Compositions and methods for enhanced drug delivery

Abstract

The present disclosure features targeted cell delivery lipid nanoparticle (LNP) compositions that enable enhanced delivery of agents, e.g., nucleic acids such as therapeutic and / or prophylactic RNA, to target cells, particularly liver cells and / or splenocytes. The LNPs comprise an effective amount of a target cell delivery-enhancing lipid such that delivery of the agent by the targeted cell delivery LNPs is enhanced when compared to LNPs that do not contain the target cell delivery-enhancing agent. Methods of using the targeted cell delivery LNPs to deliver agents, e.g., deliver nucleic acids, for modulation of protein expression and target cell activity are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 884,133, filed August 7, 2019, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on July 21, 2020, is named M2180-7000WO_SL.txt and is 12,612 bytes in size. [Background technology]

[0003] Effective targeted delivery of biologically active agents, such as small molecule drugs, proteins, and nucleic acids, is an ongoing medical challenge. In particular, delivery of nucleic acids into cells is difficult due to the relative instability and poor cellular permeability of such species. Thus, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, into cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as vehicles for transporting bioactive substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. Such compositions generally contain lipids containing one or more of: (1) "cationic" and / or amino (ionic) lipids; (2) phospholipids and / or polyunsaturated lipids (helper lipids); (3) structured lipids (e.g., sterols); and / or (4) polyethylene glycol (PEG) lipids. Optimally, lipid nanoparticle compositions contain each of: 1) amino (ionic) lipids; 2) phospholipids; 3) structured lipids or blends thereof; 4) PEG lipids; and 5) a drug. Cationic and / or ionic lipids include, for example, amine-containing lipids that can be readily protonated. Although various lipid-containing nanoparticle compositions have been demonstrated, there remains a lack of effective delivery vehicles for reaching desired cell populations while maintaining safety and efficacy. Summary of the Invention [Means for solving the problem]

[0005] In some aspects, the use of targeted cell delivery LNPs enhances delivery to target cells in vitro, while in other aspects, delivery to target cells is enhanced in vivo. When administered in vivo, in one embodiment, the targeted cell delivery LNPs demonstrate enhanced drug delivery to the liver and spleen compared to a reference LNP. In some aspects, the LNPs are contacted with target cells, e.g., liver cells (e.g., hepatocytes) or splenocytes, in vitro. In some aspects, the LNPs are contacted with target cells in vivo by administering the LNPs to a subject, e.g., a human subject. In one embodiment, the subject is one who would benefit from modulating protein expression of a target protein, e.g., in target cells. In some aspects, the LNPs are administered intravenously. In some aspects, the LNPs are administered intramuscularly. In some aspects, the LNPs are administered by a route selected from the group consisting of subcutaneous, intranodal, and intratumoral.

[0006] In one embodiment, the agent may comprise or consist of a nucleic acid molecule. In some aspects, the nucleic acid molecule is selected from the group consisting of RNA, mRNA, RNAi, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA. In some aspects, the nucleic acid molecule is RNA selected from the group consisting of shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA or miR), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the nucleic acid molecule is an siRNA molecule. In some embodiments, the nucleic acid molecule is an miR. In some embodiments, the nucleic acid molecule is an antagomir. In some aspects, the nucleic acid molecule is DNA. In some aspects, the nucleic acid molecule is mRNA.

[0007] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid 1. A targeted cell delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, compared to a reference LNP. In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte; The present invention features targeted cell delivery lipid nanoparticles (LNPs) that provide one, two, or all of the following:

[0008] In certain embodiments, the targeted cell delivery LNP results in expression and / or activity of the payload in greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75% or more of the total hepatocytes.

[0009] In certain embodiments, the targeted cell delivery LNP results in payload expression and / or activity in approximately 30-75%, 40-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, or 30-40% of total hepatocytes, e.g., as measured by the assay of Example 6. In some embodiments, the targeted cell delivery LNPs result in payload expression and / or activity in about 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of total hepatocytes. In some embodiments, the targeted cell delivery LNPs result in payload expression and / or activity in about 60% of total hepatocytes.

[0010] In some embodiments, the target cell delivery LNPs provide enhanced payload levels (e.g., expression) in hepatocytes, e.g., hepatocytes, compared to reference LNPs. In some embodiments, the target cell delivery LNPs provide an increase in hepatocyte expression, e.g., hepatocyte expression, of about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold compared to reference LNPs. In some embodiments, the target cell delivery LNPs provide an increase in hepatocyte expression, e.g., hepatocyte expression, of about 3-fold compared to reference LNPs.

[0011] In certain embodiments, the target cell delivery LNP has increased cytosolic delivery efficiency, eg, when compared to a reference LNP, eg, as described herein.

[0012] In one embodiment, the target cell delivery LNP comprises: a) a maximum blood concentration (Cmax) in the liver that is greater than in the plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater than in the plasma, or more; b) a greater half-life (t) in the liver relative to plasma, e.g., a t in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 or more times greater than in plasma; or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in the plasma; Bring on one, two, or all of the above.

[0013] In certain embodiments, the target cell delivery LNP has an improved parameter in vivo compared to a reference LNP, wherein the improved parameter is one of the following: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slow lipid metabolism in the liver compared with the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lipids remaining in the liver 24 hours after administration; or any combination thereof.

[0014] In another aspect, the present invention provides a method of enhancing payload levels (e.g., payload expression) in a subject, comprising: administering to a subject a delivery lipid nanoparticle (LNP) as described herein in an amount sufficient to enhance payload levels in the subject; The present invention is characterized by a method comprising:

[0015] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0016] In one aspect, the invention features a method of enhancing payload levels (e.g., payload expression) in a subject, the method comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to a subject a targeted cell delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., increased payload distribution, delivery, and / or expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma, e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; It is administered in an amount sufficient to produce one, two, or all of the following:

[0017] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0018] In one aspect, the invention features a method of treating or ameliorating a symptom of a disorder or disease, e.g., an orphan disease, in a subject, the method comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to a subject a targeted cell delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., increased payload distribution, delivery, and / or expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma, e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; administered in an amount sufficient to produce one, two, or all of the following: Thereby, the disorder or disease is treated or its symptoms are ameliorated.

[0019] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0020] In any of the embodiments of the methods disclosed herein, the targeted cell delivery LNPs result in payload expression and / or activity in greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, or more of the total hepatocytes. In certain embodiments, the targeted cell delivery LNPs result in payload expression and / or activity in about 30-75%, 40-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, or 30-40% of the total hepatocytes, as measured, for example, by the assay of Example 6. In some embodiments, the targeted cell delivery LNPs result in payload expression and / or activity in about 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of total hepatocytes. In some embodiments, the targeted cell delivery LNPs result in payload expression and / or activity in about 60% of total hepatocytes.

[0021] In any embodiment of the methods disclosed herein, the target cell delivery LNPs result in enhanced payload levels (e.g., expression) in hepatocytes, e.g., hepatocytes, compared to a reference LNP. In some embodiments, the target cell delivery LNPs result in an increase in hepatocyte expression, e.g., hepatocyte expression, of about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold compared to a reference LNP. In some embodiments, the target cell delivery LNPs result in an increase in hepatocyte expression, e.g., hepatocyte expression, of about 3-fold compared to a reference LNP.

[0022] In any embodiment of the methods disclosed herein, the target cell delivery LNP has increased cytosolic delivery efficiency, e.g., when compared to a reference LNP, e.g., as described herein.

[0023] In embodiments of any of the methods disclosed herein, the target cell delivery LNP comprises: a) a maximum blood concentration (Cmax) in the liver that is greater than in the plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater than in the plasma, or more; b) A longer half-life in the liver compared to plasma (t 1 / 2 ), e.g., at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more t in the liver compared to plasma 1 / 2 ;or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in the plasma; It is administered in an amount that results in one, two, or all of the following:

[0024] In embodiments of any of the methods disclosed herein, the target cell delivery LNPs are administered in an amount that results in an improved parameter in vivo compared to a reference LNP, wherein the improved parameter is one of the following: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slow lipid metabolism in the liver compared with the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lipids remaining in the liver 24 hours after administration; or any combination thereof.

[0025] In some embodiments, the method further comprises simultaneously or sequentially administering a second LNP encapsulating the same or a different nucleic acid molecule, wherein the second LNP does not comprise a target cell delivery-enhancing lipid, e.g., comprises a different ionic lipid. In other embodiments, the method further comprises simultaneously or sequentially administering a second LNP encapsulating a different nucleic acid molecule, wherein the second LNP comprises a target cell delivery-enhancing lipid, e.g., comprises the same ionic lipid.

[0026] In one embodiment of the disclosed LNPs or methods, the enhanced delivery is compared to a reference LNP, e.g., an LNP comprising a different ionic lipid, e.g., as described herein. In another embodiment of the disclosed LNPs or methods, the enhanced delivery is compared to a suitable control.

[0027] In one embodiment of the LNPs or methods of the present disclosure, the agent stimulates protein expression in target cells, e.g., as described herein, e.g., hepatocytes or splenocytes. In another embodiment of the LNPs or methods of the present disclosure, the agent inhibits protein expression in target cells, e.g., as described herein, e.g., hepatocytes or splenocytes. In another embodiment of the LNPs or methods of the present disclosure, the agent encodes a soluble protein that regulates target cell activity, e.g., hepatocyte or splenocyte activity. In another embodiment of the LNPs or methods of the present disclosure, the agent encodes an intracellular protein that regulates target cell activity, e.g., hepatocyte or splenocyte activity. In another embodiment of the LNPs or methods of the present disclosure, the agent encodes a transmembrane protein that regulates target cell activity, e.g., hepatocyte or splenocyte activity. In another embodiment of the LNPs or methods of the present disclosure, the agent enhances target cell function, e.g., hepatocyte or splenocyte function. In another embodiment of the LNPs or methods of the present disclosure, the agent inhibits target cell function, e.g., hepatocyte or splenocyte function.

[0028] In one embodiment of the LNP or method of the present disclosure, the target cell is a liver cell, eg, a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a hepatic sinusoidal cell, or a combination thereof.

[0029] In one embodiment of the LNP or method of the disclosure, the target cell is a splenocyte, eg, a non-immune splenocyte (eg, a splenocyte).

[0030] In one embodiment of the LNP or method of the disclosure, the target cell is selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell.

[0031] In one embodiment of the LNP or method of the present disclosure, the target cell is a non-immune cell.

[0032] In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise a phytosterol or a combination of a phytosterol and cholesterol. In one embodiment, the phytosterol is selected from the group consisting of β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In one embodiment, the phytosterol is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In one embodiment, the phytosterol is selected from the group consisting of Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In one embodiment, the phytosterol is a combination of Compound S-141, Compound S-140, Compound S-143, and Compound S-148. In one embodiment, the phytosterol comprises sitosterol or a salt or ester thereof. In one embodiment, the phytosterol comprises stigmasterol or a salt or ester thereof. In one embodiment, the phytosterol comprises beta-sitosterol. [ka] or a salt or ester thereof.

[0033] In one embodiment of the LNP or method of the disclosure, the LNP comprises a phytosterol, or a salt or ester thereof, and cholesterol, or a salt thereof.

[0034] In some embodiments, the target cell is a cell described herein (e.g., a hepatocyte or a splenocyte), and the phytosterol, or salt or ester thereof, is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, and brassicasterol, and combinations thereof. In one embodiment, the phytosterol is β-sitosterol. In one embodiment, the phytosterol is β-sitostanol. In one embodiment, the phytosterol is campesterol. In one embodiment, the phytosterol is brassicasterol.

[0035] In some embodiments, the target cell is a cell described herein (e.g., a hepatocyte or a splenocyte), and the phytosterol, or salt or ester thereof, is selected from the group consisting of β-sitosterol and stigmasterol, and combinations thereof. In one embodiment, the phytosterol is β-sitosterol. In one embodiment, the phytosterol is stigmasterol.

[0036] In some embodiments of the LNPs or methods of the present disclosure, the LNPs comprise a sterol, or a salt or ester thereof, and cholesterol, or a salt thereof, wherein the target cell is a cell described herein (e.g., a hepatocyte or a splenocyte), and the sterol, or a salt or ester thereof, is selected from the group consisting of β-sitosterol-d7, brassicasterol, compound S-30, compound S-31, and compound S-32.

[0037] In one embodiment, the mol% of cholesterol is between about 1% and 50% of the mol% of phytosterols present in the lipid nanoparticles. In one embodiment, the mol% of cholesterol is between about 10% and 40% of the mol% of phytosterols present in the lipid nanoparticles. In one embodiment, the mol% of cholesterol is between about 20% and 30% of the mol% of phytosterols present in the lipid nanoparticles. In one embodiment, the mol% of cholesterol is about 30% of the mol% of phytosterols present in the lipid nanoparticles.

[0038] In one embodiment of the LNP or method of the disclosure, the ionizable lipid is selected from the group consisting of: (II), (IIIA), (IIIB), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIIf), (IIIg), (IIIh), (IIIj), (IIIk), (III), (III), (III), (III-a), (III), (III), (III-a), (III), (IIIa), (IIIb-1), (IIIb-2), (IIIb-3), (IIIb-4), (IIIb-5), (IIIc), (IIId), (III), (IIIa), (IIIb ... XI-b) and / or a compound selected from the group consisting of compound I-18, compound I-48, compound I-49, compound I-50, compound I-182, compound I-184, compound I-292, compound I-301, compound I-309, compound I-317, compound I-321, compound I-326, compound I-347, compound I-348, compound I-349, compound I-350, and compound I-352.

[0039] In one embodiment, the ionizable lipid comprises a compound selected from the group consisting of Compound X, Compound I-48, Compound I-49, Compound I-50, Compound I-182, Compound I-184, Compound I-292, Compound I-301, Compound I-309, Compound I-317, Compound I-321, Compound I-326, Compound I-347, Compound I-348, Compound I-349, Compound I-350, and Compound I-352. In one embodiment, the ionizable lipid comprises a compound selected from the group consisting of Compound I-182, Compound I-292, Compound I-301, Compound I-309, Compound I-317, Compound I-321, Compound I-326, Compound I-347, Compound I-348, Compound I-349, Compound I-350, and Compound I-352. In one embodiment, the ionizable lipid comprises a compound selected from the group consisting of Compound X, Compound I-48, Compound I-49, Compound I-50, and Compound I-184. In one embodiment, the ionizable lipid comprises a compound selected from the group consisting of Compound X, Compound I-49, Compound I-182, Compound I-184, Compound I-301, and Compound I-321. In one embodiment, the ionizable lipid comprises a compound selected from the group consisting of Compound I-301 and Compound I-49. In one embodiment, the ionizable lipid comprises Compound I-301. In one embodiment, the ionizable lipid comprises Compound I-49.

[0040] In some embodiments, the target cells are cells described herein and the ionizable lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49. In other embodiments, the target cells are hepatocytes or spleenocytes and the ionizable lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49.

[0041] In any of the foregoing or related aspects, the ionizable lipid of the LNPs of the present disclosure comprises at least one compound selected from the group consisting of compound I-301 and compound I-49. In one embodiment, the ionizable lipid comprises compound I-301. In one embodiment, the ionizable lipid comprises compound I-49.

[0042] In some embodiments, the ionizable lipid comprises an enantiomer, for example, the (R) enantiomer or (S) enantiomer of the amino lipid. In some embodiments, the ionizable lipid comprises a substantially pure enantiomer, for example, at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure enantiomer. In some embodiments, the ionizable lipid comprises a substantially pure enantiomer of the amino lipid, for example, at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure enantiomer. In some embodiments, the ionizable lipid comprises a substantially pure (R) enantiomer of the amino lipid, for example, at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure (R) enantiomer. In some embodiments, the ionizable lipid comprises a substantially pure (S) enantiomer of an amino lipid, e.g., at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure (S) enantiomer.

[0043] In one embodiment, the ionic lipid comprises a racemic mixture of amino lipids, e.g., a mixture comprising the (R) and (S) enantiomers of an amino lipid. In one embodiment, the racemic mixture comprises about 1-99%, 5-99%, 10-99%, 15-99%, 20-99%, 25-99%, 30-99%, 35-99%, 40-99%, 45-99%, 50-99%, 55-99%, 60-99%, 65-99%, 70-99%, 75-99%, 80-99%, 85-99%, 90-99%, 95-99%, 1-95%, 1-90%, 1-85%, 1-85%, 1-85%, 1-9 ... Contains 0%, 1-75%, 1-70%, 1-65%, 1-60%, 1-55%, 1-50%, 1-45%, 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-5%, 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99% of the (R) enantiomer. In one embodiment, the racemic mixture is about 1-99%, 5-99%, 10-99%, 15-99%, 20-99%, 25-99%, 30-99%, 35-99%, 40-99%, 45-99%, 50-99%, 55-99%, 60-99%, 65-99%, 70-99%, 75-99%, 80-99%, 85-99%, 90-99%, 95-99%, 100-99%, 110-115%, 120-125%, 130-135%, 140-145%, 150-155%, 160-165%, 170-175%, 180-185%, 190-195%, 200-200%, 210-215%, 220-225%, 230-235%, 240-245%, 250-265%, 260-275%, 270-285%, 280-295%, 290-300%, 300-315%, 310-325%, 320-335%, 330-345%, 340-355%, 350-365%, 360-375%, 370-385%, 380-395%, 390-400%, 410-425%, 420-435%, 430-445%, 440-450%, 450-465%, 460-475%, 470-485%, 480-495%, 490-500%, 500-515%, 510 Contains 0%, 1-75%, 1-70%, 1-65%, 1-60%, 1-55%, 1-50%, 1-45%, 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-5%, 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99% of the (S) enantiomer.

[0044] In one embodiment of the LNPs or methods of the present disclosure, the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DMPE, DOPC, and compound H-409. In one embodiment of the LNPs or methods of the present disclosure, the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DPPC, DMPE, DMPC, DOPC, compound H-409, compound H-418, compound H-420, compound H-421, and compound H-422. In one embodiment, the phospholipid is DSPC. In one embodiment of the LNPs or methods of the present disclosure, the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DPPC, DMPC, compound H-418, compound H-420, compound H-421, and compound H-422.

[0045] In one embodiment of the LNP or method of the disclosure, the target cell is a cell described herein, and the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DMPE, and the compound H-409. In one embodiment, the phospholipid is DSPC. In one embodiment, the phospholipid is DMPE. In one embodiment, the phospholipid is the compound H-409.

[0046] In one embodiment of the LNP or method of the present disclosure, the target cell is a cell described herein, and the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DOPC, DMPE, and the compound H-409. In one embodiment, the phospholipid is DSPC. In one embodiment, the phospholipid is DMPE. In one embodiment, the phospholipid is the compound H-409.

[0047] In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise a PEG-lipid. In one embodiment, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In one embodiment, the PEG-lipid is selected from the group consisting of Compound P415, Compound P-416, Compound P-417, Compound P-419, Compound P-420, Compound P-423, Compound P-424, Compound P-428, Compound P-L1, Compound P-L2, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L22, and Compound P-L23. In one embodiment, the PEG lipid is selected from the group consisting of Compound 428, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L1, and Compound P-L2. In one embodiment, the PEG lipid is selected from the group consisting of Compound P415, Compound P-416, Compound P-417, Compound P-419, Compound P-420, Compound P-423, Compound P-424, Compound P-428, Compound P-L1, Compound P-L2, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L22, and Compound P-L23. Compound P-415, Compound P-416, Compound P-417, Compound P-419, Compound P-420, Compound P-423, Compound P-424, Compound P-428, Compound P-L1, Compound P-L2, Compound P-L3, Compound P-L4, Compound P-L6, Compound P-L8, Compound P-L9, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L22, Compound P-L23, and Compound P-L25. In one embodiment, the PEG lipid is selected from the group consisting of Compound P-L3, Compound P-L4, Compound P-L6, Compound P-L8, Compound P-L9, and Compound P-L25.

[0048] In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 30 mol% to about 60 mol% ionic lipid, about 0 mol% to about 30 mol% non-cationic helper lipid or phospholipid, about 18.5 mol% to about 48.5 mol% sterol or other structured lipid, and about 0 mol% to about 10 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 35 mol% to about 55 mol% ionic lipid, about 5 mol% to about 25 mol% non-cationic helper lipid or phospholipid, about 30 mol% to about 40 mol% sterol or other structured lipid, and about 0 mol% to about 10 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% ionic lipid, about 10 mol% non-cationic helper lipid or phospholipid, about 38.5 mol% sterol or other structured lipid, and about 1.5 mol% PEG lipid. In one embodiment, the mol% of sterols or other structured lipids is 18.5% phytosterols and the total mol% of structured lipids is 38.5%. In one embodiment, the mol% of sterols or other structured lipids is 28.5% phytosterols and the total mol% of structured lipids is 38.5%.

[0049] In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 41 mol% to about 50 mol% ionic lipid and about 10 mol% to about 19 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% ionic lipid and about 10 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises 50 mol% ionic lipid and 10 mol% non-cationic helper lipid or phospholipid.

[0050] In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise about 50 mol% of compound I-301 and about 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise about 50 mol% of compound I-301 and about 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise about 50 mol% of compound I-301 and 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise 50 mol% of compound I-301 and 10 mol% of a non-cationic helper lipid or phospholipid.

[0051] In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 50 mol% of compound I-49 and about 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 50 mol% of compound I-49 and about 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 50 mol% of compound I-49 and 10 mol% of a non-cationic helper lipid or phospholipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise 50 mol% of compound I-49 and 10 mol% of a non-cationic helper lipid or phospholipid.

[0052] In one embodiment of the LNP or method of the disclosure, the LNP comprises: (i) about 50 mol % of an ionic lipid, which is a compound selected from the group consisting of compound I-301 and compound I-49; (ii) approximately 10 mol % phospholipid, which is DSPC; (iii) about 38.5 mol % structural lipids selected from β-sitosterol and cholesterol; and (iv) about 1.5 mol % PEG lipid, which is compound P-428; Includes:

[0053] In some aspects, the present disclosure provides targeted cell delivery lipid nanoparticles (LNPs) for use in methods of enhancing payload levels (e.g., payload expression) in a subject, the LNPs comprising: (i) sterols or other structural lipids; (ii) ionic lipids; and (iii) an agent for delivery to a target cell in a subject; wherein one or more of (i) a sterol or other structural lipid and / or (ii) an ionic lipid comprises a target cell delivery-enhancing lipid in an amount effective to enhance payload levels in a subject or enhance delivery of the LNP to a target cell subject.

[0054] In some embodiments, enhanced delivery is a property of the LNP compared to a reference LNP. In some embodiments, the reference LNP does not include a target cell delivery-enhancing lipid. In some embodiments, the reference LNP includes an ionic lipid having a formula I-XII.

[0055] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0056] In some aspects, the present disclosure provides targeted cell delivery lipid nanoparticles (LNPs) for use in methods of enhancing payload levels (e.g., payload expression) in a subject, the LNPs comprising: (i) sterols or other structural lipids; (ii) ionic lipids; and (iii) an agent for delivery to a target cell in a subject; wherein the sterol or other structural lipid comprises a target cell delivery-enhancing lipid in an amount effective to enhance payload levels in a subject or enhance delivery of the LNP to a target cell subject; Enhanced delivery is a property of the LNP compared to a reference LNP.

[0057] In some embodiments, the reference LNP does not include a target cell delivery-enhancing lipid. In some embodiments, the reference LNP includes an ionic lipid having a formula I-XII.

[0058] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0059] In some aspects, the present disclosure provides targeted cell delivery lipid nanoparticles (LNPs) for use in methods of enhancing payload levels (e.g., payload expression) in a subject, The LNP is (i) sterols or other structural lipids; (ii) ionic lipids; and (iii) an agent for delivery to a target cell in a subject; wherein the ionic lipid comprises a target cell delivery-enhancing lipid in an amount effective to enhance delivery of the LNP to a target cell (e.g., as described herein, e.g., a hepatocyte or a splenocyte); Enhanced delivery is a property of the LNP compared to a reference LNP.

[0060] In some embodiments, the reference LNP does not include a target cell delivery-enhancing lipid. In some embodiments, the reference LNP includes an ionic lipid having a formula I-XII.

[0061] In some embodiments, the target cell is a liver cell, e.g., a hepatocyte. In some embodiments, the target cell is a hepatocyte.

[0062] In any of the preceding or related embodiments, the sterol or other structured lipid is a phytosterol or cholesterol.

[0063] In any of the foregoing or related aspects, the target cell delivery-enhancing lipid is preferentially taken up by liver cells (e.g., hepatocytes), splenocytes, ovarian cells, lung cells, intestinal cells, cardiac cells, skin cells, eye cells, or brain cells, or skeletal muscle cells, relative to the reference LNP. In certain embodiments, the reference LNP does not comprise the target cell delivery-enhancing lipid and / or is not preferentially taken up by liver cells (e.g., hepatocytes), splenocytes, ovarian cells, lung cells, intestinal cells, cardiac cells, skin cells, eye cells, or brain cells, or skeletal muscle cells.

[0064] In any of the foregoing or related embodiments, the agent for delivery to a target cell described herein is a nucleic acid molecule. In some embodiments, the agent stimulates expression of a protein of interest in the target cell. In some embodiments, the agent for delivery to a target cell is a nucleic acid molecule encoding the protein of interest. In some embodiments, the agent for delivery to a target cell is an mRNA encoding the protein of interest.

[0065] In any of the foregoing or related embodiments, expression of the protein of interest in the target cell is enhanced relative to a reference LNP that does not include the target cell delivery-enhancing lipid. In some embodiments, the agent encodes a protein that modulates target cell activity.

[0066] In any of the foregoing or related aspects, the target cells are liver cells, e.g., hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof. In some aspects, the liver cells are hepatocytes. In some aspects, the hepatocytes are hepatic stellate cells. In some aspects, the hepatocytes are Kupffer cells. In some aspects, the hepatocytes are hepatic sinusoidal cells.

[0067] In any of the foregoing or related embodiments, the target cell is a splenocyte, eg, a non-immune splenocyte (eg, a splenocyte).

[0068] In any of the foregoing or related aspects, the target cell is selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell.

[0069] In any of the foregoing or related aspects, the target cell is not an immune cell.

[0070] In any of the foregoing or related embodiments, the targeted cell delivery lipid nanoparticle (LNP) further comprises (iv) a non-cationic helper lipid or phospholipid, and / or (v) a PEG-lipid.

[0071] In some embodiments, the target cell delivery lipid nanoparticles (LNPs) further comprise a non-cationic helper lipid or phospholipid. In some embodiments, the target cell delivery LNPs further comprise a PEG-lipid. In some embodiments, the target cell delivery lipid nanoparticles (LNPs) further comprise a non-cationic helper lipid or phospholipid and a PEG-lipid.

