Novel lipids containing diketopiperazine core, lipid nanoparticle containing thereof, and methods of using thereof

Abstract

The present disclosure provides ionizable lipids containing a diketopiperazine moiety, lipid nanoparticles formed using such ionizable lipids, as well as their use in delivering nucleic acids and other therapeutic agents to certain cell types.

Description

NOVEL LIPIDS CONTAINING DIKETOPIPERAZINE CORE, LIPID NANOPARTICLE CONTAINING THEREOF, AND METHODS OF USING THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 632,354, filed April 10, 2024, the entire contents of which are incorporated herein by reference.GOVERNMENT RIGHTS

[0002] This invention was made with government support under Contract No. UG3-TR002855 awarded by the National Institutes of Health, and Contract No. HR00111920008 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure concerns ionizable lipids that can be used to form lipid nanoparticles (LNPs).BACKGROUND

[0004] Systemically administered lipid nanoparticles (LNPs) have delivered RNA in FDA approved drugs and early-stage clinical trials. The clinical impact of these LNP-RNA therapies supports the exploration of nanoparticles that deliver RNA to new tissues. One important cell type for next- generation therapies is hematopoietic stem cells (HSCs). Since all blood cells derive from HSCs, their dysfunction drives dozens of pathologies including sickle cell disease, B-thalassemia, as well as numerous anemias, immunodeficiencies, and metabolic disorders. As of April 2020, there were 48 active HSC gene therapy clinical trials6; many have reported promising results including two FDA approvals. However, these therapies typically require ex vivo HSC transduction. In this process, cells are mobilized from the bone marrow and removed from the blood. While the cells are being modified in the lab, the patient undergoes chemotherapy that depletes the immune system so the manipulated cells can engraft after theyare re-administered. This often leads to patient morbidity. As a result, there is an established need for HSC therapies that do not require ex vivo workflows.

[0005] Scientists have attempted to target HSCs in vivo. In one example, HSC delivery was achieved in non-human primates using adenoviruses engineered to express CD46. However, this required pre-treatment with granulocyte colony stimulating factor (G-CSF) to induce mobilization as well as three rounds of chemotherapy to select cells after delivery. Systemic HSC delivery has also been achieved using nanoparticles. In one example, mice were pre-treated with stem cell factor, then injected with 4 mg of nanoparticles. Assuming a 25 g mouse, this equates to a 160 mg / kg dose. For comparison, systemically administered siRNA and Cas9 mRNA drugs work robustly at 0.3 mg / kg doses, highlighting the relative difficulty of targeting HSCs. In addition, three companies have reported LNP-mediated bone marrow delivery in vivo. However, none reported all the details (the LNP chemical structures and molar ratio of the components) required to recreate the delivery system.SUMMARY

[0006] One aspect of the invention relates to a compound of Formula (I):wherein:Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl;each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', and R" each are independently H, OR14, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl;Ra, and Rbeach are independently H or branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci - C20 monovalent hydrocarbon chain, or represented by -(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(0)N(R13)-, -N(R13)-, -N(R13)C(0)-, -C(O)S-, -SC(O)-, -CH(0R14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, and R14are independently H, branched or unbranched alkyl or alkenyl, and each R13is independently H, alkyl, or OR14; provided that when Z is -C(O)-, Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. The compounds also include the a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds disclosed herein,

[0007] Another aspect of Another aspect of the invention relates to a method of making a compound of formula(i) reactingthe presence of a coupling agent, an organic base, and a solvent to form an intermediate compound, and(ii) reacting the intermediate compound formed in (i) with an acid, and then withform the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(CCR11)(R12))m-Q-(C(R1’XR12))^, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

[0008] Another aspect of the invention relates to a method of making a compound of formulaO R1(a) HO A \iX R2in the presence of an organic base,R1I(b)in the presence of an organic carbonate, orR1I(c)in the presence of an organic coupling agent to form a carbamate, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

[0009] Also disclosed are lipid nanoparticles comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein.

[0010] Another aspect of the invention relates to a pharmaceutical composition comprising the lipid nanoparticle, comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable carrier.

[0011] Another aspect of the invention relates to methods of delivering a therapeutic agent to asubject comprising administering to the subject the presently disclosed lipid nanoparticles, wherein optionally the therapeutic agent may be encapsulated within the lipid nanoparticles.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1 A-1I provide chemical and in vivo characterization of stereopure ionizable lipid nanoparticles (LNPs) according to the present disclosure.

[0013] FIGS. 2A-2I provide the results of a study concerning delivery of mRNA by a lipid nanoparticle according to the present disclosure.

[0014] FIGS. 3A-3D provide the results of an investigation of delivery of mRNA by an inventive LNP to transcriptionally defined cells in bone marrow.

[0015] FIGS. 4A-4C depict the results of a study of delivery of mRNA by an inventive LNP to HSC-specific cell types.

[0016] FIGS. 5A-5B provide scRNA-seq control data.

[0017] FIGS. 6A-6B depict the results of a study concerning delivery of mRNA by an inventive LNP to non-human primate cells.

[0018] FIG. 7 illustrates delivery of mRNA by an inventive LNP to CD34+ non-human primate cells at low doses.

