure contains an active agent. In some embodiments, an active agent is a polynucleotide. In some embodiments, a LNP is capable of delivering a polynucleotide to a target organ. A polynucleotide, in its broadest sense of the term, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, a polynucleotide is mRNA. In some embodiments, a polynucleotide is circular RNA. Any of the circular polynucleotides as taught in U.S. Patent No. 10,709,779, which is incorporated by reference herein in its entirety, may be used herein. In addition, any of the circular RNAs, methods for making circular RNAs, circular RNA compositions that are described in the following publications are contemplated herein and are incorporated by reference in their entireties as part of the instant specification: US Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; US Application Publications US 2022/0177540, US 2021/0371494, US 2022/0090137, US 2019/0345503, and US 2015/0299702; and PCT Application Publications WO 2021/226597, WO 2019/236673, WO 2017/222911, WO2016/187583, WO2014/082644 and WO 1997/007825.

[0838] In some embodiments, a polynucleotide encodes a protein. A polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0839] In other embodiments, a polynucleotide is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0840] In some embodiments, a polynucleotide is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.

[0841] A polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3 '-stabilizing region. In some embodiments, a polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide (e.g., an mRNA) may include a 5'cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and/or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5- methoxyu ridine), a 1 -substituted pseudouridine (e.g., 1 -methyl pseudouridine or 1-ethyl- pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine). In some embodiments, a polynucleotide contains only naturally occurring nucleosides.

[0842] In some cases, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.

[0843] In some embodiments, a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in W02002/098443, W02003/051401, W02008/052770, W02009/127230, WO2006/122828,

W02008/083949, WO2010/088927, W02010/037539, W02004/004743, W02005/016376,

W02006/024518, W02007/095,976, W02008/014979, W02008/077592, W02009/030481,

W02009/095226, WO2011/069586, WO2011/026641, WO2011/144358, W02012/019780,

WO2012/013326, WO2012/089338, WO2012/113513, WO2012/116811, WO2012/116810,

WO2013/113502, WO2013/113501, WO2013/113736, WO2013/143698, WO2013/143699,

W02013/143700, WO2013/120626, WO2013/120627, WO2013/120628, WO2013/120629,

WO20 13/174409, WO2014/127917, WO2015/024669, WO2015/024668, WO2015/024667,

WO20 15/024665, WO2015/024666, WO2015/024664, W02015/101415, W02015/101414,

WO20 15/024667, WO2015/062738, W02015/101416, all of which are incorporated by reference herein.

[0844] Polynucleotides, such as circular RNA, may contain an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. A polynucleotide containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multi ci stronic mRNA). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical Swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0845] In some embodiments, a polynucleotide comprises one or more microRNA binding sites. In some embodiments, a microRNA binding site is recognized by a microRNA in a nontarget organ. In some embodiments, a microRNA binding site is recognized by a microRNA in the liver. In some embodiments, a microRNA binding site is recognized by a microRNA in hepatic cells.

D. Methods and Pharmaceutical Compositions i. Methods

[0846] The pharmaceutical composition may be delivered as described in PCT Publication WO2012135805, which is incorporated herein by reference in its entirety.

[0847] The present disclosure provides methods comprising administering a pharmaceutical composition to a subject in need thereof. The pharmaceutical composition may be administered to a subject using any amount and any route of administration which may be effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition. The exact amount required will vary from subject to subject, depending on factors such as, but not limited to, the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The pharmaceutical composition may be administered to animals, such as mammals (e.g., humans, domesticated animals, cats, dogs, monkeys, mice, rats, etc.). The payload of the pharmaceutical composition is a polynucelotide.

[0848] In some embodiments, pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered to humans.

[0849] In some embodiments, the active agent is administered by one or more of a variety of routes, including, but not limited to, local, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter. [0850] In some embodiments, the active agent is administered by systemic intravenous injection. In some embodiments, the active agent is administered intravenously and/or orally.

[0851] In specific embodiments, the active agent may be administered in a way which allows the active agent to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0852] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0853] Inj ectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0854] Dosage forms for local, topical and/or transdermal administration of a pharmaceutical composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

[0855] Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0856] A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this disclosure.

[0857] In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the active agent to be delivered (e.g., its stability in the environment of the gastrointestinal tract, bloodstream, etc), the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc. The present disclosure encompasses the delivery of the active agent by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

[0858] In certain embodiments, pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic or prophylactic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administration is employed, split dosing regimens such as those described herein may be used.

[0859] According to the present disclosure, administration of active agent in split-dose regimens may produce higher levels of proteins in mammalian subjects. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the active agent of the present disclosure are administered to a subject in split doses. In some embodiments, the active agent is formulated in buffer only or in a formulation described herein. [0860] LNPs of the present disclosure may be used or administered in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

[0861] It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single pharmaceutical composition or administered separately in different pharmaceutical compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In one embodiment, the combinations, each or together may be administered according to the split dosing regimens described herein.

[0862] The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a pharmaceutical composition useful for treating cancer in accordance with the present disclosure may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects).

[0863] Pharmaceutical compositions containing LNPs disclosed herein are formulated for administration intramuscularly, transarterially, intraocularly, vaginally, rectally, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, intramuscularly, intraventricularly, intradermally, intrathecally, topically (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosally, nasal, enterally, intratumorally, by intratracheal instillation, bronchial instillation, and/or inhalation; nasal spray and/or aerosol, and/or through a portal vein catheter. [0864] The pharmaceutical compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the pharmaceutical compositions, and the like. In some embodiments, the pharmaceutical composition is formulated for extended release. In specific embodiments, the active agent and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, may be administered in a way which allows the active agent to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0865] In some aspects of the present disclosure, the active agent of the present disclosure are spatially retained within or proximal to a target tissue. Provided are methods of providing a pharmaceutical composition to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the pharmaceutical composition under conditions such that the pharmaceutical composition, in particular the active agent component(s) of the pharmaceutical composition, is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition is retained in the target tissue. Advantageously, retention is determined by measuring the amount of a component of the active agent present in the pharmaceutical composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the active agent administered to the subject are present intracellularly at a period of time following administration.

[0866] Aspects of the present disclosure are directed to methods of providing a pharmaceutical composition to a target tissue or organ of a mammalian subject, by contacting the target tissue (containing one or more target cells) or organ (containing one or more target cells) with the pharmaceutical composition under conditions such that the pharmaeutical composition is substantially retained in the target tissue or organ. The pharmaceutical composition contains an effective amount of an active agent.

[0867] Pharmaceutical compositions which may be administered intramuscularly and/or subcutaneously may include, but are not limited to, polymers, copolymers, and gels. The polymers, copolymers and/or gels may further be adjusted to modify release kinetics by adjusting factors such as, but not limited to, molecular weight, particle size, payload and/or ratio of the monomers. As a nonlimiting example, formulations administered intramuscularly and/or subcutaneously may include a copolymer such as poly(lactic-co-glycolic acid). [0868] Localized delivery of the pharmaceutical compositions described herein may be administered by methods such as, but not limited to, topical delivery, ocular delivery, transdermal delivery, and the like. The pharmaceutical composition may also be administered locally to a part of the body not normally available for localized delivery such as, but not limited to, when a subject’s body is open to the environment during treatment. The pharmaceutical composition may further be delivered by bathing, soaking and/or surrounding the body part with the pharmaceutical composition.

[0869] However, the present disclosure encompasses the delivery of an active agent disclosed herein, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, by any appropriate route taking into consideration likely advances in the sciences of drug delivery. ii. Pharmaceutical compositions

[0870] In some embodiments, a nanoparticle includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid. In a particular embodiment, the lipid component includes about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component includes about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol. The amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the active agent. For example, the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to an enzyme in a 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. The amount of a enzyme in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0871] In some embodiments, a nanoparticle composition comprising an active agent of the present disclosure is formulated to provide a specific E:P ratio. The E: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 active agent. In general, a lower E:P ratio is preferred. The one or more enzymes, lipids, and amounts thereof may be selected to provide an E:P ratio from about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the E:P ratio may be from about 2: 1 to about 8: 1. In other embodiments, the E:P ratio is from about 5: 1 to about 8:1. For example, the E:P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1.

[0872] The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composi tion including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. Nanoparticle compositions 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 nanoparticle composition. 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 nanoparticle composition, Such as particle size, poly dispersity index, and Zeta potential.

[0873] The mean size of a nanoparticle composition may be between 10s of nm and 100s of nm, 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, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition may be 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, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.

[0874] A nanoparticle composition may be relatively homogenous. A poly dispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.

[0875] The Zeta potential of a 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 nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are 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 nanoparticle composition may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 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 +15 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 +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV, to about +15 mV, or from about +5 mV to about +10 mV.

[0876] The efficiency of encapsulation of a payload describes the amount of payload that is encapsulated or otherwise associated with a 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 payload in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free payload in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and/or prophylactic may be at least 50%, for example 50%, 55%, 60%. 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0877] Lipids and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2017/117528, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO2011/141705, and WO 2001/07548 and Semple et. al, Nature Biotechnology, 2010, 28, 172-176, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0878] A nanoparticle composition may include any substance useful in pharmaceutical compositions. For example, the nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see for example Remington’s The Science and Practice of Pharmacy, 21^st Edition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006). iii. Diseases, Disorders, and Other Uses

Methods of Producing Polypeptides in Cells

[0879] The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. Methods of producing polypeptides involve contacting a cell with a formulation of the disclosure comprising an LNP including an mRNA encoding the polypeptide of interest. Upon contacting the cell with the lipid nanoparticle, the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.

[0880] In general, the step of contacting a mammalian cell with a LNP including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticle contacted with a cell, and/or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the lipid nanoparticle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors. In general, an effective amount of the lipid nanoparticle will allow for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.

[0881] The step of contacting an LNP including an mRNA with a cell may involve or cause transfection. A phospholipid including in the lipid component of a LNP may facilitate transfection and/or increase transfection efficiency, for example, by interacting and/or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell.

[0882] In some embodiments, the lipid nanoparticles described herein may be used therapeutically. For example, an mRNA included in an LNP may encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contacting and/or entry (e.g., transfection) into a cell. In other embodiments, an mRNA included in a LNP may encode a polypeptide that may improve or increase the immunity of a subject. In some embodiments, an mRNA may encode a granulocyte-colony stimulating factor or trastuzumab.

[0883] In some embodiments, an mRNA included in an LNP may encode a recombinant polypeptide that may replace one or more polypeptides that may be substantially absent in a cell contacted with the lipid nanoparticle. The one or more substantially absent polypeptides may be lacking due to a genetic mutation of the encoding gene or a regulatory pathway thereof. Alternatively, a recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. An antagonistic recombinant polypeptide may be desirable to combat deleterious effects caused by activities of the endogenous protein, such as altered activities or localization caused by mutation. In another alternative, a recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in, on the surface of, or secreted from the cell. Antagonized biological moi eties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides produced by translation of the mRNA may be engineered for localization within the cell, such as within a specific compartment such as the nucleus, or may be engineered for secretion from the cell or for translocation to the plasma membrane of the cell.

[0884] In some embodiments, contacting a cell with an LNP including an mRNA may reduce the innate immune response of a cell to an exogenous nucleic acid. A cell may be contacted with a first lipid nanoparticle including a first amount of a first exogenous mRNA including a translatable region and the level of the innate immune response of the cell to the first exogenous mRNA may be determined. Subsequently, the cell may be contacted with a second composition including a second amount of the first exogenous mRNA, the second amount being a lesser amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may include a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The steps of contacting the cell with the first and second compositions may be repeated one or more times. Additionally, efficiency of polypeptide production (e.g., translation) in the cell may be optionally determined, and the cell may be re-contacted with the first and/or second composition repeatedly until a target protein production efficiency is achieved.

Methods of Delivering Therapeutic Agents to Cells and Organs

[0885] Provided herein are methods of treating a disease or disorder, the methods comprising administering to a subject in need thereof a pharmaceutical composition of the present disclosure, such as a pharmaceutical composition comprising an LNP described herein.

[0886] The present disclosure provides methods of delivering an active agent and/or prophylactic, such as a nucleic acid, to a mammalian cell or organ. Delivery of a therapeutic and/or prophylactic to a cell involves administering a formulation of the disclosure that comprises a LNP including the therapeutic and/or prophylactic, such as a nucleic acid, to a subject, where administration of the composition involves contacting the cell with the composition. In some embodiments, a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ. In the instance that a therapeutic and/or prophylactic is an mRNA, upon contacting a cell with the lipid nanoparticle, a translatable mRNA may be translated in the cell to produce a polypeptide of interest. However, mRNAs that are substantially not translatable may also be delivered to cells. Substantially non- translatable mRNAs may be useful as vaccines and/or may sequester translational components of a cell to reduce expression of other species in the cell.

[0887] In some embodiments, an LNP may target a particular type or class of cells (e.g., cells of a particular organ or system thereof). In some embodiments, a LNP including a therapeutic and/or prophylactic of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung. “Specific delivery” to a particular class of cells, an organ, or a system or group thereof implies that a higher proportion of lipid nanoparticles including a therapeutic and/or prophylactic are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of an LNP to a mammal. In some embodiments, specific delivery may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of therapeutic and/or prophylactic per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g., the spleen). In some embodiments, the tissue of interest is selected from the group consisting of a liver, kidney, a lung, a spleen, a femur, vascular endothelium in vessels (e.g., intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g., via intratumoral injection).

[0888] As another example of targeted or specific delivery, an mRNA that encodes a proteinbinding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or a receptor on a cell surface may be included in an LNP. An mRNA may additionally or instead be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other therapeutics and/or prophylactics or elements (e.g., lipids or ligands) of an LNP may be selected based on their affinity for particular receptors (e.g., low density lipoprotein receptors) such that a LNP may more readily interact with a target cell population including the receptors. In some embodiments, ligands may include, but are not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bi-specific antibodies such as disulfide stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors, and fusion proteins. [0889] In some embodiments, a ligand may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In some embodiments, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of targeting interactions.

[0890] A ligand can be selected, e.g., by a person skilled in the biological arts, based on the desired localization or function of the cell. In some embodiments an estrogen receptor ligand, such as tamoxifen, can target cells to estrogen-dependent breast cancer cells that have an increased number of estrogen receptors on the cell surface. Other non-limiting examples of ligand/receptor interactions include CCR1 (e.g., for treatment of inflamed joint tissues or brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., to target to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin), CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., for treatment of inflammation and inflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinal mucosa targeting), and VLA-4NCAM-1 (e.g., targeting to endothelium). In general, any receptor involved in targeting (e.g., cancer metastasis) can be harnessed for use in the methods and compositions described herein.

[0891] Targeted cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0892] In some embodiments, an LNP may target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with neutral or near neutral lipid- containing lipid nanoparticles in the body, and are known to associate with receptors such as low- density lipoprotein receptors (LDLRs) found on the surface of hepatocytes. Thus, an LNP including a lipid component with a neutral or near neutral charge that is administered to a subject may acquire apoE in a subject's body and may subsequently deliver a therapeutic and/or prophylactic (e.g., an RNA) to hepatocytes including LDLRs in a targeted manner.