[0072] In some aspects, the present disclosure provides an in vitro method of delivering a drug to a target cell (e.g., as described herein, e.g., a liver cell, e.g., a hepatocyte), the method comprising contacting the target cell with a target cell delivery LNP comprising a target cell delivery-enhancing lipid. In some aspects of the in vitro method, the method results in activation or modulation of the activity of the target cell.

[0073] Additional features of any of the LNP compositions or methods of using the LNP compositions include one or more of the embodiments listed below. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the embodiments listed below.

[0074] Other embodiments of the present disclosure The present disclosure relates to the following embodiments. Throughout this section, the term embodiment will be abbreviated as "E" followed by an ordinal number. For example, E1 is equivalent to embodiment 1.

[0075] E1. In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid 1. A targeted cell delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; The present invention features targeted cell delivery lipid nanoparticles (LNPs) that provide one, two, or all of the following:

[0076] E2. The target cell delivery LNP of E1, wherein the target cell is a liver cell, e.g., a hepatocyte.

[0077] E3. The targeted cell delivery LNP of E1 or E2, which results in payload expression and / or activity in greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75% or more of total hepatocytes.

[0078] E4. The targeted cell delivery LNP of any one of the preceding embodiments, which results in payload expression and / or activity in about 30-75%, 40-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, or 30-40% of total hepatocytes, e.g., as measured by the assay of Example 6.

[0079] E5. The targeted cell delivery LNP of any one of the preceding embodiments, wherein the LNP results in payload expression and / or activity in approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of total hepatocytes.

[0080] E6. The targeted cell delivery LNP of any one of the preceding embodiments, which results in expression and / or activity of the payload in about 60% of total hepatocytes.

[0081] E7. The target cell delivery LNP of any one of the preceding embodiments, which results in enhanced payload levels (e.g., expression) in liver cells, e.g., hepatocytes, relative to a reference LNP.

[0082] E8. The target cell delivery LNP of any one of the preceding embodiments, which results in about a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold increase in hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP.

[0083] E9. The targeted cell delivery LNP of any one of the preceding embodiments, which results in a 1.5-6-fold, 1.5-5-fold, 1.5-4-fold, 1.5-3-fold, 1.5-2-fold, 2-6-fold, 3-6-fold, 4-6-fold, or 5-6-fold increase in hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP.

[0084] E10. The target cell delivery LNP of any one of the preceding embodiments, which results in about a three-fold increase in hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP.

[0085] E11. The target cell delivery LNP of any one of the preceding embodiments, having increased cytosolic delivery efficiency, e.g., when compared to a reference LNP, e.g., as described herein.

[0086] E12.a) a maximum blood concentration (Cmax) in the liver that is greater than in plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater or greater than in plasma; b) A longer half-life in the liver compared to plasma (t 1 / 2 ), e.g., at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more t in the liver compared to plasma 1 / 2 ;or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in the plasma; The target cell delivery LNP of any one of the preceding embodiments, wherein the LNP provides one, two, or all of the following:

[0087] E13. Having improved parameters in vivo compared to a reference LNP, wherein said improved parameters are: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced payload expression and / or activity in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75% or more of total hepatocytes; or 8) enhanced endosomal escape;

[0023] The target cell delivery LNP of any one of the preceding embodiments, wherein the target cell delivery LNP is selected from one, two, three, four, five, six, seven, or more (e.g., all), or any combination thereof.

[0088] E14.9) Increased response rates, e.g., as defined by a specific threshold of hepatocyte transfection; 10) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more hepatocyte transfection; 11) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 12) an increased response rate greater than the reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater response rate; The target cell delivery LNP of any one of the preceding embodiments, wherein the LNP provides one, two, or all of the following:

[0089] E15. The targeted cell delivery LNP of any one of the preceding embodiments, formulated for systemic delivery.

[0090] E16. The target cell delivery LNP of any one of the preceding embodiments, which is administered systemically, e.g., parenterally (e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally), or enterally (e.g., orally, rectally, or sublingually).

[0091] E17. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said payload is delivered to cells capable of synthesizing proteins and / or cells with high engulfment capacity.

[0092] E18. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said payload is delivered to liver cells, e.g., hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof.

[0093] E19. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said LNP delivers said payload to hepatocytes.

[0094] E20. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said LNP delivers said payload to a non-immune cell.

[0095] E21. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said payload is delivered to a splenocyte, e.g., a non-immune splenocyte (e.g., a splenocyte).

[0096] E22. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said payload is delivered to a cell selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell.

[0097] E23. The target cell delivery LNP of any one of the preceding embodiments, wherein the intracellular concentration of said nucleic acid molecule in said target cell is enhanced.

[0098] E24. The target cell delivery LNP of any one of the preceding embodiments, wherein uptake of said nucleic acid molecule by said target cell is enhanced.

[0099] E25. The target cell delivery LNP of any one of the preceding embodiments, wherein the activity of said nucleic acid molecule in said target cell is enhanced.

[0100] E26. The target cell delivery LNP of any one of the preceding embodiments, wherein expression of said nucleic acid molecule in said target cell is enhanced.

[0101] E27. The target cell delivery LNP of any one of the preceding embodiments, wherein the activity of a protein encoded by said nucleic acid molecule in said target cell is enhanced.

[0102] E28. The target cell delivery LNP of any one of the preceding embodiments, wherein expression of a protein encoded by said nucleic acid molecule in said target cell is enhanced.

[0103] E29. The targeted cell delivery LNP of any one of the preceding embodiments, wherein delivery is enhanced in vivo.

[0104] E30. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is a peptide, polypeptide, protein, or nucleic acid.

[0105] E31. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA.

[0106] E32. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is selected from a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), or a combination thereof.

[0107] E33. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is mRNA, siRNA, miR, or CRISPR.

[0108] E34. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is mRNA.

[0109] E35. The targeted cell delivery LNP of any one of the preceding embodiments, wherein said payload is an mRNA encoding a protein of interest other than an immune cell payload.

[0110] E36. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is selected from an mRNA encoding a secreted protein, a membrane-bound protein, an intracellular protein, an antibody molecule, or an enzyme.

[0111] E37. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is an mRNA encoding an antibody molecule.

[0112] E38. The target cell delivery LNP of any one of the preceding embodiments, wherein said payload is an mRNA encoding an enzyme.

[0113] E39. The targeted cell delivery LNP of E38, wherein said enzyme is associated with an orphan disease (e.g., a lysosomal storage disease).

[0114] E40. The targeted cell delivery LNP of E38, wherein said enzyme is associated with a metabolic disorder (e.g., as described herein).

[0115] E41. The targeted cell delivery LNP of E38 or E39, wherein said payload is an mRNA encoding a urea cycle enzyme.

[0116] E42. The target cell delivery LNP of any one of the preceding embodiments, which can be administered at a lower dose compared to a reference LNP, e.g., as described herein.

[0117] E43. The target cell delivery LNP of E42, administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to the dose of the reference LNP.

[0118] E44. The targeted cell delivery LNP of E42 or E43, wherein said targeted cell delivery LNP delivered at a lower dose results in similar or enhanced lipid and / or payload levels in the target cell, organ, or cellular compartment.

[0119] E45. The target cell delivery LNP of any one of the preceding embodiments, which can be administered at a reduced frequency compared to a reference LNP, e.g., as described herein.

[0120] E46. The targeted cell delivery LNP of E45, wherein the number of administrations of said targeted cell delivery LNP is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the number of administrations of a reference LNP.

[0121] E47. The targeted cell delivery LNP of E45 or E46, wherein said targeted cell delivery LNP delivered in fewer doses results in similar or enhanced lipid and / or payload levels in the target cell, organ, or cellular compartment.

[0122] E48. In some embodiments, the present invention provides a method for enhancing payload levels (e.g., payload expression) in a subject, comprising: administering to the subject the delivery lipid nanoparticle (LNP) of any one of E1 to E47 in an amount sufficient to enhance payload levels in the subject. The method is characterized in that it comprises:

[0123] E49. In some embodiments, the present invention provides a method for enhancing payload levels (e.g., payload expression) in a subject, comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to the subject a delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; administered in an amount sufficient to produce one, two, or all of the following: The method is characterized by the above.

[0124] E50. In some embodiments, the present invention provides a method of treating or ameliorating a symptom of a disorder or disease, e.g., an orphan disease, in a subject, comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to the subject a delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; and administering the compound in an amount sufficient to produce one, two, or all of the following effects, thereby treating the disorder or disease or ameliorating its symptoms. The method is characterized by the above.

[0125] The method of E49 or E50, wherein the target cells are liver cells, such as hepatocytes. In one embodiment, the target cells are hepatocytes.

[0126] E52. The method of any one of E49-E51, wherein the target cell delivery LNP results in payload expression and / or activity in greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75% or more of total hepatocytes.

[0127] E53. The method of any one of E49-E52, wherein the target cell delivery LNP results in payload expression and / or activity in about 30-75%, 40-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, or 30-40% of total hepatocytes, e.g., as measured by the assay of Example 6.

[0128] E54. The method of any one of E49-E53, wherein the target cell delivery LNP results in payload expression and / or activity in approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of total hepatocytes.

[0129] E55. The method of any one of E49-E54, wherein said targeted cell delivery LNP results in payload expression and / or activity in about 60% of total hepatocytes.

[0130] E56. The method of any one of E49-E55, wherein said target cell delivery LNP provides enhanced payload levels (e.g., expression) in liver cells, e.g., hepatocytes, relative to a reference LNP.

[0131] E57. The method of any one of E49-E56, wherein the target cell delivery LNP results in about a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold increase in hepatocyte expression, e.g., hepatocyte expression, compared to a reference LNP.

[0132] E58. The method of any one of E49-E57, wherein said target cell delivery LNP results in about a three-fold increase in hepatocyte expression, eg, hepatocyte expression, compared to a reference LNP.

[0133] E59. The method of any one of E49-E54, wherein the target cell delivery LNP has increased cytosolic delivery efficiency, e.g., when compared to a reference LNP, e.g., as described herein.

[0134] E60. The target cell delivery LNP is as follows: a) a maximum blood concentration (Cmax) in the liver that is greater than in the plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater than in the plasma, or more; b) A longer half-life in the liver compared to plasma (t 1 / 2 ), e.g., at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more t in the liver compared to plasma 1 / 2 ;or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold greater than in the plasma, or more; The method of any one of E49-E59, wherein the compound is administered in an amount that results in one, two, or all of the following:

[0135] E61. The target cell delivery LNP is administered in an amount that results in an improved parameter in vivo compared to a reference LNP, wherein the improved parameter is one of the following: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced payload expression and / or activity in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75% or more of total hepatocytes; or 8) enhanced endosomal escape; selected from one, two, three, four, five, six, seven, or more (e.g., all), or any combination of The method according to any one of E49 to E60.

[0136] E62. The target cell delivery LNP is: 1) increased response rates, e.g., as defined by a specific threshold of hepatocyte transfection; 2) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more hepatocyte transfection; 3) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 4) an increased response rate greater than the reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater response rate; The method of any one of E49 to E61, wherein the compound is administered in an amount that results in one, two, or all of the following:

[0137] E63. The method of any one of E49-E62, wherein said targeted cell delivery LNP is formulated for systemic delivery.

[0138] E64. The method of any one of E49-E63, wherein the target cell delivery LNP is administered systemically, e.g., parenterally (e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally) or enterally (e.g., orally, rectally, or sublingually).

[0139] E65. The method of any one of E49-64, wherein the target cell delivery LNP delivers the payload to cells capable of synthesizing proteins and / or cells with high engulfment capacity.

[0140] E66. The method of any one of E49-E65, wherein the target cell delivery LNP delivers the payload to liver cells, such as hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof.

[0141] E67. The method of any one of E49-E66, wherein said target cell delivery LNP delivers said payload to hepatocytes.

[0142] E68. The method of any one of E49-E67, wherein the target cell delivery LNP delivers the payload to a splenocyte, e.g., a non-immune splenocyte (e.g., a splenocyte).

[0143] E69. The method of any one of E49-E68, wherein said targeted cell delivery LNP delivers said payload to a cell selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell.

[0144] E70. The method of any one of E49-E69, wherein said targeted cell delivery LNP delivers said payload to a non-immune cell.

[0145] E71. The method of any one of E49 to E69, wherein the intracellular concentration of said nucleic acid molecule in said target cell is enhanced.

[0146] E72. The method of any one of E49-E71, wherein uptake of said nucleic acid molecule by said target cells is enhanced.

[0147] E73. The method of any one of E49 to E72, wherein the activity of said nucleic acid molecule in said target cell is enhanced.

[0148] E74. The method of any one of E49 to E73, wherein expression of said nucleic acid molecule in said target cell is enhanced.

[0149] E75. The method of any one of E49-E74, wherein the activity of the protein encoded by said nucleic acid molecule in said target cell is enhanced.

[0150] E76. The method of any one of E49 to E75, wherein expression of the protein encoded by said nucleic acid molecule in said target cell is enhanced.

[0151] E77. The method of any one of E49-E76, wherein delivery is enhanced in vivo.

[0152] E78. The method of any one of E49 to E76, wherein the payload is a peptide, polypeptide, protein, or nucleic acid.

[0153] E79. The method of any one of E49 to E78, wherein the payload is a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA.

[0154] E80. The method of any one of E49 to E79, wherein the payload is selected from a shortmer, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), or a combination thereof.

[0155] E81. The method of any one of E49 to E80, wherein the payload is mRNA, siRNA, miR, or CRISPR.

[0156] E82. The method of any one of E49 to E81, wherein said payload is an mRNA encoding a protein of interest other than an immune cell payload.

[0157] E83. The method of any one of E49 to E82, wherein said payload is selected from an mRNA encoding a secreted protein, a membrane-bound protein, an intracellular protein, or an enzyme.

[0158] E84. The method of any one of E49 to E83, wherein the payload is an mRNA encoding an antibody molecule.

[0159] E85. The method of any one of E49 to E84, wherein the payload is an mRNA encoding an enzyme.

[0160] E86. The method of any one of E49-E85, wherein the enzyme is associated with an orphan disease (e.g., a lysosomal storage disease) or a metabolic disorder (e.g., as described herein).

[0161] E87. The method of E86, wherein said payload is an mRNA encoding a urea cycle enzyme.

[0162] E88. The method of E86, wherein said disease is a metabolic disorder.

[0163] E89. The method of any one of E49-E88, wherein said target cell delivery LNP can be administered at a lower dose compared to a reference LNP, eg, as described herein.

[0164] E90. The method of any one of E49-E89, wherein said target cell delivery LNP is administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to the dose of a reference LNP.

[0165] E91. The method of E90, wherein said target cell delivery LNP delivered at a lower dose results in similar or enhanced lipid and / or payload levels in the target cell, organ, or cellular compartment.

[0166] E92. The method of E90 or E91, wherein said target cell delivery LNP can be administered at a reduced frequency compared to a reference LNP, eg, as described herein.

[0167] E93. The method of E92, wherein the number of administrations of said target cell delivery LNP is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the number of administrations of a reference LNP.

[0168] E94. The method of E92 or E93, wherein said targeted cell delivery LNP delivered less frequently results in similar or enhanced lipid and / or payload levels in the targeted cell, organ, or cellular compartment.

[0169] E95. The targeted cell delivery LNP or method of any preceding embodiment, wherein said ionic lipid comprises an amino lipid.

[0170] E96. The target cell delivery LNP or method of any of the preceding embodiments, wherein the ionizable lipid comprises a compound of any of formulas (I VI), (I VI-a), (I VII), (I VIII), (I VIIa), (I VIIIa), (I VIIIb), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIIIc), or (I VIIId).

[0171] E97. The target cell delivery LNP or method of any preceding embodiment, wherein said ionizable lipid comprises an amino lipid having a squaramide head group.

[0172] E98. The target cell delivery LNP or method of any preceding embodiment, wherein the ionizable lipid comprises a compound selected from the group consisting of Compound I-301, Compound (R)I-301, Compound (S)I-301, Compound I-49, Compound (R)I-49, Compound (S)I-49, Compound I-292, Compound I-309, Compound I-317, Compound I-326, Compound I-347, Compound I-348, Compound I-349, Compound I-350, and Compound I-352.

[0173] E99. The target cell delivery LNP or method of any preceding embodiment, wherein said ionizable lipid comprises a compound selected from compound I-301 and compound I-49.

[0174] E100. The target cell delivery LNP or method of any preceding embodiment, wherein said ionic lipid comprises compound I-301.

[0175] E101. The target cell delivery LNP or method of any of E1-E99, wherein said ionic lipid comprises compound I-49.

[0176] E102. The targeted cell delivery LNP or method of any of E1-E99, wherein the cell is a liver cell, e.g., a hepatocyte, and the ionic lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49.

[0177] E103. The target cell delivery LNP or method of any of E1-E99, wherein the cell is a splenocyte, e.g., a spleen parenchymal cell, and the ionic lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49.

[0178] E104. The target cell delivery LNP or method of any preceding embodiment, wherein the ionizable lipid comprises a racemic mixture of the amino lipid, e.g., a mixture comprising the (R) enantiomer and the (S) enantiomer of the amino lipid.

[0179] E105. The target cell delivery LNP or method of any preceding embodiment, wherein said ionizable lipid comprises an enantiomer, e.g., the (R) enantiomer or the (S) enantiomer of an amino lipid.

[0180] E106. The target cell delivery LNP or method of E105, wherein said ionic lipid comprises a substantially pure (R) enantiomer of said amino lipid, e.g., at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure enantiomer.

[0181] E107. The target cell delivery LNP or method of E105, wherein said ionizable lipid comprises a substantially pure (S) enantiomer of said amino lipid, e.g., at least 80%, 90%, 95%, 95%, 97%, 98%, 99%, or 100% pure enantiomer.

[0182] E108. The target cell delivery LNP or method of any preceding embodiment, wherein said reference LNP comprises an ionizable lipid having formula I-XII.

[0183] E109. The target cell delivery LNP or method of E108, wherein said reference LNP does not contain an ionic lipid having a chiral center.

[0184] E110. The target cell delivery LNP or method of E108, wherein said reference LNP does not contain an ionic lipid comprising multiple branched alkyl chains.

[0185] E111. The target cell delivery LNP or method of E108, wherein said reference LNP does not comprise a ring-substituted amino lipid.

[0186] E112. The target cell delivery LNP or method of E108, wherein said reference LNP does not comprise a carbocyclic-substituted ionic lipid.

[0187] E113. The target cell delivery LNP or method of E108, wherein said reference LNP does not comprise a cycloalkenyl-substituted amino lipid.

[0188] E114. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises an amino lipid having a chiral center.

[0189] E115. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises an ionizable lipid comprising multiple branched alkyl chains.

[0190] E116. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises a ring-substituted amino lipid.

[0191] E117. The targeted cell delivery LNP or method of any of E1-E114 or E116, wherein said targeted cell delivery LNP comprises a carbocyclic-substituted amino lipid.

[0192] E118. The target cell delivery LNP or method of any of E1-E114 or E116-E117, wherein said target cell delivery LNP comprises a cycloalkenyl-substituted amino lipid.

[0193] E119. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises a cyclobutenyl-substituted amino lipid.

[0194] E120. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises a cyclobutene-1,2-dione substituted amino lipid.

[0195] E121. The target cell delivery LNP or method of any preceding embodiment, wherein said target cell delivery LNP comprises a squaramide-substituted amino lipid, e.g., an amino lipid comprising a squaramide group.

[0196] E122. The target cell delivery LNP or method of any preceding embodiment, wherein said non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DPPC, DMPC, DMPE, DOPC, Compound H-409, Compound H-418, Compound H-420, Compound H-421, and Compound H-422.

[0197] E123. The target cell delivery LNP or method of E122, wherein the cell is a liver cell, e.g., a hepatocyte, and the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DMPE, and compound H-409.

[0198] E124. The target cell delivery LNP or method of E122, wherein said phospholipid is DSPC.

[0199] E125. The target cell delivery LNP or method of E122, wherein said phospholipid is DMPE.

[0200] E126. The target cell delivery LNP or method of E122, wherein said phospholipid is compound H-409.

[0201] E127. The target cell delivery LNP or method of any preceding embodiment, comprising a PEG lipid.

[0202] E128. The target cell delivery LNP or method of E127, wherein said PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0203] E129. The target cell delivery LNP or method of E127, wherein said PEG lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE lipids.

[0204] E130. The targeted cell delivery LNP or method of E127, wherein said PEG lipid is PEG-DMG.

[0205] E131. The target cell delivery LNP or method of any of E127-E130, wherein the PEG-lipid comprises a compound selected from the group consisting of Compound P-415, Compound P-416, Compound P-417, Compound P-419, Compound P-420, Compound P-423, Compound P-424, Compound P-428, Compound P-L1, Compound P-L2, Compound P-L3, Compound P-L4, Compound P-L6, Compound P-L8, Compound P-L9, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L22, Compound P-L23, and Compound P-L25.

[0206] E132. The target cell delivery LNP or method of any of E127-E130, wherein the PEG-lipid comprises a compound selected from the group consisting of compound P-428, PL-16, compound PL-17, compound PL-18, compound PL-19, compound PL-1, and compound PL-2.

[0207] E133. The target cell delivery LNP or method of any preceding embodiment, wherein said LNP comprises a molar ratio of (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid of about 50:10, 49:11, 48:12, 47:13, 46:14, 45:15, 44:16, 43:17, 42:18, or 41:19.

[0208] E134. The target cell delivery LNP or method of any preceding embodiment, wherein said LNP comprises about 41 mol% to about 50 mol% ionic lipid, and about 10 mol% to about 19 mol% non-cationic helper lipid or phospholipid.

[0209] E135. The target cell delivery LNP or method of any preceding embodiment, wherein said LNP comprises about 50 mol % ionic lipids and about 10 mol % non-cationic helper lipids or phospholipids.

[0210] E136. The target cell delivery LNP or method of any preceding embodiment, wherein the molar ratio of said (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid is about 50:10.

[0211] E137. The target cell delivery LNP or method of any preceding embodiment, comprising about 30 mol% to about 60 mol% ionic lipid, about 0 mol% to about 30 mol% non-cationic helper lipid or phospholipid, about 18.5 mol% to about 48.5 mol% sterol or other structured lipid, and about 0 mol% to about 10 mol% PEG lipid.

[0212] E138. The target cell delivery LNP or method of any preceding embodiment, comprising about 35 mol% to about 55 mol% ionic lipid, about 5 mol% to about 25 mol% non-cationic helper lipid or phospholipid, about 30 mol% to about 40 mol% sterol or other structured lipid, and about 0 mol% to about 10 mol% PEG lipid.

[0213] E139. The target cell delivery LNP or method of any preceding embodiment, comprising about 50 mol % ionic lipid, about 10 mol % non-cationic helper lipid or phospholipid, about 38.5 mol % sterol or other structured lipid, and about 1.5 mol % PEG lipid.

[0214] E140. The target cell delivery LNP or method of any preceding embodiment, wherein the mol % of said sterol or other structural lipid is 18.5% phytosterol and the total mol % of said structural lipid is 38.5%.

[0215] E141. The target cell delivery LNP or method of any preceding embodiment, wherein the mol % of said sterol or other structural lipid is 28.5% phytosterol and the total mol % of said structural lipid is 38.5%.

[0216] E142. The targeted cell delivery LNP or method of any preceding embodiment, wherein the lipid nanoparticle comprises compound I-301 as the ionic lipid, DSPC as the phospholipid, cholesterol or a cholesterol / β-sitosterol blend as the structural lipid, and compound 428 as the PEG lipid.

[0217] E143. The target cell delivery LNP or method of any preceding embodiment, wherein the ionic lipids:phospholipids:structural lipids:PEG lipids are in a ratio selected from: (i) 50:10:38:2; (ii) 50:20:28:2; (iii) 40:20:38:2; or (iv) 40:30:28:2.

[0218] E144. The target cell delivery LNP or method of E143, wherein said structural lipid is entirely cholesterol, 38% or 28%.

[0219] E145. The target cell delivery LNP or method of E143, wherein the structural lipid is cholesterol / β-sitosterol in a total percentage of 38% or 28%, and the blend comprises: (i) 20% cholesterol and 18% β-sitosterol; (ii) 10% cholesterol and 18% β-sitosterol, or (iii) 10% cholesterol and 28% β-sitosterol.

[0220] E146. The LNP is: i) about 50 mol % of an ionic lipid, which is a compound selected from the group consisting of compound I-301, compound I-321, compound I-182, or compound I-49; (ii) approximately 10 mol % phospholipid, which is DSPC; (iii) about 38.5 mol % structural lipids selected from β-sitosterol and cholesterol; and (iv) about 1.5 mol % PEG lipid, which is compound P-428; The target cell delivery LNP or method according to E143 to E145, comprising:

[0221] E147. A pharmaceutical composition comprising the delivery lipid nanoparticles of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0222] E148. A GMP-grade pharmaceutical composition comprising the delivery lipid nanoparticles of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0223] E149. Any of the pharmaceutical compositions of E147 or E148 having a purity of 95%, 96%, 97%, 98%, or greater than 99%, e.g., at least 1%, 2%, 3%, 4%, 5% or more of contaminants removed.