[0019] FIGS. 8A-8F illustrate the flow cytometry results for five high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 16 in the liver (FIG.8 A), lung (FIG. 8B), spleen (FIG. 8C), bone marrow (FIG. 8D), kidney (FIG. 8E), and heart (FIG. 8F).

[0020] FIGS. 9A-9F show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG.9A), lung (FIG. 9B), bone marrow (FIG. 9C), spleen (FIG. 9D), kidney (FIG. 9E), and heart (FIG. 9F).

[0021] FIGS. 10A-10F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 10A), lung (FIG. 10B), bone marrow (FIG. 10C), spleen (FIG. 10D), kidney (FIG. 10E), and heart (FIG. 10F).

[0022] FIGS. 11 A-l 1C show the flow cytometry results for individual LNP formulations selected from Screen 4 (discussed in FIGs. 8A-8F) in mouse using the LNP formulationscontaining Compound 16 in the liver (FIG. 11A), lung (FIG. 1 IB), and bone marrow (FIG. 11C).

[0023] FIGS. 12A-12C show the flow cytometry results for individual LNP formulations selected from Screen 5 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 12), lung (FIG. 12B), and bone marrow (FIG. 12C).

[0024] FIGS. 13A-13F illustrate the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 61 in the liver (FIG. 13 A), lung (FIG. 13B), spleen (FIG. 13C), bone marrow (FIG. 13D), kidney (FIG. 13E), and heart (FIG. 13F).

[0025] FIGS. 14A-14F show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 13A-13F) in mouse using the LNP formulations containing Compound 61 in the liver (FIG. 14A), lung (FIG. 14B), bone marrow (FIG. 14C), spleen (FIG. 14D), kidney (FIG. 14E), and heart (FIG. 14F).

[0026] FIGS. 15A-15F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 56 and Compound 62 in the liver (FIG. 15 A), lung (FIG. 15B), spleen (FIG. 15C), bone marrow (FIG. 15D), kidney (FIG. 15E), and heart (FIG. 15F).

[0027] FIGS. 16A-16C show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 15A-15F) in mouse using the LNP formulations containing Compound 56 and Compound 62 in the liver (FIG. 16A), lung (FIG. 16B), and spleen (FIG. 16C).

[0028] FIGS. 17A-17F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 68, Compound 69, and Compound 70 in the liver (FIG. 17A), lung (FIG. 17B), spleen (FIG. 17C), bone marrow (FIG. 17D), kidney (FIG. 17E), and heart (FIG. 17F).

[0029] FIGS. 18A-18F show the flow cytometry results for a LNP formulation containing Compound 16 in primate at various doses (0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg), in the liver (FIG. 18 A), lung (FIG. 18B), spleen (FIG. 18C), bone marrow (FIG. 18D), kidney (FIG. 18E), and heart (FIG. 18F).

[0030] FIGS. 19A-19F show the flow cytometry results for a screen of LNP formulations using the top performing individual LNP formulations containing Compound 18 in primate injected intravenously at 1.5 mg / kg, in the liver (FIG. 19A), lung (FIG. 19B), spleen (FIG. 19C), bonemarrow (FIG. 19D), kidney (FIG. 19E), and heart (FIG. 19F).DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031] The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0032] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

[0033] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0034] In the present disclosure the singular forms “a”, “an”, and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a certain chemical moiety “may be” X, Y, or Z, it is not necessarily intended by such usage to exclude other choices for the moiety; for example, a statement to the effect that Ri “may be alkyl, aryl, or amino” does not necessarily exclude other choices for Ri, such as halo, aralkyl, and the like.

[0035] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” may refer to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” may refer to a value of 7.2% to 8.8%, inclusive. Also, when the term “about” precedes a range, it is understood that the term modifies both recited endpoints and all points embraced within the range. For example, the phrase “about 1-10” is understood to mean “about 1 to about 10”, as well as “about x”, wherein x refers to any value between 1 and 10. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” In another example, when a listing of possible substituents including “hydrogen, alkyl, and aryl” is provided, the recited listing may be construed as including situations whereby any of “hydrogen, alkyl, and aryl” is negatively excluded; thus, a recitation of “hydrogen, alkyl, and aryl” may be construed as “hydrogen and aryl, but not alkyl”, or simply “wherein the substituent is not alkyl”.

[0036] Protective groups are abbreviated according to the system disclosed in Greene, T. W. and Wuts, P.G.M., Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons, 1991, which is incorporated in its entirety herein. For example, “CBZ” or “Cbz” or “Z” stands for carbobenzyloxy or benzyloxycarbonyl, “Boc” or “BOC” represents / -butoxycarbonyl, “Alloc” denotes allyloxycarbonyl, Bz means benzoyl, and “Fmoc” stands for 9- fluoreny 1 m ethoxy carb ony 1.

[0037] As used herein, the terms “component”, “compound”, “drug”, “pharmacologically active agent”, “active agent”, “therapeutic”, “therapeutic agent”, “therapy”, “treatment”, or “medicament” may be used herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and / or physiologic effect by local and / or systemic action.

[0038] The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. Unless otherwise indicated, “alkyl” generally refers to C1-C24 alkyl (e.g., C1-C12 alkyl, Ci-Cs alkyl, or C1-C4 alkyl).

[0039] The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Unless otherwise indicated, “alkenyl” generally refers to C2-C8 alkenyl (e.g., C2-C6 alkenyl, C2-C4 alkenyl, or C2- C3 alkenyl). Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups.