Methods of Treating Diseases and Disorders

[0893] Lipid nanoparticles are useful for treating a disease, disorder, or condition. In particular, such compositions are useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. In some embodiments, a formulation of the disclosure that comprises an LNP including an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. Because translation may occur rapidly, the methods and compositions may be useful in the treatment of acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. A therapeutic and/or prophylactic included in an LNP may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.

[0894] Diseases, disorders, and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a composition may be administered include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases. Multiple diseases, disorders, and/or conditions may be characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, or they may be essentially non-functional. A specific example of a dysfunctional protein is the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a dysfunctional protein variant of CFTR protein, which causes cystic fibrosis. The present disclosure provides a method for treating such diseases, disorders, and/or conditions in a subject by administering a LNP including an RNA and a lipid component including a PEGylated lipid compound disclosed herein, a phospholipid (optionally unsaturated), optionally a second PEGylated lipid, and a structural lipid, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes an aberrant protein activity present in the cell of the subject.

INCORPORATION BY REFERENCE

[0895] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present disclosure. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control. EXAMPLES

[0896] The following are examples of methods and compositions of the present disclosure. It is understood that various other embodiments may be practiced, given the general description provided herein.

Example 1 : Synthesis of Compounds

Scheme 1: Synthesis of Compound PL-1

Synthesis of benzyl bis(2-hydroxyethyl)carbamate (L58-2):

[0897] A mixture of DOWEX-50 resin (190 mg) and benzyl chloroformate (1.0 g, 9.51 mmol) in a 20 mL vial was stirred for 5 mins. L58-1 (1.62 g, 9.51 mmol) was added to the mixture and stirring continued for 20 mins. To the reaction mixture was added ethyl acetate and the solution phase was separated and evaporated. The crude material was then purified by column chromatography (40 g SiO2: 0 to 10% methanol in DCM gradient) to give compound L58-2 as a colorless liquid (1.2 g, 53%). ¹H-NMR (300 MHz, CDCl3) δ 7.33-7.25 (m, 5H), 5.19 (s, 2H), 3.82 (m, 4H), 3.51 (m, 4H), 3.17-2.90 (m, 2H); CIMS m/z [M-H]’ 237.7. Synthesis of (((benzyloxy)carbonyl)azanediyl)bis(ethane-2,l-diyl) ditetradecanoate (L58-3)

[0898] A solution of compound L58-2 (1.20 g, 5.02 mmol) in dichloromethane (15 mL) and pyridine (2.02 mL, 25.08 mmol, 5.0 eq) was stirred for 5 mins. Myristoyl chloride (3.22 g, 13.04 mmol, 2.6 eq) was added. The reaction mixture was stirred at room temperature for 16 h and then evaporated under vacuum. The crude material was purified by column chromatography (Gold 40 g SiO2: 0 to 50% EtOAc: Hexanes) to obtain compound L58-3 as a colorless liquid (2.17 g, 65%). ¹H-NMR (300 MHz, CDCl3) δ 7.34-7.33 (m, 5H), 5.13 (s, 2H), 4.22-4.16 (m, 4H), 3.59-3.55 (m, 4H), 2.28-2.24 (m, 4H), 1.59-1.49 (m, 6H), 1.38-1.11 (m, 45H), 0.87 (t, 6H); CIMS m/z [M+H]⁺ 660.6.

Synthesis of azanediylbis(ethane-2,l-diyl) ditetradecanoate (L58-4)

[0899] To a solution of L58-3 (2.00 g, 3.03 mmol) in methanol (10 mL) and EtOAc (2 mL), was added Pd(OH)2 (200 mg) and the resulting mixture was stirred at room temperature for 18 h under hydrogen gas (1 atm). The reaction mixture was then filtered and evaporated to get the desired product L58-4 which was used in the next step without any further purification. CIMS m/z [M+H]⁺ 526.6.

Synthesis of Compound PL-1

Compound PL-1 [0900] To a solution of compound L58-4 (0.125 g, 0.238 mmol) in dichloromethane (5 mL) was added DIPEA (92 mg, 0.714 mmol) and triphosgene (0.085 g, 0.286 mmol), followed by mPEG-NH2 2K (0.404 g, 0.202 mmol). The resulting mixture was stirred at room temperature for 16 h and then evaporated under vacuum. The crude material was purified by column chromatography (40 g SiO2: 0 to 10% MeOH in DCM) to obtain compound PL-1 as a white solid (127 mg, 22%); ¹H-NMR (300 MHz, CDCl3) δ 4.25-4.14 (m, 4H), 3.89 (t, 1H), 3.72-3.47 (m, 190H), 3.43-3.34 (m, 6H), 2.29 (t, 2H), 1.36-1.19 (m, 45H), 0.87 (t, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 12.57 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, Cl 8, 1.7 pm, 3. Ox 150 mm, (Part No. 186005302), mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: CAD, fe= 13.50 min, purity: > 99%.

Scheme 2: Synthesis of Compound PL-2

Compound PL-2 Synthesis of 3,5-dihydroxycyclohexyl 4-methylbenzenesulfonate (L60-2):

[0901] To a 250 mL round bottom flask was added L60-1 (5.2 g, 39.34 mmol) and anhydrous pyridine (50 mL), and the reaction mixture was cooled in an ice-water bath. /?-tosyl sulphonyl chloride (2.5 g, 13.11 mmol) was added to the reaction mixture and was stirred at 0 °C for 2h. The reaction mixture was allowed to warm to room temperature and stirred for an additional 18h. After completion of the reaction, about 20 g of Flash silica was added and the contents were stirred well to yield a uniform mixture. Solvent was removed from this mixture under vacuum. The residue was purified by flash chromatography ( SiO2: ethyl acetate/hexane 20-100%) to get compound L60-2 (3.7 g , 83%); ¹H NMR (300 MHz, CDCl3): δ ppm 7.86 (d, J =8.0 Hz, 2H), 7.36 (d, J =8.0 Hz, 2H), 4.89-4.79 (m, 0.3H), 4.56-4.44(m, 0.7H), 4.19-4.09 (m, 1H), 3.76-3.61 (m, 1H), 2.49 (s, 3H), 2.21-2.1 (m, 2H), 2.03 (s, lH),1.75(d, J =8.0 Hz, 1H), 1.69-1.46 (m, 3H), 1.39-1.31 (m, 1H), 1.28-1.23 (m, 1H); MS (CI) : m/z [M+H]⁺ 287.38.

Synthesis of 5-azidocyclohexane-l,3-diol (L60-3):

[0902] To a 25mL round bottom flask was added L60-2 (1.08 g, 3.77 mmol), sodium azide (981mg, 15.08 mmol), potassium carbonate (1.04 g, 7.45 mmol) and anhydrous DMF (3 mL), and the reaction mixture was stirred at 70 °C for 18h. After completion of the reaction, the reaction mixture was loaded onto a silica cartridge which was then attached to flash purification system loaded with 40g flash silica column and purified by flash chromatography (SiO2: ethyl acetate/hexane 20-100%) to get Compound L60-3 (300 mg, 51%); ¹H NMR (300 MHz, CDCl3): δ ppm 4.29-4.19 (m, 2H), 4.17-4.12 (m, 1H), 2.19-2.08 (m, 2H), 2.00-1.88 (s, 1H), 1.78-1.58 (m, 6H); MS (CI) : m/z [M+H]⁺ 158.19. Synthesis of 5-azidocyclohexane-l,3-diyl ditetradecanoate (L60-4):

[0903] To a 100 mL round bottom flask was added L60-3 (300 mg, 3.77 mmol), potassium carbonate (686 mg, 4.96 mmol) and myristoyl chloride (1.41mg, 5.73 mmol) and anhydrous pyridine (20 mL). The reaction mixture was stirred at room temperature for 18h. After completion of the reaction the reaction contents were loaded onto a silica cartridge which was then attached to flash purification system loaded with 40 g flash silica column and purified by flash chromatography (SiO2: ethyl acetate/hexane 0-60%) to give L60-4 (284 mg, 26%). ¹H NMR (300 MHz, CDCl3): δ ppm 5.19-5.08 (m, 2H), 4.08-4.00(m, 1H), 2.29-2.22 (m, 4H), 2.10-1.58 (m, 6H), 1.34-1.18 (s, 36H), 0.86 (t, J =7.0 Hz, 6H); MS (CI): m/z [M+H]⁺ 578.82.

Synthesis of 5-aminocyclohexane-l,3-diyl ditetradecanoate (L60-5):

L60-5

[0904] To a 100 mL round bottom flask was added L60-4 (268 mg, 0.26 mM), 10% Pd/C (50 mg) and anhydrous toluene (20 mL). The reaction flask was degassed using high vacuum and flushed with hydrogen gas (1 atm, balloon). The reaction mixture was stirred at room temperature for 65h. After completion of the reaction, the Pd/C was removed using syringe filter and the filtrate was concentrated under vacuum. The residue was loaded on to a silica cartridge which was then attached to flash purification system loaded with 20 g flash silica column and purified by flash chromatography (SiO2: ethyl acetate/hexane 0-60%) to give L60-5 (220 mg, 86%). ¹H NMR (300 MHz, CDCl3): δ ppm ¹H NMR (300 MHz, CDCl3): δ ppm 5.29-5.18 (m, 2H), 3.48- 3.34 (m, 1H), 2.29-2.22 (m, 4H), 2.10-1.58 (m, 8H), 1.34-1.18 (s, 44H), 0.89 (t, J =7.0 Hz, 6H); MS (CI): m/z [M+H]⁺ 552.96. Synthesis of 4-((3,5-bis(tetradecanoyloxy)cyclohexyl)amino)-4-oxobutanoic acid (L60-6):

[0905] To a 50 mL round bottom flask was added L60-5 (200 mg, 0.36 mmol) and anhydrous THF (20 mL). To this was added DIPEA (281 mg, 2.17 mmol) and succinic anhydride (36.98 mg, 0.37 mmol) and the reaction mixture was stirred at room temperature for 18h. After completion of the reaction the volatiles were removed under high vacuum. Residue was acidified to pH 4 and extracted with dichloromethane. Solvent was removed from this mixture under vacuum. The residue was loaded onto 20g flash column and was purified by flash chromatography (SiO2: ethyl acetate (5% AcOH)/hexane 20-100%) to give L60-6 (220 mg, 93%). ¹HNMR (300 MHz, CDCl3): δ ppm 6.31 (d, =7.0 Hz, 1H), 5.17-5.02 (m, 2H), 4.49-4.38 (m, 1H), 2.74 (t, J =7.0 Hz, 2H), 2.55 (t, J =7.0 Hz, 2H), 2.28 (t, J =7.0 Hz, 4H), 2.21-2.08 (m, 2H), 1.99-1.89 (m, 2H), 1.76-1.55 (m, 6H), 1.34-1.18 (s, 44H), 0.87 (t, J=7.0 Hz, 6H); MS (CI): m/z [M+H]⁺ 652.06.

Synthesis of Compound PL-2:

[0906] To a 50 mL round bottom flask was added L60-6 (212 mg, 0.32 mmol) and anhydrous dichloromethane (15 mL). To this was added DIPEA (129.25 mg, 1.0 mmol), EDC (95.85 mg, 0.5 mmol), DMAP (30.5 mg, 0.2 mmol), followed by mPEG-NH2 2K (0.66 g, 0.32 mmol). The reaction mixture was stirred at room temperature for 18h. After completion of the reaction the volatiles were removed under high vacuum. Residue was dissolved in minimal dichloromethane and was loaded onto 20 g flash column and purified by flash chromatography (SiO2: Diehl oromethane/Methanol 0-100%) to give Compound PL-2 (144 mg , 22%) ¹H NMR (300 MHz, CDCl3): δ ppm 6.77-6.52 (m, 1H), 5.07-5.02 (m, 1H), 4.47-4.32 (m, 1H), 3.90-3.62 (bs, 180H), 3.52 (s, 3H), 2.54-2.45 (m, 2H), 2.40-2.32 (m, 2H), 1.90-1.70 (m, 6H), 1.45-1.18 (bs, 44H), 0.89(t, ./=7.0 Hz, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 10.7 min, purity: 96.9%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3.0x 150 mm, (Part No. 186005302) , mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: CAD,

11.0 min, purity: > 99%.

Scheme 3: Synthesis of Compound PL-3

Synthesis of 4-(bis(2-(tetradecanoyloxy)ethyl)amino)-4-oxobutanoic acid (L61-1)

L61-1

[0907] To a solution of L58-4 (0.92 g, 1.61 mmol, see synthesis of Compound 1) in THF (10 mL) was added DIPEA (1.68 mL, 9.6 mmol), followed by succinic anhydride (0.164 g, 1.64 mmol). The reaction mixture was stirred at room temperature for 2.5 h and then evaporated under vacuum. The crude material was purified by column chromatography (80 g SiO2: 0 to 10% methanol in DCM gradient) to give compound L61-1 as a white solid (700 mg, 70%). ^-NMR (300 MHz, CDCl3) δ 4.20-4.16 (m, 4H), 3.64-3.60 (m, 4H), 2.66.41-2.63 (m, 4H), 2.30.41-2.27 (m, 4H), 1.61-1.56 (m, 3H), 1.36-1.24 (m, 44H), 0.87 (t, 6H); CIMS m/z [M+H]⁺ 626.5. Synthesis of Compound PL-3

Compound PL-3

[0908] To a solution of compound L61-1 (0.203 g, 0.33 mmol) in dichloromethane (5 mL) was added DMAP (33 mg, 0.27 mmol), EDC (0.191 g, 0.90 mmol) and DIPEA (0.26 mL), followed by mPEG-NEE 2K (0.5 g, 0.24 mmol). The resulting reaction mixture was stirred at room temperature for 12 h and then evaporated under vacuum. The crude material was purified by column chromatography (Gold 40 g SiO2: 0 to 20% MeOH in DCM) to obtain compound PL- 3 as a white solid (73 mg). ¹H-NMR (300 MHz, CDCl3) δ 6.42-6.35 (m, 1H), 4-21-4.14 (m, 5H), 3.77-3.51 (m, 186H), 3.43-3.38 (m, 3H), 3.35 (s, 3H), 2.71-2.67 (m, 2H), 2.52-2.48 (m, 2H), 2.32-2.24 (m, 6H), 1.59-1.55 (m, 5H), 1.31-1.09 (m, 49H), 0.85 (t, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 8.23 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, Cl 8, 1.7 pm, 3.0x 150 mm, (Part No. 186005302) , mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: CAD, fe= 12.84 min, purity: > 98.9%.

Scheme 4: Synthesis of Compound PL-4

Synthesis of benzyl bis(2-(tetradecyloxy)ethyl)carbamate (L62-1)

[0909] To a suspension of NaH (60% in mineral oil, 379 mg, 9.47 mmol) in DMF (10 mL) and THF (2 mL) was added compound L58-2 (0.74 g, 3.79 mmol, see synthesis of Compound 1). After stirring for 30 mins, tetradecyl bromide (2.10 g, 7.57 mmol) was added and the reaction mixture was stirred at room temperature for an additional 18 h. The reaction mixture was then diluted with brine and extracted with ethyl acetate. The organic layer was washed with water, separated, and dried over MgSCU. After evaporation under vacuum, the crude material was purified by column chromatography (40 g SiO2: 0 to 100% EtOAc in Hexanes gradient) to obtain compound L62-1 as a colorless liquid (875 mg, 39%). ¹H-NMR (300 MHz, CDCl3) δ 7.32-7.26 (m, 5H), 3.71 (s, 1H), 3.50 (t, 4H), 3.37 (t, 4H), 2.73 (t, 4H), 1.55-1.49 (m, 4H), 1.38-1.11 (m, 46H), 0.87 (t, 6H).