[0224] E150. The pharmaceutical composition according to E147 to E149, which is large scale, for example, at least 20 g, 30 g, 40 g, 50 g, 100 g, 200 g, 300 g, 400 g, or more. In an embodiment of the present invention, for example, the following items are provided: (Item 1) (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid 1. A targeted cell delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; Targeted cell delivery lipid nanoparticles (LNPs) that provide one, two, or all of the following: (Item 2) 2. The delivery LNP of item 1, wherein the target cells are liver cells, e.g., hepatocytes. (Item 3) 3. The delivery LNP of item 1 or 2, which results in expression and / or activity of the payload in greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75% or more of total hepatocytes. (Item 4) 4. The delivery LNP of item 3, which results in expression and / or activity of the payload in approximately 60% of total hepatocytes. (Item 5) The delivery LNP of any preceding item, which results in enhanced payload levels (e.g., expression) in liver cells, e.g., hepatocytes, relative to a reference LNP. (Item 6) The delivery LNP of any of the preceding items, which results in about a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold increase in hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP. (Item 7) For example, a delivery LNP of any of the preceding items that has increased cytosolic delivery efficiency when compared to a reference LNP, e.g., as described herein. (Item 8) a) a maximum blood concentration (Cmax) in the liver that is greater than in the plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater than in the plasma, or more; b) a greater half-life (t) in the liver relative to plasma, e.g., a t in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 or more times greater than in plasma; or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in the plasma; The delivered LNP of any of the preceding items, resulting in one, two, or all of: (Item 9) have improved parameters in vivo compared to a reference LNP, wherein said improved parameters are: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slow lipid metabolism in the liver compared with the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lipids remaining in the liver 24 hours after administration; The delivery LNP of any preceding item, selected from one, two, three, four, five, six, seven, or more (e.g., all), or any combination thereof. (Item 10) 13) increased response rates, e.g., as defined by a specific threshold of hepatocyte transfection; 14) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more hepatocyte transfection; 15) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 16) An increased response rate greater than the reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater response rate; The delivery LNP of any one of the preceding items, which results in one, two, three, or all of: (Item 11) The delivery LNP of any one of the preceding items, formulated for systemic delivery. (Item 12) The delivered LNP of any one of the preceding items, which is administered systemically, e.g., parenterally (e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally) or enterally (e.g., orally, rectally, or sublingually). (Item 13) The delivery LNP of any one of the preceding items, which delivers the payload to cells capable of synthesizing proteins and / or cells with high engulfment capacity. (Item 14) The delivery LNP of any one of the preceding items, wherein the payload is delivered to liver cells, such as hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof. (Item 15) The delivery LNP of any one of the preceding items, which delivers the payload to hepatocytes. (Item 16) The delivery LNP of any one of the preceding items, which delivers the payload to a non-immune cell. (Item 17) The delivery LNP of any one of the preceding items, which delivers the payload to a splenocyte, e.g., a non-immune splenocyte (e.g., a splenocyte). (Item 18) The delivery LNP of any one of the preceding items, wherein the delivery LNP delivers the payload to a cell selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell. (Item 19) The delivery LNP of any one of the preceding items, wherein the intracellular concentration of the nucleic acid molecule in the target cell is enhanced. (Item 20) The delivery LNP of any one of the preceding items, wherein uptake of the nucleic acid molecule by the target cell is enhanced. (Item 21) The delivery LNP of any one of the preceding items, wherein the activity of the nucleic acid molecule in the target cell is enhanced. (Item 22) The delivery LNP of any one of the preceding items, wherein expression of the nucleic acid molecule in the target cell is enhanced. (Item 23) The delivery LNP of any one of the preceding items, wherein the activity of a protein encoded by the nucleic acid molecule in the target cell is enhanced. (Item 24) The delivery LNP of any one of the preceding items, wherein expression of a protein encoded by the nucleic acid molecule in the target cell is enhanced. (Item 25) The delivery LNP of any one of the preceding items, wherein delivery is enhanced in vivo. (Item 26) The delivery LNP of any one of the preceding items, wherein the payload is a peptide, polypeptide, protein, or nucleic acid. (Item 27) The delivery LNP of any one of the preceding items, wherein the payload is a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA. (Item 28) The delivery LNP of any one of the preceding items, wherein the payload is selected from a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a dicer substrate RNA (dsRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), or a combination thereof. (Item 29) The delivery LNP of any one of the preceding items, wherein the payload is mRNA, siRNA, miR, or CRISPR. (Item 30) The delivery LNP of any one of the preceding items, wherein the payload is mRNA. (Item 31) The delivery LNP of any one of the preceding items, wherein the payload is an mRNA encoding a protein of interest other than an immune cell payload. (Item 32) The delivery LNP of any one of the preceding items, wherein the payload is selected from an mRNA encoding a secreted protein, a membrane-bound protein, an intracellular protein, an antibody molecule, or an enzyme. (Item 33) The delivery LNP of any one of the preceding items, wherein the payload is an mRNA encoding an antibody molecule. (Item 34) The delivery LNP of any one of the preceding items, wherein the payload is an mRNA encoding an enzyme. (Item 35) 35. The delivery LNP of item 34, wherein the enzyme is associated with an orphan disease (e.g., a lysosomal storage disease). (Item 36) 35. The delivery LNP of item 34, wherein the enzyme is associated with a metabolic disorder (e.g., as described herein). (Item 37) 35. The delivery LNP of item 34, wherein the payload is an mRNA encoding a urea cycle enzyme. (Item 38) The delivery LNP of any one of the preceding items, which can be administered at a lower dose compared to a reference LNP, e.g., as described herein. (Item 39) 39. The delivered LNP of item 38, administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to the dose of a reference LNP. (Item 40) 1. A method of enhancing payload levels (e.g., expression of a payload) in a subject, comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to the subject a delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) administered in an amount sufficient to result in one, two, or all of enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, compared to a reference LNP. (Item 41) 1. A method of treating or ameliorating a symptom of a disorder or disease, e.g., an orphan disease, in a subject, comprising: (i) ionic lipids, e.g., amino lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) the payload; and (v) optionally, a PEG lipid administering to the subject a delivery lipid nanoparticle (LNP) comprising: (a) enhanced payload levels (e.g., expression) in a target cell, organ, cellular compartment, or body fluid compartment, e.g., liver or plasma (e.g., increased payload distribution, delivery, and / or expression), e.g., enhanced payload levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (b) enhanced lipid levels (e.g., increased lipid distribution, delivery, or exposure) in a target cell, organ, cellular compartment, or fluid compartment, e.g., liver or plasma, e.g., enhanced lipid levels compared to a different target cell, organ, or cellular compartment, or compared to a reference LNP; (c) expression and / or activity of the payload in greater than 30%, 40%, 50%, 60%, 65%, 70%, 75%, or more of the total hepatocytes, e.g., in about 60% of the total hepatocytes; or (d) enhanced payload levels (e.g., expression) and / or lipid levels, e.g., about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold (e.g., about 3-fold) hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; and administering to said subject a therapeutic agent in an amount sufficient to produce one, two, or all of the following: method. (Item 42) The target cell delivery LNP is: a) a maximum blood concentration (Cmax) in the liver that is greater than in the plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater than in the plasma, or more; b) A longer half-life in the liver compared to plasma (t 1 / 2 ), e.g., at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more t in the liver compared to plasma 1 / 2 ;or c) an extrapolated area (%) under the concentration-time curve in the liver that is greater than in the plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in the plasma; 42. The method of claim 40 or 41, wherein the compound is administered in an amount that results in one, two, or all of the following: (Item 43) The target cell delivery LNPs are administered in an amount that results in an improved parameter in vivo compared to a reference LNP, the improved parameter being one of the following: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more lipid remaining for 24 hours after administration to a subject, e.g., a mouse, e.g., after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slow lipid metabolism in the liver compared with the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more lipids remaining in the liver 24 hours after administration; 43. The method of any one of items 40 to 42, wherein the method comprises one, two, three, four, five, six, seven, or more (e.g., all) of the above, or any combination thereof. (Item 44) The target cell delivery LNP is: 1) increased response rates, e.g., as defined by a specific threshold of hepatocyte transfection; 2) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more hepatocyte transfection; 3) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 4) an increased response rate greater than the reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater response rate; 44. The method according to any one of items 40 to 43, which results in one, two, three or all of: (Item 45) 45. The method of any one of items 40 to 44, wherein the targeted cell delivery LNP is formulated for systemic delivery. (Item 46) 46. ​​The method of any one of items 40 to 45, wherein the target cell delivery LNP is administered systemically, e.g., parenterally (e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally) or enterally (e.g., orally, rectally, or sublingually). (Item 47) 47. The method of any one of items 40 to 46, wherein the target cell delivery LNP delivers the payload to cells capable of synthesizing proteins and / or cells with high engulfment ability. (Item 48) 48. The method of any one of items 40 to 47, wherein the target cell delivery LNP delivers the payload to liver cells, such as hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof. (Item 49) 49. The method of any one of items 40 to 48, wherein the target cell delivery LNP delivers the payload to hepatic parenchymal cells. (Item 50) 50. The method of any one of items 40 to 49, wherein the target cell delivery LNP delivers the payload to a splenocyte, e.g., a non-immune splenocyte (e.g., a splenocyte). (Item 51) 51. The method of any one of items 40 to 50, wherein the target cell delivery LNP delivers the payload to a cell selected from an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell or a brain cell, or a skeletal muscle cell. (Item 52) 52. The method of any one of items 40 to 51, wherein the target cell delivery LNP delivers the payload to a non-immune cell. (Item 53) 53. The method according to any one of items 40 to 52, wherein the intracellular concentration of the nucleic acid molecule in the target cell is enhanced. (Item 54) 54. The method of any one of items 40 to 53, wherein uptake of the nucleic acid molecule by the target cells is enhanced. (Item 55) 55. The method of any one of items 40 to 54, wherein the activity of the nucleic acid molecule in the target cell is enhanced. (Item 56) 56. The method according to any one of items 40 to 55, wherein expression of the nucleic acid molecule in the target cell is enhanced. (Item 57) 57. The method according to any one of items 40 to 56, wherein the activity of a protein encoded by the nucleic acid molecule in the target cell is enhanced. (Item 58) 58. The method according to any one of items 40 to 57, wherein expression of the protein encoded by the nucleic acid molecule in the target cell is enhanced. (Item 59) 59. The method of any one of items 40 to 58, wherein delivery is enhanced in vivo. (Item 60) 60. The method according to any one of items 40 to 59, wherein the payload is a peptide, polypeptide, protein, or nucleic acid. (Item 61) 61. The method of any one of items 40 to 60, wherein the payload is a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA. (Item 62) 62. The method of any one of items 40 to 61, wherein the payload is selected from a shortmir, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), or a combination thereof. (Item 63) 63. The method of any one of items 40 to 62, wherein the payload is mRNA, siRNA, miR, or CRISPR. (Item 64) 64. The method according to any one of items 40 to 63, wherein the payload is an mRNA encoding a protein of interest other than an immune cell payload. (Item 65) 65. The method according to any one of items 40 to 64, wherein the payload is selected from an mRNA encoding a secreted protein, a membrane-bound protein, an intracellular protein, or an enzyme. (Item 66) 66. The method according to any one of items 40 to 65, wherein the payload is an mRNA encoding an antibody molecule. (Item 67) 67. The method according to any one of items 40 to 66, wherein the payload is an mRNA encoding an enzyme. (Item 68) 68. The method of any one of items 40 to 67, wherein the enzyme is associated with an orphan disease (e.g., a lysosomal storage disease) or a metabolic disorder (e.g., as described herein). (Item 69) 69. The method of claim 68, wherein the payload is an mRNA encoding a urea cycle enzyme. (Item 70) 69. The method of claim 68, wherein the disease is a metabolic disorder. (Item 71) 71. The method of any one of items 40 to 70, wherein the target cell delivery LNP can be administered at a lower dose compared to a reference LNP, such as one described herein. (Item 72) 72. The method of any one of items 40-71, wherein the target cell delivery LNP is administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to the dose of a reference LNP. (Item 73) 73. The method of claim 72, wherein the target cell delivery LNP delivered at a lower dose results in similar or enhanced lipid and / or payload levels in the target cell, organ, or cellular compartment. (Item 74) 73. The method of claim 71 or 72, wherein the target cell delivery LNP can be administered at a reduced frequency compared to a reference LNP, such as one described herein. (Item 75) The delivery LNP or method of any preceding item, wherein the ionic lipid comprises an amino lipid. (Item 76) The delivery LNP or method of any of the preceding items, wherein the ionic lipid comprises a compound of any of formulas (I VI), (I VI-a), (I VII), (I VIII), (I VIIa), (I VIIIa), (I VIIIb), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIIIc), or (I VIIId). (Item 77) The delivery LNP or method of any preceding item, wherein the ionic lipid comprises an amino lipid having a squaramide head group. (Item 78) The delivery LNP or method of any of the preceding items, wherein the ionic lipid comprises a compound selected from the group consisting of Compound I-301, Compound (R)I-301, Compound (S)I-301, Compound I-49, Compound (R)I-49, Compound (S)I-49, Compound I-292, Compound I-309, Compound I-317, Compound I-326, Compound I-347, Compound I-348, Compound I-349, Compound I-350, and Compound I-352. (Item 79) The delivery LNP or method of any of the preceding items, wherein the ionic lipid comprises a compound selected from compound I-301 and compound I-49. (Item 80) The delivery LNP or method of any of the preceding items, wherein the ionic lipid comprises compound I-301. (Item 81) 80. The delivery LNP or method according to any one of items 1 to 79, wherein the ionic lipid comprises compound I-49. (Item 82) The delivery LNP or method of any of the preceding items, wherein the cells are hepatocytes, e.g., hepatocytes, and the ionic lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49. (Item 83) The delivery LNP or method of any of the preceding items, wherein the cells are splenocytes, e.g., splenocytes, and the ionic lipid comprises a compound selected from the group consisting of compound I-301 and compound I-49. (Item 84) The delivery LNP or method of any of the preceding items, wherein the ionic lipid comprises a racemic mixture of the amino lipid, e.g., a mixture comprising the (R) enantiomer and the (S) enantiomer of the amino lipid. (Item 85) The delivery LNP or method of any of the preceding items, wherein the reference LNP comprises an ionic lipid having formula I-XII. (Item 86) 86. The delivery LNP or method of claim 85, wherein the reference LNP does not contain an ionic lipid having a chiral center. (Item 87) 86. The delivery LNP or method of claim 85, wherein the reference LNP does not contain an ionic lipid comprising multiple branched alkyl chains. (Item 88) 86. The delivery LNP or method of claim 85, wherein the reference LNP does not contain a ring-substituted amino lipid. (Item 89) 86. The target cell delivery LNP or method of claim 85, wherein the reference LNP does not contain a carbocyclic-substituted ionic lipid. (Item 90) 86. The target cell delivery LNP or method of claim 85, wherein the reference LNP does not contain a cycloalkenyl-substituted amino lipid. (Item 91) The delivery LNP or method of any of the preceding items, wherein the target cell delivery LNP comprises an amino lipid having a chiral center. (Item 92) The delivery LNP or method of any preceding item, wherein the target cell delivery LNP comprises an ionic lipid comprising multiple branched alkyl chains. (Item 93) The delivery LNP or method of any preceding item, wherein the target cell delivery LNP comprises a ring-substituted amino lipid. (Item 94) 93. The delivery LNP or method of any of items 1 to 92, wherein the target cell delivery LNP comprises a carbocyclic-substituted amino lipid. (Item 95) 93. The delivery LNP or method of any of items 1 to 92, wherein the target cell delivery LNP comprises a cycloalkenyl-substituted amino lipid. (Item 96) The delivery LNP or method of any of the preceding items, wherein the target cell delivery LNP comprises a cyclobutenyl-substituted amino lipid. (Item 97) The delivery LNP or method of any preceding item, wherein the target cell delivery LNP comprises a cyclobutene-1,2-dione substituted amino lipid. (Item 98) The delivery LNP or method of any preceding item, wherein the target cell delivery LNP comprises a squaramide-substituted amino lipid, e.g., an amino lipid comprising a squaramide group. (Item 99) The delivery LNP or method of any of the preceding items, wherein the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DPPC, DMPC, DMPE, DOPC, Compound H-409, Compound H-418, Compound H-420, Compound H-421, and Compound H-422. (Item 100) 99. The delivery LNP or method of claim 98, wherein the cell is a liver cell, e.g., a hepatocyte, and the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DMPE, and compound H-409. (Item 101) 99. The delivery LNP or method of claim 99, wherein the phospholipid is DSPC. (Item 102) 99. The delivery LNP or method of claim 99, wherein the phospholipid is DMPE. (Item 103) 99. The delivery LNP or method of claim 99, wherein the phospholipid is compound H-409. (Item 104) The delivery LNP or method of any preceding item, comprising a PEG-lipid. (Item 105) 105. The delivery LNP or method of claim 104, wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. (Item 106) 105. The delivery LNP or method of claim 104, wherein the PEG lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE lipids. (Item 107) 107. The delivery LNP or method of any one of items 104 to 106, wherein the PEG lipid is PEG-DMG. (Item 108) 105. The delivery LNP or method of item 104, wherein the PEG-lipid comprises a compound selected from the group consisting of Compound P-415, Compound P-416, Compound P-417, Compound P-419, Compound P-420, Compound P-423, Compound P-424, Compound P-428, Compound P-L1, Compound P-L2, Compound P-L3, Compound P-L4, Compound P-L6, Compound P-L8, Compound P-L9, Compound P-L16, Compound P-L17, Compound P-L18, Compound P-L19, Compound P-L22, Compound P-L23, and Compound P-L25. (Item 109) 109. The target cell delivery LNP or method of item 104 or 108, wherein the PEG-lipid comprises a compound selected from the group consisting of compound P-428, PL-16, compound PL-17, compound PL-18, compound PL-19, compound PL-1, and compound PL-2. (Item 110) The delivery LNP or method of any of the preceding items, wherein the LNP comprises a molar ratio of (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid of about 50:10, 49:11, 48:12, 47:13, 46:14, 45:15, 44:16, 43:17, 42:18, or 41:19. (Item 111) 10. The delivery LNP or method of any preceding item, wherein the LNP comprises about 41 mol% to about 50 mol% ionic lipid, and about 10 mol% to about 19 mol% non-cationic helper lipid or phospholipid. (Item 112) The delivery LNP or method of any of the preceding items, wherein the LNP comprises about 50 mol % ionic lipid and about 10 mol % non-cationic helper lipid or phospholipid. (Item 113) The delivery LNP or method of any preceding item, wherein the molar ratio of (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid is about 50:10. (Item 114) The delivery LNP or method of any of the preceding items, wherein the lipid nanoparticle comprises compound I-301 as the ionic lipid, DSPC as the phospholipid, cholesterol or a cholesterol / β-sitosterol blend as the structural lipid, and compound 428 as the PEG lipid. (Item 115) The delivery LNP or method of any of the preceding items, wherein the ionic lipid:phospholipid:structural lipid:PEG lipid is in a ratio selected from: (i) 50:10:38:2; (ii) 50:20:28:2; (iii) 40:20:38:2; or (iv) 40:30:28:2. (Item 116) The LNP comprises: i) about 50 mol % of an ionic lipid, which is a compound selected from the group consisting of compound I-301, compound I-321, compound I-182, or compound I-49; (ii) approximately 10 mol % phospholipid, which is DSPC; (iii) about 38.5 mol % structural lipids selected from β-sitosterol and cholesterol; and (iv) about 1.5 mol % PEG lipid, which is compound P-428; 116. The delivery LNP or method of item 115, comprising: (Item 117) 117. A pharmaceutical composition comprising the delivery lipid nanoparticles according to any of items 1 to 40 or 75 to 116 and a pharmaceutically acceptable carrier. (Item 118) 117. A GMP-grade pharmaceutical composition comprising the delivery lipid nanoparticles according to any one of items 1 to 40 or 75 to 116 and a pharmaceutically acceptable carrier. (Item 119) 119. The pharmaceutical composition of item 117 or 118, having a purity of more than 95%, 96%, 97%, 98%, or 99%, e.g., at least 1%, 2%, 3%, 4%, 5% or more of contaminants removed. [Brief explanation of the drawings]

[0225] [Figure 1] A series of graphs showing the concentration of Compound 301-containing LNPs in the liver, spleen, or plasma on day 1 (left) or day 15 (right). Rats were intravenously administered 2 mg / kg of LNPs encapsulating NPI-Luc mRNA, and lipid levels were assessed at the indicated time points. [Figure 2]A series of graphs showing NPI-Luc mRNA expression in the liver, spleen, or plasma on day 1 (left) or day 15 (right). Rats were intravenously administered 2 mg / kg of LNPs encapsulating NPI-Luc mRNA, and mRNA levels were assessed at the indicated time points. [Figure 3] 1 is a graph showing lipid metabolism of LNPs containing Compound 301, Compound 18, or Compound 50 in the liver and spleen of mice. [Figure 4A] 1 shows NPI-Luc expression in animals administered Compound 301 LNPs encapsulating NPI-Luc mRNA or Compound 18 LNPs encapsulating NPI-Luc mRNA, showing NPI-Luc expression relative to total hepatocytes in the liver. [Figure 4B] 1 shows NPI-Luc expression in animals administered Compound 301 LNPs encapsulating NPI-Luc mRNA or Compound 18 LNPs encapsulating NPI-Luc mRNA. NPI-Luc expression in the spleen is shown relative to total spleen parenchymal cells. [Figure 5] FIG. 1 shows the results of immunohistochemical analysis of NPI-Luc protein expression in liver samples from animals administered compound 301 LNPs encapsulating NPI-Luc mRNA or compound 18 LNPs encapsulating NPI-Luc mRNA. [Figure 6] 1 is a graph showing NPI-Luc protein levels in liver samples from animals administered Compound 301 LNPs encapsulating NPI-Luc mRNA or Compound 18 LNPs encapsulating NPI-Luc mRNA. NPI-Luc protein expression was quantified using an ELISA from Meso Scale Discovery (MSD). [Figure 7]1 shows human EPO protein concentrations in the plasma of animals administered LNPs encapsulating human EPO mRNA. (A) Human EPO protein levels in animals administered LNPs containing compound 18 encapsulating human EPO mRNA. (B) Human EPO protein levels in animals administered LNPs containing compound 301. [Figure 8] 1 shows human EPO levels in plasma of animals administered various LNP formulations as indicated. A shows human EPO levels in plasma at 3 hours post-dose, B shows human EPO levels in plasma at 6 hours post-dose, and C shows human EPO levels in plasma at 24 hours post-dose. [Figure 9] FIG. 1 shows the development of human EPO levels over time in the plasma of animals administered various LNP formulations as indicated. [Figure 10] 1 shows the physical properties of the indicated compound 301-containing LNP formulations. A shows the diameter of the LNP. B shows the surface polarity of the LNP. [Figure 11] FIG. 1 shows the optimal composition ratio of ionic lipid:DSPC:cholesterol for in vivo expression. DETAILED DESCRIPTION OF THE INVENTION

[0226] The present disclosure provides improved lipid-based compositions, specifically delivery lipid nanoparticles (LNPs), which exhibit increased delivery of a drug(s) to target cells, e.g., hepatocytes or splenocytes, when compared to LNPs that contain a lipid and do not contain a target cell delivery-enhancing lipid. In various aspects, the present disclosure provides improved LNPs containing a target cell delivery-enhancing lipid, which LNPs contain a drug(s) for delivery to a target cell or population of target cells, methods for enhancing delivery of a drug (e.g., a nucleic acid molecule) to a target cell or population of target cells, methods for delivering such LNPs to subjects who would benefit from modulation of target cell activity, and methods for treating such subjects. The present disclosure is based, at least in part, on the discovery that when certain lipid components of LNPs are present in LNPs, the lipid components enhance association of the LNPs with target cells and delivery of the drug into the target cells, as demonstrated, for example, by expression of a nucleic acid molecule by the target cells. Although the LNPs of the present disclosure have demonstrated enhanced delivery to target cells (e.g., hepatocytes or splenocytes) by measuring increased mRNA expression in said target cells, the same approach can be demonstrated using knockdown (i.e., reduction) of pre-existing expression, depending on the nucleic acid molecule being delivered.

[0227] In addition, those skilled in the art will recognize that, having demonstrated enhanced delivery to target cells, such as hepatocytes and / or splenocytes, in this model system using mRNA, the subject target cell delivery LNPs can now be used to deliver other agents to target cells. Such agents are known in the art, and in one embodiment, the agent comprises or consists of a nucleic acid molecule. In particular, certain potentially therapeutic nucleic acid molecules are known, and in some cases, proteins encoded by such nucleic acid molecules or the nucleic acid molecules themselves are currently used therapeutically. Given the advances made by LNPs that enhance targeted delivery to target cells of interest (e.g., hepatocytes or splenocytes), improved therapies are possible. In some aspects, the agent is a nucleic acid molecule selected from the group consisting of mRNA, RNAi, dsRNA, siRNA, miR, antagomir, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, and DNA.

[0228] In certain embodiments, target cell delivery LNPs enhance delivery of agents (e.g., nucleic acid molecules) to target cells such as liver cells (e.g., hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof) or splenocytes (e.g., splenocytes) compared to LNPs that do not contain a target cell delivery-enhancing lipid, e.g., an LNP comprising an amino lipid of Formulas I-XII. In one embodiment, it has been demonstrated that when mRNA is delivered by target cell delivery LNPs that contain a target cell delivery-enhancing lipid, expression of mRNA encoding a protein of interest is enhanced in the target cells compared to LNPs that do not contain a target cell delivery-enhancing lipid, e.g., an LNP comprising an amino lipid of Formulas I-XII. Delivery of agents associated with (e.g., encapsulated within) target cell delivery-enhancing LNPs to target cells (e.g., liver cells or splenocytes) has been demonstrated in vitro and in vivo.

[0229] As demonstrated herein, LNPs that enhance target cell delivery have been shown to increase protein expression in target cells (e.g., hepatocytes or splenocytes) by at least about two-fold. Target cell delivery has also been demonstrated in vivo. In vivo delivery of encapsulated mRNA has been demonstrated to be delivered to at least about 30% of hepatocytes after a single intravenous injection of LNPs of the present disclosure. Delivery of encapsulated mRNA to greater than 20% of splenocytes has also been demonstrated. The level of delivery demonstrated herein using LNPs containing target cell delivery-enhancing lipids enables in vivo therapy. The present disclosure provides methods for modulating various proteins (including upregulating and downregulating protein expression and / or activity) in a wide variety of clinical settings, including cancer, infectious diseases, vaccinations, and autoimmune diseases.

[0230] The LNPs of the present disclosure are particularly useful for targeting liver cells or splenocytes. The LNPs can include a nucleic acid molecule (e.g., mRNA) that encodes a protein that is an intracellular or secreted protein.

[0231] Without intending to be bound by any particular mechanism or theory, it is believed that the enhanced delivery of nucleic acid molecules to target cells (e.g., liver cells or splenocytes) by the LNPs of the present disclosure results from the presence of an effective amount of a target cell delivery-enhancing lipid, such as a cholesterol analog or an amino lipid, or a combination thereof, which, when present in the LNP, can function by enhancing cell association and / or uptake, internalization, intracellular trafficking and / or processing, and / or endosomal escape, and / or can enhance recognition by and / or binding to the target cell, relative to LNPs that do not contain the target cell delivery-enhancing lipid.

[0232] Thus, without intending to be bound by any particular mechanism or theory, in one embodiment, the target cell delivery-enhancing lipids of the present disclosure are preferentially taken up by liver cells, splenocytes, ovarian cells, lung cells, intestinal cells, cardiac cells, skin cells, eye cells, or brain cells, or skeletal muscle cells, relative to a reference LNP. In certain embodiments, the reference LNP does not comprise a target cell delivery-enhancing lipid and / or is not preferentially taken up by liver cells, splenocytes, ovarian cells, lung cells, intestinal cells, cardiac cells, skin cells, eye cells, or brain cells, or skeletal muscle cells.

[0233] The ability to efficiently deliver agents (e.g., nucleic acid molecules, including mRNA) to target cells is useful for modulating protein expression and / or activity in the target cells. Furthermore, LNPs can alter cellular activity and / or function in the cells to which they are delivered, or in cells that interact with and / or are affected by such cells (e.g., in an autocrine or paracrine manner).