[0040] The term “alkoxy” refers to an -O-alkyl radical. The term “alkylene” refers to adivalent alkyl (z.e., -R-). The term “aminoalkyl” refers to an alkyl substituted with an amino. The term “mercapto” refers to an -SH radical. The term “thioalkoxy” refers to an -S-alkyl radical.

[0041] The term “alkylene” refers to abivalent form of an alkyl group. An “alkylene chain” is a polymethylene group, i.e., — (CH2)n — , wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below.

[0042] The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below.

[0043] The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term “aryl” may be used interchangeably with the term “aryl ring.” Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

[0044] The term “cycloalkyl” or “cyclyl” as employed herein includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0045] The term “heteroaryl” or “heteroar-” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring maybe substituted by a substituent. The term also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one and the like.

[0046] The term “heterocyclyl,” “heterocycle,” “heterocyclic radical,” or “heterocyclic ring” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0047] A divalent radical of an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocyclyl is formed by removal of a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by removal of two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).

[0048] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo,alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, ami noalkyl ami no, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

[0049] “Isomers.” The compounds described herein or their pharmaceutically acceptable salts may include all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like For instance, the compounds can contain one or more stereocenters and may thus give rise to geometic isomers (e.g., double bond causing geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- such as for sugar anomers, or as (D)- or (L)- such as for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0050] A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are non-superimposablemirror images of one another.

[0051] The term “pharmaceutically acceptable salt” include the salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bi sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI alkylAsalts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.

[0052] Disclosed herein are diketopiperazine-containing ionizable lipids that can, among other things, preferentially deliver mRNA to immune cells or stem cells in various tissues in vivo without targeting ligands. The inventive lipids were synthesized and characterized, and high- throughput DNA barcoding was used to quantify how a collection of chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in multiple cell types directly in vivo. By analyzing the relationships between lipid structure and cellular targeting, lipid traits that increase delivery in vivo were identified. In addition, LNPs that preferentially delivers mRNA to liver or non-liver cells at clinically relevant doses wereprepared and characterized. The obtained data highlighted inventive nanoparticles with natural non-hepatocyte tropism and demonstrated that the presently disclosed lipids with bioactive small-molecule motifs successfully deliver mRNA in vivo.The novel lipids

[0053] Accordingly, disclosed are compounds of Formula (I)wherein:Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl;wherein: each t is independently 1, 2, 3, or 4; each Z is absent, -C(O)-, -C(O)O-, -C(O)N(R")-, or -S(O)(O)-;X is -O-, -S-, or -N(R")-; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', and R'' each are independently H, OR14, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl;Ra, and Rbeach are independently H or branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci - C20 monovalent hydrocarbon chain, or represented by -(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10,each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-,-OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-,-CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, and R14are independently H, branched or unbranched alkyl or alkenyl, and each R13is independently H, alkyl, or OR14; provided that when Z is -C(O)-, Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. The compounds also include the a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds disclosed herein,

[0054] In some embodiments, the lipid compound has the formula of:

[0055] In some embodiments, in any of the formulas disclosed herein (each of the below variables, if present):Y is H; aryl; branched or unbranched Ci-Ce alkyl; Ci-Ce alkyl interrupted with a N, O, oreach R, R', Ra, and Rbare H; each t is independently 1 or 2; each Z is independently absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-;X is -O-, -S-, -NH-, or -N(OH)-; each L is independently a C1-C4 alkylene optionally substituted by OH; andRi and R2 are each independently represented by -(CH2)m-Q-(CH2)nH, wherein Q is absent, -CH=CH-, -C=C-, -S-S-, -N(OH)-, -C(O)NH-, -C(O)N(OH)-, -NHC(O)-,

[0056] In some embodiments, the compound has the formula ofeach u is independently 2, 3, or 4. All the other variables have the same definitions as those defined above.

[0057] In some embodiments, the compound the formula of:variables have the same definitions as those defined above.

[0058] In some embodiments, in any of the formulas disclosed herein (if Z is present), at least one Z is absent, -C(O)-, -C(O)O-, -C(O)N(R")-, or -S(O)(O)-. In some embodiments, at least one Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some embodiments, at least one Z is -C(O)-. In some embodiments, at least one Z is absent. In some embodiments, at least one Z is -C(O)O-. In some embodiments, at least one Z is -C(O)NH-. In some embodiments, at least one Z is -S(O)(O)-.

[0059] In some embodiments, each R, R', and R" are independently H, OR14, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl. In some embodiments, R, R', and R” each may be independently H or C1-C3 alkyl. In some embodiments, R, R', and R" each may be independently a C3-C7 cycloalkyl. In some embodiments, R” is OR14(e.g., OH, or OCH3). In some embodiments, each R, R', and R" are H.

[0060] In some embodiments, in any of the formulas disclosed herein (if L is present), each L is independently a C2-C4 alkylene optionally substituted by OH. In some embodiments, each Lis independently a C2-C4 alkylene, or -CH2-CH(OH)-CH2- (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the Z variable next to the L variable is -O-. In one embodiment, L is independently a C2-C4 alkylene (e.g., a C2, C3, or C4 alkylene). In one embodiment, L is independently -CH2-CH(OH)-CH2- (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the Z variable next to the L variable is -O-.