Synthesis of bis(2-(tetradecyloxy)ethyl)amine (L62-2)

L62-2

[0910] To a solution of L62-1 (870 mg, 1.48 mmol) in methanol (10 mL) and EtOAc (2 mL), was added Pd(OH)2 (31 mg) and stirred for 18 h under hydrogen gas (1 atm, balloon). The reaction mixture was then filtered and evaporated to get the desired product which was used in the next step without any further purification. ¹H-NMR (300 MHz, CDCl3) δ 3.53 (t, 4H), 3.42 (t, 4H), 2.79 (t, 4H), 1.58-1.53 (m, 4H), 1.38-1.11 (m, 45H), 0.87 (t, 6H); CIMS m/z [M+H]⁺ 498.6.

Synthesis of 4-(bis(2-(tetradecyloxy)ethyl)amino)-4-oxobutanoic acid (L62-3)

[0911] To a solution of compound L62-2 (0.69 g, 1.39 mmol) in DCM (10 mL), was added succinic anhydride (0.208 g, 2.08 mmol). The reaction mixture was stirred at room temperature for 16 h and evaporated under vacuum. The crude was purified by column chromatography (40 g SiO2: 0 to 10% methanol in DCM gradient) to obtain compound L62-3 as a white solid (730 mg, 88%). ¹H-NMR (300 MHz, CDCl3) δ 3.59-3.53 (m, 7H), 3.38 (t, 4H), 2.86-2.82 (m, 2H), 2.67-2.63 (m, 2H), 1.59-1.46 (m, 6H), 1.36-1.14 (m, 42H), 0.87 (t, 6H); CIMS m/z [M+H]⁺ 598.1.

Synthesis of Compound 4

Compound 4

[0912] To a solution of compound L62-3 (0.15 g, 0.25 mmol) in dichloromethane (15 mL) was added DMAP (31 mg, 0.25 mmol) and EDC (0.19 g, 1.0 mmol), followed by mPEG-NEE 2K (0.45 g, 0.24 mmol). The reaction mixture was stirred at room temperature for 16 h and evaporated under vacuum. The crude material was purified by column chromatography (Gold 12 g SiO2: 0 to 10% MeOH in DCM) to obtain compound PL-4 as a white solid (320 mg, 49%). ¹H- NMR (300 MHz, CDCl3) δ 6.42-6.35 (m, 1H), 3.89 (t, 1H), 3.65-3.52 (m, 186H), 3.43-3.38 (m, 10H), 2.73 (t, 2H), 2.50 (t, 2H), 1.59-1.55 (m, 5H), 1.31-1.09 (m, 45H), 0.87 (t, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 10.63 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3. Ox 150 mm, (Part No. 186005302), mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: CAD, fe= 13.68 min, purity: > 99%.

Scheme 5: Synthesis of Compound PL-5

Synthesis of tert-butyl (3,4-dihydroxycyclohexyl)carbamate (L63-2)

[0913] L63-1 (3 g, 15.2 mmol) was dissolved in acetone/^O (40/8 mL), followed by addition of potassium osmate dihydrate (56 mg, 0.15 mmol). A solution of NMO (2.67 g, 22.8 mmol) in H2O (2.67 g) was prepared and added to the reaction mixture. The reaction mixture was then stirred at room temperature for 5 hours. When TLC (10% MeOH in DCM, Rf = 0.3) showed completion of the reaction, sat. TsfeSC (30 mL) was added. After stirring the mixture for 30 min, the product was extracted with DCM (30 mL x 3). The organic fraction was evaporated to obtain crude product which was then subjected to silica gel column using 0 - 10% MeOH in DCM as eluent to afford L63-2 as white solid (3.17 g, 91%). ¹H-NMR (300 MHz, CDCl3) δ 4.05-3.51 (m, 3H), 2.80-2.05 (m, 2H), 1.96-1.46 (m, 4H), 1.42 (s, 9H), 1.25-1.05 (m, 2H).

Synthesis of 4-((tert-butoxycarbonyl)amino)cyclohexane-l,2-diyl ditetradecanoate (L63-3)

[0914] L63-2 (3.17 g, 13.7 mmol) was dissolved in DCM/DMF (100/10 mL), followed by addition of TEA (8.4 mL, 60.4 mmol). To this clear solution was added myristoyl chloride (8.2 mL, 30.2 mmol), slowly. The resulting pink cloudy suspension was stirred at room temperature for 20 hours. The reaction mixture was then washed with water (100 mL x 2). The organic layer was dried and evaporated to give crude product which was subjected to silica gel column using 0 - 20% EA in hexane as eluent to afford L63-3 as colorless semi-solid (1.6 g, 18%). ¹H-NMR (300 MHz, CDCl3) δ 5.32-5.11 (m, 1H), 4.97-4.64 (m, 1H), 4.42-4.29 (m, 1H), 3.88-3.57 (m, 1H), 2.36-2.25 (m, 2H), 2.25-2.19 (m, 2H), 2.18-1.68 (m, 4H), 1.68-1.45 (m, 6H), 1.43 (s, 9H), 1.40-1.14 (m, 44H), 0.87 (t, 6H).

Synthesis of 4-aminocyclohexane-l,2-diyl ditetradecanoate (L63-4)

[0915] L63-3 (300 mg, 0.46 mmol) was dissolved in DCM (8 mL) followed by addition of

TFA (2 mL) slowly. The reaction mixture was then stirred at room temperature for 2 hours. A solution of sat. NaHCOs (30 mL) was used to neutralize the reaction. The mixture was then extracted with DCM (30 mL x 2). The organic fraction was evaporated to obtain product L63-4 as colorless oil (230 mg, 91%). ¹H-NMR (300 MHz, CDCl3) δ 5.34-5.18 (m, 1H), 4.88-4.75 (m, 1H), 3.19-2.76 (m, 1H), 2.36-2.25 (m, 2H), 2.25-2.19 (m, 2H), 2.18-1.77 (m, 3H), 1.71-1.49 (m, 11H), 1.49-1.36 (m, 1H), 1.35-1.14 (m, 40H), 0.87 (t, 6H).

Synthesis of 4-((3,4-bis(tetradecanoyloxy)cyclohexyl)amino)-4-oxobutanoic acid (L63-5)

[0916] L63-4 (220 mg, 0.39 mmol) and succinic anhydride (40 mg, 0.39 mmol) were each separately dissolved in DCM (1 mL). The solution of L63-4 was then added to the solution of succinic anhydride slowly at 0 °C. The resulting mixture was stirred at room temperature for 4 hours. When TLC (20% MeOH in DCM, Rf = 0.8) showed completion of the reaction, the solvent was evaporated to give crude product which was subjected to silica gel column using 0 - 20% MeOH in DCM as eluent to afford L63-5 as white solid (240 mg, 92%). ¹H-NMR (300 MHz, CDCl3) δ 5.85 (bs, 0.5H), 5.67-5.55 (m, 0.5H), 5.29 (bs, 0.5H), 5.17 (bs, 0.5H), 5.05-4.95 (m, 0.5H), 4.88-4.78 (m, 0.5H), 4.14 (bs, 0.5H), 3.99 (bs, 0.5H), 2.74-2.59 (m, 2H), 2.51-2.38 (m, 2H), 2.37-2.16 (m, 4H), 2.14-1.68 (m, 4H), 1.68-1.45 (m, 6H), 1.35-1.14 (m, 36H), 0.87 (t, 6H). Synthesis of Compound PL-5

Compound PL-5

[0917] A mixture of L63-5 (230 mg, 0.35 mmol), EDC (270 mg, 1.4 mmol) and DMAP (47 mg, 0.39 mmol) in DCM (10 mL) was stirred at room temperature for 5 minutes to form a clear solution. mPEG-NH22K (705 mg, 0.35 mmol) was added to the reaction solution and the reaction mixture was stirred for 2 hours at room temperature. When TLC (15% MeOH in DCM, Rf = 0.6) showed completion of the reaction, the solvent was evaporated. The crude product was then subjected to silica gel column using 0 - 20% MeOH in DCM as eluent to afford Compound PL- 5 as white solid (683 mg, 72%). ¹H-NMR (300 MHz, CDCl3) δ 6.51 (bs, 1H), 6.45-6.34 (m, 1H), 5.28 (bs, 0.5H), 5.18 (bs, 0.5H), 4.98-4.88 (m, 0.5H), 4.88-4.80 (m, 0.5H), 4.08 (bs, 0.5H), 3.92 (bs, 0.5H), 3.87 (dd, 2H), 3.77-3.46 (m, 180H), 3.44-3.38 (m, 4H), 3.36 (s, 3H), 2.57-2.43 (m, 4H), 2.35-2.18 (m, 4H), 2.14-1.68 (m, 4H), 1.68-1.45 (m, 6H), 1.35-1.14 (m, 40H), 0.87 (t, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 10 min. Flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 7.17 min, purity: > 99.9%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 15 min. Flow rate: ImL/min, column temperature: 20±2 °C, detector: CAD, fe= 8.71 min, purity: > 99.9%.

Scheme 6: Synthesis of Compound PL-6

Synthesis of tert-butyl (3,4-dihydroxycyclopentyl)carbamate (L59-2)

[0918] Starting material L59-1 (2 g, 11 mmol) was dissolved in acetone/JLO (30/6 mL), followed by addition of potassium osmate dihydrate (40 mg, 0.11 mmol). A solution of NMO (1.92 g, 16 mmol) in H2O (1.92 g) was prepared and added to above mixture. The reaction mixture was then stirred at room temperature for 5 hours. When TLC (10% MeOH in DCM, Rf = 0.3) showed the completion of reaction, sat. Na2SO4 (25 mL) was added. After stirring the mixture for 30 min, DCM (25 mL x 3) was used to extract the product. The organic fraction was evaporated to obtain crude product which was then subjected to silica gel column using 0 - 10% MeOH in DCM as eluent to afford L59-2 as white solid (1.55 g, 66%). ¹H-NMR (300 MHz, CDCL) δ 4.32-3.75 (m, 3H), 2.28-2.15 (m, 2H), 1.76-1.67 (m, 2H), 1.43 (s, 9H).

Synthesis of 4-((tert-butoxycarbonyl)amino)cyclopentane-l,2-diyl ditetradecanoate (L59- 3)

[0919] L59-2 (500 mg, 2.3 mmol) was dissolved in DCM/DMF (25/5 mL), followed by addition of pyridine (0.8 mL, 10.2 eq). To this clear solution was added myristoyl chloride (1.38 mL, 5.1 mmol), slowly. The resulting mixture was stirred at room temperature for 20 hours. When TLC (20% EA in hexane, Rf = 0.2) showed completion of the reaction, the reaction mixture was then washed by water (50 mL x 2). The organic fraction was evaporated to obtain crude product which was then subjected to silica gel column using 0 - 20% EA in hexane as eluent to afford L59-3 as colorless semi-solid (1.2 g, 82%). ¹H-NMR (300 MHz, CDCl3) δ 5.37-5.33 (m, 2H), 4.82-4.02 (m, 1H), 2.47-2.12 (m, 7H), 1.83-1.73 (m, 1H), 1.68-1.49 (m, 5H), 1.44 (s, 9H), 1.34-1.08 (m, 42H), 0.87 (t, 6H).

Synthesis of 4-aminocyclopentane-l,2-diyl ditetradecanoate (L59-4)

[0920] L59-3 (500 mg, 0.78 mmol) was dissolved in DCM (8 mL) followed by addition of

TFA (2 mL) slowly. The reaction mixture was then stirred at room temperature for 2 hours. A solution of sat. NaHCCh (30 mL) was used to neutralize the reaction. The mixture was then extracted by DCM (30 mL x 2). The organic fraction was evaporated to obtain product L59-4 as colorless oil (405 mg, 96%). ¹H-NMR (300 MHz, CDCl3) δ 5.37-5.02 (m, 2H), 3.73-3.22 (m, 1H), 2.41-2.03 (m, 6H), 1.89-1.66 (m, 5H), 1.65-1.41 (m, 5H), 1.29-1.03 (m, 40H), 0.87 (t, 6H). CIMS m/z [M+H]⁺ 538.1.

Synthesis of 4-((3,4-bis(tetradecanoyloxy)cyclopentyl)amino)-4-oxobutanoic acid (L59-5)

L59-5

[0921] L59-4 (400 mg, 0.74 mmol) and succinic anhydride (82 mg, 0.81 mmol) were dissolved in DCM (3 mL) respectively. The solution of L59-4 was then added to the solution of succinic anhydride slowly at 0 °C. The resulting mixture was then stirred at room temperature for 4 hours. When TLC (20% MeOH in DCM, Rf = 0.8) showed the completion of reaction, the solvent was evaporated to obtain crude product which was then subjected to silica gel column using 0 - 20% MeOH in DCM as eluent to afford L59-5 as white solid (356 mg, 75%). ¹H-NMR (300 MHz, CDCl3) δ 6.11-5.86 (m, 1H), 5.37-5.02 (m, 2H), 4.54-4.31 (m, 1H), 2.78-2.58 (m, 2H), 2.50-2.36 (m, 3H), 1.92-1.77 (m, 1H), 1.70-1.1.46 (m, 5H), 1.36-1.01 (m, 34H), 0.87 (t, 6H). CIMS m/z [M+H]⁺ 638.1.

Synthesis of Compound PL-6

Compound PL-6

[0922] L59-5 (200 mg, 0.31 mmol), EDC (220 mg, 0.8 mmol) and DMAP (38 mg, 0.31 mmol) were dissolved in DCM (10 mL). After stirring for 5 minutes the solution became clear. mPEG-NH2 2K (570 mg, 0.28 mmol) was then added to the solution and the reaction mixture was stirred for 2 hours at room temperature. When TLC (15% MeOH in DCM, Rf = 0.6) showed the completion of reaction, the solvent was evaporated. The obtained crude product was then subjected to silica gel column twice using 0 - 20% MeOH in DCM as eluent to afford mPEG2000-DMCPA Compound PL-6 as white solid (310 mg, 37%). ¹H-NMR (300 MHz, CDCl3) δ 6.78-6.43 (m, 2H), 5.37-5.07 (m, 2H), 4.58-4.28 (m, 1H), 3.87 (t, 2H), 3.77-3.46 (m, 180H), 3.45-3.40 (m, 4H), 3.37 (s, 3H), 2.57-2.39 (m, 5H), 2.36-2.16 (m, 5H), 1.90-1.75 (m, 1H), 1.70-1.1.50 (m, 5H), 1.36-1.13 (m, 40H), 0.87 (t, 6H). Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% tri fluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 10 min. Flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe= 7.11 min, purity: >99.9%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 15 min. Flow rate: 1 mL/min, column temperature: 20±2 °C, detector: CAD, fe= 9.14 min, purity: 96.7%. Scheme 7: Synthesis of Compound PL-9A

PL-9A

Synthesis of ((tert-butoxycarbonyl)azanediyl)bis(ethane-2,l-diyl) distearate

[0923] A mixture of stearic acid (2A) (5 g, 24.36 mmol), RWL-4 1 (13.86 g, 48.72 mmol), EDCI (17.28 g, 90.13 mmol), DIEA (12.59 g, 97.44 mmol) and DMAP (893 mg, 7.31 mmol) in THF (20 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 20 °C for 12 h under N2 atmosphere. TLC (eluted with petroleum ether: ethyl acetate = 4: 1, PMA) indicated one major new spot (Rf = 0.42) was detected. The reaction mixture was partitioned between H2O (30 mL) and ethyl acetate (30 mL). The organic phase was separated, washed with sat. NaCl aqueous solution (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (Petroleum ether :Ethyl acetate = 1 : 100 to 7: 100) to give ((tertbutoxy carbonyl)azanediyl)bis(ethane-2,l -diyl) distearate (RWL-5 2) (14 g, 77.85% yield) as a white solid. ¹H NMR: ET59539-1-P1A1 (400 MHz CDCl3) δ = 4.24 - 4.12 (m, 4H), 3.57 - 3.42 (m, 4H), 2.31 (t, J= 8.0 Hz, 4H), 1.68 - 1.55 (m, 5H), 1.47 (s, 9H), 1.37 - 1.20 (m, 55H), 0.89 (t, J= 4.0 Hz, 6H).