[0234] Targeted cell delivery LNPs are useful, for example, for delivery of nucleic acid molecules that modulate the expression of naturally occurring or engineered molecules. In one embodiment, the expression of a soluble / secreted protein (e.g., a naturally occurring soluble molecule or one that has been modified or engineered to promote improved function / half-life / and / or stability) is modulated. In another embodiment, the expression of an intracellular protein (e.g., a naturally occurring intracellular protein or an engineered or modified intracellular protein with altered function) is modulated. In another embodiment, the expression of a transmembrane protein (e.g., a naturally occurring soluble molecule or one that has been modified or engineered to have altered function) is modulated.

[0235] In one embodiment, the nucleic acid molecule can encode a protein that is not naturally expressed in the target cell (e.g., a heterologous protein or a modified protein). In one embodiment, the nucleic acid molecule can encode or knock down a protein that is naturally expressed in the target cell.

[0236] For example, in some embodiments, the LNPs of the present disclosure are useful for enhancing the delivery and expression of mRNA encoding soluble / secreted proteins, transmembrane proteins, or intracellular proteins into target cells. Exemplary transmembrane proteins can confer new binding specificity to target cells. Exemplary intracellular molecules can regulate cell signaling or cell fate.

[0237] The present disclosure also provides methods for using multiple LNPs in combination to deliver the same (e.g., within different LNPs) or different (e.g., within the same LNP or within different LNPs (e.g., LNPs that enhance target cell delivery and those that do not) agents, e.g., nucleic acid molecules, for delivery of the same agent (e.g., within different LNPs) or different (e.g., within the same LNP or within different LNPs (e.g., LNPs that enhance target cell delivery and those that do not) for delivery of nucleic acid molecules to target cells or different cell populations).

[0238] Targeted cell delivery LNP Target cell delivery LNPs can be characterized as providing increased delivery of a drug to target cells (e.g., liver cells or splenocytes) when compared to a reference LNP (e.g., an LNP not containing a target cell delivery-enhancing lipid). In particular, in one embodiment, the target cell delivery LNPs provide an increased (e.g., a two-fold or greater increase) percentage of LNPs associated with target cells when compared to a reference LNP (e.g., an LNP comprising an amino lipid of Formula I-XII). In another embodiment, the target cell delivery LNPs provide an increased (e.g., a two-fold or greater increase) percentage of target cells expressing the drug carried by the LNP (e.g., expressing a protein encoded by an mRNA associated / encapsulated in the LNP) when compared to a reference LNP (e.g., an LNP comprising an amino lipid of Formula I-XII). In another embodiment, the target cell delivery LNPs provide preferential uptake by liver cells, splenocytes, ovarian cells, lung cells, intestinal cells, heart cells, skin cells, eye cells, or brain cells, or skeletal muscle cells when compared to a reference LNP. In certain embodiments, the reference LNP does not contain a target cell delivery-enhancing lipid and / or is not preferentially taken up by liver cells, splenocytes, ovarian cells, lung cells, intestinal cells, heart cells, skin cells, eye cells or brain cells, or skeletal muscle cells.

[0239] In another embodiment, the target cell delivery LNPs provide increased delivery of an agent (e.g., a nucleic acid molecule) to a target cell when compared to a reference LNP (e.g., an LNP comprising an amino lipid of Formula I-XII). In one embodiment, the target cell delivery LNPs provide increased delivery of a nucleic acid molecule agent to a hepatocyte when compared to a reference LNP. In one embodiment, the target cell delivery LNPs provide increased delivery of a nucleic acid molecule agent to a hepatic parenchymal cell when compared to a reference LNP. In one embodiment, the target cell delivery LNPs provide increased delivery of a nucleic acid molecule agent to a hepatic stellate cell when compared to a reference LNP. In one embodiment, the target cell delivery LNPs provide increased delivery of a nucleic acid molecule agent to a Kupffer cell when compared to a reference LNP. In one embodiment, the target cell delivery LNPs provide increased delivery of a nucleic acid molecule agent to a hepatic sinusoidal cell when compared to a reference LNP.

[0240] In one embodiment, when the nucleic acid molecule is mRNA, increased delivery of the nucleic acid agent to a target cell can be measured by the ability of the LNP to result in at least about two-fold greater expression of a protein molecule encoded by the mRNA in the target cell (e.g., a liver cell or splenocyte) when compared to a reference LNP.

[0241] The target cell delivery LNP comprises (i) an ionic lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; (iv) a PEG lipid, and (v) a drug (e.g., a nucleic acid molecule) encapsulated and / or associated with the LNP, wherein one or more of the (i) ionic lipid or (ii) structural lipid or sterol in the target cell delivery LNP comprises an effective amount of a target cell delivery-enhancing lipid.

[0242] In another embodiment, the targeted cell delivery lipid nanoparticles of the present disclosure comprise: (i) ionic lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) an agent for delivery to a target cell; and (v) optionally, a PEG lipid wherein one or more of (i) an ionizable lipid or (ii) a sterol or other structural lipid comprises a target cell delivery-enhancing lipid in an amount effective to enhance delivery of the lipid nanoparticle to a target cell. In one embodiment, the enhanced delivery is compared to a lipid nanoparticle that does not comprise a target cell delivery-enhancing lipid. In another embodiment, the enhanced delivery is compared to a suitable control, e.g., a reference LNP.

[0243] In another embodiment, the targeted cell delivery lipid nanoparticles of the present disclosure comprise: (i) ionic lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) an agent for delivery to a target cell; and (v) optionally, a PEG lipid wherein one or more of: (i) an ionic lipid, or (ii) a sterol or other structural lipid, or (iii) a non-cationic helper lipid or phospholipid, or (v) a PEG-lipid are preferentially taken up by target cells (e.g., hepatocytes or splenocytes) when compared to a reference LNP.

[0244] In another embodiment, the targeted cell delivery lipid nanoparticles of the present disclosure comprise: (i) ionic lipids; (ii) sterols or other structural lipids; (iii) noncationic helper lipids or phospholipids; (iv) an agent for delivery to a target cell; and (v) optionally, a PEG lipid wherein one or more of (i) an ionic lipid, or (ii) a sterol or other structural lipid is preferentially taken up by target cells (e.g., hepatocytes or splenocytes) when compared to a reference LNP.

[0245] Lipid content of LNPs As noted above, with respect to lipids, target cell delivery LNPs include (i) ionic lipids; (ii) sterols or other structural lipids; (iii) non-cationic helper lipids or phospholipids; and (iv) PEG-lipids, wherein one or more of the (i) ionic lipids or (ii) structural lipids or sterols in the target cell delivery LNPs includes an effective amount of a target cell delivery-enhancing lipid. These categories of lipids are described in more detail below.

[0246] (i) Ionic lipids The lipid nanoparticles of the present disclosure comprise one or more ionic lipids.In certain embodiments, the ionic lipids of the present disclosure comprise a central amine moiety and at least one biodegradable group.The ionic lipids described herein can be advantageously used in the lipid nanoparticles of the present disclosure to deliver nucleic acid molecules to mammalian cells or organs.The structures of the ionic lipids described below include the prefix I to distinguish them from other lipids of the present invention.

[0247] In a first aspect of the present invention, the compounds described herein are of formula (II): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring, R 4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH) n N(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)R 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9)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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9 )R, -C(O)N(R)OR, and -C(R)N(R)C(O)OR, each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, OC(O)-M"-C(O)O-, -C(O)N(R')-, is selected from —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an aryl group, and a heteroaryl group, wherein M″ is a bond, C 1-13 Alkyl, or C 2-13 is alkenyl, R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 are H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6selected from the group consisting of carbocycles and heterocycles; R 10 are H, OH, and C 1-3 Alkyl, and C 2-3 alkenyl, Each R is independently C 1-3 Alkyl, C 2-3 Alkenyl, (CH2) q OR*, and H; each q is independently selected from 1, 2, and 3; Each R' is independently C 1-18 Alkyl, C 2-18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl, Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, where R 4 Ga-(CH2) n Q, -(CH2) n If CHQR, -CHQR, or -CQ(R), then (i) when n is 1, 2, 3, 4, or 5, then Q is not -N(R), or (ii) when n is 1 or 2, then Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0248] Another aspect of the present disclosure is a compound of formula (III): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: R 1 is C 5-30 Alkyl, C5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring, R 4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH) n N(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)R 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9 )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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9)R, -C(O)N(R)OR, and -C(R)N(R)C(O)OR, each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; R x is C 1-6 Alkyl, C 2-6 Alkenyl, -(CH2) v OH, and -(CH2) v N(R)2, where v is selected from 1, 2, 3, 4, 5, and 6; Each R 5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S), —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an alkyl group, and a heteroaryl group, wherein M″ is a bond, C 1-13 Alkyl, or C 2-13 is alkenyl, R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 are H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 10 are H, OH, and C 1-3Alkyl, and C 2-3 alkenyl, Each R is independently C 1-3 Alkyl, C 2-3 Alkenyl, (CH2) q OR*, and H; each q is independently selected from 1, 2, and 3; Each R' is independently C 1-18 Alkyl, C 2-18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl, Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0249] In certain embodiments, a subset of compounds of formula (I) includes those of formula (IA): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M′; and R 4 is hydrogen, unsubstituted C 1-3 Alkyl, -(CH2) o C(R 10 )2(CH2) n-o Q, or -(CH2) n Q, where Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R 8 , -NHC(=NR9 )N(R)2, -NHC(=CHR 9 )N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 For example, Q is selected from the group consisting of alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.

[0250] In certain embodiments, a subset of compounds of formula (I) includes those of formula (IB): [ka] or an N-oxide thereof, or a salt or isomer thereof, where all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl. For example, m is 5, 7, or 9. In certain embodiments, a subset of compounds of formula (I) includes those of formula (II): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M is a bond or M′; and R4 is hydrogen, unsubstituted C 1-3 Alkyl, -(CH2) o C(R 10 )2(CH2) n-o Q, or -(CH2) n Q, where 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)R 8 , -NHC(=NR 9 )N(R)2, -NHC(=CHR 9 )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)—, —OC(O)—M″—C(O)O—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0251] Another aspect of the present disclosure is a compound of formula (I VI): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring, Each R5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S), —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an alkyl group, and a heteroaryl group, wherein M″ is a bond, C 1-13 Alkyl, or C 2-13 is alkenyl, R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R is independently H, C 1-3 Alkyl, and C 2-3 alkenyl, R N is H or C 1-3 is alkyl, Each R' is independently C 1-18 Alkyl, C 2-18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl, Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; X a and X b are each independently O or S, R 10 is H, halo, -OH, R, -N(R)2, -CN, -N3, -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O)2OR, -NO2, -S(O)2N(R)2, -N(R)S(O)2R, -NH(CH2) t1 N(R)2, -NH(CH2) p1 O(CH2) q1 N(R)2, -NH(CH2) s1 OR, -N((CH2) s1 OR)2, carbocycle, heterocycle, aryl, and heteroaryl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; r is 0 or 1, t 1 is selected from 1, 2, 3, 4, and 5; p 1 is selected from 1, 2, 3, 4, and 5; q 1 is selected from 1, 2, 3, 4, and 5; s 1 is selected from 1, 2, 3, 4, and 5.

[0252] In one embodiment, a subset of compounds of formula (VI) includes those of formula (VI-a): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: R 1a and R 1b independently, C 1-14 Alkyl and C 2-14 alkenyl, R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring.

[0253] In another embodiment, a subset of compounds of formula (VI) includes those of formula (VII): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0254] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIII): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R a’ and R b’ independently, C 1-14 Alkyl and C 2-14 alkenyl, R 2 and R 3 independently, C 1-14 Alkyl, and C 2-14 alkenyl.

[0255] The compounds of any one of formula (II), (I IA), (I VI), (I VI-a), (I VII), or (I VIII), as applicable, include one or more of the following features:

[0256] In some embodiments, M1 is M'.

[0257] In some embodiments, M and M' are independently -C(O)O- or -OC(O)-.

[0258] In some embodiments, at least one of M and M' is -C(O)O- or -OC(O)-.

[0259] In certain embodiments, at least one of M and M' is -C(O)O-.

[0260] In certain embodiments, M is -OC(O)- and M' is -C(O)O-. In some embodiments, M is -C(O)O- and M' is -OC(O)-. In certain embodiments, M and M' are each -OC(O)-. In some embodiments, M and M' are each -C(O)O-.

[0261] In certain embodiments, at least one of M and M′ is —OC(O)—M″—C(O)O—.

[0262] In some embodiments, M and M' are independently -SS-.

[0263] In some embodiments, at least one of M and M' is -SS-.

[0264] In some embodiments, one of M and M' is -C(O)O- or -OC(O)- and the other is -SS-. For example, M is -C(O)O- or -OC(O)- and M' is -SS-, or M' is -C(O)O- or -OC(O)- and M is -SS-.

[0265] In some embodiments, one of M and M' is -OC(O)-M"-C(O)O-, where M" is a bond, C 1-13 Alkyl, or C 2-13 In other embodiments, M" is C 1-6 Alkyl or C 2-6 In certain embodiments, M" is C 1-4 Alkyl or C 2-4 For example, in some embodiments, M" is a C1 alkyl. For example, in some embodiments, M" is a C2 alkyl. For example, in some embodiments, M" is a C3 alkyl. For example, in some embodiments, M" is a C4 alkyl. For example, in some embodiments, M" is a C2 alkenyl. For example, in some embodiments, M" is a C3 alkenyl. For example, in some embodiments, M" is a C4 alkenyl.

[0266] In some embodiments, 1 is 1, 3, or 5.

[0267] In some embodiments, R 4 is hydrogen.

[0268] In some embodiments, R 4 is not hydrogen.

[0269] In some embodiments, R 4 is unsubstituted methyl or -(CH2) n Q, where Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, or —N(R)S(O)R.

[0270] In some embodiments, Q is OH.

[0271] In some embodiments, Q is —NHC(S)N(R) 2 .

[0272] In some embodiments, Q is —NHC(O)N(R) 2 .

[0273] In some embodiments, Q is —N(R)C(O)R.

[0274] In some embodiments, Q is —N(R)S(O)R.

[0275] In some embodiments, Q is —O(CH) n N(R)2.

[0276] In some embodiments, Q is —O(CH) n It is OR.

[0277] In some embodiments, Q is —N(R)R 8 is.

[0278] In some embodiments, Q is —NHC(═NR 9 )N(R)2.

[0279] In some embodiments, Q is —NHC(═CHR 9 )N(R)2.

[0280] In some embodiments, Q is —OC(O)N(R) 2 .

[0281] In some embodiments, Q is —N(R)C(O)OR.

[0282] In some embodiments, n is 2.

[0283] In some embodiments, n is 3.

[0284] In some embodiments, n is 4.

[0285] In some embodiments, M1 is absent.

[0286] In some embodiments, at least one R 5 is hydroxyl. For example, one R 5 is hydroxyl.

[0287] In some embodiments, at least one R 6 is hydroxyl. For example, one R 6 is hydroxyl.

[0288] In some embodiments, R 5 and R 6 One of the R is hydroxyl. For example, 5 is hydroxyl, and each R 6 is hydrogen. For example, one R 6 is hydroxyl, and each R 5 is hydrogen.

[0289] In some embodiments, R x is C 1-6 In some embodiments, R x is C 1-3 alkyl. For example, R x is methyl. For example, R x is ethyl. For example, R x is propyl.

[0290] In some embodiments, R x Ha-(CH2) v OH and v is 1, 2, or 3. For example, R x is methanoyl. For example, R x is ethanoyl. For example, R x is propanoyl.

[0291] In some embodiments, R x Ha-(CH2) v N(R)2, where v is 1, 2, or 3, and each R is H or methyl. For example, R x is methaneamino, methylmethaneamino, or dimethylmethaneamino. For example, R x is aminomethanil, methylaminomethanil, or dimethylaminomethanil. For example, R xis aminoethanyl, methylaminoethanyl, or dimethylaminoethanyl. For example, R x is aminopropanyl, methylaminopropanyl, or dimethylaminopropanyl.

[0292] In some embodiments, R' is C 1-18 Alkyl, C 2-18 alkenyl, -R*YR'', or -YR''.

[0293] In some embodiments, R 2 and R 3 independently, C 3-14 Alkyl or C 3-14 It is alkenyl.

[0294] In some embodiments, R 1b is C 1-14 In some embodiments, R 1b is C 2-14 In some embodiments, R 1b is C 3-14 In some embodiments, R 1b is C 1-8 In some embodiments, R 1b is C 1-5 In some embodiments, R 1b is C 1-3 In some embodiments, R 1b is selected from C alkyl, C alkyl, C alkyl, C alkyl, and C alkyl. For example, in some embodiments, R 1b is C alkyl. For example, in some embodiments, R 1b is C alkyl. For example, in some embodiments, R 1b is a C alkyl. For example, in some embodiments, R 1b is C4 alkyl. For example, in some embodiments, R 1b is a C5 alkyl.

[0295] In some embodiments, R 1is -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 is different.

[0296] In some embodiments, -CHR 1a R 1b - is -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 is different.

[0297] In some embodiments, R 7 is H. In some embodiments, R 7 is C 1-3 For example, in some embodiments, R 7 is C alkyl. For example, in some embodiments, R 7 is C alkyl. For example, in some embodiments, R 7 is C alkyl. In some embodiments, R 7 is C4 alkyl, C4 alkenyl, C5 alkyl, C5 alkenyl, C6 alkyl, C6 alkenyl, C7 alkyl, C7 alkenyl, C9 alkyl, C9 alkenyl, C 11 Alkyl, C 11 Alkenyl, C 17 Alkyl, C 17 Alkenyl, C 18 Alkyl, and C 18 alkenyl.

[0298] In some embodiments, R b’ is C 1-14 In some embodiments, R b’ is C 2-14 In some embodiments, R b’ is C 3-14 In some embodiments, R b’ is C 1-8 In some embodiments, Rb’ is C 1-5 In some embodiments, R b’ is C 1-3 In some embodiments, R b’ is selected from C alkyl, C alkyl, C alkyl, C alkyl, and C alkyl. For example, in some embodiments, R b’ is C alkyl. For example, in some embodiments, R b’ is C alkyl. For example, in some embodiments, R b’ is a C alkyl. For example, in some embodiments, R b’ is a C4 alkyl.

[0299] Another aspect of the present disclosure is a compound of formula (I XI): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: Q is selected from -OR, -OC(O)R, or -OC(O)N(R); R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring, Each R 5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3selected from the group consisting of alkenyl, and H; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S), —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an alkyl group, and a heteroaryl group, wherein M″ is a bond, C 1-13 Alkyl, or C 2-13 is alkenyl, R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R is independently H, C 1-3 Alkyl, and C 2-3 alkenyl, Each R' is independently C 1-18 Alkyl, C 2-18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl, Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0300] In another embodiment, a subset of compounds of formula (I XI) includes those of formula (I XI-a): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: Q is -OR, l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl, n is selected from 1, 2, and 3.

[0301] In another embodiment, a subset of compounds of formula (I XI) includes those of formula (I XI-b): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein: l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R a’ and R b’ independently, C 1-14 Alkyl and C 2-14 alkenyl, R 2 and R 3 independently, C 1-14 Alkyl and C 2-14 alkenyl.

[0302] Compounds of any one of formula (I XI), (I XI-a), or (I XI-b) include one or more of the following features, where applicable.

[0303] In some embodiments, M1 is M'.

[0304] In some embodiments, M and M' are independently -C(O)O- or -OC(O)-.

[0305] In some embodiments, at least one of M and M' is -C(O)O- or -OC(O)-.

[0306] In certain embodiments, at least one of M and M' is -OC(O)-.

[0307] In certain embodiments, M is -OC(O)- and M' is -C(O)O-. In some embodiments, M is -C(O)O- and M' is -OC(O)-. In certain embodiments, M and M' are each -OC(O)-. In some embodiments, M and M' are each -C(O)O-.

[0308] In certain embodiments, at least one of M and M′ is —OC(O)—M″—C(O)O—.

[0309] In some embodiments, M and M' are independently -SS-.

[0310] In some embodiments, at least one of M and M' is -SS-.

[0311] In some embodiments, one of M and M' is -C(O)O- or -OC(O)- and the other is -SS-. For example, M is -C(O)O- or -OC(O)- and M' is -SS-, or M' is -C(O)O- or -OC(O)- and M is -SS-.

[0312] In some embodiments, one of M and M' is -OC(O)-M"-C(O)O-, where M" is a bond, C 1-13 Alkyl, or C 2-13 In other embodiments, M" is C 1-6 Alkyl or C 2-6 In certain embodiments, M" is C 1-4 Alkyl or C 2-4For example, in some embodiments, M" is a C1 alkyl. For example, in some embodiments, M" is a C2 alkyl. For example, in some embodiments, M" is a C3 alkyl. For example, in some embodiments, M" is a C4 alkyl. For example, in some embodiments, M" is a C2 alkenyl. For example, in some embodiments, M" is a C3 alkenyl. For example, in some embodiments, M" is a C4 alkenyl.

[0313] In some embodiments, 1 is 1, 3, or 5.

[0314] In some embodiments, Q is —OR.

[0315] In some embodiments, n is 2.

[0316] In some embodiments, n is 3.

[0317] In some embodiments, n is 4.

[0318] In some embodiments, M1 is absent.

[0319] In some embodiments, R is H.

[0320] In some embodiments, at least one R 5 is hydroxyl. For example, one R 5 is hydroxyl.

[0321] In some embodiments, at least one R 6 is hydroxyl. For example, one R 6 is hydroxyl.

[0322] In some embodiments, R 5 and R 6 One of the R is hydroxyl. For example, 5 is hydroxyl, and each R 6is hydrogen. For example, one R 6 is hydroxyl, and each R 5 is hydrogen. In some embodiments, R and R 6 Each of is hydrogen.

[0323] In some embodiments, R' is C 1-18 Alkyl, C 2-18 alkenyl, -R*YR'', or -YR''.

[0324] In some embodiments, R 2 and R 3 independently, C 3-14 Alkyl or C 3-14 It is alkenyl.

[0325] In some embodiments, R 7 is H. In some embodiments, R 7 is C 1-3 For example, in some embodiments, R 7 is C alkyl. For example, in some embodiments, R 7 is C alkyl. For example, in some embodiments, R 7 is C alkyl. In some embodiments, R 7 is C4 alkyl, C4 alkenyl, C5 alkyl, C5 alkenyl, C6 alkyl, C6 alkenyl, C7 alkyl, C7 alkenyl, C9 alkyl, C9 alkenyl, C 11 Alkyl, C 11 Alkenyl, C 17 Alkyl, C 17 Alkenyl, C 18 Alkyl, and C 18 alkenyl.

[0326] In some embodiments, R b’ is C 1-14 In some embodiments, R b’ is C 2-14 In some embodiments, R b’ is C 3-14In some embodiments, R b’ is C 1-8 In some embodiments, R b’ is C 1-5 In some embodiments, R b’ is C 1-3 In some embodiments, R b’ is selected from C alkyl, C alkyl, C alkyl, C alkyl, and C alkyl. For example, in some embodiments, R b’ is C alkyl. For example, in some embodiments, R b’ is C alkyl. For example, in some embodiments, R b’ is a C alkyl. For example, in some embodiments, R b’ is a C4 alkyl.

[0327] In some embodiments, M1 is M'. In some embodiments, M and M' are each -C(O)O-. In some embodiments, l is 5. In some embodiments, Q is -OH. In some embodiments, n is 2. In some embodiments, R 5 and R 6 In some embodiments, R' is C 1-18 In some embodiments, R' is C 11 In some embodiments, R 2 and R 3 independently, C 3-14 In some embodiments, R 2 and R 3 is independently C alkyl. In some embodiments, R 7 is H. In some embodiments, R a’ is C 1-14 In some embodiments, R a’ is C alkyl. In some embodiments, R b’ is C 1-3 In some embodiments, R b’ is a C2 alkyl.

[0328] In one embodiment, the compound of formula (I) is of formula (IIa): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0329] In another embodiment, the compound of formula (I) is of formula (IIb): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0330] In another embodiment, the compound of formula (I) is of formula (IIc) or (IIe): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0331] In another embodiment, the compound of formula (I) is of formula (IIIh): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0332] In another embodiment, the compound of formula (I) is of formula (IIIj): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4is as described herein.

[0333] In another embodiment, the compound of formula (I) is of formula (IIIk): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0334] In another embodiment, the compound of formula (II) is of formula (IIIf): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, M is —C(O)O— or —OC(O)—, and M″ is C 1-6 Alkyl or C 2-6 alkenyl, and R 2 and R 3 independently, C 5-14 Alkyl and C 5-14 alkenyl, wherein n is selected from 2, 3, and 4.

[0335] In a further embodiment, the compound of formula (II) is of formula (IId): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein n is 2, 3, or 4; m, R′, R″, and R 2 ~R6 is as described herein. For example, R 2 and R 3 Each of the 5-14 Alkyl and C 5-14 It may be selected from the group consisting of alkenyl.

[0336] In a further embodiment, the compound of formula (I) is of formula (IIg): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M′; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)—M″—C(O)O—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 For example, M" is selected from the group consisting of C 1-6 Alkyl (e.g., C 1-4 alkyl) or C 2-6 Alkenyl (e.g., C 2-4 alkenyl). For example, R 2 and R 3 independently, C 5-14 Alkyl and C 5-14 alkenyl.

[0337] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIa): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0338] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIIa): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0339] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIIb): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0340] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIb-1): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0341] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIb-2): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0342] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIb-3): [ka] or an N-oxide thereof, or a salt or isomer thereof. In another embodiment, a subset of compounds of formula (VI) is that of formula (VIIc): [ka] Includes:

[0343] In another embodiment, a subset of compounds of formula (IV) includes those of formula (VIId): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0344] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIIc): [ka] Includes:

[0345] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIId): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0346] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIb-4): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0347] In another embodiment, a subset of compounds of formula (I VI) includes those of formula (I VIIb-5): [ka] or an N-oxide thereof, or a salt or isomer thereof.

[0348] Formula (II), (I IA), (I IB), (I II), (I IIa), (I IIb), (I IIc), (I IId), (I IIe), (I IIf), (I IIg), (I IIh), (I IIj), (I IIk), (I III), (I VI), (I VI-a), (I VII), (I VIII), (I VIIa), (I VIIIa), (I VIIIb), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIIIc), (I VIIId), (I XI), (I XI-a), or (I Any one of the compounds of XI-b) comprises one or more of the following characteristics, if applicable:

[0349] In some embodiments, R 4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o -Q, -CHQR, and -CQ(R), wherein Q is C 3-6 Carbocycle, 5-14 membered aromatic or non-aromatic heterocycle having one or more heteroatoms selected from N, O, S, and P, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -N(R)S(O)2R 8 , -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, and -C(R)N(R)2C(O)OR, where each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5.