[0061] In some embodiments, in any of the formulas disclosed herein (if Y is present), Y is H; aryl; branched or unbranched Ci-Ce alkyl; Ci-Ce alkyl interrupted with a N, O, or S atom; or Ci- C3 alkyl substituted with an aryl. In one embodiment, Y is H. In one embodiment, Y is branched or unbranched C1-C4 alkyl, a C1-C3 alkyl interrupted with an S atom, or benzyl.

[0062] In some embodiment,some embodiments, X is -O-,-S-, -NH-, or -N(OH)-. In some embodiments, X is O or S. In some embodiments, X is -N(R")- (e g., -NH- or -N(OH)-.

[0063] In some embodiment,

[0064] In some embodiments, in any of the formulas disclosed herein (if Ra / Rbis present), Raand Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl. In some embodiments, Raand Rbeach are independently H, or C1-C3 alkyl. In some embodiments, Raand / or Rbis C3-C7 cycloalkyl. In one embodiment, Raand Rbare H.

[0065] In some embodiments, in any of the formulas disclosed herein (if t is present), each t is independently 1, 2, 3, or 4. In some embodiments, each t is independently 1 or 2.

[0066] In some embodiments, the compound has the formula of:(IVB); wherein each u is independently 2, 3, or 4.All the other variables have the same definitions as those defined above.

[0067] In some embodiments, the compound has the formula of:All the variables have the same definitions as those defined above.

[0068] In some embodiments, Y may be -(CH2)tO-Z-L-N(Ri)(R2). In certain instances wherein Y is -(CH2)tO-Z-L-N(Ri)(R2), each of the Z groups within the compound are the same, each of the Ri groups within the compound are the same, and each of the R2 groups within the compound are the same. In other embodiments in which Y is -(CH2)tO-Z-L-N(Ri)(R2), the Z groups within the compound may be independently selected, i.e., may be different, the Ri groups within the compound may be independently selected, i.e., may be different, and the R2 groups within the compound may be independently selected, i.e., may be different.

[0069] In some embodiments, in any of the formulas disclosed herein (if Ri / R2is present), each Ri and R2 may be the same, e.g., each Ri may be the same as each R2. In some embodiments, at least one, two, or three of the Ri and R2 variables within the compound are different than the other(s). In some embodiments, a particular Ri group may be the same as one or two R2 groups within the compound. In other instances, the compound contains two Ri groups, and both Ri groups are the same as one or two R2 groups within the compound.

[0070] In some embodiments, each Ri and R2 is independently branched or unbranched C1-20 alkyl or branched or unbranched C2 -C20 alkenyl. For example, each Ri and R2 may independently be C1.20 linear or branched alkyl; each Ri and R2 may independently be C1-20 linear or branched alkenyl. In some embodiments, each Ri and R2 are independently C9-C16 alkyl.In some embodiments, each Ri and R2 are independently C9-C12 alkyl. In some embodiments, each Ri and R2 may independently be C9 alkyl, Cwalkyl, Cualkyl, Cnalkyl, Cisalkyl, Cualkyl, Cisalkyl, or Cualkyl. In some embodiments, each Ri and R2 are independently C9-C16 alkenyl. In some embodiments, each Ri and R2 are independently C9-C 12 alkenyl.

[0071] In some embodiments, Ri and / or R2 may be represented by -(C(Ru)(R12))m-Q- (C(Rn)(R12))nH. In this formula, each m is independently 0 to 10; each n is independently 1 to 10; each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl or heterocyclic; each R11, R12, and R14are independently H, branched or unbranched alkyl or alkenyl; and each R13is independently H, alkyl, or OR14. In some embodiments, when X is O or S or when Z is C(O), Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-.

[0072] In some embodiments, Q in each Ri and R2 variable is independently -CH=CH-, -C=C-, or -S-S-. In some embodiments, Q in each Ri and R2 variable is independently -C(O)O- or -OC(O)-. In some embodiments, Q in each Ri and R2 variable is independently -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or-NHC(O)-.

[0073] In some embodiments, one of Ri and R2 is Ci -Ci6 branched or unbranched alkyl; and in the other Ri or R2, Q is -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or -NHC(O)-.

[0074] Non-limiting examples of the lipid compounds are set forth below.

[0075] In some embodiments, exemplary lipid compounds are:.

[0076] In some embodiments, these exemplary lipid compounds provide targeted delivery to specific cells, tissues, and / or organs such as the lung, heart, kidney, liver, splenic, lymphaticcells, or marrow cells of a subject. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of lung. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen.Preparation of the novel lipids

[0077] Certain aspects of the invention relate to methods of making the novel lipids described herein.

[0078] All above descriptions and all embodiments regarding the novel lipid compounds discussed in the above aspects of the invention are applicable to these aspect of the invention relating to the method of making the compounds.

[0079] Accordingly, one aspect of the invention relates to a method of making a compound of formulacomprising: ing(i) reactingpresence of a coupling agent, an organic base, and a solvent to form an intermediate compound, and(ii) reacting the intermediate compound formed in (i) with an acid, and then withform the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(CCRi^CRi^^-Q-CCCRi^CR12)),^, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C~, -S-S-, -C(O)O-, -OC(O)-, -C(0)N(R13)-, -N(R13)C(0)-, -C(O)S-, -SC(O)-, -CH(0R14)-, -CH(RU)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

[0080] In some embodiments, the coupling agent is EDCI. Other coupling agents can be used herein. In some embodiments, the organic base is DIPEA. Other organic bases are suitable for use herein. In some embodiments, the solvent is DMF, DCM, or a mixture thereof.