Synthesis of azanediylbis(ethane-2,l-diyl) distearate

[0924] To a solution of RWL-5 2 (1.5 g, 2.03 mmol) in DCM (15 mL) was added TFA (7.70 g, 67.53 mmol) at 20 oC. The mixture was stirred at 20 °C for 2 h under N2 atmosphere. TLC (eluted with petroleum etherethyl acetate =3: 1, PMA) showed RWL-5 2 (Rf = 0.18) was consumed completely and one new spot (Rf = 0.28) was formed. The reaction mixture was concentrated under reduced pressure to remove solvent to give azanediylbis(ethane-2,l-diyl) distearate (RWL-5 3) (2.09 g, crude) as a white solid. ¹H NMR: ET62978-5-P1A (400 MHz CDCl3 - d) δ = 4.39 (m, 4H), 3.44 (m, 4H), 2.35 (t, J = 7.6 Hz, 4H), 1.60 (t, J= 6.8 Hz, 4H), 1.27- 1.25 (m, 56H), 0.88 (t, J= 6.4 Hz, 6H).

Synthesis of 4-(bis(2-(stearoyloxy)ethyl)amino)-4-oxobutanoic acid

RWL-5 3

[0925] To a solution of RWL-5_3 (2.09 g, 3.28 mmol) in DCM (20 mL) was added TEA (994 mg, 9.83 mmol) and tetrahydrofuran-2,5-dione (4A) (983 mg, 9.83 mmol) at 20 °C. The mixture was stirred at 20 °C for 12 h. TLC (eluted with petroleum ether: ethyl acetate = 1 : 1, PMA) showed RWL-5_3 (Rf = 0.46) was consumed completely and one new spot (Rf = 0.67) was formed. The residue was diluted with H2O (20 mL) and extracted with DCM (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 4-(bis(2-(stearoyloxy)ethyl)amino)-4-oxobutanoic acid (RWL-5 4) (1.08 g, 44.67% yield) as a white solid. ¹H NMR: ET62978-6-P1 A (400 MHz CDCl3) 6 = 4.30-4.20 (m, 4H), 3.68 - 3.60 (m, 4H), 2.72 (s, 4H), 2.35 - 2.27 (m, 4H), 1.36 - 1.19 (m, 60H), 0.92 - 0.86 (t,

J= 6.8 H_Z, 6H).

Synthesis of PL-9A

PL-9A

[0926] To a solution of RWL-5 4 (1.40 g, 0.68 mmol) and mPEG-(CH₂)3-NH₂ (5A) (1.40 g, 0.68 mmol) in DCM (10 mL) was added HATU (309 mg, 0.81 mmol) and DIEA (219 mg, 1.69 mmol) at 20 °C. The mixture was stirred at 20 °C for 12 h, at which point TLC (eluted with Dichloromethane:Methanol =10: 1, PMA) showed RWL-5 4 (Rf = 0.90) was consumed completely and two new spots (Rf = 0.39) were formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate: Petroleum ether and 0-30% methyl alcohohdichloromethane @ 80 mL/min) to give RWL 5 (600 mg, 31.73% yield) as green oil. LCMS Method: (The column used for chromatography was a XSelect CSH phenyl-hexyl 2.1 ><50mm, 3.5um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD). MS mode was positive electrospray ionization. MS range was 100-1500. Mobile Phase A: 0.04% TFA in water and mobile phase B was 0.02 % TFA in HPLC grade acetonitrile. The gradient 50%-100% B in 4.0 minutes and holding at 100% for 1.5 minutes The flow rate was 1 mL/min. LCMS: ET62978-18-P1E1; r.t. = 4.624 min, m/z = 2790.87 (M+H)⁺. ' H NMR: ET62978-18-P1E (400 MHz CD₃C1) δ = 6.50 (br t, J= 5.4 Hz, 1H), 4.26 - 4.13 (m, 4H), 3.70-3.53 (m, 170H), 3.37 (s, 3H), 3.33 (br d, J= 6.0 Hz, 2H), 3.12 (q, J= 7.2 Hz, 2H), 2.70 (br t, J= 6.4 Hz, 2H), 2.49 (br t, J= 6.4 Hz, 2H), 2.35 - 2.24 (m, 6H), 1.81 - 1.71 (m, 2H), 1.64 - 1.56 (m, 4H), 1.56 - 1.47 (m, 9H), 1.43 (br d, J= 6.6 Hz, 6H), 1.25 (s, 58H), 0.87 (br t, J= 6.0 Hz, 6H). Scheme 8: Synthesis of Compound PL-10A

PL-10A

Synthesis of N-benzyl-2-(octadecyloxy)-N-(2-(octadecyloxy)ethyl)ethan-l-amine

RWL-4_5 RWL-4 6

[0927] To a solution of RWL-4 5 (6 g, 30.73 mmol) in THF (200 mL) was added NaH (9.83 g, 245.83 mmol, 60% purity) in portions at 0 °C under N2. After addition, the mixture was stirred at 20 °C for 30 min, and then a solution of 1A (60.78 g, 159.79 mmol) in THF (200 mL) was added dropwise at 20 °C. The resulting mixture was stirred at 20 °C for 48 h. TLC (eluted with petroleum etherethyl acetate =5: 1, I2, Rf = 0.5) indicated RWL-4 5 was consumed, and one major new spot was detected. The reaction mixture was poured into ice water (400 mL) at 0 °C, and then extracted with ethyl acetate (3 x 150 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Petroleum etherEthyl acetate = 100: 1 to 10: 1) to give (RWL-4 6) (6.2 g, 28.82% yield) as a white solid. ¹H NMR: ET59539- 49 (400 MHz CDCl3) δ = 7.37 - 7.20 (m, 5H), 3.72 (s, 2H), 3.51 (t, J= 6.0 Hz, 4H), 3.38 (t, J= 6.0 Hz, 4H), 2.75 (t, J= 6.0 Hz, 4H), 1.55 (m, J= 6.8 Hz, 4H), 1.36-1.25 (m, 60H), 0.93 - 0.85

(t, J = 8.0 Hz, 6H)

Synthesis of bis(2-(octadecyloxy)ethyl)amine

RWL-4_6 RWL-4 3

[0928] To a solution of RWL-4 6 (5 g, 7.14 mmol) in toluene (30 mL) was added 1- chloroethyl carb onochlori date (3.06 g, 21.42 mmol). The mixture was stirred at 130 °C for 1 h, and then MeOH (30 mL) was added. The mixture was stirred at 130 °C for another 1 h after which LC-MS showed that the desired compound was detected. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water (50 mL) and adjusted to pH = 2 with aq. HC1 (2M). The aqueous phase was extracted with MTBE (20 mL) to remove the impurity. The aqueous phase was adjusted to pH = 10 with Na2CO3 solid and then extracted with ethyl acetate (2 x 30 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give bis(2- (octadecyloxy)ethyl)amine (RWL-4 3) (3.8 g, 87.23% yield) as a white solid. ' H NMR: ET59539-63-P1N1 (400 MHz CDCl3) δ = 3.80 (t, J= 4.8 Hz, 4H), 3.51 (t, J= 6.8 Hz, 4H), 3.28 - 3.17 (m, 4H), 1.60 - 1.53 (m, 4H), 1.38 - 1.16 (m, 61H), 0.88 (t, J= 8.0 Hz, 6H).

Synthesis of 4-(bis(2-(octadecyloxy)ethyl)amino)-4-oxobutanoic acid

[0929] To a solution of RWL-4_3 (500 mg, 0.82 mmol) in DCM (5 mL) was added TEA (249 mg, 2.46 mmol) and tetrahydrofuran-2, 5-dione (4A) (246 mg, 2.46 mmol) at 0 °C. The mixture was stirred at 20 °C for 2 h. TLC (eluted with petroleum ether: ethyl acetate =1 : 1, PMA, Rf = 0.6) showed RWL-4 3 was consumed completely and one new spot was formed. The reaction mixture was diluted with H2O (5 mL) and extracted with DCM (3 x 5 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 4-(bis(2-(octadecyloxy)ethyl)amino)-4-oxobutanoic acid (RWL-4 4) (530 mg, 72.85% yield) as a white solid. ¹H NMR: ET62978-12-P1A (400 MHz CDCl3) δ = 3.63 - 3.53 (m, 8H), 3.40 (t, J = 6.4 Hz, 4H), 2.87 - 2.81 (m, 2H), 2.69 - 2.64 (m, 2H), 1.60-1.50 (m, 4H), 1.26 (s, 60H), 0.97 - 0.82 (m, 6H).

Synthesis of PL-10A

PL-10A

[0930] To a solution of RWL-4_4 (583 mg, 0.28 mmol) in DCM (4 mL) was added mPEG- (CH₂)₃-NH₂ (5A), HATU (129 mg, 0.34 mmol) and DIEA (91 mg, 0.70 mmol) at 20 °C. The mixture was stirred at 20 °C for 12 h. TLC (eluted with dichloromethane:methanol = 10: 1, PMA, Rf = 0.48) showed RWL-4 4 was consumed completely and one new spot was formed. The residue was dissolved in DCM (10 mL), and 1 g of silica gel was added. The resulting mixture was concentrated to give a dry flowing solid, and then it was loaded to Biotage using a 12 g Agela flash silica gel column, eluted with 0% to 10% methanol in di chloromethane to give PL- 10A (522 mg, 80.00% yield) as white oil. LCMS Method: (The column used for chromatography was a XSelect CSH phenyl-hexyl 2.1 ><50mm, 3.5um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD). MS mode was positive electrospray ionization. MS range was 100-1500. Mobile Phase A: 0.04% TFA in water and mobile phase B was 0.02 % TFA in HPLC grade acetonitrile. The gradient 50%-100% B in 4.0 minutes and holding at 100% for 1.5 minutes The flow rate was 1 mL/min. LCMS: ET62978-26-P1A; r.t. = 5.045 min, m/z = 2762.91 (M+H)⁺. ¹H NMR: ET62978-26-P1A (400 MHz CD₃C1) δ = 6.87 - 6.30 (m, 1H), 3.85 - 3.79 (m, 1H), 3.91 - 3.44 (m, 186H), 3.43 - 3.27 (m, 10H), 2.78 - 2.69 (m, 2H), 2.56 - 2.47 (m, 2H), 1.77 (t, J= 6.4 Hz, 2H), 1.59 -1.47 (m, 4H), 1.25 (s, 60H), 0.93 - 0.83 (m, 6H). Scheme 9: Synthesis of Compound PL-12B

Synthesis of tert-butyl ((3R,4S)-3,4-dihydroxycyclopentyl)carbamate

RWL-3_1 RWL-3_2

[0931] To a solution of tert-butyl cyclopent-3 -en-l-ylcarbamate (RWL-3 1) (2 g, 10.91 mmol) in acetone (64 mL) and H2O (8 mL) was added N-Methylmorpholine N-oxide (NMO) (2.56g, 21.83 mmol) at 20 °C. After 2 min, dipotassium;dioxido(dioxo)osmium;dihydrate (402 mg, 1.09 mmol) was added and the resulting mixture was stirred at 20 °C for 12 h. The reaction mixture was poured into sat. Na2SO4 (50 mL) at 0 °C and stirred for 10 min, then extracted with dichloromethane (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give tert-butyl ((3R,4S)- 3,4-dihydroxycyclopentyl)carbamate (RWL-3 2) (1.4 g, 59.04% yield) as a white solid, which was used directly in the next step. ¹H NMR: ET82990-9 (400 MHz CDCl3) δ = 4.38 - 4.62 (m, 1H), 4.13 - 4.30 (m, 3H), 2.11 - 2.21 (m, 2H), 1.68 - 1.81 (m, 2H), 1.44 (s, 9H). Synthesis of (lR,2S)-4-((tert-butoxycarbonyl)amino)cyclopentane-l,2-diyl distearate

[0932] To a solution of stearic acid (2A) (288 mg, 1.01 mmol) in DCM (2 mL) was added DMAP (12 mg, 0.09 mmol), EDCI (194 mg, 1.01 mmol) and DIEA (0.4 mL, 2.30 mmol) at 20 °C. After 1 min, RWL-3 2 (100 mg, 0.46 mmol) was added and the resulting mixture was stirred at 20 °C for 12 h. The reaction mixture was poured into water (10 mL) at 0 °C, then extracted with DCM (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum etherethyl acetate = 100:0 to 10: 1) to give (lR,2S)-4-((tert-butoxycarbonyl)amino)cyclopentane-l,2-diyl distearate (RWL- 3 3) (100 mg, 28.96% yield) as a white solid. ' H NMR: ET82990-11 (400 MHz CDCl3) δ = 5.32-5.28 (m, J = 4.0 Hz, 1H), 5.17 (br t, J = 4.4 Hz, 1H), 4.87 - 4.45 (m, 1H), 4.35 - 4.10 (m, 1H), 4.22 - 4.07 (m, 1H), 2.48 - 2.38 (m, 1H), 2.32 - 2.18 (m, 5H), 1.87 - 1.79 (m, 1H), 1.70 - 1.59 (m, 5H), 1.48 - 1.43 (m, 9H), 1.26 (s, 59H), 0.94 - 0.84 (m, 6H).