[0350] In another embodiment, R 4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2)o C(R 10 )2(CH2) n-o -Q, -CHQR, and -CQ(R), wherein Q is C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)S(O)2R 8 , —N(R)C(O)R, —N(R)S(O)R, —N(R)C(O)N(R), —N(R)C(S)N(R), —C(R)N(R)C(O)OR, and 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S, including oxo (═O), OH, amino, and C 1-3 wherein each o is independently selected from 1, 2, 3, and 4; and each n is independently selected from 1, 2, 3, 4, and 5.

[0351] In another embodiment, R 4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o -Q, -CHQR, and -CQ(R), wherein Q is C 3-6 Carbocycle, 5-14 membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)S(O)2R 8, —N(R)C(O)R, —N(R)S(O)R, —N(R)C(O)N(R), —N(R)C(S)N(R), —C(R)N(R)C(O)OR; each o is independently selected from 1, 2, 3, and 4; each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5-14 membered heterocycle; and (i) R 4 Ga-(CH2) n Q (wherein n is 1 or 2), or (ii) R 4 Ga-(CH2) n CHQR (wherein n is 1), or (iii) R 4 When is -CHQR and -CQ(R)2, Q is either a 5- to 14-membered heterocycle or an 8- to 14-membered heterocycloalkyl.

[0352] In another embodiment, R 4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o -Q, -CHQR, and -CQ(R), wherein Q is C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)S(O)2R 8 , -N(R)C(O)R, -N(R)S(O)R, -N(R)C(O)N(R), -N(R)C(S)N(R), -C(R)N(R)C(O)OR, where each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5.

[0353] In another embodiment, R 4 Ha-(CH2) n Q, where Q is -N(R)S(O)R 8and n is selected from 1, 2, 3, 4, and 5. In a further embodiment, R 4 Ha-(CH2) n Q, where Q is -N(R)S(O)R 8 where R 8 is C 3-6 Carbocyclic rings, e.g., C 3-6 cycloalkyl, where n is selected from 1, 2, 3, 4, and 5. For example, R 4 -(CH2)3NHS(O)2R 8 and R 8 is cyclopropyl.

[0354] In another embodiment, R 4 Ha-(CH2) o C(R 10 )2(CH2) n-o Q, where Q is —N(R)C(O)R, n is selected from 1, 2, 3, 4, and 5, and o is selected from 1, 2, 3, and 4. In a further embodiment, R 4 Ha-(CH2) o C(R 10 )2(CH2) n-o Q, where Q is —N(R)C(O)R, where R is C-C alkyl, n is selected from 1, 2, 3, 4, and 5, and o is selected from 1, 2, 3, and 4. In another embodiment, R 4 Ha-(CH2) o C(R 10 )2(CH2) n-o Q, where Q is —N(R)C(O)R, where R is C-C alkyl, n is 3, and o is 1. In some embodiments, R 10 are H, OH, and C 1-3 Alkyl, or C 2-3 alkenyl. For example, R 4 is 3-acetamido-2,2-dimethylpropyl.

[0355] In some embodiments, one R 10 is H and one R 10 is C 1-3 Alkyl or C2-3 In another embodiment, each R 10 is C 1-3 Alkyl or C 2-3 In another embodiment, each R 10 is C 1-3 alkyl (e.g., methyl, ethyl, or propyl). For example, one R 10 is methyl and one R 10 is ethyl or propyl. For example, one R 10 is ethyl and one R 10 is methyl or propyl. For example, one R 10 is propyl and one R 10 is methyl or ethyl. For example, each R 10 is methyl. For example, each R 10 is ethyl. For example, each R 10 is propyl.

[0356] In some embodiments, one R 10 is H and one R 10 is OH. In another embodiment, each R 10 is OH.

[0357] In another embodiment, R 4 Ha-(CH2) n Q, where Q is -OR and n is selected from 1, 2, 3, 4, and 5. In a further embodiment, R 4 Ha-(CH2) n Q, where Q is -OR, where R is H, and n is selected from 1, 2, and 3. For example, R 4 is -(CH2)2OH.

[0358] In another embodiment, R 4 is the unsubstituted C 1-4 Alkyl, for example, unsubstituted methyl.

[0359] In another embodiment, R 4 is hydrogen.

[0360] In certain embodiments, the present disclosure provides compounds having formula (I), wherein R 4 Ha-(CH2) n Q or -(CH2) n CHQR, where Q is —N(R) 2 and n is selected from 3, 4, and 5.

[0361] In certain embodiments, the present disclosure provides compounds having formula (I), wherein R 4 is -(CH2) n Q, -(CH2) n and n is selected from the group consisting of -CHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2 and n is selected from 1, 2, 3, 4, and 5.

[0362] In certain embodiments, the present disclosure provides compounds having formula (I), wherein R 2 and R 3 independently, C 2-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring, and R 4 Ha-(CH2) n Q or -(CH2) n CHQR, where Q is —N(R) 2 and n is selected from 3, 4, and 5.

[0363] In certain embodiments, R 2 and R 3 independently, C 2-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocycle or a carbocycle. 2 and R 3 independently, C 2-14Alkyl and C 2-14 In some embodiments, R is selected from the group consisting of alkenyl. 2 and R 3 are independently selected from the group consisting of -R*YR", -YR", and -R*OR". In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring.

[0364] In some embodiments, R 1 is C 5-20 Alkyl and C 5-20 In some embodiments, R is selected from the group consisting of alkenyl. 1 is a hydroxyl-substituted C 5-20 is.

[0365] In other embodiments, R 1 is selected from the group consisting of -R*YR", -YR", and -R"M'R'.

[0366] In certain embodiments, R 1 is selected from -R*YR" and -YR". In some embodiments, Y is a cyclopropyl group. In some embodiments, R* is a C8 alkyl or C8 alkenyl. In certain embodiments, R" is a C 3-12 For example, R" may be a C alkyl. For example, R" may be a C 4-8 It may be alkyl (e.g., C4, C5, C6, C7, or C8 alkyl).

[0367] In some embodiments, R is (CH) q OR*, where q is selected from 1, 2, and 3, and R* is a C substituted with one or more substituents selected from the group consisting of amino, C1-C6 alkylamino, and C1-C6 dialkylamino. 1-12 For example, R is (CH2) q OR*, q is selected from 1, 2, and 3, and R* is a C substituted with C-C dialkylamino. 1-12For example, R is (CH2) q OR*, q is selected from 1, 2, and 3, and R* is a C substituted with C-C dialkylamino. 1-3 For example, R is (CH2) q OR*, q is selected from 1, 2, and 3, and R* is a C substituted with dimethylamino (e.g., dimethylaminoethanyl). 1-3 It is alkyl.

[0368] In some embodiments, R 1 is C 5-20 In some embodiments, R 1 is C alkyl. In some embodiments, R 1 is C alkyl. In other embodiments, R 1 is C alkyl. In certain embodiments, R 1 is C 14 In another embodiment, R 1 is C 18 It is alkyl.

[0369] In some embodiments, R 1 is C 21-30 In some embodiments, R 1 is C 26 In some embodiments, R 1 is C 28 In certain embodiments, R 1 teeth [ka] is.

[0370] In some embodiments, R 1 is C 5-20 In certain embodiments, R is alkenyl. 1 is C 18 In some embodiments, R is alkenyl. 1 is linoleyl.

[0371] In certain embodiments, R1 is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, or heptadec-9-yl). 1 teeth [ka] is.

[0372] In certain embodiments, R 1 is the unsubstituted C 5-20 Alkyl or C 5-20 In certain embodiments, R' is a substituted C 5-20 Alkyl or C 5-20 Alkenyl (e.g., C 3-6 substituted with a carbocycle, e.g., 1-cyclopropylnonyl, or substituted with OH or alkoxy). For example, R 1 teeth [ka] is.

[0373] In other embodiments, R 1 is -R"M'R'. In certain embodiments, M' is -OC(O)-M"-C(O)O-. For example, R 1 teeth [ka] where x 1 is an integer between 1 and 13 (e.g., selected from 3, 4, 5, and 6), and x 2 is an integer between 1 and 13 (e.g., selected from 1, 2, and 3), and x 3 is an integer between 2 and 14 (e.g., selected from 4, 5, and 6). For example, x 1 is selected from 3, 4, 5, and 6, and x 2is selected from 1, 2, and 3, and x 3 is selected from 4, 5, and 6.

[0374] In other embodiments, R 1 is -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 is different.

[0375] In some embodiments, R' is selected from -R*YR" and -YR". In some embodiments, Y is C 3-8 In some embodiments, Y is C 6-10 In some embodiments, Y is aryl. In some embodiments, Y is a cyclopropyl group. In some embodiments, Y is a cyclohexyl group. In certain embodiments, R* is C alkyl.

[0376] In some embodiments, R″ is C 3-12 Alkyl and C 3-12 In some embodiments, R" is selected from the group consisting of alkenyl. In some embodiments, R" is C8 alkyl. In some embodiments, R" adjacent to Y is C1 alkyl. In some embodiments, R" adjacent to Y is C 4-9 alkyl (e.g., C4, C5, C6, C7, or C8, or C9 alkyl).

[0377] In some embodiments, R″ is a substituted C 3-12 (e.g., hydroxyl-substituted C 3-12 For example, R" is [ka] is.

[0378] In some embodiments, R' is selected from C4 alkyl and C4 alkenyl. In certain embodiments, R' is selected from C5 alkyl and C5 alkenyl. In some embodiments, R' is selected from C6 alkyl and C6 alkenyl. In some embodiments, R' is selected from C7 alkyl and C7 alkenyl. In some embodiments, R' is selected from C9 alkyl and C9 alkenyl.

[0379] In some embodiments, R' is a C4 alkyl, a C4 alkenyl, a C5 alkyl, a C5 alkenyl, a C6 alkyl, a C6 alkenyl, a C7 alkyl, a C7 alkenyl, a C9 alkyl, a C9 alkenyl, a C 11 Alkyl, C 11 Alkenyl, C 17 Alkyl, C 17 Alkenyl, C 18 Alkyl, and C 18 alkenyl, each of which is linear or branched.

[0380] In some embodiments, R' is linear. In some embodiments, R' is branched.

[0381] In some embodiments, R' is [ka] In some embodiments, R' is [ka] and M' is -OC(O)-. In other embodiments, R' is [ka] and M' is -C(O)O-.

[0382] In other embodiments, R' is C 11 Alkyl and C 11 In other embodiments, R' is selected from C 12 Alkyl, C12 Alkenyl, C 13 Alkyl, C 13 Alkenyl, C 14 Alkyl, C 14 Alkenyl, C 15 Alkyl, C 15 Alkenyl, C 16 Alkyl, C 16 Alkenyl, C 17 Alkyl, C 17 Alkenyl, C 18 Alkyl, and C 18 In certain embodiments, R' is selected from linear C 4-18 Alkyl or C 4-18 In certain embodiments, R' is branched (e.g., decan-2-yl, undecane-3-yl, dodecane-4-yl, tridecane-5-yl, tetradecane-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, or heptadec-9-yl). In certain embodiments, R' is [ka] is.

[0383] In certain embodiments, R' is an unsubstituted C 1-18 In certain embodiments, R' is a substituted C 1-18 Alkyl (e.g., substituted C 1-15 alkyl, for example, alkoxy, for example, methoxy, or C 3-6 For example, R' is a carbocycle, such as 1-cyclopropylnonyl, or substituted with C(O)O-alkyl or OC(O)-alkyl, such as C(O)OCH3 or OC(O)CH3. [ka] is.

[0384] In certain embodiments, R' is a branched C 1-18For example, R' is: [ka] is.

[0385] In some embodiments, R″ is C 3-15 Alkyl and C 3-15 In some embodiments, R" is selected from the group consisting of C alkyl, C alkyl, C alkyl, C alkyl, C alkyl, or C alkyl. In some embodiments, R" is selected from the group consisting of C alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, or C 15 It is alkyl.

[0386] In some embodiments, M' is -C(O)O-. In some embodiments, M' is -OC(O)-. In some embodiments, M' is -OC(O)-M"-C(O)O-.

[0387] In some embodiments, M' is -C(O)O-, -OC(O)-, or -OC(O)-M"-C(O)O-. In some embodiments where M' is -OC(O)-M"-C(O)O-, M" is C 1-4 Alkyl or C 2-4 It is alkenyl.

[0388] In other embodiments, M' is an aryl or heteroaryl group. For example, M' may be selected from the group consisting of phenyl, oxazole, and thiazole.

[0389] In some embodiments, M is -C(O)O-. In some embodiments, M is -OC(O)-. In some embodiments, M is -C(O)N(R')-. In some embodiments, M is -P(O)(OR')O-. In some embodiments, M is -OC(O)-M"-C(O)O-.

[0390] In some embodiments, M is -C(O). In some embodiments, M is -OC(O)- and M' is -C(O)O-. In some embodiments, M is -C(O)O- and M' is -OC(O)-. In some embodiments, M and M' are each -OC(O)-. In some embodiments, M and M' are each -C(O)O-.

[0391] In other embodiments, M is an aryl or heteroaryl group. For example, M may be selected from the group consisting of phenyl, oxazole, and thiazole.

[0392] In some embodiments, M is the same as M'. In other embodiments, M is different from M'.

[0393] In some embodiments, M" is a bond. In some embodiments, M" is C 1-13 Alkyl or C 2-13 In some embodiments, M" is C 1-6 Alkyl or C 2-6 In certain embodiments, M" is a linear alkyl or alkenyl. In certain embodiments, M" is branched, e.g., -CH(CH3)CH2-.

[0394] In some embodiments, each R 5 is H. In some embodiments, each R 6 is H. In certain such embodiments, each R 5 and each R 6 is H.

[0395] In some embodiments, R 7 is H. In other embodiments, R 7 is C 1-3 alkyl (for example, methyl, ethyl, propyl, or I-propyl).

[0396] In some embodiments, R 2 and R3 independently, C 5-14 Alkyl or C 5-14 It is alkenyl.

[0397] In some embodiments, R 2 and R 3 are the same. In some embodiments, R 2 and R 3 is C alkyl. In certain embodiments, R 2 and R 3 is C alkyl. In other embodiments, R 2 and R 3 is a C alkyl. In some embodiments, R 2 and R 3 is C4 alkyl. In certain embodiments, R 2 and R 3 is C5 alkyl. In other embodiments, R 2 and R 3 is C alkyl. In some embodiments, R 2 and R 3 is a C7 alkyl.

[0398] In other embodiments, R 2 and R 3 In certain embodiments, R 2 is C alkyl. In some embodiments, R 3 is C 1-7 (e.g., C1, C2, C3, C4, C5, C6, or C7 alkyl) or C9 alkyl.

[0399] In some embodiments, R 3 is C alkyl. In some embodiments, R 3 is C alkyl. In some embodiments, R 3 is C alkyl. In some embodiments, R 3 is C4 alkyl. In some embodiments, R 3 is a C5 alkyl. In some embodiments, R 3 is C alkyl. In some embodiments, R 3is a C7 alkyl. In some embodiments, R 3 is a C9 alkyl.

[0400] In some embodiments, R 7 and R 3 is H.

[0401] In certain embodiments, R 2 is H.

[0402] In some embodiments, m is 5, 6, 7, 8, or 9. In some embodiments, m is 5, 7, or 9. For example, in some embodiments, m is 5. For example, in some embodiments, m is 7. For example, in some embodiments, m is 9.

[0403] In some embodiments, R 4 is -(CH2) n Q and -(CH2) n Selected from CHQR.

[0404] In some embodiments, Q is —OR, —OH, —O(CH) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N( R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -C(R)N(R)2C(O)OR, -N(R)S(O)2R 8 , carbocycle, and heterocycle.

[0405] In certain embodiments, Q is —N(R)R 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9)N(R)2, -OC(O)N(R)2, or -N(R)C(O)OR.

[0406] In certain embodiments, Q is —N(OR)C(O)R, —N(OR)S(O)R, —N(OR)C(O)OR, —N(OR)C(O)N(R), —N(OR)C(S)N(R), —N(OR)C(═NR 9 )N(R)2, or -N(OR)C(=CHR 9 )N(R)2.

[0407] In certain embodiments, Q is a thiourea or an isostere thereof, e.g., [ka] or -NHC(=NR 9 )N(R)2.

[0408] In certain embodiments, Q is —C(═NR 9 )N(R)2. For example, Q is -C(=NR 9 )N(R)2, n is 4 or 5. For example, R 9 is -S(O)2N(R)2.

[0409] In certain embodiments, Q is —C(═NR 9 )R or —C(O)N(R)OR, for example, —CH(═N—OCH3), —C(O)NH—OH, —C(O)NH—OCH3, —C(O)N(CH3)—OH, or —C(O)N(CH3)—OCH3.

[0410] In certain embodiments, Q is —OH.

[0411] In certain embodiments, Q is a substituted or unsubstituted 5-10 membered heteroaryl, e.g., Q is triazole, imidazole, pyrimidine, purine, 2-amino-1,9-dihydro-6H-purin-6-on-9-yl (or guanin-9-yl), adenin-9-yl, cytosin-1-yl, or uracil-1-yl, each of which is optionally substituted with one or more substituents selected from alkyl, OH, alkoxy, -alkyl-OH, and -alkyl-O-alkyl, which may be further substituted. In certain embodiments, Q is substituted with a 5-14 membered heterocycloalkyl, e.g., oxo (=O), OH, amino, mono- or di-alkylamino, and C 1-3 and alkyl. For example, Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, isoindolin-2-yl-1,3-dione, pyrrolidin-1-yl-2,5-dione, or imidazolidin-3-yl-2,4-dione.

[0412] In certain embodiments, Q is —NHR 8 where R 8 is oxo (=O), amino (NH2), mono- or di-alkylamino, C 1-3 C optionally substituted with one or more substituents selected from alkyl and halo 3-6 Cycloalkyl. For example, R 8 is cyclobutenyl, for example, 3-(dimethylamino)-cyclobut-3-en-4-yl-1,2-dione. In a further embodiment, R 8 is oxo (=O), thio (=S), amino (NH2), mono- or di-alkylamino, C 1-3 C optionally substituted with one or more substituents selected from alkyl, heterocycloalkyl, and halo 3-6 cycloalkyl, where mono- or di-alkylamino, C 1-3 Alkyl and heterocycloalkyl may be further substituted. For example, R 8is cyclobutenyl substituted with one or more of oxo, amino, and alkylamino, where alkylamino is, for example, C 1-3 Further substituted with one or more of alkoxy, amino, mono- or di-alkylamino, and halo. For example, R 8 is 3-(((dimethylamino)ethyl)amino)cyclobut-3-enyl-1,2-dione. For example, R 8 is cyclobutenyl substituted with one or more of oxo and alkylamino. For example, R 8 is 3-(ethylamino)cyclobut-3-ene-1,2-dione. For example, R 8 is cyclobutenyl substituted with one or more of oxo, thio, and alkylamino. For example, R 8 is 3-(ethylamino)-4-thioxocyclobut-2-en-1-one or 2-(ethylamino)-4-thioxocyclobut-2-en-1-one. For example, R 8 is cyclobutenyl substituted with one or more of thio and alkylamino. For example, R 8 is 3-(ethylamino)cyclobut-3-ene-1,2-dithione. For example, R 8 is cyclobutenyl substituted with one or more of oxo and dialkylamino. For example, R 8 is 3-(diethylamino)cyclobut-3-ene-1,2-dione. For example, R 8 is cyclobutenyl substituted with one or more of oxo, thio, and dialkylamino. For example, R 8 is 2-(diethylamino)-4-thioxocyclobut-2-en-1-one or 3-(diethylamino)-4-thioxocyclobut-2-en-1-one. For example, R 8 is cyclobutenyl substituted with one or more of thio and dialkylamino. For example, R 8 is 3-(diethylamino)cyclobut-3-ene-1,2-dithione. For example, R 8is cyclobutenyl substituted with one or more of oxo and alkylamino or dialkylamino, where the alkylamino or dialkylamino is further substituted, for example, with one or more alkoxy. For example, R 8 is 3-(bis(2-methoxyethyl)amino)cyclobut-3-ene-1,2-dione. For example, R 8 is cyclobutenyl substituted with one or more of oxo and heterocycloalkyl. For example, R 8 is cyclobutenyl substituted with one or more of oxo, piperidinyl, piperazinyl, or morpholinyl. For example, R 8 is cyclobutenyl substituted with one or more of oxo and heterocycloalkyl, where heterocycloalkyl is, for example, one or more C 1-3 Further substituted with alkyl. For example, R 8 is cyclobutenyl substituted with one or more of oxo and heterocycloalkyl, where the heterocycloalkyl (eg, piperidinyl, piperazinyl, or morpholinyl) is further substituted with methyl.

[0413] In certain embodiments, Q is —NHR 8 where R 8 is amino (NH2), mono- or di-alkylamino, C 1-3 and heteroaryl optionally substituted with one or more substituents selected from alkyl, and halo. For example, R 8 is thiazole or imidazole.

[0414] In certain embodiments, Q is —NHC(═NR 9 )N(R)2, where R 9 CN, C 1-6 alkyl, NO, -S(O)N(R), -OR, -S(O)R, or H. For example, Q is -NHC(=NR 9 )N(CH3)2, -NHC(=NR 9 )NHCH3, -NHC(=NR 9)NH2. In some embodiments, Q is -NHC(=NR 9 )N(R)2, where R 9 is CN, and R is C substituted with mono- or di-alkylamino. 1-3 In some embodiments, Q is -NHC(=NR 9 )N(R)2, where R 9 is C 1-6 alkyl, NO, -S(O)N(R), -OR, -S(O)R, or H, where R is a C substituted with mono- or di-alkylamino. 1-3 alkyl, for example, R is ((dimethylamino)ethyl)amino.

[0415] In certain embodiments, Q is —NHC(═CHR 9 )N(R)2, where R 9 are NO2, CN, C 1-6 alkyl, -S(O)2N(R)2, -OR, -S(O)2R, or H. For example, Q is -NHC(=CHR 9 )N(CH3)2, -NHC(=CHR 9 )NHCH3, or -NHC(=CHR 9 )NH2.

[0416] In certain embodiments, Q is -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)OR, e.g., -OC(O)NHCH, -N(OH)C(O)OCH, -N(OH)C(O)CH, -N(OCH)C(O)OCH, -N(OCH)C(O)CH, -N(OH)S(O)CH, or -NHC(O)OCH.

[0417] In certain embodiments, Q is —N(R)C(O)R, where R is C 1-3 Alkoxy or S(O) z C 1-3 alkyl optionally substituted with alkyl, where z is 0, 1, or 2.

[0418] In certain embodiments, Q is unsubstituted or substituted C 6-10 Aryl (e.g., phenyl) or C 3-6 It is cycloalkyl.

[0419] In some embodiments, n is 1. In other embodiments, n is 2. In further embodiments, n is 3. In certain embodiments, n is 4. For example, R 4 may be -(CH2)2OH. For example, R 4 may be -(CH2)3OH. For example, R 4 may be -(CH2)4OH. For example, R 4 may be benzyl. For example, R 4 may be 4-methoxybenzyl.

[0420] In some embodiments, R 4 is C 3-6 In some embodiments, R 4 is C 3-6 Cycloalkyl. For example, R 4 For example, OH, halo, C 1-6 It may be cyclohexyl optionally substituted with alkyl, etc. For example, R 4 may be 2-hydroxycyclohexyl.

[0421] In some embodiments, R is H.

[0422] In some embodiments, R is a C substituted with mono- or di-alkylamino. 1-3 alkyl, for example, R is ((dimethylamino)ethyl)amino.

[0423] In some embodiments, R is C 1-3 C substituted with one or more substituents selected from the group consisting of alkoxy, amino, and C1-C3 dialkylamino 1-6 It is alkyl.

[0424] In some embodiments, R is unsubstituted C 1-3 Alkyl or unsubstituted C 2-3 alkenyl. For example, R 4 may be —CH2CH(OH)CH3, —CH(CH3)CH2OH, or —CH2CH(OH)CH2CH3.

[0425] In some embodiments, R is a substituted C 1-3 alkyl, for example, CHOH. For example, R 4 may be -CH2CH(OH)CH2OH, -(CH2)3NHC(O)CH2OH, -(CH2)3NHC(O)CH2OBn, -(CH2)2O(CH2)2OH, -(CH2)3NHCH2OCH3, -(CH2)3NHCH2OCH2CH3, CH2SCH3, CH2S(O)CH3, CH2S(O)2CH3, or -CH(CH2OH)2.

[0426] In some embodiments, R 4 is selected from any of the following groups: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0427] In some embodiments, [ka] is selected from any of the following groups: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0428] In some embodiments, R 4 is selected from any of the following groups: [ka] [ka]

[0429] In some embodiments, [ka] is selected from any of the following groups: [ka]

[0430] In some embodiments, the compound of Formula (III) further comprises an anion. As described herein, the anion can be any anion capable of reacting with an amine to form an ammonium salt. Examples include, but are not limited to, chloride, bromide, iodide, fluoride, acetate, formate, trifluoroacetate, difluoroacetate, trichloroacetate, and phosphate.

[0431] In some embodiments, compounds of any of the formulae described herein are suitable for making nanoparticle compositions for intramuscular administration.

[0432] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a heterocycle or a carbocycle. 2 and R 3 together with the atoms to which they are attached form a 5-14 membered aromatic or non-aromatic heterocycle having one or more heteroatoms selected from N, O, S, and P. In some embodiments, R 2 and R 3 together with the atoms to which they are attached, can be either aromatic or non-aromatic, optionally substituted C 3-20 Carbocyclic rings (e.g., C 3-18 carbocycle, C 3-15 carbocycle, C 3-12 Carbocyclic, or C 3-10 In some embodiments, R 2 and R 3 together with the atoms to which they are attached, C 3-6 In another embodiment, R 2 and R 3taken together with the atoms to which they are attached form a C6 carbocyclic ring, such as a cyclohexyl or phenyl group. In certain embodiments, a heterocycle or C 3-6 The carbocycle may be substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms). For example, R 2 and R 3 may, together with the atom to which they are attached, form a cyclohexyl or phenyl group having one or more C alkyl substitutions. In certain embodiments, R 2 and R 3 or C 3-6 The carbocycle is substituted with a carbocyclic group, e.g., R 2 and R 3 may be taken together with the atoms to which they are attached to form a cyclohexyl or phenyl group substituted with cyclohexyl. 2 and R 3 together with the atoms to which they are attached form a C group such as a cycloheptyl, cyclopentadecanyl, or naphthyl group. 7-15 Forms a carbocyclic ring.