[0081] In some embodiments, the reactant iform correspondingthe reagents in step (i) further comprises a benzotriazole or its derivative. In some embodiments, the reactantform correspondingthe reagents in step (i) further comprises a benzotriazole or its derivative.

[0082] The use of the benzotriazole or its derivative could eliminate or avoid the racemization of single-enantiomer chiral molecules and to result in a chiral enantiomer. In some embodiments, the benzotriazole or its derivative is HOBt, HATU, or a mixture thereof.

[0083] In some embodiments, the acid in step (ii) is HC1 in dioxane, or TFA; and the reagents in step (ii) that reacts withfurther comprise sodium triacetoxyhydroborate (NaBH(OAc)3).

[0084] Another aspect of the invention relates to a method of making a compound of formulain the presence of an organic base, the presence of an organic carbonate, orthe presence of an organic coupling agent to form a carbamate, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(CCRi^CRi^^-Q-CCCRi^CR12)),^, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C~, -S-S-, -C(O)O-, -OC(O)-, -C(0)N(R13)-, -N(R13)C(0)-, -C(O)S-, -SC(O)-, -CH(0R14)-, -CH(RU)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

[0085] In some embodiments, the reaction reactsin the presence of a couple agent (e.g., EDCI). In some embodiments, the organic base is a pyridine or its derivative (e.g., DMAP).

[0086] In some embodiments, the reaction reactsin the presence of triphosgene (bis(trichloromethyl carbonate)) and an organic base such as pyridine.

[0087] In some embodiments, the reaction reactsR1in the presence of 4-nitrophenyl chloroformate, pyridine, and DMAP.

[0088] These above aspects of the invention relating to the synthesis of the compounds are further illustrated in Examples 1 and 7.Lipid Nanoparticles

[0089] Also disclosed herein are lipid nanoparticles comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any one of the embodiments described herein. The lipid nanoparticles may further comprise one or more of a helper lipid, a sterol, and a polyethylene glycol (PEG)-modified lipid. Advantageously, the lipid nanoparticles according to the present disclosure that include a compound of Formula (I) deliver a therapeutic agent, such as a nucleic acid, preferentially to certain cell types within the liver cells, lung cells, spleen cells, cells, lung cells, lymphatic cells, or marrow cells of the subject, including stem cells. Such preferential delivery occurs without the requirement for a specific targeting ligand.

[0090] For example, the presently disclosed lipid nanoparticles can deliver a therapeutic agent preferentially to liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, or other immune cells within the liver.

[0091] In other embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to spleen dendritic cells, spleen macrophages, spleen B cells, spleen T cells, or other immune cells within the spleen.

[0092] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, lymphatic natural killer (NK) cells, or other immune cells within the lymph nodes.

[0093] In some embodiments, the lipid nanoparticles can deliver a therapeutic agentpreferentially to lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung macrophages, lung T cells, lung natural killer (NK) cells, or other immune cells within the lung.

[0094] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to heart endothelial cells, heart epithelial cells, heart dendritic cells, heart macrophages, heart B cells, heart T cells, heart natural killer (NK) cells, or other immune cells within the heart.

[0095] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to kidney endothelial cells, kidney epithelial cells, kidney macrophages, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, or other immune cells within the kidney.

[0096] In other embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to marrow dendritic cells, marrow macrophages, marrow B cells, marrow T cells, other immune cells within marrow, or marrow stem-like cells, or marrow hematopoietic stem cells (HSCs).

[0097] As used herein, preferential delivery to a particular class of cells or cell type refers to delivery at a higher rate than to non-targeted cells. For example, the preferential delivery can mean delivery at or above a rate that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than to non-targeted cells, z'.e., cells not within the particular class of cells or of the particular cell type. In some instances, the delivery is to a particular targeted class of cells or cell type, and there is no delivery or only minimal delivery to non-targeted cells. As a result, the preferential delivery can be at a rate that is hundreds of times, thousands of times, or, theoretically infinitely greater than to the non-targeted cells.

[0098] The role and identity of exemplary helper lipids for lipid nanoparticles are known among those skilled in the art and may be any compound that contributes to the stability and delivery efficiency of the LNP, or to the stable encapsulation of a therapeutic agent within the LNP.

[0099] In certain embodiments, the helper lipid is a cationic lipid.

[0100] In certain embodiments, the helper lipid is DDAB (Didodecyldimethylammonium bromide), DOTAP (l,2-Dioleoyl-3-trimethylammonium propane), or DOTMA (1, 2-di-O- octadecenyl-3 -trimethylammonium propane).

[0101] In certain embodiments, the helper lipid is a phospholipid. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the nonlimiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0102] Particular phospholipids can facilitate fusion to a membrane. In some embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0103] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0104] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0105] In certain embodiments, the phospholipid is selected from the group consisting of 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC),1.2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC),1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-di-O-octadecenyl-5«-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-s7z-glycero-3-phosphocholine (OChemsPC),1-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC),1.2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3 -phosphocholine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1.2-distearoyl-sn-glycero-3 -phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine,1.2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphoethanol amine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, and derivatives thereof.