Synthesis of (lR,2S)-4-aminocyclopentane-l,2-diyl distearate

[0933] To a solution of RWL-3 3 (500 mg, 0.67 mmol) in DCM (6 mL) was added TFA (1 mL) at 25 °C. The resulting mixture was stirred at 30 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give (lR,2S)-4-aminocyclopentane-l,2-diyl distearate (RWL-3 4) (430 mg, crude) as yellow solid, which was used in the next step. ¹H NMR: ET82990-16 (400 MHz CDCl3) δ = 5.42 - 5.31 (m, 1H), 5.18-5.16 (m, 1H), 2.47 - 2.36 (m, 1H), 2.32 - 2.26 (m, 3H), 2.23 - 2.12 (m, 1H), 2.06 - 1.76 (m, 7H), 1.73 - 1.56 (m, 5H), 1.44 (s, 4H), 1.26 (s, 50H), 0.93 - 0.85 (m, 6H). Synthesis of 4-(((3R,4S)-3,4-bis(stearoyloxy)cyclopentyl)amino)-4-oxobutanoic acid

[0934] To a solution of RWL-3 4 (500 mg, 0.65 mmol) in DCM (10 mL) was added DLEA (0.6 mL, 3.27 mmol) and tetrahydrofuran-2, 5-dione (197 mg, 1.96 mmol) at 25 °C. The resulting mixture was stirred at 25 °C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography

(petroleum etherethyl acetate = 100:0 to 1 : 1) to give 4-(((3R,4S)-3,4- bis(stearoyloxy)cyclopentyl)amino)-4-oxobutanoic acid (RWL-3 5) (520 mg, 63.56% yield) as yellow solid. ¹H NMR: ET82990-24-P1A1 (400 MHz CDCl3) δ = 5.31 (t, J= 4.4 Hz, 1H), 5.18

(t, J = 4.4 Hz, 1H), 4.57-4.35 (m, 1H), 3.65 (t, J= 6.0 Hz, 2H), 2.71-2.66 (m, 2H), 2.59 (t, J = 6.8 Hz, 3H), 2.47 (dt, J = 4.0, 6.4 Hz, 3H), 2.32-2.25 (m, 4H), 1.85 (qd, J = 6.0, 12.8 Hz, 5H), 1.64-1.56 (m, 4H), 1.43-1.39 (m, 4H), 1.25 (s, 47H), 0.88 (t, J= 6.8 Hz, 6H).

[0935] To a solution of mPEG-(CH2)3-NH2 (5A) (670 mg, 0.89 mmol) in DCM (10 mL) was added DIEA (288 mg, 2.23 mmol), HATU (407 mg, 1.07 mmol) and RWL-3 5 (1.85 g, 0.89 mmol) at 20 °C. The mixture was stirred at 20 °C for 12 h, after which TLC (eluted with dichloromethane:methanol = 10: 1, PMA, Rf = 0.48) showed RWL-3 5 was consumed completely and one new spot was formed. The residue was purified by flash silica gel chromatography (petroleum ether: ethyl acetate = 100:1 to 0: 1) and slurried in ethyl acetate:petroleum ether = 1 : 1. The mixture was filtered and the filter cake was dried under reduced pressure to give PL-12B (258.6 mg, 17.24% yield) as a white solid. LCMS Method: (The column used for chromatography was a XSelect CSH phenyl-hexyl 2.1 x50mm, 3.5um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD). MS mode was positive electrospray ionization. MS range was 100-1500. Mobile Phase A: 0.04% TFA in water and mobile phase B was 0.02 % TFA in HPLC grade acetonitrile. The gradient 50%-100% B in 4.0 minutes and holding at 100% for 1.5 minutes The flow rate was 1 mL/min. LCMS: ET62972-44-P1C; r.t. = 4.743 min, m/z = 2802.87 (M+H)⁺. ¹H NMR: ET62972-44-P1B (400 MHz CD3CI) δ = 6.97-6.57 (m, 2H), 5.31 (br t, J= 4.0 Hz, 1H), 5.16 (br t, J= 4.0 Hz, 1H), 4.57- 4.30 (m, 1H), 4.05-3.43 (m, 184H), 3.40-3.32 (m, 5H), 2.48 (s, 4H), 2.35-2.20 (m, 4H), 1.89- 1.71 (m, 4H), 1.65-1.56 (m, 4H), 1.33-1.22 (m, 56H), 0.88 (t, J= 8.0 Hz, 6H).

Scheme 10: Synthesis of Compound PL-16A

Compound PL-16A

Synthesis of 142-oxo-2,5,8,l 1,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,

68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137- hexatetracontaoxa-141-azapentatetracontahectan- 145-oic acid (L93-6)

[0936] To a solution of compound L93-5 (1.4 g, 0.67 mmol) in DCM (10 mL) was added succinic anhydride (0.34 g, 3.37 mmol). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated under vacuum. The crude was purified by column chromatography (80 g SiO2: 0 to 20% methanol in DCM gradient) to obtain compound L93-6 as white solid (0.7 g, 48%); ¹H-NMR (300 MHz, CDCI3) δ 3.87-3.83 (m, 1H), 3.65-3.52 (m, 184H), 3.45-3.35 (m, 4H), 3.36 (s, 3H), 2.65-2.49 (m, 4H).

Synthesis of tert-butyl(2S,4R)-4-(stearoyloxy)-2-((stearoyloxy) methyl) pyrrolidine-1- carboxylate (L93-3)

[0937] A solution of stearic acid L93-1 (3.27 g, 11.52 mmol), EDC (2.7 g, 13.82 mmol) and DMAP (285 mg, 2.3 mmol) was stirred at room temperature for 30 min. Tert-butyl (2S,4R)-4- hydroxy-2-(hydroxymethyl)pyrrolidine-l -carboxylate (commercially available, L93-2) (1.0 g, 4.6 mmol) was added in. The reaction mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel 80 g, solvent 0-5% MeOH/DCM) to provide L93-3 (3.2 g, 97%); ¹H NMR(CDCl3): 0.85 - 0.87, (t, 6H), 1.23-1.24 (m, 56H), 1.46 (s, 9H), 1.51-1.69 (m, 4H), 2.02- 2.20 (m, 2H), 2.22-2.35 (m, 4H), 3.41-3.73 (m, 2H), 4.01- 4.32 (m, 3H), 5.22 (m, 1H); CIMS m/z [M-Boc+H]⁺ 650.1.

Synthesis of (3R,5S)-5-((stearoyloxy)methyl)pyrrolidin-3-yl stearate (L93-4)

L93-4

[0938] To a solution of L93-3 (0.35 g, 0.46 mmol) in DCM (5 mL), cooled in an ice-water bath under nitrogen, was added CF3CO2H (5 mL) and maintained internal temperature between 0- 5 °C. The resulting mixture was allowed to warm to room temperature and stirred for 8 h. The solvent and excess CF3CO2H were removed in vacuo and the residue was taken up in di chloromethane (100 mL), washed with 10% aq. NaHCO3 and brine, dried with Na2SO4. Filtration and concentration in vacuo provided crude product which was purified by column chromatography (silica gel with hexane/ EtOAc 75:25) (150 mg, 49%); ^-NMR (300 MHz, CDCl3) δ 5.40 (t, 1H), 4.48-4.01 (m, 3H), 3.69-3.42 (m, 2H), 2.33-2.18 (m, 5H), 1.61-1.57 (m, 4H), 1.26-1.08 (m, 58H), 0.86 (t, 6H); CIMS m/z [M+H]⁺ 650.6.

Synthesis of ((2S,4R)-l-(142-oxo-2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56 ,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,13 4,137-hexatetracontaoxa-141-azapentatetracontahectan-145-oyl)-4- (stearoyloxy)pyrrolidin-2-yl)methyl stearate (Compound PL-16A)

PL-16A

[0939] To a solution of L93-6 (0.35 g, 0.16 mmol), DMAP (21 mg, 0.17 mmol) and EDC 123 mg, 0.64 mmol) in dichloromethane (10 mL) was added L93-4 (136 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 12 h and then concentrated under reduced pressure. The crude was purified by column chromatography (Gold 40 g SiO2: 0 to 20% MeOH in DCM) to obtain compound PL-16A as white solid (101 mg, 23%); ¹H-NMR (300 MHz, CDCl3) δ 6.34- 6.28 (m, 1H), 5.35-5.28 (m, 1H), 4.50-4.10 (m, 3H), 3.91-3.38 (m, 182H), 3.36 (s, 3H), 2.71- 2.45 (m, 3H), 2.35-2.02 (m, 8H), 1.68-1.50 (m, 4H), 1.38-1.15 (m,61H), 0.86 (t, 6H); Analytical HPLC column: Agilent Zorbax SB-C4, 5 pm, 4.6x150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 60 °C, detector: ELSD, fe = 13.25 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 55±2 °C, detector: CAD, fe = 16.95 min, purity: 97.02%. Scheme 11: Synthesis of Compound PL-15A

Compound PL-15A

Synthesis of t-butyl (2S, 4R)-4-(tetradeconoyloxy)-2-((tetradecanoyloxy) methyl)pyrrolidne-l-carboxylate (L94-3)

[0940] A solution of myristic acid L94-1 (2.7 g, 11.97 mmol), EDC (2.63 g, 13.81 mmol) and DMAP (1.7 g, 13.81 mmol) was stirred at room temperature for 30 min. L93-2 (1.0 g, 4.6 mmol) was added in. The reaction mixture was stirred at room temperature for 20 h and concentrated. The residue was purified by column chromatography (silica gel 80 gm, solvent 0-5% MeOH/DCM) to provide L94-3 (2.8 g, 95%); ¹H NMR (CDCl3): 0.85 - 0.89, (t, 6H), 1.12 - 1.4 (m, 40H), 1.46 (9H), 1.53-1.69 (m, 4H), 2.02- 2.20 (m, 2H), 2.22-2.35 (m, 4H), 3.43-3.72 (m, 2H), 4.01- 4.33 (m, 3H), 5.23 (m, 1H); CIMS m/z [M-Boc+H]⁺ 538.2. Synthesis of (3R,5S)-5-((stearoyloxy)methyl)pyrrolidin-3-yl stearate (L94-4)

L94-4

[0941] To an ice-water bath cooled solution of L94-3 (0.35 g, 0.46 mmol) in DCM (5 mL) under nitrogen was added CF3CO2H (5 mL) and maintained internal temperature between 0- 5 °C. The resulting mixture was allowed to warm to room temperature and stirred for 8 h. The solvent and excess CF3CO2H were removed in vacuo and the residue was taken up in di chloromethane (100 mL), washed with 10% aq. NaHCOs and brine, dried with anhydrous Na2SO4. Filtration and concentration in vacuo provided crude product which was purified by column chromatography (40 g SiO2: hexane/ EtOAc 75:25) to obtain L94-4 (200 mg, 66%); ¹H- NMR (300 MHz, CDCl3) δ 5.42 (t, 1H), 4.49-4.01 (m, 3H), 3.68-3.41 (m, 2H), 2.32-2.17 (m, 5H), 1.62-1.55 (m, 4H), 1.23-1.10 (m, 42H), 0.86 (t, 6H); CIMS m/z [M+H]⁺ 537.8.

Synthesis of ((2S,4R)-l-(142-oxo-2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59, 62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,13

7-hexatetracontaoxa- 141-azapentatetracontahectan- 145-oyl)-4-(tetradecanoyloxy) pyrrolidin-2-yl)methyl tetradecanoate (Compound PL-15A)

Compound PL-15A

[0942] To a solution of L93-6 (0.48 g, 0.22 mmol), DMAP (29 mg, 0.24 mmol) and EDC (169 mg, 0.88 mmol,) in di chloromethane (10 mL) was added L94-4 (154 mg, 0.28 mmol). The reaction mixture was stirred at room temperature for 12 h and then concentrated under vacuum. The crude was purified by column chromatography (Gold 40 g SiO2: 0 to 20% MeOH in DCM) to obtain compound PL-15A as white solid (320 mg, 53%); ¹H-NMR (300 MHz, CDCl3) δ 6.51- 6.32 (m, 1H), 5.41-5.33 (m, 1H), 4.51-3.43 (m, 189H), 3.36 (s, 3H), 2.63-2.12 (m, 9H), 1.93- 1.86 (m, 4H), 1.68-1.52 (m, 4H), 1.36-1.08 (m,41H), 0.86 (t, 6H); Analytical HPLC column: Agilent Zorbax SB-C4, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 60±2 °C, detector: ELSD, fe = 13.85 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 55±2 °C, detector: CAD, fe = 13.53 min, purity: > 99%.

Scheme 12: Synthesis of Compound PL-13A

Compound PL-13A

Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-((trityloxy)methyl)pyrrolidine-l-carboxylate (L95-1)

Boc

L95-1

[0943] A mixture of L93-2 (5.00 g, 23.01 mmol), trityl chloride (8.98 g, 32.21 mmol) and pyridine (50 mL) was stirred at RT for 65h. The reaction mixture was then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (120 mL) and washed with water (60 mL X 3) and brine (60 mL X 3). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration provided a residue which was purified using flash chromatography (SiO2: 0-40% ethyl acetate in hexanes gradient) to yield L95-1 as white powder (8.88 g, 84%); ¹HNMR (CDCl3) δ 7.43 - 7.29 (m, 15H), 4.53 (m, 1H), 4.12 (m, 1H), 3.56 (m, 2H), 3.17 (m, 3H), 2.20 (m, 2H), 1.31 (s, 9H).

Synthesis of tert-butyl (2S,4R)-4-(benzyloxy)-2-((trityloxy)methyl)pyrrolidine-l- carboxylate (L95-2)

[0944] To an ice bath cooled solution of L95-1 (6.30 g, 13.71 mmol) in anhydrous THF (50 mL) was added sodium hydride (0.82 g, 60% in mineral oil, 20.50 mmol) under nitrogen atmosphere. The resulting mixture was stirred at room temperature for Ih. TBAI (0.51 g, 1.37 mmol) and benzyl bromide (2.81 g, 16.45 mmol) were added, and the mixture was left to stir overnight. The reaction mixture was concentrated under reduced pressure and the crude was mixed with ethyl acetate (100 mL). The organic layer was washed with saturated ammonium chloride solution (50 mL X 3), water (50 mL X 3) and brine (50 mL X 3). The organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure to give a residue which was purified with flash chromatography (SiO2: 0-15% ethyl acetate in hexanes) to afford L95-2 as creamy white solid (6.50 g, 86%). ¹HNMR (CDCl3) δ 7.43 - 7.16 (m, 20H), 4.53 (s, 2H), 4.15 - 4.09 (m, IH), 3.59 - 3.53 (m, 2H), 3.25 - 2.87 (m, 3H), 2.20 (dt, J= 11.8, 5.6 Hz, 2H), 1.31 (s, 9H).

Synthesis of ((2S,4R)-4-(benzyloxy)pyrrolidin-2-yl)methanol (L95-3)

[0945] A mixture of L95-2 (6.50 g, 11.82 mmol) and TFA (10 mL) in DCM (40 mL) was stirred at room temperature for 6h. The reaction mixture was then concentrated under reduced pressure. To the residue was added water (100 mL) and the pH of the solution was adjusted to 9. The aqueous solution was extracted with 10% MeOH in DCM (300 mL X 3). The combined organic layers were dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure to afford L95-3 as yellow oil (1.51 g, 62%) which was used for the next step without further purification; ¹HNMR (CDCl3) δ 7.39 - 7.27 (m, 5H), 4.47 (s, 2H), 4.11 (ddt, J = 5.7, 3.8, 1.7 Hz, 1H), 3.62 - 3.43 (m, 2H), 3.35 - 3.18 (m, 1H), 3.11 (dt, J = 12.4, 1.8 Hz, 1H), 2.83 (dd, J = 12.4, 3.9 Hz, 1H), 2.10 - 1.91 (m, 1H), 1.67 - 1.45 (m, 1H); CIMS m/z [M+H]⁺ 208.2

Synthesis of ((2S,4R)-4-(benzyloxy)-l-tetradecanoylpyrrolidin-2-yl)methyl tetradecanoate

(L95-4)

[0946] A solution of L94-1 (1.65 g, 7.22 mmol), EDC (2.76 g, 14.40 mmol) and DMAP (0.30 g, 2.46 mmol) in DCM (30 mL) was stirred at room temperature for 30 min. A solution of L95- 3 (0.50, 2.41 mmol) in DCM (10 mL) was added in. The resulting mixture was stirred at room temperature for 24h and concentrated under reduced pressure to give a residue which was purified by flash chromatography (SiO2: 0-20% ethyl acetate in hexanes gradient) to afford L95-4 as white solid (1.41 g, 93%); ¹HNMR (CDCl3) δ 7.37 - 7.27 (m, 5H), 4.59 - 4.37 (m, 3H), 4.33 - 3.90 (m, 3H), 3.65 - 3.35 (m, 2H), 2.41 - 1.94 (m, 6H), 1.59 (s, 4H), 1.40 - 1.00 (m, 40H), 0.87 (d, J = 13 Hz, 6H); CIMS m/z [M+H]⁺ 628.5.