[0433] In some embodiments, R 4 is -(CH2) n Q and -(CH2) n In some embodiments, Q is selected from -OR, -OH, -O(CH) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(R)S(O)2R 8 , -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), and heterocycle. In other embodiments, Q is selected from the group consisting of imidazole, pyrimidine, and purine.

[0434] In some embodiments, R2 and R 3 together with the atoms to which they are attached form a heterocycle or a carbocycle. 2 and R 3 together with the atoms to which they are attached, C 3-6 In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a C6 carbocyclic ring. 2 and R 3 together with the atoms to which they are attached form a phenyl group. In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a cyclohexyl group. In some embodiments, R 2 and R 3 taken together with the atoms to which they are attached form a heterocycle. In certain embodiments, a heterocycle or C 3-6 The carbocycle may be substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms). For example, R 2 and R 3 may be taken together with the atom to which they are attached to form a phenyl group having one or more C5 alkyl substitutions.

[0435] In some embodiments, R 5 and R 6 At least one occurrence of C 1-3 In some embodiments, the R adjacent to M is an alkyl, such as methyl. 5 and R 6 One of them is C 1-3 alkyl, e.g., methyl, and the other is H. In some embodiments, the R adjacent to M is 5 and R 6 One of them is C 1-3 alkyl, such as methyl, and the other is H, and M is -OC(O)- or -C(O)O-.

[0436] In some embodiments, R5 and R 6 At most one occurrence of C 1-3 In some embodiments, the R adjacent to M is an alkyl, such as methyl. 5 and R 6 One of them is C 1-3 alkyl, e.g., methyl, and the other is H. In some embodiments, the R adjacent to M is 5 and R 6 One of them is C 1-3 alkyl, such as methyl, and the other is H, and M is -OC(O)- or -C(O)O-.

[0437] In some embodiments, R 5 and R 6 At least one occurrence of is methyl.

[0438] The compounds of any one of formula (VI), (VI-a), (VII), (VIIa), (VIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIII), (VIIIa), (VIIIb), (VIIIc), or (VIIId) comprise one or more of the following features, as applicable:

[0439] In some embodiments, r is 0. In some embodiments, r is 1.

[0440] In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 4. In some embodiments, n is not 3.

[0441] In some embodiments, R N is H. In some embodiments, R N is C 1-3 For example, in some embodiments, R N is C alkyl. For example, in some embodiments, R N is C alkyl. For example, in some embodiments, R Nis a C2 alkyl.

[0442] In some embodiments, X a is O. In some embodiments, X a is S. In some embodiments, X b is O. In some embodiments, X b is S.

[0443] In some embodiments, R 10 is N(R)2, -NH(CH2) t1 N(R)2, -NH(CH2) p1 O(CH2) q1 N(R)2, -NH(CH2) s1 OR, -N((CH2) s1 OR)2, and heterocycles.

[0444] In some embodiments, R 10 is -NH(CH2) t1 N(R)2, -NH(CH2) p1 O(CH2) q1 N(R)2, -NH(CH2) s1 OR, -N((CH2) s1 OR)2, and heterocycles.

[0445] R 10 -NH(CH2) o In some embodiments where N(R)2, o is 2, 3, or 4.

[0446] -NH(CH2) p1 O(CH2) q1 In some embodiments where N(R)2, p 1 is 2. -NH(CH2) p1 O(CH2) q1 In some embodiments where N(R), q 1 is 2.

[0447] R 10 -N((CH2) s1 In some embodiments, s1 is 2.

[0448] R 10 -NH(CH2) o N(R)2, -NH(CH2) p O(CH2) q N(R)2, -NH(CH2) s OR, or -N((CH2) s In some embodiments where R is (OR), R is H or C1-C3 alkyl. For example, in some embodiments, R is C1 alkyl. For example, in some embodiments, R is C2 alkyl. For example, in some embodiments, R is H. For example, in some embodiments, R is H and one R is C1-C3 alkyl. For example, in some embodiments, R is H and one R is C1 alkyl. For example, in some embodiments, R is H and one R is C2 alkyl. R 10 -NH(CH2) t1 N(R)2, -NH(CH2) p1 O(CH2) q1 N(R)2, -NH(CH2) s1 OR, or -N((CH2) s1 In some embodiments where OR)2, each R is C2-C4 alkyl.

[0449] For example, in some embodiments, one R is H and one R is C-C alkyl. 10 is a heterocycle. For example, in some embodiments, R 10 is morpholinyl. For example, in some embodiments, R 10 is methylpiperazinyl.

[0450] In some embodiments, R 5 and R 6 is H. In some embodiments, the compound of Formula (I) is selected from the group consisting of: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0451] In a further embodiment, the compound of formula (II) is selected from the group consisting of: [Table 2]

[0452] In some embodiments, the compound of Formula (II) or Formula (III) is selected from the group consisting of: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17]

[0453] In some embodiments, the lipids of the present disclosure comprise I-340A: [ka]

[0454] The central amine moiety of a lipid according to formula (II), (I IA), I (IB), I (II), (I IIa), (I IIb), (I IIc), (I IId), (I IIe), (I IIf), (I IIg), (I IIh), (I IIj), (I IIk), (I III), (I VI), (I VI-a), (I VII), (I VIIa), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIII), (I VIIIa), (I VIIIb), (I VIIIc), (I VIIId), (I XI), (I XI-a), or (I XI-b) can be protonated at physiological pH. Thus, lipids can have a positive or partial positive charge at physiological pH. Such lipids can be called cationic or ionic (amino) lipids. Lipids can also be zwitterions, i.e., neutral molecules that have both positive and negative charges.

[0455] Ionizable lipids can contain a single enantiomer or a mixture of enantiomers in a specific ratio. In some embodiments, the ionizable lipids contain a substantially pure enantiomer. In some embodiments, a substantially pure enantiomer is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In some embodiments, the "S" form of the ionizable lipid is substantially free of the "R" form of the ionizable lipid and is thus in enantiomeric excess relative to the "R" form. In some embodiments, the "R" form of the ionizable lipid is substantially free of the "S" form of the ionizable lipid and is thus in enantiomeric excess relative to the "S" form. In some embodiments, "substantially free" refers to (i) an aliquot of the "R" form of the compound containing less than 2% of the "S" form, or (ii) an aliquot of the "S" form of the compound containing less than 2% of the "R" form. In some embodiments, the substantially pure enantiomer comprises more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, or more than 99.9% by weight of a single enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound. In one embodiment, the ionic lipid comprises a racemic mixture of the "S" and "R" forms.

[0456] In some embodiments, the ionic lipid comprises a racemic mixture of amino lipids. In some embodiments, the ionic lipid comprises a substantially pure enantiomer of the amino lipid. In some embodiments, the ionic lipid comprises a substantially pure (R) enantiomer of the amino lipid. In some embodiments, the ionic lipid comprises a substantially pure (S) enantiomer of the amino lipid. In some embodiments, the ionic lipid is selected from the group consisting of: (II), (I IA), (I IB), (I II), (I IIa), (I IIb), (I IIc), (I IId), (I IIe), (I IIf), (I IIg), (I IIh), (I IIj), (I IIk), (I III), (I VI), (I VI-a), (I VII), (I VIII), (I VIIa), (I VIIIa), (I VIIIb), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIIIc), (I VIIId), (I XI), (I XI-a), or (I XI-b) and / or a substantially pure enantiomer of a compound selected from the group consisting of compound I-49 and compound I-301.

[0457] In some embodiments, the ionic lipid comprises a substantially pure enantiomer of compound I-49. In some embodiments, the ionic lipid comprises substantially pure compound (S)I-49. [ka]

[0458] In some embodiments, the ionic lipid comprises substantially pure compound (R)I-49. [ka]

[0459] In some embodiments, the ionic lipid comprises a substantially pure enantiomer of compound I-301. In some embodiments, the ionic lipid comprises substantially pure compound (S)I-301. [ka]

[0460] In some embodiments, the ionic lipid comprises substantially pure compound (R)I-301. [ka]

[0461] In some aspects, the ionic lipids of the present disclosure comprise one or more compounds of formula (I XII): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 40 is not a squaramide-substituted group, but a hydrogen, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is selected from the group consisting of -OR, -O(CH2), n N(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, -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9)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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9 )R, -C(O)N(R)OR, and -C(R)N(R)C(O)OR, each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; Each R is independently C 1-3 Alkyl, C 2-3 Alkenyl, (CH2) q R* and H, where q is independently selected from 1, 2, and 3; R* is independently selected from C 1-12 Alkyl, and C 2-12 alkenyl, Each R 9 are independently H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, or C 2-6 is selected from the group consisting of alkyl, R 10 are H, OH, and C 1-3 Alkyl, and C 2-3 alkenyl, X is independently selected from the group consisting of F, Cl, Br, and I.

[0462] In some embodiments, R 40 is not a squaramide substituted group. In some embodiments, R 40 is hydrogen, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2, and unsubstituted C 1-6alkyl, where Q is selected from the group consisting of -OR, -O(CH2), n N(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, -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9 )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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9 )R, —C(O)N(R)OR, and —C(R)N(R)C(O)OR, where each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5.

[0463] In some aspects, the ionic lipids of the present disclosure comprise one or more compounds of formula (I IX): [ka] or a salt or isomer thereof, wherein: W is [ka] and Ring A is [ka] and t is 1 or 2, A1 and A2 are each independently selected from CH or N; Z is CH2 or absent, where when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is absent, the dashed lines (1) and (2) are both absent; R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; R X1 and R X2 are each independently H or C alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group; M* is C1-C6 alkyl; W 1 and W 2 are each independently selected from the group consisting of —O— and —N(R6)—; Each R6 is independently H and C 1-5 is selected from the group consisting of alkyl, X 1 , X 2 , and X 3 are independently a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, or -(CH2) n -C(O)-, -C(O)-(CH2) n -, -(CH2) n -C(O)O-, -OC(O)-(CH2) n -, -(CH2) n -OC(O)-, -C(O)O-(CH2) n is selected from the group consisting of -, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3-6 is a carbocyclic ring, Each R* is independently C 1-12Alkyl and C 2-12 alkenyl, Each R is independently C 1-3 Alkyl and C 3-6 is selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 selected from the group consisting of alkenyl, and H; Each R" is independently C 3-12 Alkyl, C 3-12 alkenyl, and -R*MR'; n is an integer from 1 to 6, Here, ring A is [ka] When i)X 1 , X 2 , and X 3 at least one of is not -CH2-, and / or ii) At least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0464] In some embodiments, the compound has any of formulas (I IXa1)-(I IXa8): [ka] is.

[0465] In some embodiments, the ionizable lipid is one or more of the compounds described in U.S. Application Nos. 62 / 271,146, 62 / 338,474, 62 / 413,345, and 62 / 519,826, and PCT Application No. PCT / US2016 / 068300.

[0466] In some embodiments, the ionizable lipid is selected from compounds 1-156 described in US Application No. 62 / 519,826.

[0467] In some embodiments, the ionic lipid is selected from compounds 1-16, 42-66, 68-76, and 78-156 described in US Application No. 62 / 519,826.

[0468] In some embodiments, the ionic lipid is [ka] (Compound I-356 (also referred to herein as Compound M), or a salt thereof.

[0469] In some embodiments, the ionic lipid is [ka] [Compound IN], or a salt thereof.

[0470] In some embodiments, the ionic lipid is [ka] [Compound IO], or a salt thereof.

[0471] In some embodiments, the ionic lipid is [ka] [Compound IP], or a salt thereof.

[0472] In some embodiments, the ionic lipid is [ka] [Compound IQ], or a salt thereof.

[0473] The central amine moiety of a lipid according to any of the formulae herein, for example, a compound having any of formulas (II), (IIIA), (IIIB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), can be protonated at physiological pH. Thus, lipids can have a positive or partial positive charge at physiological pH. Such lipids can be called cationic or ionic (amino) lipids. Lipids can also be zwitterions, i.e., neutral molecules that have both positive and negative charges.

[0474] In some embodiments, the amount of ionizable amino lipids of the present invention, e.g., compounds having any of formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), is in the range of about 1 mol% to 99 mol% in the lipid composition.

[0475] In one embodiment, the amount of an ionizable amino lipid of the present invention, e.g., a compound having any of formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), is At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 mol%.

[0476] In one embodiment, the ionic amino lipids of the present invention are selected from the group consisting of ionic amino lipids of formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIb-6), (VIIb-7), (VIIb-8), (VIIb-9), (VIIb-10), (VIIb-11), (VIIb-12), (VIIb-13), (VIIb-14), (VIIb-15), (VIIb-16), (VIIb-17), (VIIb-18), (VIIb-19), (VIIb-20), (VIIb-21), (VIIb-22), (VIIb-23), (VIIb-24), (VIIb-25), (VIIb-26), (VIIb-27), (VIIb-28), (VIIb-29 ... The amount of compounds having any of (IIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity) in the lipid composition ranges from about 30 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 40 mol% to about 60 mol%, and about 45 mol% to about 55 mol%.

[0477] In one embodiment, the amount of an ionizable amino lipid of the present invention, e.g., a compound having any of the formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (I VIIb-4), (I VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), is about 45 mol% in the lipid compound.

[0478] In one embodiment, the amount of an ionizable amino lipid of the present invention, e.g., a compound having any of the formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), is about 40 mol% in the lipid compound.

[0479] In one embodiment, the amount of an ionizable amino lipid of the present invention, e.g., a compound having any of the formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity), is about 50 mol% in the lipid compound.

[0480] Ionic aminolipids disclosed herein, e.g., formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk) , (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VI In addition to compounds having any of the following structures (each of which is preceded by the letter I for clarity): (Ic), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b), the lipid-based compositions (e.g., lipid nanoparticles) disclosed herein can include additional components such as cholesterol and / or cholesterol analogs, non-cationic helper lipids, structural lipids, PEG-lipids, and any combination thereof.

[0481] Additional ionic lipids of the present invention include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMAC ... ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), (13Z ,165Z)-N,N-dimethyl-3-nonidocosa-13-16-dien-1-amine (L608), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl The ionic amino lipid may be selected from the non-limiting group consisting of N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)). In addition, the ionic amino lipid may also be a lipid containing a cyclic amine group.

[0482] The ionic amino lipids of the present invention can also be compounds disclosed in International Publication No. WO2017 / 075531A1, which is incorporated herein by reference in its entirety. For example, ionic amino lipids include, but are not limited to: [ka] and any combination thereof.

[0483] The ionic amino lipids of the present invention can also be compounds disclosed in International Publication No. WO2017 / 199952A1, which is incorporated herein by reference in its entirety.For example, ionic amino lipids include, but are not limited to: [ka] [ka] and any combination thereof.

[0484] In any of the foregoing or related embodiments, the ionizable lipids of the LNPs of the present disclosure are included in any compound having any of the following formulas: (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (III), (VI), (VI-a), (VII), (VIII), (VIIa), (VIIIa), (VIIIb), (VIIb-1), (VIIb-2), (VIIb-3), (VIIb-4), (VIIb-5), (VIIc), (VIId), (VIIIc), (VIIId), (XI), (XI-a), or (XI-b) (each of which is preceded by the letter I for clarity).

[0485] In any of the foregoing or related embodiments, the ionizable lipids of the LNPs of the present disclosure include compounds including any of Compound Nos. I 1-356.

[0486] In any of the foregoing or related aspects, the ionizable lipids of the LNPs of the present disclosure comprise at least one compound selected from the group consisting of Compound Nos. I 18 (also referred to as Compound X), I 48, I 49, I 50, I 182, I 184, I 292, I 301, I 309, I 317, I 321, I 326, I 347, I 348, I 349, I 350, and I 352. In another embodiment, the ionizable lipids of the LNPs of the present disclosure comprise a compound selected from the group consisting of Compound Nos. I 18 (also referred to as Compound X), I 49, I 182, I 184, I 301, and I 321. In another embodiment, the ionizable lipids of the LNPs of the present disclosure comprise a compound selected from the group consisting of Compound Nos. I 49 and I 301.

[0487] In any of the foregoing or related embodiments, synthesis of compounds of the invention, e.g., compounds including any of Compound Nos. 1-356, follows the synthesis instructions in U.S. Provisional Patent Application No. 62 / 733,315, filed September 19, 2018. In some embodiments, formulas (II), (I IA), (I IB), (I II), (I IIa), (I IIb), (I IIc), (I IId), (I IIe), (I IIf), (I IIg), (I IIh), (I IIj), (I IIk), (I III), (I VI), (I VI-a), (I VII), (I VIIa), (I VIIb-1), (I VIIb-2), (I VIIb-3), (I VIIb-4), (I VIIb-5), (I VIIc), (I VIId), (I VIII), (I VIIIa), (I VIIIb), (I VIIIc), (I VIIId), (I XI), (I XI-a), or (I The synthesis of any of the compounds XI-b) (e.g., compound I-49 or compound I-301) may be prepared according to the general procedures described on pages 181, 190, and 191 of PCT / US2018 / 022717, which is incorporated herein by reference in its entirety.

[0488] Representative synthesis routes: Compound I-182: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione [ka] To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in 100 mL of diethyl ether, 2 M methylamine solution in THF (3.8 mL, 7.6 mmol) was added, resulting in almost instantaneous formation of a precipitate. The mixture was stirred at room temperature for 24 hours, then filtered, and the filter solid was washed with diethyl ether and air-dried. The filter solid was dissolved in hot EtOAc, filtered, and the filtrate was allowed to cool to room temperature and then 0.05 g of ethyl acetate. o Cooling to C gave a precipitate which was isolated by filtration, washed with cold EtOAc, air-dried, and then dried under vacuum to give 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.70 g, 5 mmol, 73%) as a white solid. 1 H NMR (300 MHz, DMSO-d6) δ: ppm 8.50 (br. d, 1H, J = 69 Hz); 4.27 (s, 3H); 3.02 (sdd, 3H, J = 42 Hz, 4.5 Hz).

[0489] Heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate [ka] To a solution of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.28 mmol) in 10 mL of ethanol was added 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (39 mg, 0.28 mmol), and the resulting colorless solution was stirred at room temperature for 20 h, after which time no starting amine remained by LC / MS. The solution was concentrated in vacuo and the residue was purified by silica gel chromatography (0-100% in dichloromethane (mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (138 mg, 0.17 mmol, 60%) as a sticky white solid. UPLC / ELSD: RT=3 min. MS (ES): C 51 H 95 m / z (MH + ) 833.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 7.86 (br. s., 1H);4.86 (quintet, 1H, J = 6 Hz);4.05 (t, 2H, J = 6 Hz);3.92 (d, 2H, J = 3 Hz);3.20 (s, 6H);2.63 (br. s, 2H);2.42 (br. s, 3H);2.28 (m, 4H);1.74 (br. s, 2H);1.61 (m, 8H);1.50 (m, 5H);1.41 (m, 3H);1.25 (br. m, 47H);0.88 (t, 9H, J = 7.5 Hz).

[0490] Compound I-301: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate [ka] Compound I-301 was prepared similarly to compound 182, except that heptadecan-9-yl 8-((3-aminopropyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (500 mg, 0.66 mmol) was used instead of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate. After aqueous workup, the residue was purified by silica gel chromatography (0-50% in dichloromethane (mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (180 mg, 32%) as a waxy white solid. HPLC / UV (254 nm): RT = 6.77 min. MS (CI): C 52 H 97 m / z (MH + ) 860.7. 1 H NMR (300 MHz, CDCl3): δ ppm 4.86-4.79 (m, 2H);3.66 (bs, 2H);3.25 (d, 3H, J = 4.9 Hz);2.56-2.52 (m, 2H);2.42-2.37 (m, 4H);2.28 (dd, 4H, J = 2.7 Hz, 7.4 Hz);1.78-1.68 (m, 3H);1.64-1.50 (m, 16H);1.48-1.38 (m, 6H);1.32-1.18 (m, 43H);0.88-0.84 (m, 12H).

[0491] Compound I-49: Heptadecan-9-yl 8-((2-hydroxyethyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate [ka] Compound I-49 may be prepared according to the general procedures described on pages 181, 190, and 191 of PCT / US2018 / 022717, which is incorporated herein by reference in its entirety. UPLC / ELSD: RT = 3.68 min. MS (ES): C 46 H 91 m / z (MH + ) 739.21. 1 H NMR (300 MHz, CDCl3): δ ppm 4.89 (m, 2H); 3.56 (br. m, 2H); 2.68-2.39 (br. m, 5H); 2.30 (m, 4H); 1.71-1.19 (m, 66H); 0.90 (m, 12H).

[0492] (ii) Cholesterol / structural lipids The target cell delivery LNPs described herein comprise one or more structural lipids.

[0493] As used herein, the term "structured lipid" refers to sterol and also to lipids containing a sterol moiety. Incorporating structured lipids into lipid nanoparticles can help reduce aggregation of other lipids within the particles. Structured lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

[0494] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol. Examples of structured lipids include, but are not limited to, the following: [ka]

[0495] The target cell delivery LNPs described herein comprise one or more structural lipids.

[0496] As used herein, the term "structured lipid" refers to a sterol and also to a lipid containing a sterol moiety. Incorporation of a structured lipid into a lipid nanoparticle can help reduce aggregation of other lipids within the particle. In certain embodiments, the structured lipid comprises cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

[0497] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. Structured lipids can include, but are not limited to, sterols (e.g., phytosterols or zoosterols).

[0498] In certain embodiments, the structured lipid is a steroid. For example, the sterol can include, but is not limited to, cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, or any one of compounds S1-148 in Tables 1-16 herein.

[0499] In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog.

[0500] In certain embodiments, the structured lipid is alpha-tocopherol.

[0501] In one embodiment, the structured lipids of the present invention have the formula SI: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H, optionally substituted C1-C6 alkyl, or [ka] and R b1 , R b2 and R b3 each independently represents an optionally substituted C-C alkyl or an optionally substituted C-C 10 is aryl, R 2 is H or OR A where R Ais H or optionally substituted C1-C6 alkyl, R 3 is H or [ka] and each [ka] independently represent a single bond or a double bond; W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming L 1a does not exist or [ka] or [ka] and L 1b does not exist or [ka] or [ka] and m is 1, 2, or 3; L 1c does not exist or [ka] or [ka] and R 6 is an optionally substituted C3-C 10 Cycloalkyl, optionally substituted C-C 10 Cycloalkenyl, optionally substituted C-C 10 aryl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heteroaryl.

[0502] In some embodiments, the compound has formula SIa: [ka] or a pharmaceutically acceptable salt thereof.

[0503] In some embodiments, the compound has the formula SIb: [ka] or a pharmaceutically acceptable salt thereof.

[0504] In some embodiments, the compound has the formula SIc: [ka] or a pharmaceutically acceptable salt thereof.

[0505] In some embodiments, the compound has the formula SId: [ka] or a pharmaceutically acceptable salt thereof.

[0506] In some embodiments, L 1a is absent. In some embodiments, L 1a teeth, [ka] In some embodiments, L 1a teeth, [ka] is.

[0507] In some embodiments, L 1b is absent. In some embodiments, L 1b teeth, [ka] In some embodiments, L 1b teeth, [ka] is.

[0508] In some embodiments, m is 1 or 2. In some embodiments, m is 1. In some embodiments, m is 2.

[0509] In some embodiments, L 1c is absent. In some embodiments, L 1c teeth, [ka] In some embodiments, L 1c teeth, [ka] is.

[0510] In some embodiments, R 6 is an optionally substituted C6-C 10 It is aryl.

[0511] In some embodiments, R 6 teeth, [ka] where: n1 is 0, 1, 2, 3, 4, or 5; Each R 7 is independently halo, or optionally substituted C1-C6 alkyl.

[0512] In some embodiments, each R 7 is, independently, [ka] is.

[0513] In some embodiments, n1 is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n1 is 1. In some embodiments, n1 is 2.

[0514] In some embodiments, R 6 is an optionally substituted C3-C 10 It is cycloalkyl.

[0515] In some embodiments, R 6 is an optionally substituted C3-C 10 It is a monocycloalkyl.

[0516] In some embodiments, R 6 teeth, [ka] where: n2 is 0, 1, 2, 3, 4, or 5; n3 is 0, 1, 2, 3, 4, 5, 6, or 7; n4 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n5 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, n6 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, Each R 8 is independently halo, or optionally substituted C1-C6 alkyl.

[0517] In some embodiments, each R 8 is, independently, [ka] is.

[0518] In some embodiments, R 6 is an optionally substituted C3-C 10 It is a polycycloalkyl.

[0519] In some embodiments, R 6 teeth, [ka] is.

[0520] In some embodiments, R 6 is an optionally substituted C3-C 10 It is a cycloalkenyl.

[0521] In some embodiments, R 6 teeth, [ka] where: n7 is 0, 1, 2, 3, 4, 5, 6, or 7, n8 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, n9 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, Each R 9 is independently halo, or optionally substituted C1-C6 alkyl.

[0522] In some embodiments, R 6 teeth, [ka] is.

[0523] In some embodiments, each R 9 is, independently, [ka] is.

[0524] In some embodiments, R 6 is an optionally substituted C2-C9 heterocyclyl.

[0525] In some embodiments, R 6 teeth, [ka] where: n10 is 0, 1, 2, 3, 4, or 5, n11 is 0, 1, 2, 3, 4, or 5, n12 is 0, 1, 2, 3, 4, 5, 6, or 7; n13 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; Each R 10 is independently halo, or optionally substituted C-C alkyl; Y 1 and Y 2 each independently represents O, S, NR B , or CR 11a R 11b and where R B is H or optionally substituted C1-C6 alkyl; R 11a and R 11b each is independently H, halo, or optionally substituted C-C alkyl; Y 2 is CR 11a R 11b If so, then Y 1 is O, S, or NR B is.

[0526] In some embodiments, Y 1 is O.

[0527] In some embodiments, Y 2 is O. In some embodiments, Y 2 is CR 11a R 11b is.

[0528] In some embodiments, each R 10 is, independently, [ka]

[0529] In some embodiments, R 6 is an optionally substituted C2-C9 heteroaryl.

[0530] In some embodiments, R 6 teeth, [ka] where: Y 3 is NR C , O, or S; n14 is 0, 1, 2, 3, or 4; R C is H or optionally substituted C1-C6 alkyl; Each R 12is independently halo, or optionally substituted C1-C6 alkyl.

[0531] In some embodiments, R 6 teeth, [ka] In some embodiments, R 6 teeth, [ka] is.

[0532] In one embodiment, the structured lipids of the present invention have the formula SII: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4bwhere if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming L 1 is an optionally substituted C1-C6 alkylene; R 13a , R 13b , and R 13c each independently represents an optionally substituted C-C alkyl, or an optionally substituted C-C 10 It is aryl.

[0533] In some embodiments, the compound has formula SIIa: [ka] or a pharmaceutically acceptable salt thereof.