[0106] In some embodiments, the lipid nanoparticle further comprises one or more helper lipids selected from the group consisting of l,2-dioeoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),2-distearoyl-sn-glycero-3-phosphocholine (DSPC), didodecyldimethylammonium bromide (DDAB), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleoyl-3- trimethylammonium propane (DOTAP), 18: 1 PA, N-methyldioctadecylamine (MDOA), N,N- dicotadecylaniline, sn-(3-myristoyl-2-hydroxy)-glycerol-l-phospho-sn-3'-(r,2'-dimyristoyl)- glycerol (14:0 Hemi BMP), l,2-dimyristoyl-sn-glycero-3-phosphate (DMPA; 14:0 PA),

[0107] l,2-dioleoyl-sn-glycero-3-phosphate (DOPA; 18: 1 PA), l,2-distearoyl-sn-glycero-3- phosphate (DSP A; 18:0 PA), and a combination thereof.

[0108] In some embodiments, the one or more helper lipids are l,2-dioeoyl-sn-glycero-3- phosphoethanolamine (DOPE), dimethyldioctadecylammonium (DDAB), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), or a combination thereof.

[0109] The lipid nanoparticles may also include a sterol or derivative thereof. In some embodiments, the sterol or derivative thereof is cholesterol or derivative thereof. In some embodiments, a combination of two or more cholesterols or derivatives thereof are used. The inclusion of a cholesterol in nanoparticle formulations can improve efficacy, potentially due to enhanced membrane fusion. Exemplary cholesterol or derivatives thereof include cholesterol (C27H46O), 20a-OH cholesterol, 20a-hydroxycholesterol (5-cholestene-3p,20a-diol), and DC- Cholesterol (N,N-dimethyl- N-ethylcarboxamidocholesterol). Any natural sterols may also be used for the cholesterol component. Examples of natural sterols include, for example, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin, DHEA sulfate, DHEA, sitosterol, lanosterol-95, cholesterol (plant), dihydro FF-MAS-d6, dihydro T-MAS-d6, zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, dihydro T-MAS, delta 5- avenasterol, brassicasterol, dihydro FF-MAS, and 24-methylene cholesterol. A large diversity of structural analogs of cholesterol exist as natural products (e.g., phytosterols that are plant-based sterols, which provide stability to the plant cell wall). Exemplary cholesterol analogs include, for example, Vitamin D derivatives (such as 9,10-secosteroids, Vitamin D2, Vitamin D3, Calcipotriol), alkyl-substituted steroids (such as C-24 alkyl steroids), and cholesterol analogs wherein the tail is modified into a fifth ring (such as pentacyclic steroids).

[0110] The lipid nanoparticles may also include a PEG-lipid. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. 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 dialkylamines and PEG- modified l,2-diacyloxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0111] In some embodiments, the PEG lipid includes, but are not limited to, 1,2-dimyristoyl- sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0112] In some embodiment, the PEG lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0113] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about Cn to about C22. In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14 to about Ci6. In some embodiments, a PEG moiety, for example an mPEG-NFE, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG lipid is PEG2k-DMG.

[0114] In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

[0115] In some embodiment, the PEG-modified lipid is PEG-DMG or PEG-PE.

[0116] In some embodiment, the PEG-modified lipid is l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DMPE-PEG2K; 14:0 PEG2K PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DSPE-PEG2K; 18:0 PEG2K PE), l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG 2K), 2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), distearoyl-rac-glycerol-PEG2K (DSG-PEG 2K), or a combination thereof.

[0117] In some embodiments, in the presently disclosed LNPs, in addition to the compound of Formula (I), the LNP also comprises a helper lipid (e.g., DOPE, DDAB, DOTAP, DSPC, or a mixture thereof), a sterol (e.g., cholesterol (C27H46O), 20a-OH cholesterol, DC-cholesterol, or a mixture thereof), and a PEG lipid (e.g., 14:0PEG2K PE, 18:0PEG2K PE, or a mixture thereof).

[0118] The molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid in the lipid nanoparticles may be about 20-60 : 7-50 : 5-70 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid is about 25-55 : 10-50 : 15-50 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid is about 30-55 : 10-20 : 30-50 : 1-3.In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 32-50 : 12-18 : 32-48 : 1-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 35-50 : 12-16 : 35-47 : 2-3.

[0119] In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid is about 25 : 33 : 40 : 2; is about 30 :22.5 : 45 : 2.5; is about 30 : 39 : 30 : 1; is about 30 : 50 : 18 : 2; is about 35 : 35 : 27.5 : 2.5; is about 35 : 43 : 20 : 2; is about 35 : 44.5 : 18 : 2.5; is about 35 : 16 : 46.5 : 2.5; is about 45 : 13 :39.5 : 2.5; is about 50 : 40 : 7.5 : 2.5; is about 50 : 17.5 : 30 : 2.5; is about 50 : 12.5 : 35 : 2.5; is about 50 : 10 : 38.5 : 1.5; or is about 35 : 15 : 47.5 : 2.5.

[0120] In some embodiments, the sterol component (e.g., cholesterol or its derivative) may be absent from the LNP. In these embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid in the lipid nanoparticles may be about 20-60 : 10- 60 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 25-55 : 15-60 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 30-55 : 30-55 : 1-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 32-50 : 40-55 : 1.5-2.5.