Synthesis of ((2S,4R)-4-hydroxy-l-tetradecanoylpyrrolidin-2-yl)methyl tetradecanoate (L95-5)

[0947] A mixture of L95-4 (1.34 g, 2.13 mmol) and Pd(OH)₂/C (700 mg, 20 wt. %, 1.00 mmol) in ethyl acetate (35 mL) was stirred under hydrogen atmosphere (balloon) for 40h. The mixture was filtered through Celite, concentrated under reduced pressure to afford L95-5 as white solid (1.00 g, 87%); ¹HNMR (CDCl3) δ 4.58 - 4.41 (m, 2H), 4.37 - 3.92 (m, 2H), 3.66 - 3.35 (m, 2H), 2.41 - 2.19 (m, 4H), 2.12 - 1.99 (m, 2H), 1.60 (d, J= 9.4 Hz, 4H), 1.25 (s, 40H), 0.92 - 0.82 (t, J = 6.3 Hz, 6H); CIMS m/z [M+H]⁺ 538.4. Synthesis of (3R,5S)-l-tetradecanoyl-5-((tetradecanoyloxy)methyl)pyrrolidin-3-yl 142-oxo-

2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,

74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137- hexatetracontaoxa-141-azapentatetracontahectan-145-oate (Compound PL-13A)

Compound PL-13A

[0948] A solution of L93-6 (0.40 g, 0.18 mmol), DMAP (22 mg, 0.18 mmol) and EDC (103 mg, 0.53 mmol) in DCM (15 mL) was stirred at room temperature for 30 min. A solution of L95- 5 (116 mg, 0.22 mmol) in DCM (5 mL) was added in. The reaction mixture was stirred at room temperature for 16h and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2: 0-100% ethyl acetate in hexane, then 0-10% methanol in dichloromethane gradient) to afford Compound PL-13A as white solid (0.34 g, 59%); ¹HNMR (CDC1₃) δ 6.44 (m, 1H), 5.31 (m, 1H), 4.46 (m, 1H), 4.33 (m, lH), 4.17 (m, 1H), 3.92 - 3.32 (m, 182H),3.37 (s, 3H), 2.63 - 2.41 (m, 4H), 2.34 - 2.06 (m, 6H), 1.94 (m, 6H), 1.59 (m, 4H), 1.24 (m, 40H), 0.88 (t, J = 6.3 Hz, 6H); Analytical HPLC column: Agilent Zorbax SB-C18, 5 pm, 4.6* 150 mm, mobile phase A: acetonitrile with 0.1% tri fluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 95% in 15 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, fe = 10.2 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, Cl 8, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 55±2 °C, detector: CAD, fe= 13.2 min, purity: > 99 %.

Scheme 13: Synthesis of Compound PL-14A

Compound PL-14A

45

Synthesis of ((2S,4R)-4-(benzyloxy)-l-stearoylpyrrolidin-2-yl)methyl stearate (L96-1)

[0949] A solution of stearic acid (2.06 g, 7.24 mmol), EDC (2.76 g, 14.40 mmol) and DMAP (0.30 g, 2.46 mmol) in DCM (30 mL) was stirred at room temperature for 30 min at room temperature. A solution of L95-3 (0.50 g, 2.41 mmol) in DCM (10 mL) was added in. The resulting mixture was stirred at room temperature for 20h and concentrated under reduced pressure. The residue was dissolved in DCM (200 mL) and the solution was washed with water (100 mL x 3) and brine (100 mL x 3). After drying with anhydrous sodium sulfate, the organic phase was filtered and concentrated under reduced pressure. The crude was purified by flash chromatography (SiO2: 0-20% ethyl acetate in hexanes gradient) to afford L96-1 as white solid (1.34 g, 75%); ¹HNMR (CDCl3) δ 7.40 - 7.26 (m, 5H), 4.61 - 4.37 (m, 3H), 4.34 - 4.05 (m, 3H), 3.70 - 3.32 (m, 2H), 2.32 - 1.94 (m, 6H), 1.61 (s, 4H), 1.25 (s, 56H), 0.87 (d, J = 6.9 Hz, 6H); CIMS m/z [M+H]⁺ 740.7. Synthesis of ((2S,4R)-4-hydroxy-l-stearoylpyrrolidin-2-yl)methyl stearate (L96-2)

[0950] A mixture of L96-1 (1.30 g, 1.76 mmol) and Pd(OH)2/C (700 mg, 20 wt. %, 1.00 mmol) in ethyl acetate (40 mL) and DCM (10 mL) was stirred under hydrogen atmosphere (balloon) for 16h. The reaction mixture was filtered through Celite and concentrated under reduced pressure to afford L96-2 as white solid (1.10 g, 96%); ¹HNMR (CDCl3) δ 4.58 - 4.45 (m, 2H), 4.36 - 3.96 (m, 2H), 3.72 - 3.29 (m, 2H), 2.38 - 2.19 (m, 4H), 2.11 - 1.95 (m, 2H), 1.60 (s, 4H), 1.25 (s, 56H), 0.92 - 0.82 (d, J= 6.9 Hz, 6H). CIMS m/z [M+H]⁺ 650.6.

Synthesis of (3R,5S)-l-stearoyl-5-((stearoyloxy)methyl) pyrrolidine-3-yl 142-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137-hexatetracontaoxa-

141-azapentatetracontahectan-145-oate (Compound PL-14A)

Compound PL-14A

[0951] To a stirring solution of L93-6 (500 mg, 0.23 mmol) in DCM (10 ml) was added EDC (131 mg 0.69 mmol) and DMAP (0.028 g, 0.23 mmol). After stirring for 30 min, L96-2 (180 mg, 0.27 mmol) was added in. The reaction mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. The residue was purified by column chromatography (80 g silica gel, solvent 0-5% MeOH in DCM) to provide Compound PL-14A (463 mg, 72%); ¹HNMR (CDCl3): 6.46 (m, 1H), 5.31 (m, 1H), 4.46 (m, 1H), 4.33 (m, 1H), 4.18 (m, 1H), 3.92 - 3.32 (m, 182H), 3.37 (s, 3H), 2.63 - 2.41 (m, 4H), 2.34 - 2.06 (m, 6H), 1.94 (m, 6H), 1.59 (m, 4H), 1.50-1.00 (m, 56H), 0.88 (t, J = 6.3 Hz, 6H); Analytical HPLC column: Agilent Zorbax SB- C4, 5 pm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 60±2 °C, detector: ELSD,

14.9 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, Cl 8, 1.7 pm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 19 min, flow rate: 0.5 mL/min, column temperature: 55±2 °C, CAD, fe= 13.8 min, purity: 98.2%.

Example 2: Production of nanoparticle compositions

[0952] A nanoparticle composition may be produced as described in US patent application US20170210697A1, which is incorporated herein by reference in its entirety.

[0953] In order to investigate safe and efficacious nanoparticle compositions for use in the delivery of various payloads, including but not limited to mRNA and siRNA therapeutics, a range of formulations are prepared and tested. Specifically, the particular elements and ratios thereof in the lipid component of nanoparticle compositions are optimized.

[0954] Nanoparticles can be made with mixing processes such as microfluidics and T- junction mixing of two fluid streams, one of which contains the active agent and the other has the lipid components.

[0955] Lipid compositions are prepared by combining an ionizable lipid, a phospholipid (such as DOPE or DSPC, obtainable from Avanti Polar Lipids, Alabaster, Ala.), a PEG lipid (such as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, Ala.), and a structural lipid (such as cholesterol, obtainable from Sigma-Aldrich, Taufkirchen, Germany, or a cholesterol analog) in ethanol. Lipids are combined to yield desired molar ratios and diluted with water and ethanol.

[0956] Nanoparticle compositions may be prepared by combining a lipid solution with a solution including the active agent. The lipid solution is rapidly injected using, for example, a NanoAssemblr® microfluidic based system, into the active agent solution.

[0957] Solutions of the active agent in deionized water may be diluted in citrate buffer to form a stock solution.

[0958] Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations are dialyzed against a buffer such as phosphate buffered saline (PBS), Tris-HCl, or sodium citrate, using, for example, Slide- A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill.). The resulting nanoparticle suspension is filtered through sterile filters (Sarstedt, Numbrecht, Germany) into glass vials and sealed with crimp closures. Alternatively, a Tangential Flow Filtration (TFF) system, such as a Spectrum KrosFlo system, may be used.

[0959] The method described above induces nano-precipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nano-precipitation. Example 2A: Exemplary Nanoparticle Formulation Procedure

[0960] Ionizable lipids, phospholipids, structural lipids (eg. Cholesterol or other sterols), and PEGlipids are dissolved in ethanol. The ionizable lipids mol % can be from 30-70%, phospholipids mol % can be 5-20%, sterols mol % can be 20-60%, and PEG lipid mol % can be 0.1-10%. The lipid solution is mixed with an acidic buffer containing mRNA on a mixing device, such as a NanoAssemblr® microfluidic systems, to form LNPs. To adjust LNP particle size, the volume ratio of lipid solution to mRNA solution can be varied from 1 : 1 to 20: 1, mRNA concentration in aqueous buffer can be 0.01 mg/mL to 10 mg/mL, N/P ratio can be 1 to 50 and different identities of PEG lipids or other polymers can be used. After the LNP is formed from the mixing device, aqueous buffer is added to reduce the ethanol concentration. The volume of aqueous buffer can be 0. 1 to 100 volume of LNP volume coming out of the mixing device. The LNPs are further dialyzed against aqueous and concentrated to a desired concentration. The particle size of LNPs is measured by dynamic light scattering (DLS), for example, by using a Zetasizer Ultra (Malvern Panalytical). RNA encapsulation efficiency is determined, for example, by Quant-it™ RiboGreen assay.

Example 3: Characterization of nanoparticle compositions

[0961] A nanoparticle composition may be characterized as described in US patent application US20170210697A1, which is incorporated herein by reference in its entirety.

[0962] Particle size, poly dispersity index (PDI), and the zeta potential of a nanoparticle composition can be determined using, for example, a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK), or a Wyatt DynaPro plate reader.

[0963] Ultraviolet-visible spectroscopy can be used to determine the concentration of active agent in nanoparticle compositions. The formulation may be diluted in PBS then added to a mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). The concentration of active agent in the nanoparticle composition can be calculated based on the extinction coefficient of the active agent used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[0964] For nanoparticle compositions including an RNA, a QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) can be used to evaluate the encapsulation of an RNA by the nanoparticle composition. The samples are diluted in a TE buffer solution. Portions of the diluted samples are transferred to a polystyrene 96 well plate and either TE buffer or a 2% Triton X-100 solution is added to the wells. The plate is incubated at, for example, a temperature of 37° C for 15 minutes. The RIBOGREEN® reagent is diluted in TE buffer, and this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

Example 4: In vivo studies including protein expression by organ

[0965] Delivery to a target organ may be assessed as described in US patent application US20170210697A1, which is incorporated herein by reference in its entirety.

[0966] In order to monitor how effectively various nanoparticle compositions deliver polynucleotides to targeted cells, different nanoparticle compositions including a particular polynucleotide are prepared and administered to rodent populations. Mice are intravenously, intramuscularly, intraarterially, or intratumorally administered a single dose of a nanoparticle composition. In some instances, mice may be made to inhale doses. Dose sizes may range from 0.001 mg/kg to 10 mg/kg, where 10 mg/kg describes a dose including 10 mg of polynucleotide in a nanoparticle composition for each 1 kg of body mass of the mouse. A control composition including PBS may also be employed.

[0967] Upon administration of nanoparticle compositions to mice, dose delivery profiles, dose responses, and toxicity of particular formulations and doses thereof can be measured by enzyme-linked immunosorbent assays (ELISA), bioluminescent imaging, or other methods. Time courses of protein expression can also be evaluated. Samples collected from the rodents for evaluation may include blood, sera, and tissue (for example, muscle tissue from the site of an intramuscular injection and internal tissue); sample collection may involve sacrifice of the animals.

[0968] For example, LNP formulations including RNA encoding a detectable protein such as luciferase may be administered intravenously to mice at a dosage of, for example, 0.5 mg/kg. A standard MC3 formulation and a PBS control may also be tested. Bioluminescence in various organs, such as the liver, lung, spleen, and femur, may be measured after 6 hours.

[0969] Nanoparticle compositions including protein coding RNA are useful in the evaluation of the efficacy and usefulness of various formulations for the delivery of polynucleotides. Higher levels of protein expression induced by administration of a composition including protein coding RNA will be indicative of higher RNA translation and/or nanoparticle composition RNA delivery efficiencies. As the non-RNA components are not thought to affect translational machineries themselves, a higher level of protein expression is likely indicative of a higher efficiency of delivery of the RNA by a given nanoparticle composition relative to other nanoparticle compositions or the absence thereof.

Example 5: Toxicity, cytokine induction, and complement activation

[0970] Toxicity of the LNP compositions of the disclosure may be analyzed as described by international patent application WO2016118724 and/or US20170210697A1, which are incorporated herein by reference in its entirety.

Example 5A: Liver toxicity

[0971] RNA encoding a detectable protein is generated and loaded into lipid nanoparticles. The nanoparticles are administered to mice, and expression of the detectable protein as well as levels of certain liver enzymes are measured. Additional mice may be dosed with a reference LNP formulation, such as one containing MC3, as a comparison. To assess dose response, mice may be given varying levels of the LNP formulations. Liver enzymes, such as alanine transaminase (ALT) and aspartate transaminase (AST), may be measured to assess liver toxicity. In some embodiments, creatine phosphokinase (CPK) may also be measured to assess cardiac or muscular toxicity. In some embodiments, a pharmaceutical composition described herein provides a safer toxicity profile than a reference pharmaceutical composition, such as one containing MC3.

Example 5B: Cytokine Induction

[0972] The introduction of foreign material into a mammalian body induces an innate immune response that promotes cytokine production. Such immune responses to, for example, nanoparticle compositions including an active agent of the disclosure, are undesirable. The induction of certain cytokines is thus measured to evaluate the efficacy of nanoparticle compositions and the inflammatory response. The concentrations of various cytokines in mice upon intravenous administration of nanoparticle compositions at a dosage of 0.5 mg/kg are measured at 6 hours. The standard MC3 formulation and a PBS control may also be tested. Cytokines including TNF-a, IFN-y, IP-10, MCP-1 , IFN-a, IL-6, and IL-5 may be measured. In some embodiments, IP-10 and IL-6 are measured. In some embodiments, histamine levels may also be measured. In some embodiments, a pharmaceutical composition described herein provides an improved inflammatory profile than a reference pharmaceutical composition, such as one containing MC3. Example 5C: Complement Activation

[0973] Complement activation assists in the clearance of pathogens from an organism. As it is undesirable that a subject's body recognize a nanoparticle composition as a foreign invader, low complement system activation upon administration of such a composition is preferred. The complex sC5b-9 is a marker for the activation of the complement system. Thus, human cells are contacted in vitro with nanoparticle compositions and are evaluated for sC5b-9 levels.