[0534] In some embodiments, the compound has formula SIIb: [ka] or a pharmaceutically acceptable salt thereof.

[0535] In some embodiments, L 1 teeth, [ka] is.

[0536] In some embodiments, R 13a , R 13b , and R 13c each of which independently [ka] is.

[0537] In one embodiment, the structured lipids of the present invention have the formula SIII: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H, or [ka] and each [ka] independently represent a single bond or a double bond; W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4aIf there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each independently represents H, halo, hydroxyl, optionally substituted C-C alkyl, —OS(O)R 4c where R 4c is an optionally substituted C1-C6 alkyl or an optionally substituted C6-C 10 is aryl, R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 14 is H or C1-C6 alkyl, R 15 teeth, [ka] where: R 16 is H or optionally substituted C1-C6 alkyl; R 17b H, OR 17c , optionally substituted C6-C 10 aryl, or optionally substituted C1-C6 alkyl; R 17c is H or optionally substituted C1-C6 alkyl; o1 is 0, 1, 2, 3, 4, 5, 6, 7, or 8, p1 is 0, 1, or 2; p2 is 0, 1, or 2; Z is CHO, S, or NR D where R Dis H or optionally substituted C1-C6 alkyl; Each R 18 is independently halo, or optionally substituted C1-C6 alkyl.

[0538] In some embodiments, the compound has formula SIIIa: [ka] or a pharmaceutically acceptable salt thereof.

[0539] In some embodiments, the compound has formula SIIIb: [ka] or a pharmaceutically acceptable salt thereof.

[0540] In some embodiments, R 14 H, [ka] is.

[0541] In some embodiments, R 14 teeth, [ka] is.

[0542] In some embodiments, R 15 teeth, [ka] In some embodiments, R 15 teeth, [ka] is.

[0543] In some embodiments, R 16is H. In some embodiments, R 16 teeth, [ka] is.

[0544] In some embodiments, R 17a is H. In some embodiments, R 17a is an optionally substituted C1-C6 alkyl.

[0545] In some embodiments, R 17b is H. In some embodiments, R 17b is an optionally substituted C1-C6 alkyl. In some embodiments, R 17b is OR 17c is.

[0546] In some embodiments, R 17c H, [ka] or [ka] In some embodiments, R 17c is H. In some embodiments, R 17c teeth, [ka] is.

[0547] In some embodiments, R 15 teeth, [ka] is.

[0548] In some embodiments, each R 18 is, independently, [ka] is.

[0549] In some embodiments, Z is CH. In some embodiments, Z is O. In some embodiments, Z is NR D is.

[0550] In some embodiments, o1 is 0, 1, 2, 3, 4, 5, or 6.

[0551] In some embodiments, o1 is 0. In some embodiments, o1 is 1. In some embodiments, o1 is 2. In some embodiments, o1 is 3. In some embodiments, o1 is 4. In some embodiments, o1 is 5. In some embodiments, o1 is 6.

[0552] In some embodiments, p1 is 0 or 1. In some embodiments, p1 is 0. In some embodiments, p1 is 1.

[0553] In some embodiments, p2 is 0 or 1. In some embodiments, p2 is 0. In some embodiments, p2 is 1.

[0554] In one embodiment, the structured lipids of the present invention have the formula SIV: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R Ais H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming s is 0 or 1, R 19 is H or C1-C6 alkyl, R 20 is C1-C6 alkyl, R 21 is H or C1-C6 alkyl.

[0555] In some embodiments, the compound has the formula SIVa: [ka] or a pharmaceutically acceptable salt thereof.

[0556] In some embodiments, the compound has the formula SIVb: [ka] or a pharmaceutically acceptable salt thereof.

[0557] In some embodiments, R 19 H, [ka] is.

[0558] In some embodiments, R 19 teeth, [ka] is.

[0559] In some embodiments, R 20 teeth, [ka] is.

[0560] In some embodiments, R 21 H, [ka] is.

[0561] In one embodiment, the structured lipids of the present invention have the formula SV: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 22 is H or C1-C6 alkyl, R 23 is halo, hydroxyl, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl.

[0562] In some embodiments, the compound has the formula SVa: [ka] or a pharmaceutically acceptable salt thereof.

[0563] In some embodiments, the compound has formula SVb: [ka] or a pharmaceutically acceptable salt thereof.

[0564] In some embodiments, R 22 H, [ka] is.

[0565] In some embodiments, R 22 teeth, [ka] is.

[0566] In some embodiments, R 23 teeth, [ka] is.

[0567] In one embodiment, the structured lipids of the present invention have the formula SVI: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 24 is H or C1-C6 alkyl, R 25a and R 25b Each of is C1-C6 alkyl.

[0568] In some embodiments, the compound has formula SVIa: [ka] or a pharmaceutically acceptable salt thereof.

[0569] In some embodiments, the compound has formula SVIb: [ka] or a pharmaceutically acceptable salt thereof.

[0570] In some embodiments, R 24 H, [ka] is.

[0571] In some embodiments, R 24 teeth, [ka] is.

[0572] In some embodiments, R 25a and R 25b each of which independently [ka] is.

[0573] In one embodiment, the structured lipids of the present invention have the formula SVII: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, or [ka] where R1c , R 1d , and R 1e each independently represents an optionally substituted C-C alkyl, or an optionally substituted C-C 10 is aryl, X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming q is 0 or 1, R 26aand R 26b is independently H or an optionally substituted C-C alkyl, or R 26a and R 26b are each bonded to the atom, [ka] where R 26c and R 26 each is independently H or optionally substituted C1-C6 alkyl; R 27a and R 27b Each of is H, hydroxyl, or optionally substituted C1-C6 alkyl.

[0574] In some embodiments, the compound has formula SVIIa: [ka] or a pharmaceutically acceptable salt thereof.

[0575] In some embodiments, the compound has formula SVIIb: [ka] or a pharmaceutically acceptable salt thereof.

[0576] In some embodiments, R 26a and R 26b are independently H, [ka] is.

[0577] In some embodiments, R 26a and R 26b are each bonded to the atom, [ka] Form.

[0578] In some embodiments, R 26a and R 26b are each bonded to the atom, [ka] In some embodiments, R 26a and R 26b are each bonded to the atom, [ka] Form.

[0579] In some embodiments, R 26c and R 26 are independently H, [ka] is.

[0580] In some embodiments, R 27a and R 27b Each of is H, hydroxyl, or an optionally substituted C1-C3 alkyl.

[0581] In some embodiments, R 27a and R 27b each independently represents H, hydroxyl, [ka] is.

[0582] In one embodiment, the structured lipids of the present invention have the formula SVIII: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1ais an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 28 is H or optionally substituted C1-C6 alkyl; r is 1, 2, or 3; Each R 29 are independently H or optionally substituted C1-C6 alkyl; R 30a , R 30b , and R 30c Each of is C1-C6 alkyl.

[0583] In some embodiments, the compound has formula SVIIIa: [ka] or a pharmaceutically acceptable salt thereof.

[0584] In some embodiments, the compound has formula SVIIIb: [ka] or a pharmaceutically acceptable salt thereof.

[0585] In some embodiments, R 28 H, [ka] is.

[0586] In some embodiments, R 28 teeth, [ka] is.

[0587] In some embodiments, R 30a , R 30b , and R 30c each of which independently [ka] is.

[0588] In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3.

[0589] In some embodiments, each R 29 are independently H, [ka] is.

[0590] In some embodiments, each R 29 are independently H or [ka] is.

[0591] In one embodiment, the structured lipids of the present invention have the formula SIX: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 1b is H or optionally substituted C1-C6 alkyl; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 31 is H or C1-C6 alkyl, R 32a and R 32b Each of is C1-C6 alkyl.

[0592] In some embodiments, the compound has formula SIXa: [ka] or a pharmaceutically acceptable salt thereof.

[0593] In some embodiments, the compound has formula SIXb: [ka] or a pharmaceutically acceptable salt thereof.

[0594] In some embodiments, R 31 H, [ka] is.

[0595] In some embodiments, R 31 teeth, [ka] is.

[0596] In some embodiments, R 32a and R 32b each of which independently [ka] is.

[0597] In one embodiment, the structured lipids of the present invention have the formula SX: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4aIf there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 33a is an optionally substituted C1-C6 alkyl, or [ka] where R 35 is an optionally substituted C1-C6 alkyl or an optionally substituted C6-C 10 is aryl, R 33b is H or optionally substituted C1-C6 alkyl, or R 35 and R 33b each taken together with the atom to which it is attached forms an optionally substituted C3-C9 heterocyclyl; R 34 is optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl.

[0598] In some embodiments, the compound has the formula SXa: [ka] or a pharmaceutically acceptable salt thereof.

[0599] In some embodiments, the compound has the formula SXb: [ka] or a pharmaceutically acceptable salt thereof.

[0600] In some embodiments, R 33a teeth, [ka] is.

[0601] In some embodiments, R 35 teeth, [ka] is.

[0602] In some embodiments, R 35 teeth, [ka] where: t is 0, 1, 2, 3, 4, or 5; R 36 is independently halo, hydroxyl, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl.

[0603] In some embodiments, R 34 teeth, [ka] where u is 0, 1, 2, 3, or 4.

[0604] In some embodiments, u is 3 or 4.

[0605] In one embodiment, the structured lipids of the present invention have the formula SXI: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R 3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming R 37a and R 37bEach of is independently an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, halo, or hydroxyl.

[0606] In some embodiments, the compound has formula SXIa: [ka] or a pharmaceutically acceptable salt thereof.

[0607] In some embodiments, the compound has formula SXIb: [ka] or a pharmaceutically acceptable salt thereof.

[0608] In some embodiments, R 37a is a hydroxyl.

[0609] In some embodiments, R 37b teeth, [ka] is.

[0610] In one embodiment, the structured lipids of the present invention have the formula SXII: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1a is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl; X is O or S; R 2 is H or OR A where R A is H or optionally substituted C1-C6 alkyl; R3 is H or [ka] and [ka] represents a single or double bond, W is CR 4a or CR 4a R 4b where if there is a double bond between W and the adjacent carbon, W is CR 4a If there is a single bond between W and the adjacent carbon, W is CR 4a R 4b and R 4a and R 4b each is independently H, halo, or optionally substituted C-C alkyl; R 5a and R 5b each independently represents H or OR A or R 5a and R 5b are each bonded to the atom, [ka] Forming Q is O, S, or NR E where R E is H or optionally substituted C1-C6 alkyl; R 38 is an optionally substituted C1-C6 alkyl.

[0611] In some embodiments, the compound has formula SXIIa: [ka] or a pharmaceutically acceptable salt thereof.

[0612] In some embodiments, the compound has formula SXIIb: [ka] or a pharmaceutically acceptable salt thereof.

[0613] In some embodiments, Q is NR E is.

[0614] In some embodiments, R E is H, or [ka] is.

[0615] In some embodiments, R E is H. In some embodiments, R E teeth, [ka] is.

[0616] In some embodiments, R 38 teeth, [ka] where u is 0, 1, 2, 3, or 4.

[0617] In some embodiments, X is O.

[0618] In some embodiments, R 1a is H or optionally substituted C1-C6 alkyl.

[0619] In some embodiments, R 1a is H.

[0620] In some embodiments, R 1b is H or optionally substituted C1-C6 alkyl.

[0621] In some embodiments, R 1b is H.

[0622] In some embodiments, R 2 is H.

[0623] In some embodiments, R 4a is H.

[0624] In some embodiments, R 4b is H.

[0625] In some embodiments, [ka] represents a double bond.

[0626] In some embodiments, R 3 is H. In some embodiments, R 3 teeth, [ka] is.

[0627] In some embodiments, R 5a is H.

[0628] In some embodiments, R 5b is H.

[0629] In one aspect, the invention features a compound having the structure of any one of compounds S-1 to S-42, S-150, S-154, S-162 to S-165, S-169 to S-172, and S-184 in Table 1, or any pharmaceutically acceptable salt thereof. As used herein, "CMPD" refers to "compound." [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0630] In one aspect, the invention features a compound having the structure of any one of compounds S-43 to S-50 and S-175 to S-178 in Table 2, or any pharmaceutically acceptable salt thereof. [Table 5]

[0631] In one aspect, the invention features a compound having the structure of any one of compounds S-51 to 67, S-149, and S-153 in Table 3, or any pharmaceutically acceptable salt thereof. [Table 6-1] [Table 6-2]

[0632] In one aspect, the invention features a compound having the structure of any one of compounds S-68 to S-73 in Table 4, or any pharmaceutically acceptable salt thereof. [Table 7]

[0633] In one aspect, the invention features a compound having the structure of any one of compounds S-74 to S-78 in Table 5, or any pharmaceutically acceptable salt thereof. [Table 8]

[0634] In one aspect, the invention features a compound having the structure of any one of compounds S-79 or S-80 in Table 6, or any pharmaceutically acceptable salt thereof. [Table 9]

[0635] In one aspect, the invention features a compound having the structure of any one of compounds S-81 to 87, S-152, and S-157 in Table 7, or any pharmaceutically acceptable salt thereof. [Table 10]

[0636] In one aspect, the invention features a compound having the structure of any one of compounds S-88 to S-97 in Table 8, or any pharmaceutically acceptable salt thereof. [Table 11]

[0637] In one aspect, the invention features a compound having the structure of any one of compounds S-98 to 105 and S-180 to 182 in Table 9, or any pharmaceutically acceptable salt thereof. [Table 12]

[0638] In one aspect, the invention features a compound having the structure of compound S-106 in Table 10, or any pharmaceutically acceptable salt thereof. [Table 13]

[0639] In one aspect, the invention features a compound having the structure of compound S-107 or S-108 in Table 11, or any pharmaceutically acceptable salt thereof. [Table 14]

[0640] In one aspect, the invention features a compound having the structure of compound S-109 in Table 12, or any pharmaceutically acceptable salt thereof. [Table 15]

[0641] In one aspect, the invention features a compound having the structure of any one of compounds S-110 to S-130, S-155, S-156, S-158, S-160, S-161, S-166 to S-168, S-173, S-174, and S-179 in Table 13, or any pharmaceutically acceptable salt thereof. [Table 16-1] [Table 16-2] [Table 16-3]

[0642] In one aspect, the invention features a compound having the structure of any one of compounds S-131-133 in Table 14, or any pharmaceutically acceptable salt thereof. [Table 17]

[0643] In one aspect, the invention features a compound having the structure of any one of compounds S-134 to 148, S-151, and S-159 in Table 15, or any pharmaceutically acceptable salt thereof. [Table 18-1] [Table 18-2]

[0644] The one or more structured lipids of the lipid nanoparticles of the present invention can be a structured lipid composition (e.g., a mixture of two or more structured lipids, a mixture of three or more structured lipids, a mixture of four or more structured lipids, or a mixture of five or more structured lipids). The structured lipid composition can include, but is not limited to, any combination of sterols (e.g., cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, or any one of compounds 134-148, 151, and 159 in Table 15). For example, the one or more structured lipids of the lipid nanoparticles of the present invention can be composition 183 in Table 16. [Table 19]

[0645] Composition S-183 is a mixture of compounds S-141, S-140, S-143, and S-148. In some embodiments, composition S-183 contains about 35% to about 45% of compound S-141, about 20% to about 30% of compound S-140, about 20% to about 30% of compound S-143, and about 5% to about 15% of compound S-148. In some embodiments, composition S-183 contains about 40% of compound S-141, about 25% of compound S-140, about 25% of compound S-143, and about 10% of compound S-148.

[0646] In some embodiments, the structured lipid is a phytosterol. In some embodiments, the phytosterol is sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartenol, Δ5-avenaserol, Δ7-avenaserol, or Δ7-stigmasterol (including analogs, salts, or esters thereof), alone or in combination. In some embodiments, the phytosterol component of the LNPs of the present disclosure is a single phytosterol. In some embodiments, the phytosterol component of the LNPs of the present disclosure is a mixture of different phytosterols (e.g., 2, 3, 4, 5, or 6 different phytosterols). In some embodiments, the phytosterol component of the LNPs of the present disclosure is a blend of one or more phytosterols with one or more zoosterols, for example, a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) with cholesterol.

[0647] Compound ratio The lipid nanoparticles of the present invention can comprise a structural component as described herein, which can be any one of compounds S-1 to 148, a mixture of one or more structural compounds of the present invention, and / or any one of compounds S-1 to 148 in combination with cholesterol and / or phytosterols.

[0648] For example, the structural component of the lipid nanoparticles can be a mixture of one or more structural compounds of the present invention (e.g., any of compounds S-1 to S-148) with cholesterol. The mol% of the structural compound present in the lipid nanoparticles can be 0 to 99 mol% relative to cholesterol. The mol% of the structural compound present in the lipid nanoparticles can be about 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol% relative to cholesterol.

[0649] In one aspect, the invention features a composition including two or more sterols, where the two or more sterols include at least two of β-sitosterol, sitostanol, campesterol, stigmasterol, and brassicasterol. The composition may additionally include cholesterol. In one embodiment, the β-sitosterol comprises about 35-99% of the non-cholesterol sterols in the composition, e.g., greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0650] In another aspect, the invention features a composition that includes two or more sterols, the two or more sterols including β-sitosterol and campesterol, wherein the β-sitosterol comprises 95-99.9% of the sterols in the composition and the campesterol comprises 0.1-5% of the sterols in the composition.

[0651] In some embodiments, the composition further comprises sitostanol. In some embodiments, β-sitosterol comprises 95-99.9% of the sterols in the composition, campesterol comprises 0.05-4.95%, and sitostanol comprises 0.05-4.95%.

[0652] In another aspect, the invention features a composition that includes two or more sterols, the two or more sterols including β-sitosterol and sitostanol, wherein the β-sitosterol comprises 95-99.9% of the sterols in the composition and the sitostanol comprises 0.1-5% of the sterols in the composition.

[0653] In some embodiments, the composition further comprises campesterol. In some embodiments, β-sitosterol comprises 95-99.9% of the sterols in the composition, campesterol comprises 0.05-4.95%, and sitostanol comprises 0.05-4.95%.

[0654] In some embodiments, the composition further comprises campesterol. In some embodiments, β-sitosterol comprises 75-80% of the sterols in the composition, campesterol comprises 5-10%, and sitostanol comprises 10-15%.

[0655] In some embodiments, the composition further comprises additional sterols, such as β-sitosterol comprising 35-45% of the sterols in the composition, stigmasterol comprising 20-30%, campestanol comprising 20-30%, and brassicasterol comprising 1-5%.

[0656] In another aspect, the invention features a composition that includes a plurality of lipid nanoparticles, the plurality of lipid nanoparticles including an ionic lipid and two or more sterols, where the two or more sterols include β-sitosterol and campesterol, where the β-sitosterol comprises 95-99.9% of the sterols in the composition and the campesterol comprises 0.1-5% of the sterols in the composition.

[0657] In some embodiments, the two or more sterols further comprise sitostanol, ie, β-sitosterol comprises 95-99.9% of the sterols in the composition, campesterol comprises 0.05-4.95%, and sitostanol comprises 0.05-4.95%.

[0658] In another aspect, the invention features a composition that includes a plurality of lipid nanoparticles, the plurality of lipid nanoparticles including an ionic lipid and two or more sterols, the two or more sterols including β-sitosterol and sitostanol, wherein the β-sitosterol comprises 95-99.9% of the sterols in the composition and the sitostanol comprises 0.1-5% of the sterols in the composition.

[0659] In some embodiments, the two or more sterols further include campesterol. In some embodiments, β-sitosterol comprises 95-99.9% of the sterols in the composition, campesterol comprises 0.05-4.95%, and sitostanol comprises 0.05-4.95%.

[0660] (iii) Non-cationic helper lipids / phospholipids In some embodiments, the lipid-based compositions (e.g., LNPs) described herein comprise one or more non-cationic helper lipids. In certain embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a substitute or alternative for a phospholipid.

[0661] As used herein, the term "non-cationic helper lipid" refers to a lipid comprising at least one fatty acid chain at least 8 carbons in length and at least one polar head group moiety. In one embodiment, the helper lipid is not phosphatidylcholine (PC). In one embodiment, the non-cationic helper lipid is a phospholipid or phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute may be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0662] The phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0663] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0664] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include sphingophospholipids such as sphingomyelin.

[0665] In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0666] In some embodiments, the non-cationic helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-i77-glycero-3-phosphocholine (DSPC) substitute.

[0667] phospholipids The lipid composition of the pharmaceutical composition disclosed herein may contain one or more non-cationic helper lipids. In some embodiments, the non-cationic helper lipid is a phospholipid, such as one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties. As used herein, a "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid or its analog or derivative may comprise choline. A phospholipid or its analog or derivative may not comprise choline. Certain phospholipids can facilitate membrane fusion. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). When a phospholipid fuses with a membrane, it can allow one or more components of a lipid-containing composition to cross the membrane, thereby, for example, allowing the one or more components to be delivered to a cell.

[0668] The phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0669] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0670] Certain phospholipids can be easily fused to membranes.For example, cationic phospholipids can interact with one or more negatively charged phospholipids in membranes (for example, cell membranes or intracellular membranes).When phospholipids are fused to membranes, they can allow one or more components (for example, therapeutic agents) of lipid-containing compositions (for example, LNPs) to pass through the membrane, thereby, for example, allowing one or more components to be delivered to target tissues.

[0671] The lipid component of the lipid nanoparticle of the present disclosure can comprise one or more phospholipids, for example, one or more (poly)unsaturated lipids. The phospholipid can be assembled into one or more lipid bilayers. Generally, the phospholipid can comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid can be (H III): [ka] wherein R prepresents a phospholipid moiety, and R1 and R2 represent fatty acid moieties, with or without unsaturation, which may be the same or different. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species are also contemplated, including naturally occurring species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds have been replaced with triple bonds). Under appropriate reaction conditions, the alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions can be useful for functionalizing the lipid bilayer of LNPs to facilitate membrane permeation or cell recognition, or for conjugating LNPs to useful components such as targeting or imaging moieties (e.g., dyes). Each possibility represents a separate embodiment of the present invention.

[0672] Phospholipids useful in the compositions and methods described herein include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3(cis)PC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6(cis)PC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (PE(18:2 / 18:2), 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (PE 18:3(9Z,12Z,15Z), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE The glycerol-containing ester may be selected from the non-limiting group consisting of 18:3(9Z,12Z,15Z), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6(cis)PE), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment of the present invention.

[0673] In some embodiments, the LNPs comprise DSPC. In certain embodiments, the LNPs comprise DOPE. In some embodiments, the LNPs comprise DMPE. In some embodiments, the LNPs comprise both DSPC and DOPE.

[0674] In one embodiment, the non-cationic helper lipid for use in the target cell delivery LNP is selected from the group consisting of DSPC, DMPE, and DOPC, or a combination thereof.

[0675] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include sphingophospholipids such as sphingomyelin.

[0676] Examples of structured lipids include, but are not limited to: [ka] [ka] [ka] [ka] [ka]

[0677] In certain embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine). In certain embodiments, phospholipids useful or potentially useful in the present invention are compounds of formula (H IX): [ka] or a salt thereof, wherein Each R 1 are independently optionally substituted alkyl, or optionally two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] It is of L 2 each instance of independently represents a bond or an optionally substituted C 1-6 alkylene, wherein optionally substituted C 1-6 One methylene unit of the alkylene is optionally -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- and R 2 In each case, independently, optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, wherein optionally R 2one or more methylene units in the formula (I) are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2; However, the compound has the following formula: [ka] wherein R 2 Each instance of is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0678] i) Modification of phospholipid head groups In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, the phospholipid with a modified head group is DSPC or an analog thereof with a modified quaternary amine. For example, in embodiments of formula (IX), R 1 At least one of R is not methyl. 1 In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof, wherein each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each v is independently 1, 2, or 3.

[0679] In certain embodiments, the compound of formula (H IX) has the following formula: [ka] or a salt thereof.

[0680] In certain embodiments, the compound of formula (H IX) is: [ka] [ka] [ka] or a salt thereof.

[0681] In one embodiment, the target cell delivery LNP comprises the compound H-409 as a non-cationic helper lipid.

[0682] (ii) Modification of phospholipid tails In certain embodiments, phospholipids useful or potentially useful in the present invention include modified tails. In certain embodiments, phospholipids useful or potentially useful in the present invention are DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine) or analogs thereof with modified tails. As described herein, a "modified tail" can be a tail with a shorter or longer aliphatic chain, a branched aliphatic chain, a substituted aliphatic chain, an aliphatic chain in which one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. For example, in certain embodiments, the compound of (H IX) is of formula (H IX-a) or a salt thereof, wherein R 2 In each case, optionally replaced C 1-30 alkyl, where R 2 one or more methylene units in the formula (I) are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by

[0683] In certain embodiments, the compound of formula (H IX) has formula (H IX-c): [ka] or a salt thereof, wherein each x is independently an integer between 0 and 30, inclusive; Each instance of G is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(RN )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-. Each possibility represents a separate embodiment of the present invention.

[0684] In certain embodiments, the compound of formula (H IX-c) has the formula (H IX-c-1): [ka] or a salt thereof, wherein Each instance of v is independently 1, 2, or 3.

[0685] In certain embodiments, the compound of formula (H IX-c) has formula (H IX-c-2): [ka] or a salt thereof.

[0686] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0687] In certain embodiments, the compound of formula (H IX-c) is: [ka] or a salt thereof.

[0688] In certain embodiments, the compound of formula (H IX-c) has formula (H IX-c-3): [ka] or a salt thereof.

[0689] In certain embodiments, the compound of formula (H IX-c) has the following formula: [ka] or a salt thereof.

[0690] In certain embodiments, the compound of formula (H IX-c) is: [ka] or a salt thereof.

[0691] In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified phosphocholine moiety, wherein the alkyl chain connecting the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids useful or potentially useful in the present invention are compounds of formula (H IX), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, compounds of formula (H IX) have the following formula: [ka] or a salt thereof.

[0692] In certain embodiments, the compound of formula (H IX) is: [ka] [ka] or a salt thereof.

[0693] In certain embodiments, alternative lipids are used in place of the phospholipids of the present invention. Non-limiting examples of such alternative lipids include: [ka]

[0694] Modification of phospholipid tails In certain embodiments, the phospholipid useful in the present invention comprises modified tail.In certain embodiments, the phospholipid useful in the present invention is DSPC or its analogue with modified tail.As described herein, "modified tail" can be a tail with shorter or longer aliphatic chain, aliphatic chain with branching, aliphatic chain with substituent, aliphatic chain with one or more methylene replaced by cyclic or heteroatom group, or any combination thereof.For example, in certain embodiments, the compound of (HI) is of formula (HIa) or its salt, wherein R 2 In each case, optionally replaced C 1-30 alkyl, where R 2 one or more of the methylene units in N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(RN )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by

[0695] In certain embodiments, the compound of formula (H Ia) has formula (H Ic): [ka] or a salt thereof, wherein each x is independently an integer between 0 and 30, inclusive; Each instance of G is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-. Each possibility represents a separate embodiment of the present invention.