[0121] In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 45 : 52.5 : 2.5; is about 45 : 53: 2; or is about 50 : 47.5 : 2.5.

[0122] The molar concentration of the compound according to Formula (I) in the LNPs may be about 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, or 55 percent of the total lipids in the LNP. The molar concentration of the helper lipid in the LNPs may be about 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, or 50 percent of the total lipids in the LNP. The molar concentration of the cholesterol or its derivative in the LNPs may be about 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, or 50 percent of the total lipids in the LNP. The molar concentration of the PEG lipid may be about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 percent of the total lipids in the LNP.

[0123] The lipid nanoparticle may have a diameter of about 20-250, 20-225, 20-200, 30-250,30-225, 20-300, 40-250, 40-225, 40-200, 45-250, 45-225, 45-200, 50-200, 50-180, or 75-170 nm. For example, the diameter of the lipid nanoparticle may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm. In a given population of lipid nanoparticles according to the present disclosure, the population may include individual members of respectively different sizes. The particle size distribution of a given population of LNPs according to the present disclosure may be characterized by a D90 of about 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, or 60 nm, and / or a D10 of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0124] The lipid nanoparticles according to the present disclosure can be used for the delivery of therapeutic agents to a living organism, such as to a human subject. The therapeutic agent may be encapsulated within the lipid nanoparticle. In some embodiments, the therapeutic agent may be a nucleic acid molecule (e.g., oligonucleotide), protein or peptide, carbohydrate or glycoprotein, lipid, small molecule, or any combination thereof.

[0125] In some embodiments, the therapeutic agent is a small molecule.

[0126] In some embodiments, the therapeutic agent is a protein or peptide.

[0127] In some embodiment, the therapeutic agent is a nucleic acid molecule (e.g., DNA or RNA).

[0128] In some embodiments, the therapeutic agent is a DNA, e.g., in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.

[0129] In some embodiments, the therapeutic agent is an RNA, e.g., in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis- NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi- interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satelliteRNA, or derivatives of these groups.

[0130] In some embodiments, the therapeutic agent is an mRNA.

[0131] In some embodiments, the lipid nanoparticle (LNP) formulations include (i) a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any one of the embodiments described herein, (ii) a helper lipid, (iii) a PEG lipid, and optionally (iv) a sterol (e.g., cholesterol).

[0132] In some embodiments, in the exemplary LNP formulations, compound (i) is compound 73, A-10, compound 117, or compound 94; the helper lipid (ii) is DDAB; the PEG lipid (iii) is C14PEG2K PE, C18PEG2K PE, or DSG-PEG2K; and the sterol (iv) is cholesterol. The ratios for these lipid components are:

[0133] In some embodiment, exemplary LNP formulations include:

[0134] In some embodiments, the above exemplary LNP formulations provide targeted delivery to specific cells, tissues, and / or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, the above exemplary lipid compounds provide targeted delivery to the cells or tissues of lung.

[0135] In some embodiments, in the exemplary LNP formulations, compound (i) is compound 94; the helper lipid (ii) is DSPC or DOPE; the PEG lipid (iii) is C14PEG2K PE or DMG-PEG2K; and the sterol (iv) is cholesterol. The ratios for these lipid components are

[0136] In some embodiment, exemplary LNP formulations include:

[0137] In some embodiments, the above exemplary LNP formulations provide targeted delivery to specific cells, tissues, and / or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, the above exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen.

[0138] Additional non-limiting examples of the lipid nanoparticles formulations are further illustrated in Examples 2-3 and 8-9.

[0139] Aspects of the invention also relates to a pharmaceutical composition comprising the lipid nanoparticle, comprising a compound of Formula (I), a compound of the subgenus formulasof formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable carrier.

[0140] All above descriptions and all embodiments regarding the novel lipid compounds and lipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the pharmaceutical composition.

[0141] The present disclosure also provides a pharmaceutical composition comprising a lipid nanoparticle comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any of the embodiments described herein, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” preferably refers to a material that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition. Such pharmaceutically acceptable materials typically have met the required standards of toxicological and manufacturing testing, and include those materials identified as suitable inactive ingredients by the U.S. Food and Drug Administration.

[0142] Thus, the LNPs according to the present disclosure may be provided in a composition that is formulated for any type of administration. For example, the compositions may be formulated for administration orally, topically, parenterally, enterally, or by inhalation (e.g., intranasally). The active agent may be formulated for neat administration, or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing / dispersing agent, tablet-disintegrating agent, encapsulating material, film former or coating, flavoring agent, or printing ink. Any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the LNPs may be incorporated into sustained-release preparations and formulations. Administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.

[0143] In powders, the carrier, diluent, or excipient may be a finely divided solid that is inadmixture with the finely divided active ingredient. In tablets, the LNPs are mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the LNPs may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of LNP in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.

[0144] Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The LNPs may be suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators.

[0145] Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.

[0146] Suitable examples of liquid carriers, diluents, and excipients, for example, for oral, topical, or parenteral administration, include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.

[0147] For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration. Solutions of the LNPs can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations may contain a preservative to prevent the growth of microorganisms.