Example 6: LNP optimization

[0974] LNP compositions may be optimized as described by US patent application US20170210697A1, which is incorporated herein by reference in its entirety.

Example 6A: Optimization of Ionizable Lipid

[0975] As smaller particles with higher encapsulation efficiencies are generally desirable, the relative amounts of various elements in lipid components of nanoparticle compositions are optimized according to these parameters.

[0976] An ionizable lipid is selected for optimization. The relative amount of the ionizable lipid is varied between 30 mol % and 60 mol % in compositions that can include DOPE or DSPC as phospholipids to determine the optimal amount of the ionizable lipid in the formulations. Formulations are prepared using a standardized process with a water to ethanol ratio in the lipid- mRNA solution of, for example, 3: 1 and a rate of injection of the lipid solution into the mRNA solution of, for example, 12 mL/min on a NanoAssemblr® microfluidic based system. These parameters may be altered depending on, for example, the lipids used and the target particle size. This method induces nano-precipitation and particle formation. Alternative processes including, but not limited to, T-junction or direct injection, may also be used to achieve the same nanoprecipitation.

[0977] Formulations producing the smallest particles with the highest encapsulation efficiencies are generally preferred. Compositions are also evaluated for their detectable protein expression levels and cytokine profiles.

Example 6B: Optimization of Phospholipid

[0978] The relative amount of phospholipid in a lipid component of a nanoparticle composition is varied to further optimize the formulation. An ionizable lipid is selected for use in the nanoparticle composition and a phospholipid such as DOPE and DSPC are selected. Additional phospholipids can also be evaluated. Nanoparticle compositions are prepared with the relative phospholipid content varying between 0 mol % and 30 mol %. Compositions are evaluated for their size, encapsulation efficiency, detectable protein expression levels, and cytokine profiles.

Example 6C: Optimization of Structural Lipid

[0979] The relative amount of structural lipid in a lipid component of a nanoparticle composition is varied to further optimize the formulation. An ionizable lipid is selected for use in the nanoparticle composition and cholesterol or a cholesterol analog is selected as a structural lipid. Additional structural lipids can also be evaluated. Nanoparticle compositions are prepared with the relative structural lipid content varying between 18.5 mol % and 48.5 mol %. Compositions are evaluated for their size, encapsulation efficiency, detectable protein expression levels, and cytokine profiles.

Example 6D Optimization of PEG Lipid

[0980] The relative amount of PEG lipid in a lipid component of a nanoparticle composition is varied to further optimize the formulation. An ionizable lipid is selected for use in the nanoparticle composition and a PEG lipid such as PEG-DMG or PEG-DSPE is selected. Additional PEG lipids can also be evaluated. Nanoparticle compositions are prepared with the relative PEG lipid content varying between 0 mol % and 10 mol %. Compositions are evaluated for their size, encapsulation efficiency, detectable protein expression levels, and cytokine profiles. For formulations wherein the PEG lipid is conjugated to one or more targeting moi eties, the ratio of conjugated and non-conjugated PEG lipid can also be optimized in order to increase or decrease the relative amount of targeting moiety present on the outer surface of the nanoparticles.

Example 6E: Optimization of Particle Sizes

[0981] The fenestration sizes for different bodily organs often vary; for example, the kidney is known to have a smaller fenestration size than the liver. Thus, targeting delivery of an active agent (e.g., specifically delivering) to a particular organ or group of organs may require the administration of nanoparticle compositions with different particle sizes. In order to investigate this effect, nanoparticle compositions are prepared with a variety of particle sizes using a Nanoassemblr® instrument. Nanoparticle compositions include an RNA encoding Luc. Each differently sized nanoparticle composition is subsequently administered to mice to evaluate the effect of particle size on delivery selectivity. Luc expression in two or more organs or groups of organs can be measured using bioluminescence to evaluate the relative expression in each organ. [0982] A number of parameters can be adjusted in order to optimize the particle size of the nanoparticles. Exemplary parameters include, but are not limited to, the identity of the PEG lipid, mol% of the PEG lipid in the LNP formulation, the identity of the structural lipid, mol% of the structural lipid in the LNP formulation, the identity of the phospholipid, mol% of the phospholipid in the LNP formulation, the identity of the ionizable lipid, mol% of the ionizable lipid in the LNP formulation, identity of lipid components covalently bound to one or more targeting moieties, mol% of said targeting moiety bound lipids in the LNP formulation, flow rate of the Nanoassemblr® instrument in the preparation of the formulation, concentration of the mixing solutions used in the formulation, buffers used in the preparation of the formulation, and duration of formulation mixing.

Example 7: Nanoparticle Formulation Procedure - hEPO and fLuc

[0983] Ionizable lipids (“IL”) 78 or C66 (see PCT Application Publication WO2023044343A1 and PCT Application PCT/US2022/082276), DSPC, cholesterol, and designated PEG lipid were dissolved in pure ethanol at a 48.5: 10:39:2.5 mol% ratio with a total lipid concentration of 10.8 mM. See, e.g., Qiu et al., PNAS 77S:e2020401118 (2021). The lipid solution was mixed at a 3 : 1 volume ratio with an acidic sodium citrate buffer (pH 4.0) containing mRNA encoding human erythropoietin (hEPO; SEQ ID NO: 2) and firefly luciferase (fLUC; SEQ ID NO: 1) in a 1 :2 ratio using the NanoAssemblr microfluidic system at a 12 mL/min total flow rate resulting in rapid mixing and self-assembly of LNPs. Formulations were further dialyzed against PBS (pH 7.4) overnight at 4 °C, concentrated using centrifugal filtration and filtered (0.2 pm pore size). The particle size and poly dispersity index (PDI) of formulations was measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 7-1: Lipid nanoparticle formulations

Table 7-2: mRNA used in LNP formulations

Example 8A: Method for lipid screening

[0984] LNP/mRNA formulations F-l through F-6 (Table 7-1, prepared as described in Example 7) are dosed in Balb/cAnNCrl (female, 6-8 weeks) via tail vein IV infusion (0.2 mg/kg fLuc). 6, 24, and 48 hrs after LNP injection, 150 mg/kg D-luciferin are injected intraperitoneally and the whole-body bioluminescence signal is acquired ~10 minutes after injection of the D- luciferin using an IVIS Lumina III LT system (PerkinElmer).

Example 8B: hEPO and fLUC in vivo reporter assays

[0985] Balb/cAnNCrl (female, 6-8 weeks) were administrated with LNPs (formulated for a 0.3 mg/kg total hEPO/fLuc mRNA dose, see formulations in Example 7) by intravenous injection (5 mL/. Plasma samples were harvested at 5, 23 and 48 hours post dose for hEPO analysis. Bioluminescence imaging (BLI) of the mice was taken at 6, 24 and 48 hours post-dosing using an IVIS Lumina III LT system (PerkinElmer) after injection of D-luciferin solution (150 mg/kg, intraperitoneal injection (IP)). hEPO concentrations were measured using an ELISA kit (DEP00, R&D Systems). The maximal concentration or BLI signal (Cmax) and area under concentration vs time curve (AUC) of the individual mouse plasma hEPO or whole body BLI data was calculated using a non-compartment analysis (NCA) program (WinNonlin®, Version 8.3.4 [Pharsight Corp (Mountain View, CA, USA)]).

[0986] Table 8-1 reports the hEPO concentration maximum (Cmax) and the AUC over the 48 hour period after dosing. Table 8-1 also reports the luciferase bioluminescence imaging Cmax and the AUC0-48hr over the 48 hour period after dosing, for each formulation tested.

Data keys: hEPO Cmax (lU/mL): £ = <100 lU/mL; 100 lU/mL < ££ < 500 lU/mL; 500 lU/mL < £££ < 1,000 lU/mL hEPO AUC (hr*IU/mL): + = <1,000 hr*IU/mL; 1,000 hr*IU/mL < ++ < 5,000 hr*IU/mL; 5,000 hr* lU/mL < +++ < 10,000 hr*IU/mL; 10,000 hr*IU/mL < ++++ < 50,000 hr*IU/mL Luciferase BLI Cmax (photons/sec): # = <10 billion p/s; 10 billion p/s < ## < 50 billion p/s; 50 billion p/s < ### < 100 billion p/s; 100 billion p/s < #### < 500 billion p/s; 500 billion p/s < ##### < 1 trillion p/s

Luciferase BLI AUC48hr (hr*photons/sec): $ < 100 billion hr*p/s; 100 billion hr*p/s < $$ < 500 billion hr*p/s; 500 billion hr*p/s < $$$ < 1 trillion hr*p/s; 1 trillion hr*p/s < $$$$ < 5 trillion hr*p/s; 5 trillion hr*p/s < $$$$$ < 10 trillion hr*p/s; ; 10 trillion hr*p/s < $$$$$ < 50 trillion hr*p/s Table 8-1: In Vivo Assay Data

Example 9: Nanoparticle Formulation Procedure - Organ Tropism

[0987] Ionizable lipids, DSPC, cholesterol, and designated PEG lipid were dissolved in pure ethanol at a 48.5: 10:40: 1.5 mol% ratio with a total lipid concentration of 10.8 mM. A 0.10 mg/mL mRNA solution was prepared using acidic buffer (pH 4.0-5.0) containing mRNAs encoding firefly luciferase (fLuc). The nucleotide and lipid solutions were mixed at a 3 : 1 volume ratio using the NanoAssemblr microfluidic system at a 12 mL/min total flow rate resulting in rapid mixing and self-assembly of LNPs. Formulations were further dialyzed against PBS (pH 7.4) overnight at 4 °C, concentrated using centrifugal filtration and filtered (0.2 pm pore size). The particle size and poly dispersity index (PDI) of formulations was measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 9-1: Lipid nanoparticle formulations

Example 10: Organ Tropism via IV injection

[0988] Balb/cAnNCrl (female, 6-8 weeks) were dosed with LNP formulations (formulated for a 0.2 mg/kg fLuc mRNA dose, see Example 9) by IV injection. At 6 hour post LNP dose, the mice were injected with D-luciferin solution (150 mg/kg, intraperitoneal (IP)). 10 minutes post D-luciferin dosing, mice were sacrificed and organs (liver, spleen, lung, kidney, and heart or brain) were harvested. Bioluminescence imaging of the organs from each dosing groups were taken simultaneously using an IVIS Lumina III LT system (PerkinElmer).

[0989] The sum of the bioluminescence of all organs from each individual mouse were summed as the total flux (photons/second). The percentage of bioluminescence of each individual organ was calculated to determine the organ tropism of the LNP formulations.

[0990] Luciferase BLI C6hr (photons/sec): # = <1 million p/s; 1 million p/s < ## < 10 million p/s; 10 million p/s < ### < 100 million p/s; 100 million p/s < #### < 1 billion p/s; 1 billion p/s < ##### < 10 billion p/s

Table 10-1. Total Flux of organs and percentage flux in each organ

Example 11: Nanoparticle Formulation Procedure - HSC Delivery

[0991] Ionizable lipids, DSPC, cholesterol, and a PEG lipid were dissolved in pure ethanol at the specified mol% ratios with a total lipid concentration of 10.8 mM. A 0.10 mg/mL mRNA solution was prepared using acidic buffer (pH 4.0-5.0) containing mRNA encoding Cre recombinase (SEQ ID NO: 3). The nucleotide and lipid solutions were mixed at a 3: 1 volume ratio using the NanoAssemblr microfluidic system at a 12 mL/min total flow rate resulting in rapid mixing and self-assembly of LNPs. Formulations were further dialyzed against PBS (pH 7.4) overnight at 4 °C, concentrated using centrifugal filtration and filtered (0.2 pm pore size). The particle size and poly dispersity index (PDI) of formulations was measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table 11-1: Lipid nanoparticle formulations

Table 11-2: mRNA used in LNP formulations

Example 12: Immune Cell and HSC Delivery

[0992] LNP/mRNA formulations were prepared as described in Example 11. Ail4 mice (B6.Cg- Gt(ROSA)26Sortml4(CAG-tdTomato)Hze/J (Jackson Laboratory 007914)), were injected via tail vein with 0.5 mg/kg ere mRNA (TriLink Biotechnologies) formulated in LNP formulations F-27 through F-31 in a total volume of 5mL/kg. Each formulation was dosed in 2- 3 mice and an additional 1-2 mice were dosed with either PBS or fLuc mRNA LNP to serve as a control. 72 h post injection, animals were euthanized by CO2 inhalation, and spleens, femurs, and tibiae/fibulae were harvested. Harvested spleens were dissociated into single cell suspension of splenocytes using the gentleMACS Octo Dissociator with Heaters (Miltenyi 130-096-427) with the Mouse Spleen Dissociation Kit (Miltenyi 130-095-926) per manufacturer’s instructions. Dissociated splenocytes were then passed through a 70pm filter (Miltenyi 130-098-462) and washed with lx PBS (ThermoFisher 10010049) containing 2mM EDTA (ThermoFisher 15575- 020) and 0.5% BSA (Miltenyi 130-091-376). Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher A1049201) and washed twice with lx PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70pm filter prior to the last wash. Following final wash, cells were resuspended in lx PBS + 2mM EDTA + 0.5% BSA and counted (ViCell XR, Beckman Coulter 731196). Cells were diluted, plated (5,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Bone marrow (BM) was harvested from the bones by using a 25G needle (BD 305122) to flush the marrow cavity with lx PBS + 2mM EDTA + 0.5% BSA. Cells were collected, passed through a 70pm filter, and washed with lx PBS + 2mM EDTA + 0.5% BSA. Red blood cells were lysed using ACK Lysing Buffer and washed twice with lx PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70pm filter prior to the last wash. Following final wash, cells were resuspended in lx PBS + 2mM EDTA + 0.5% BSA and counted. Cells were diluted, plated (~10 million per well) in a 96-well bottom plate, and stained for flow cytometry. Briefly, cells were stained in lx PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block (splenocytes) or labeled CD16/32 antibody (bone marrow) for 5min at 4°C and surface antibody stains either in full or FMO master mixes (panel and dilutions shown below in Table 12-1 and 12-2) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed three times with Cell Staining Buffer and fixed with Cytofix (BD 554655) at 4°C for 30min. Cells were washed twice with lx PBS and filtered through a 30-40 pm filter (Pall 8027) and acquired on cytometer (ThermoFisher Attune NXT with a laser configuration of Blue(3)/Red(3)/Violet(4)/Yellow(4)) equipped with a high-throughput autosampler (ThermoFisher CytKick). Compensation was performed using UltraComp eBeads (ThermoFisher 01-3333-41), ArC Amine Reactive Compensation Bead Kit (ThermoFisher A10346), and Luciferase/tdTomato Dual-Reporter HEK293 cells (Alstem LRL01). Analysis performed using Flowjo (BD V10.8.1). Cells were identified with markers in Tables 12-3 and 12-4 and tdTomato gates placed so that the negative control would be <0.5%⁺.

[0003] The percentage of cells of the noted varieties expressing ere mRNA is reported below in Table 12-5.