[0696] In certain embodiments, the compound of formula (H Ic) has the formula (H Ic-1): [ka] or a salt thereof, wherein Each instance of v is independently 1, 2, or 3.

[0697] In certain embodiments, the compound of formula (H Ic) has formula (H Ic-2): [ka] or a salt thereof.

[0698] In certain embodiments, the compound of formula (Ic) has the following formula: [ka] or a salt thereof.

[0699] In certain embodiments, the compound of formula (H Ic) is: [ka] or a salt thereof.

[0700] In certain embodiments, the compound of formula (H Ic) has formula (H Ic-3): [ka] or a salt thereof.

[0701] In certain embodiments, the compound of formula (H Ic) has the following formula: [ka] or a salt thereof.

[0702] In certain embodiments, the compound of formula (H Ic) is: [ka] or a salt thereof.

[0703] Modification of the phosphocholine linker In certain embodiments, phospholipids useful in the present invention comprise a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids useful in the present invention are compounds of formula (HI), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, compounds of formula (HI) have the following formula: [ka] or a salt thereof.

[0704] In certain embodiments, the compound of formula (HI) is: [ka] [ka] or a salt thereof.

[0705] As demonstrated in the Examples that follow, numerous LNP formulations with phospholipids other than DSPC were prepared and tested for activity.

[0706] Phospholipid substitutes or replacements In some embodiments, the lipid-based composition (e.g., lipid nanoparticles) comprises oleic acid or an oleic acid analog instead of a phospholipid. In some embodiments, the oleic acid analog comprises a modified oleic acid tail, a modified carboxylic acid moiety, or both. In some embodiments, the oleic acid analog is a compound in which the carboxylic acid moiety of oleic acid is replaced with a different group.

[0707] In some embodiments, the lipid-based composition (eg, lipid nanoparticles) comprises a different zwitterionic group in place of the phospholipid.

[0708] Exemplary phospholipid substitutes and / or replacements are provided in published PCT application WO2017 / 099823, which is incorporated herein by reference.

[0709] Exemplary phospholipid substitutes and / or replacements are provided in published PCT application WO2017 / 099823, which is incorporated herein by reference.

[0710] (iv) PEG lipid Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, and PEG-modified 1,2-diacyloxypropan-3-amine.Such lipids are also called PEGylated lipids.For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0711] In some embodiments, PEG lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0712] In one embodiment, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0713] In some embodiments, the lipid portion of the PEG-lipid has a molecular weight of about C 14 ~About C 22 , preferably about C 14 ~About C 16In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG lipid is 2k -DMG.

[0714] In one embodiment, the lipid nanoparticles described herein can include a PEG lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[0715] PEG lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584A2, which are incorporated herein by reference in their entireties.

[0716] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein may be synthesized as described in International Patent Application No. PCT / US2016 / 000129, entitled "Compositions and Methods for Delivery of Therapeutic Agents," filed December 10, 2016, which is incorporated by reference in its entirety.

[0717] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0718] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. PEG-DMG has the following structure: [ka]

[0719] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety. Any of these exemplary PEG lipids described herein may be modified to include a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0720] In some embodiments, the PEG lipid is a compound of formula (PI): [ka] or a salt or isomer thereof, wherein: r is an integer between 1 and 100; R 5PEG is C 10-40 Alkyl, C 10-40 Alkenyl, or C 10-40 alkynyl, and optionally R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4- to 10-membered heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(RN )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by R N In each case, independently, hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0721] For example, R 5PEG is C 17 For example, a PEG lipid may be represented by the formula (PI-a): [ka] or a salt or isomer thereof, wherein r is an integer between 1 and 100.

[0722] For example, a PEG lipid may have the following formula: [ka] or a salt or isomer thereof.

[0723] The PEG lipid has the formula (PII): [ka] or a salt or isomer thereof, wherein s is an integer between 1 and 100, R" is hydrogen, C1 -10 alkyl, or oxygen protecting group, R 7PEG is C 10-40 Alkyl, C 10-40 Alkenyl, or C 10-40 alkynyl, and optionally R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4- to 10-membered heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N)-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by R N In each case, independently, hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0724] In some embodiments, R 7PEG is C 10-60 alkyl, and R 7PEG One or more of the methylene groups in R is replaced with -C(O)-. For example, R 7PEG is C 31 alkyl, and R 7PEG Two of the methylene groups are replaced with -C(O)-.

[0725] In some embodiments, R″ is methyl.

[0726] In some embodiments, the PEG lipid has the formula (PII-a): [ka] or a salt or isomer thereof, wherein s is an integer between 1 and 100.

[0727] For example, a PEG lipid may have the following formula: [ka] or a salt or isomer thereof.

[0728] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (PIII): Provided herein is a compound of formula (PIII): [ka] or a salt thereof, wherein: R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is an optionally substituted C 1-10 alkylene, wherein optionally substituted C 1-10 At least one methylene of the alkylene is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ) and D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] It is of L 2 each instance of independently represents a bond or an optionally substituted C 1-6 alkylene, wherein optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ) and R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, optionally where R 2 one or more methylene units of are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O, R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2.

[0729] In certain embodiments, the compound of formula (PIII) is a PEG-OH lipid (i.e., R 3 HA-OR O and R O is hydrogen). In certain embodiments, the compound of formula (PIII) has the formula (PIII-OH): [ka] or a salt thereof.

[0730] In certain embodiments, D is a moiety obtained by click chemistry (e.g., a triazole). In certain embodiments, the compound of formula (PIII) has formula (PIII-a-1) or (PIII-a-2): [ka] or a salt thereof.

[0731] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0732] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0733] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0734] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0735] In certain embodiments, D is a moiety that is cleavable under physiological conditions (e.g., an ester, an amide, a carbonate, a carbamate, a urea). In certain embodiments, the compound of formula (PIII) has formula (PIII-b-1) or (PIII-b-2): [ka] or a salt thereof.

[0736] In certain embodiments, the compound of formula (PIII) has the formula (PIII-b-1-OH) or (PIII-b-2-OH): [ka] or a salt thereof.

[0737] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0738] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0739] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0740] In certain embodiments, the compound of formula (PIII) has the following formula: [ka] or a salt thereof.

[0741] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (PIV). Provided herein is a compound of formula (PIV): [ka] or a salt thereof, wherein R 3 -OR O and R Ois hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is an optionally substituted C 10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl, and optionally R 5 One or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N), or N(R N )S(O)2O, R N Each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0742] In certain embodiments, the compound of formula (PIV) has the formula (PIV-OH): [ka] or a salt thereof. In some embodiments, r is 40 to 50. In some embodiments, r is 45.

[0743] In certain embodiments, the compound of formula (PIV) has the following formula: [ka] or a salt thereof. In some embodiments, r is 40 to 50. In some embodiments, r is 45.

[0744] In yet another embodiment, the compound of formula (PIV) is [ka] or a salt thereof.

[0745] In one embodiment, the compound of formula (PIV) is: [ka]

[0746] In one aspect, provided herein is a compound of formula (PV): [ka] or a pharmaceutically acceptable salt thereof, wherein: L 1 is a bond, optionally substituted C 1-3Alkylene, optionally substituted C 1-3 Heteroalkylene, optionally substituted C 2-3 Alkenylene, optionally substituted C 2-3 is alkynylene, R 1 is an optionally substituted C 5-30 Alkyl, optionally substituted C 5-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer between 2 and 100, inclusive.

[0747] In certain embodiments, the PEG lipid of formula (PV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Y 1 is a bond, -CR2-, -O-, -NR N -, or -S-, each instance of R is independently hydrogen, halogen, or optionally substituted alkyl; R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group.

[0748] In certain embodiments, the PEG lipid of formula (PV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Each instance of R is independently hydrogen, halogen, or optionally substituted alkyl.

[0749] In certain embodiments, the PEG lipid of formula (PV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein s is an integer between 5 and 25 (inclusive).

[0750] In certain embodiments, the PEG lipid of formula (PV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0751] In certain embodiments, the PEG lipid of formula (PV) is: [ka] [ka] and pharmaceutically acceptable salts thereof.

[0752] In another aspect, provided herein is a compound of formula (PVI): [ka] or a pharmaceutically acceptable salt thereof, wherein: R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer between 2 and 100 (inclusive). m is an integer from 5 to 15 (inclusive) or an integer from 19 to 30 (inclusive).

[0753] In certain embodiments, the PEG lipid of formula (PVI) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0754] In certain embodiments, the PEG lipid of formula (PVI) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0755] In another aspect, provided herein is a compound of formula (PVII): [ka] or a pharmaceutically acceptable salt thereof, wherein: Y 2 -O-, -NR N -, or -S-, R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 5-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group; r is an integer between 2 and 100, inclusive.

[0756] In certain embodiments, the PEG lipid of formula (PVII) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0757] In certain embodiments, the PEG lipid of formula (PVII) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Each occurrence of s is an integer between 5 and 25, inclusive.

[0758] In certain embodiments, the PEG lipid of formula (PVII) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0759] In certain embodiments, the PEG lipid of formula (PVII) is: [ka] and pharmaceutically acceptable salts thereof.

[0760] In another aspect, provided herein is a compound of formula (PVIII): [ka] or a pharmaceutically acceptable salt thereof, wherein: L 1 is a bond, optionally substituted C 1-3 Alkylene, optionally substituted C 1-3 Heteroalkylene, optionally substituted C 2-3 Alkenylene, optionally substituted C 2-3 is alkynylene, R 1 Each instance of is independently an optionally substituted C 5-30 Alkyl, optionally substituted C 3-30 alkenyl, or optionally substituted C 5-30 is alkynyl, R O is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; r is an integer between 2 and 100, inclusive; However, L 1 is -CH2CH2- or -CH2CH2CH2-, R O is not methyl.

[0761] In certain embodiments, L 1 When is an optionally substituted C2 or C3 alkylene, R O is not optionally substituted alkyl. In certain embodiments, L 1 When is an optionally substituted C2 or C3 alkylene, R O is hydrogen. In certain embodiments, L 1 is -CH2CH2- or -CH2CH2CH2-, R O is not optionally substituted alkyl. In certain embodiments, L 1 is -CH2CH2- or -CH2CH2CH2-, R O is hydrogen.

[0762] In certain embodiments, the PEG lipid of formula (PVIII) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein Y 1 is a bond, -CR2-, -O-, -NR N -, or -S-, each instance of R is independently hydrogen, halogen, or optionally substituted alkyl; R N is hydrogen, optionally substituted alkyl, optionally substituted acyl, or a nitrogen protecting group; However, Y 1 When is a bond or -CH2-, R O is not methyl.

[0763] In certain embodiments, L 1 When is -CR2-, R Ois not optionally substituted alkyl. In certain embodiments, L 1 When is -CR2-, R O is hydrogen. In certain embodiments, L 1 When is -CH2-, R O is not optionally substituted alkyl. In certain embodiments, L 1 When is -CH2-, R O is hydrogen.

[0764] In certain embodiments, the PEG lipid of formula (PVIII) has the following formula: [ka] [ka] or a pharmaceutically acceptable salt thereof, wherein Each instance of R is independently hydrogen, halogen, or optionally substituted alkyl.

[0765] In certain embodiments, the PEG lipid of formula (PVIII) has the following formula: [ka] [ka] or a pharmaceutically acceptable salt thereof, wherein each instance of R is independently hydrogen, halogen, or optionally substituted alkyl; Each s is independently an integer from 5 to 25, inclusive.

[0766] In certain embodiments, the PEG lipid of formula (PVIII) has the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0767] In certain embodiments, the PEG lipid of formula (PVIII) is: [ka] [ka] and pharmaceutically acceptable salts thereof.

[0768] In any of the foregoing or related embodiments, the PEG-lipid of the invention is characterized in that r is 40-50.

[0769] In certain embodiments, the LNPs provided herein exhibit increased PEG release compared to existing LNP formulations containing PEG-lipids. "PEG release," as used herein, refers to the cleavage of the PEG group from the PEG-lipid. Often, the cleavage of the PEG group from the PEG-lipid occurs through serum-driven esterase cleavage or hydrolysis. In certain embodiments, the PEG-lipids provided herein are designed to control the rate of PEG release. In certain embodiments, the LNPs provided herein exhibit greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 50% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 60% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 70% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs exhibit greater than 80% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs exhibit greater than 90% PEG release after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibi...

Claims

1. 1. A composition for use in enhancing delivery of a payload to liver or spleen cells, said composition comprising a targeted cell delivery lipid nanoparticle (LNP), said LNP comprising: (i) an ionic lipid comprising compound I-301; (ii) sterols or other structured lipids; (iii) a non-cationic helper lipid or phospholipid; (iv) the payload; and (v) optionally, a PEG lipid A composition comprising:

2. The composition for use according to claim 1 , wherein the liver cells are hepatocytes.

3. (a) resulting in expression and / or activity of the payload in approximately 60% of total hepatocytes; (b) results in enhanced payload levels (e.g., expression) in liver cells, e.g., hepatocytes, relative to a reference LNP; (c) results in about a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold increase in hepatocyte expression, e.g., hepatocyte expression, relative to a reference LNP; (d) have increased cytosolic delivery efficiency, e.g., when compared to a reference LNP; and / or (e) i) a greater maximum blood concentration (Cmax) in the liver compared to plasma, e.g., a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater or more than in plasma; ii) a greater half-life (t½) in the liver relative to plasma, e.g., a t½ in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 or more times greater than in plasma; or iii) a greater extrapolated area (%) under the concentration-time curve in the liver compared to plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in plasma; bring about one, two, or all of the following: A composition for use according to claim 1 or 2.

4. have improved parameters in vivo compared to a reference LNP, said improved parameters being: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more lipid remaining after administration to a subject, e.g., a mouse, e.g., 24 hours after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more increased bioavailability, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slower lipid metabolism in the liver compared to the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the lipids remaining in the liver 24 hours after administration; 4. The composition for use according to any one of claims 1 to 3, selected from one, two, three, four, five, six, seven or more (e.g. all), or any combination thereof.

5. 1) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; 2) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more hepatocyte transfection; 3) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 4) an increased response rate greater than that of a reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater; 5. The composition for use according to any one of claims 1 to 4, which provides one, two, three or all of the following:

6. 6. The composition for use of any one of claims 1 to 5, wherein the target cell delivery LNP is formulated for systemic delivery or is administered systemically, e.g., parenterally (e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally) or enterally (e.g., orally, rectally, or sublingually).

7. The composition for use according to any one of claims 1 to 6, for delivering said payload to cells capable of synthesizing proteins and / or cells with a high engulfment capacity.

8. The payload (a) liver cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof; or (b) splenocytes that are non-immune splenocytes (e.g., splenocytes) The composition for use according to any one of claims 1 to 7, wherein the composition is delivered to

9. (a) the intracellular concentration of the nucleic acid molecule in the target cell is enhanced; (b) the uptake of the nucleic acid molecule by the target cell is enhanced; (c) the activity of the nucleic acid molecule in the target cell is enhanced. (d) expression of the nucleic acid molecule in the target cell is enhanced; (e) the activity of the protein encoded by the nucleic acid molecule in the target cell is enhanced; (f) the expression of the protein encoded by the nucleic acid molecule in the target cell is enhanced; and / or (g) delivery is enhanced in vivo; A composition for use according to any one of claims 1 to 8.

10. The payload is (a) is a peptide, polypeptide, protein, or nucleic acid; (b) a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, or DNA; or (c) selected from shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), or a combination thereof; A composition for use according to any one of claims 1 to 9.

11. The payload is (a) a protein of interest other than an immune cell payload; (b) a secreted protein, a membrane-bound protein, an intracellular protein, an antibody molecule, or an enzyme; (c) an antibody molecule; or (d) enzyme The composition for use according to any one of claims 1 to 10, wherein the composition is an mRNA encoding the

12. (a) the enzyme is associated with a lysosomal storage disease; (b) the enzyme is associated with a metabolic disorder, and / or (c) the payload is an mRNA encoding a urea cycle enzyme; A composition for use according to claim 11.

13. The target cell delivery LNP is (a) can be administered at a lower dose compared to a reference LNP; and / or (b) administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to said dose of a reference LNP; A composition for use according to any one of claims 1 to 12.

14. 1. A pharmaceutical composition for use in enhancing payload levels (e.g., expression of a payload) in liver cells or spleen cells, comprising a delivery lipid nanoparticle (LNP), wherein the delivery lipid nanoparticle (LNP) is: (i) an ionic lipid comprising compound I-301; (ii) sterols or other structured lipids; (iii) a non-cationic helper lipid or phospholipid; (iv) the payload; and (v) optionally, a PEG lipid A pharmaceutical composition for use comprising:

15. The target cell delivery LNP is as follows: a) a maximum blood concentration (Cmax) in the liver that is greater than in plasma, for example, a Cmax in the liver that is at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 times greater or more than in plasma; b) A longer half-life in the liver compared to plasma (t １／２ ), e.g., at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more t in the liver compared to plasma １／２ ;or c) A greater extrapolated area (%) under the concentration-time curve in the liver compared to plasma (AUC%Extrap), e.g., an AUC%Extrap in the liver that is at least 5, 10, 15, 20, 25, 30, 35, 40-fold or more greater than in plasma; 15. The pharmaceutical composition for use according to claim 14, formulated for administration in an amount that results in one, two, or all of:

16. The target cell delivery LNP is formulated for administration in an amount that results in an improved parameter in vivo compared to a reference LNP, the improved parameter being one of the following: 1) enhanced payload levels in the liver, e.g., increased payload mRNA or payload protein levels in the liver, e.g., increased delivery, transfection, and / or expression, by at least 1, 2, 3, 4, 5, 6, 7, 8, or more fold after administration to a subject, e.g., after IV administration to a non-human primate; 2) enhanced serum stability with at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more lipid remaining after administration to a subject, e.g., a mouse, e.g., 24 hours after IV administration; 3) reduced immunogenicity, e.g., reduced levels of IgM or IgG that recognize the LNP, e.g., at least 1.2-5 fold reduced IgM clearance; 4) increased bioavailability after administration to a subject, e.g., after IV administration to a non-human primate, e.g., at least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more increased bioavailability, as observed by increased AUC after administration to a subject, e.g., after administration to a non-human primate; 5) enhanced liver distribution, e.g., enhanced hepatocyte positivity, e.g., at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, relative to a reference LNP, after administration to a subject, e.g., after administration to a non-human primate; 6) enhanced lipid and / or payload tissue concentrations in the liver for, e.g., at least 6 hours, at least 12 hours, or at least 24 hours after administration to a subject; 7) enhanced endosomal escape; or 8) slower lipid metabolism in the liver compared to the spleen, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the lipids remaining in the liver 24 hours after administration; 16. The pharmaceutical composition for use according to claim 14 or 15, wherein the pharmaceutical composition comprises one, two, three, four, five, six, seven or more (e.g. all) of the following, or any combination thereof:

17. The target cell delivery LNP is as follows: 1) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; 2) at least 5%, 10%, 15%, 20%, 25%, 30%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more hepatocyte transfection; 3) an increased response rate, e.g., as defined by a specific threshold of hepatocyte transfection; or 4) an increased response rate greater than that of a reference LNP, e.g., at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold or greater; 17. The pharmaceutical composition for use according to any one of claims 14 to 16, formulated for administration in an amount to provide one, two, three or all of:

18. The pharmaceutical composition for use according to any one of claims 14 to 17, wherein the target cell delivery LNP is formulated for systemic administration, e.g., formulated for parenteral (e.g., intravenous, intramuscular, subcutaneous, intrathecal, or intradermal) or enteral (e.g., oral, rectal, or sublingual) administration.

19. The target cell delivery LNP is (a) delivering said payload to cells capable of synthesizing proteins and / or cells with high engulfment capacity; (b) delivering the payload to liver cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal cells, or a combination thereof; (c) delivering said payload to hepatocytes; or (d) delivering the payload to splenocytes that are non-immune splenocytes (e.g., splenocytes); A pharmaceutical composition for use according to any one of claims 14 to 18.

20. (a) the intracellular concentration of the nucleic acid molecule in the target cell is enhanced; (b) the uptake of the nucleic acid molecule by the target cell is enhanced; (c) the activity of the nucleic acid molecule in the target cell is enhanced. (d) expression of the nucleic acid molecule in the target cell is enhanced; (e) the activity of the protein encoded by the nucleic acid molecule in the target cell is enhanced; (f) the expression of the protein encoded by the nucleic acid molecule in the target cell is enhanced; and / or (g) delivery is enhanced in vivo; A pharmaceutical composition for use according to any one of claims 14 to 19.

21. The payload is (a) is a peptide, polypeptide, protein, or nucleic acid; (b) a nucleic acid molecule selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR / Cas9, ssDNA, or DNA; (c) selected from a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a dicer substrate RNA (dsRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), or a combination thereof; or (d) mRNA, siRNA, miR, or CRISPR; A pharmaceutical composition for use according to any one of claims 14 to 20.

22. The payload is (a) a protein of interest other than an immune cell payload; (b) a secreted protein, a membrane-bound protein, an intracellular protein, or an enzyme; (c) an antibody molecule; or (d) enzyme The pharmaceutical composition for use according to any one of claims 14 to 21, wherein the composition is an mRNA encoding the

23. (a) the enzyme is associated with a lysosomal storage disease or metabolic disorder; (b) the payload is an mRNA encoding a urea cycle enzyme, and / or (c) the disease is a metabolic disorder; A pharmaceutical composition for use according to any one of claims 14 to 22.

24. The target cell delivery LNP is (a) at a lower dose compared to the reference LNP; (b) said target cell delivery LNP is administered at a dose that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to said dose of a reference LNP; and / or (c) at a reduced frequency compared to the reference LNP The pharmaceutical composition for use according to any one of claims 14 to 23, which can be administered.

25. 25. The pharmaceutical composition for use according to claim 24, wherein the target cell delivery LNP delivered at a lower dose results in similar or enhanced lipid and / or payload levels in the target cell, organ, or cellular compartment.

26. The ionic lipid containing the compound I-301 is (a) Compound (R) I-301, or Compound (S) I-301, or (b) a racemic mixture comprising compound (R)I-301 and compound (S)I-301; A composition for use according to any one of claims 1 to 13 or a pharmaceutical composition for use according to any one of claims 14 to 25.

27. The reference LNP is (a) comprising an ionic lipid having formula I-XII; (b) does not contain ionic lipids with chiral centers; (c) does not contain ionic lipids containing multiple branched alkyl chains; (d) does not contain ring-substituted amino lipids; (e) does not contain carbocyclic-substituted amino lipids, and / or (f) does not contain cycloalkenyl-substituted amino lipids; A composition for use according to any one of claims 3 to 5 or 13, or a pharmaceutical composition for use according to any one of claims 16, 17 or 24.

28. 28. The composition for use according to any one of claims 1 to 13, 26 or 27, or the pharmaceutical composition for use according to any one of claims 14 to 27, wherein the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DPPC, DMPC, DMPE, DOPC, compound H-409, compound H-418, compound H-420, compound H-421 and compound H-422.

29. 29. The composition for use according to any one of claims 1 to 13 or 26 to 28, or the pharmaceutical composition for use according to any one of claims 14 to 28, wherein the liver cells are hepatocytes and the non-cationic helper lipid or phospholipid comprises a compound selected from the group consisting of DSPC, DMPE, and compound H-409.

30. A composition for use according to any one of claims 1 to 13 or 26 to 29 or a pharmaceutical composition for use according to any one of claims 14 to 29, comprising a PEG-lipid.

31. The PEG-lipid is (a) selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof; (b) selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE lipids; (c) comprising a compound selected from the group consisting of compound P-415, compound P-416, compound P-417, compound P-419, compound P-420, compound P-423, compound P-424, compound P-428, compound P-L1, compound P-L2, compound P-L3, compound P-L4, compound P-L6, compound P-L8, compound P-L9, compound P-L16, compound P-L17, compound P-L18, compound P-L19, compound P-L22, compound P-L23, and compound P-L25; or (d) A compound selected from the group consisting of compound P-428, compound PL-16, compound PL-17, compound PL-18, compound PL-19, compound PL-1, and compound PL-2; 31. A composition for use or a pharmaceutical composition for use according to claim 30.

32. The LNP is (a) a molar ratio of (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid of about 50:10, 49:11, 48:12, 47:13, 46:14, 45:15, 44:16, 43:17, 42:18, or 41:19; (b) about 41 mol% to about 50 mol% of an ionic lipid and about 10 mol% to about 19 mol% of a non-cationic helper lipid or phospholipid; or (c) about 50 mol % ionic lipid and about 10 mol % non-cationic helper lipid or phospholipid A composition for use according to any one of claims 1 to 13 or 26 to 31 or a pharmaceutical composition for use according to any one of claims 14 to 31, comprising:

33. (a) the molar ratio of (i) ionic lipid:(iii) non-cationic helper lipid or phospholipid is about 50:10; or (b) the ionic lipid:phospholipid:structural lipid:PEG lipid ratio is selected from: (i) 50:10:38:2; (ii) 50:20:28:2; (iii) 40:20:38:2; or (iv) 40:30:28:

2. A composition for use according to any one of claims 1 to 13 or 26 to 32 or a pharmaceutical composition for use according to any one of claims 14 to 32.

34. 34. A composition for use according to any one of claims 1 to 13 or 26 to 33 or a pharmaceutical composition for use according to any one of claims 14 to 33, wherein the lipid nanoparticles comprise DSPC as the phospholipid, cholesterol or a cholesterol / β-sitosterol blend as the structural lipid, and compound P-428 as the PEG lipid.

35. The LNP comprises: (i) about 50 mol % ionic lipid; (ii) about 10 mol% phospholipid that is DSPC; (iii) about 38.5 mol% structural lipids selected from β-sitosterol and cholesterol; and (iv) about 1.5 mol % PEG lipid, which is compound P-428; A composition for use according to any one of claims 1 to 13 or 26 to 33 or a pharmaceutical composition for use according to any one of claims 14 to 33, comprising:

36. A pharmaceutical composition comprising the delivery LNP of any one of claims 1 to 13 or 26 to 35 and a pharmaceutically acceptable carrier.

37. A GMP-grade pharmaceutical composition comprising the delivery LNP of any one of claims 1 to 13 or 26 to 35 and a pharmaceutically acceptable carrier.

38. 38. The pharmaceutical composition of claim 36 or 37, having a purity of greater than 95%, 96%, 97%, 98%, or 99%, e.g., at least 1%, 2%, 3%, 4%, 5%, or more contaminants removed.

Images