Table 12-1: Splenocyte surface antibody stains

For clarity, in later panels NKp46 was replaced with AF700 NK-1.1 clone S17016D used at a

1 :500 dilution (Biolegend 156512)

Table 12-2: Bone marrow surface antibody stains

Table 12-3: Definitions of Cell Subsets in Mouse Spleen by Flow Cytometry

Table 12-4: Definition of Cell Subsets in Mouse BM by Flow Cytometry

*Lineage is defined as including CD4, CD8a, CD1 lb, B220, GR1, Teri 19.

I HSC8 gated two different ways, alternative denotes second strategy for gating specific HSC population.

Table 12-5: Percentage of cells of various types expressing ere mRNA

Claims

1. A compound of formula PL-I’ :

PL-F or a pharmaceutically acceptable salt thereof, wherein:

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N₂, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

R⁴ is Ci-4 alkyl;

2. The compound of claim 1, wherein X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-.

3. The compound of claim 1 or 2, wherein A¹ is a saturated 5-6 membered carbocyclic ring substituted with t occurrences of R⁴.

4. The compound of claim 1 or 2, wherein A¹ is cyclopentyl.

5. The compound of claim 1 or 2, wherein A¹ is cyclohexyl.

6. The compound of claim 1 or 2, wherein A¹ is a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the heterocyclic ring is substituted with t occurrences of R⁴.

7. The compound of claim 1 or 2, wherein A¹ is selected from pyrrolidinylene, tetrahydrofuranylene, tetrahydrothiophenylene, imidazolidinylene, thiazolidinylene, oxazolidinylene, piperidinylene, tetrahydro-2H-pyranylene, tetrahydro-2H-thiopyranylene, piperazinylene, morpholinylene, and hexahydropyrimidinylene.

8. The compound of claim 1 or 2, wherein the compound is of formula PL-Ia, PL-Ib, or PL-Ic:

PL-Ic or a pharmaceutically acceptable salt thereof.

9. The compound of claim 1 or 2, wherein the compound is of formula PL-Id or PL-Ie:

PL-Ie or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1 or 2, wherein the compound is of formula PL-If:

PL-If or a pharmaceutically acceptable salt thereof.

11. The compound of claim 1 or 2, wherein the compound is of formula PL-Ig:

PL-Ig or a pharmaceutically acceptable salt thereof.

12. The compound of claim 1 or 2, wherein the compound is of formula PL-Ih or PL-Ii:

or a pharmaceutically acceptable salt thereof.

13. The compound of claim 1 or 2, wherein the compound is of formula PL-Ij or PL-Ik:

_/L²-R²

PL-Ik or a pharmaceutically acceptable salt thereof.

14. The compound of claim 1 or 2, wherein the compound is of formula PL-11, PL-Im, or In:

PL-In or a pharmaceutically acceptable salt thereof.

15. The compound of claim 1, wherein the compound is of formula PL-Io, PL-Ip, or PL-Iq:

PL-Io PL-Ip

PL-Iq or a pharmaceutically acceptable salt thereof.

16. The compound of claim 1, wherein the compound is of formula PL-Ir or PL-Is:

PL-Is or a pharmaceutically acceptable salt thereof.

17. The compound of claim 1, wherein the compound is of formula PL-It:

PL-It or a pharmaceutically acceptable salt thereof.

18. The compound of claim 1, wherein the compound is of formula PL-Iu:

PL-Iu or a pharmaceutically acceptable salt thereof.

19. The compound of claim 1, wherein the compound is of formula PL-Iv or PL-Iw:

PL-Iv PL-Iw or a pharmaceutically acceptable salt thereof.

20. The compound of claim 1, wherein the compound is of formula PL-Ix or PL-Ixx:

PL-Ixx or a pharmaceutically acceptable salt thereof.

21. The compound of claim 1, wherein the compound is of formula PL-Iy, PL-Iyy, or PL- iyyy

PL-Iyy

PL-Iyyy or a pharmaceutically acceptable salt thereof.

22. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is -C(O)(Ci- 6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)2-, or - C(O)(C1-6 aliphatic)C(O)-.

23. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is -C(O)(Ci- 6 aliphatic)C(O)-N(R)-.

24. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is - C(O)CH₂CH₂C(O)-N(R)-.

25. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is -C(O)(Ci- 6 aliphatic)C(O)-.

26. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is - C(O)CH₂CH₂C(O)-.

27. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is -C(O)(Ci- 6 aliphatic)C(O)₂-.

28. The compound of any one of claims 1-8, 11, 12, 15, 18, 19, or 21, wherein L¹ is - C(O)CH₂CH₂C(O)₂-.

29. A compound of Formula PL-II’:

PL-II’ or a pharmaceutically acceptable salt thereof, wherein:

X¹ is -N(H)-, -N(C1-6 alkyl)-, -C1-6 aliphatic-N(H)-, -C1-6 aliphatic-N(C1-6 alkyl)-, -O- or -C1-6 aliphatic-O-; L¹ is -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)-, or -C(O)-;

R¹ is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl;

30. The compound of claim 29, wherein X¹ is -N(H)-, -N(C1-6 alkyl)-, or -O-.

31. The compound of claim 29 or 30, wherein L¹ is -C(O)(C1-6 aliphatic)C(O)-.

32. The compound of claim 29 or 30, wherein L¹ is -C(O)CH2CH2C(O)-.

33. The compound of claim 29 or 30, wherein L¹ is -C(O)-.

34. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are the same.

35. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are independently a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is optionally replaced with -O-, -NR-, -C(O)O-, -OC(O)-, or -OC(O)N(R)-.

36. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are independently a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with - OC(O)-.

37. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are independently a C1-6 alkylene wherein one methylene unit of the C1-6 alkylene is replaced with - O-.

38. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are independently selected from -O-, -OC(O)-, -C(O)O-, -OC(O)O-, -CH2O-, -CH2OC(O)-, and - CH₂OC(O)O-.

39. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² andL³ are -OC(O)-

40. The compound of any one of claims 1-13, 15-20, or 29-33, wherein L² and L³ are -O-.

41. The compound of any one of claims 1-40, wherein R¹ is C1-6 alkyl.

42. The compound of any one of claims 1-40, wherein R¹ is methyl.

43. The compound of any one of claims 1-42, wherein R² and R³ are the same.

44. The compound of any one of claims 1-14 or 29-33, wherein X¹ is -N(H)-.

45. The compound of claim 29, wherein the compound is of formula PL-IIc or PL-IId:

PL-IId or a pharmaceutically acceptable salt thereof.

46. The compound of claim 29, wherein the compound is of formula PL-IIe:

PL-IIe or a pharmaceutically acceptable salt thereof.

47. The compound of claim 29, wherein the compound is of formula PL-IIf:

PL-IIf or a pharmaceutically acceptable salt thereof.

48. The compound of claim 29, wherein the compound is of formula PL-IIg or PL-IIh:

PL-IIh or a pharmaceutically acceptable salt thereof.

49. The compound of claim 29, wherein the compound is of formula PL-IIa or PL-IIb:

PL-IIa

PL-IIb or a pharmaceutically acceptable salt thereof.

50. The compound of claim 29, wherein the compound is of formula PL-IIk:

PL-IIk or a pharmaceutically acceptable salt thereof.

51. The compound of claim 29, wherein the compound is of formula PL-IIm or PL-IIn:

PL-IIn or a pharmaceutically acceptable salt thereof.

52. The compound of any one of claims 1-51, wherein R² and R³ are independently a straight or branched C6-30 alkyl substituted with m instances of R^x.

53. The compound of any one of claims 1-51, wherein R² and R³ are independently a straight or branched C6-25 alkyl substituted with m instances of R^x.

54. The compound of any one of claims 1-51, wherein R² and R³ are -(CH2)C6-2.5

55. The compound of any one of claims 1-51, wherein R² and R³ are -(CH2)10-25.

56. The compound of any one of claims 1-51, wherein R² and R³ are -(CH2)i0-i4.

57. The compound of any one of claims 1-51, wherein R² and R³ are -(CH2)14-16.

58. The compound of any one of claims 1-51, wherein R² and R³ are -(CH2)18-20.

59. The compound of any one of claims 1-51, wherein R² and R³ are independently a straight or branched C6-30 alkenyl substituted with m instances of R^x.

60. The compound of any one of claims 1-53 or 59, wherein m is 1.

61. The compound of any one of claims 1-53 or 59, wherein m is 0.

62. The compound of any one of claims 1-61, wherein n is an integer from 30-55, inclusive.

63. The compound of any one of claims 1-61, wherein n is an integer from 40-50, inclusive.

64. The compound of any one of claims 1-61, wherein n is 44, 45, or 46.

65. The compound of any one of claims 1-61, wherein n is 45.

66. A compound in Table 1, or a pharmaceutically acceptable salt thereof.

67. A lipid nanoparticle (LNP) comprising a compound of any one of claims 1-66, or a pharmaceutically acceptable salt thereof.

68. The LNP of claim 67, comprising:

(a) an ionizable lipid;

(b) a structural lipid; and

(c) a non-ionizable lipid and/or a zwitterionic lipid.

69. The LNP of claim 68, wherein the ionizable lipid comprises an ionizable amino lipid.

70. The LNP of claim 68, wherein the ionizable lipid is selected from 3-

(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl)oxy)-N,N-dimethylpropan-l-amine;

(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate;

(6Z,16Z)- 12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate;

(6Z,16Z)-12-((Z)- dec-4-en-l-yl)docosa-6,16-dien-l l-yl 6-(dimethylamino)hexanoate; N,N- dimethyl-4-(tris(((Z)- dec-4-en-l-yl)oxy)silyl)butan-l -amine; N,N-dimethyl-5-(tris(((Z)-dec-4- en- 1 - yl)oxy)silyl)pentan- 1 -amine; N,N-dimethyl-6-(tris(((Z)-dec-4-en- 1 -yl)oxy)silyl)hexan- 1 - amine; 2-(methyl(4-(tris(((Z)-dec-4-en-l-yl)oxy)silyl)butyl)amino)ethan-l-ol; (6Z,16Z)-12-((6- (dimethylamino)hexanoyl)oxy)docosa-6,16-dien-l 1-yl (Z)-undec-5-enoate; Nl,N3-bis(4- (bis(((Z)-dec-4-en-l-yl)oxy)(methyl)silyl)butyl)-Nl,N3-dimethylpropane-l,3-diamine; N1,N3- dimethyl-Nl,N3-bis(4-(tris(((Z)-hept-3-en-l-yl)oxy)silyl)butyl)propane-l,3-diamine; (lr,4r)- Nl,N4-bis(4-(bis(((Z)-dec-4-en-l-yl)oxy)(methyl)silyl)butyl)-Nl,N4-dimethylcyclohexane- 1,4- diamine; 2,8-bis(4-(bis(((Z)-dec-4-en-l-yl)oxy)(methyl)silyl)butyl)-2,8- diazaspiro[4.5]decane; or bis(2 -butyloctyl) 10-(N-(3-(dimethylamino)propyl) nonanamido)- nonadecanedioate; di(tridecan-7-yl) 10-(N-(3-(dimethylamino)propyl)octanamido)- nonadecanedioate; di (tridecan- 7-yl) 10-(N-decyl-4- (dimethylamino)butanamido)nonadecanedioate; ((4- hydroxybutyl)azanediyl)bis(nonane-9, 1- diyl) bis(2 -butyl octanoate); heptadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8- oxooctyl)amino)octanoate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- ((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-di enoate or 3,6- bi s(4-(bi s(2-hy droxy dodecyl)amino)butyl)piperazine-2, 5 -di one; 3 ,6-bi s(4-(bi s((9Z, 12Z)-2- hydroxyoctadeca-9,12-dien-l-yl)amino)butyl)piperazine-2, 5-dione; 1, l'-((2-(l-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidin-4- yl)ethyl)azanediyl)bis(dodecan-2-ol); tetratridecyl 3,3',3",3"'-((azanediylbis(propane-3, 1- diyl))bis(azanetriyl))tetrapropionate; nonyl 8-((8,8-bis(octyloxy)octyl)(2- hy droxy ethyl)amino)octanoate; or di((Z)-non-2-en-l-yl) 8,8'-((((2- (dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate.

71. The LNP of claim 68, wherein the ionizable lipid is selected from those compounds disclosed in Table 2B, Table (I), Table (II), Table (III), Table (IV), and Table (V).

72. The LNP of any one of claims 68-71, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

73. The LNP of any one of claims 68-72, wherein the non-ionizable lipid is a phospholipid selected from the group consisting of l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-diarachidonoyl-sn-gly cero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1,2-dioleoyl-sn-gly cero-3 - phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2-dilinoleoyl-sn-gly cero-3 - phosphoethanolamine, 1,2-dilinolenoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2- diarachidonoyl-sn-gly cero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-gly cero-3 - phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin.

74. The LNP of any one of claims 68-73, further comprising a targeting moiety.

75. The LNP of claim 74, wherein the targeting moiety is an antibody or a fragment thereof.

76. The LNP of any one of claims 68-75, further comprising a PEGylated lipid.

77. The LNP of claim 76, wherein the PEGylated lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a PEG- modified dialkylglycerol.

78. The LNP of any one of claims 67-77, further comprising an active agent.

79. The LNP of claim 78, wherein the active agent is a nucleic acid.

80. The LNP of claim 79, wherein the nucleic acid is a ribonucleic acid.

81. The LNP of claim 80, wherein the ribonucleic acid is at least one ribonucleic acid selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA).

82. The LNP of claim 79, wherein the nucleic acid is a messenger RNA (mRNA) or a circular RNA.

83. The LNP of claim 82, wherein the mRNA includes an open reading frame encoding a cancer antigen.

84. The LNP of claim 82, wherein the mRNA includes an open reading frame encoding an immune checkpoint modulator.

85. The LNP of any one of claims 82-84, wherein the mRNA includes at least one motif selected from the group consisting of a stem loop, a chain terminating nucleoside, a poly A sequence, a polyadenylation signal, and a 5' cap structure.

86. The LNP of claim 79, wherein the nucleic acid is suitable for a genome editing technique.

87. The LNP of claim 86, wherein the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nuclease (TALEN).

88. The LNP of claim 87, wherein the nucleic acid is at least one nucleic acid suitable for a genome editing technique selected from the group consisting of a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template.

89. The LNP of any one of claims 82-88, wherein the mRNA is at least 30 nucleotides in length.

90. The LNP of any one of claims 82-88, wherein the mRNA is at least 300 nucleotides in length.

91. A pharmaceutical composition comprising an LNP of any one of claims 67-90, and a pharmaceutically acceptable carrier.

92. The pharmaceutical composition of claim 91 , formulated for intravenous or intramuscular administration.

93. The pharmaceutical composition of claim 91, which is formulated for intravenous administration.

94. A method for delivering a nucleic acid to a cell comprising contacting the cell with an LNP of any one of claims 79-90.

95. A method for treating a disease characterized by a deficiency of a functional protein, the method comprising administering to a subject having the disease, an LNP formulation comprising an LNP of any one of claims 82-88, wherein the mRNA encodes the functional protein or a protein having the same biological activity as the functional protein.

96. A method for treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject having the disease an LNP formulation comprising an LNP of any one of claims 82-88, wherein the siRNA targets expression of the overexpressed polypeptide.