[0148] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some instances, the antimicrobial peptides themselves may be sufficient to prevent contamination by microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0149] Sterile injectable solutions may be prepared by incorporating the LNPs in the pharmaceutically appropriate amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the LNPs into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and freeze drying techniques that yield a powder of the LNPs or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0150] Also provided herein are methods of delivering a therapeutic agent to a subject, comprising administering to the subject a lipid nanoparticle according to any of the presently disclosed embodiments, wherein the lipid nanoparticle comprises a therapeutic agent.

[0151] All above descriptions and all embodiments regarding the novel lipid compounds andlipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the methods of delivering a therapeutic agent to a subject.

[0152] It has surprisingly been discovered that the inventive nanoparticles preferentially target certain human lung, heart, kidney, liver, splenic, lymphatic, or marrow cells, and can thereby preferentially deliver the therapeutic agent to such cells. The cells to which the present LNPs deliver the therapeutic agent can include, for example, lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung macrophages, lung natural killer (NK) cells, or other immune cells within the lung; heart endothelial cells, heart epithelial cells, heart dendritic cells, heart B cells, heart T cells, heart natural killer (NK) cells, heart macrophages, or other immune cells within the heart; kidney endothelial cells, kidney epithelial cells, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, kidney macrophages, or other immune cells within the kidney; liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, other immune cells within the liver; spleen dendritic cells, spleen macrophages, spleen B cells, spleen T cells, other immune cells within the spleen; lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, or lymphatic natural killer (NK) cells; marrow dendritic cells, marrow macrophages, marrow B cells, marrow T cells, other immune cells within marrow, or marrow stem-like cells, , or marrow hematopoietic stem cells (HSCs).

[0153] Accordingly, the present disclosure also provides methods for delivering a therapeutic agent to lung, heart, kidney, liver, splenic, lymphatic, or marrow cells of a subject, comprising administering to the subject a lipid nanoparticle according to any of the embodiments disclosed herein.

[0154] Beneficially, the lipid nanoparticles can deliver the therapeutic agent to the subject at clinically relevant doses. In some embodiments, that dose at which the present LNPs deliver the therapeutic agent is about 0.01 to about 3.0 mg / kg. For example the dose at which the therapeutic agent is delivered may be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, 2.9, or 3.0 mg / kg.EXAMPLES

[0155] The present invention is further defined in the following Examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and should not be construed as limiting the appended claims. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.Example 1 - Design of Ionizable Lipids

[0156] Ionizable lipids were synthesized by reacting two cyclic serine-like cores, denoted A and B, with lipids consisting of 10 or 12 saturated carbons via esterification followed by a reductive amination:This produced four lipids that were designated A10, A12, BIO, and B12 (FIG. 1A). Details of the synthetic process are as follows.

[0157] Intermediate synthesis. To a mixture of 1 (1.5 mmol) in 20 mL DMF / DCM (1 : 1) solution was added DIPEA (6 mmol), acid 2 or 3 (4.5 mmol), followed by EDCI (4.5 mmol) and HOBt (4.5 mmol). The resulting mixture was stirred at room temperature for 12 hr and quenched by the addition of saturated NaHCCL solution (20 mL). The aqueous phase was extracted with CH2CI2 (20 mL) three times and concentrated in vacuo. The crude product was then purified by column chromatography using eluent CFLCb / MeOH (10: 1) to obtain intermediate 4 or 5.

[0158] Final compounds synthesis. To the intermediate 4 or 5 (0.1 mmol) was added 3 mL 4N HC1 in dioxane at 0°C and the mixture was stirred at room temperature for 1 hr. Solvent was removed in vacuo and the crude product was dissolved in 1 mL CH2CI2. Aldehyde RCHO (0.6 mmol) was added followed by NaBH(OAc)3 (0.5 mmol). The resulting mixture was stirred at room temperature for 12 hr and then quenched by saturated NaHCCL solution (10 mL). The aqueous phase was extracted by CH2O2 (3X10 mL) and concentrated in vacuo, followed by purification by flash column chromatography using CILCh / MeOH (100: 1 to 10: 1).

[0159] Intermediate 4: white solid, 44% yield. 'H NMR (500 MHz, MeOD) 5 6.65 (t, J= 6.1 Hz, 2H), 4.43 (d, J= 4.6 Hz, 4H), 4.32 (t, J= 4.3 Hz, 2H), 3.36 - 3.29 (m, 4H), 2.59 (t, J= 6.6 Hz, 4H), 1.45 (s, 18H); 13C NMR (125 MHz, MeOD) 5 171.59, 165.95, 156.94, 78.84, 64.99, 54.09, 35.97, 34.06, 27.38; HRMS (ESI) m / z calculated for C22H36N4O10 [M+H]+= 517.25041, found = 517.25085.

[0160] Intermediate 5: white solid, 47% yield. ’H NMR (500 MHz, CDCI3) 5 7.98 (s, 2H), 5.06 - 4.73 (m, 2H), 4.52 - 4.17 (m, 4H), 3.21 - 3.03 (m, 4H), 2.49 - 2.23 (m, 4H), 1.77 (t, J= 7.1 Hz, 5H), 1.40 (s, 18H);13C NMR (125 MHz, CDCI3) 8 172.79, 165.16, 156.29, 79.37, 65.57, 54.43, 39.83, 30.85, 28.40, 25.30; HRMS (ESI) m / z calculated for C24H40N4010 [M+H]+= 545.28171, found = 545.28241.

[0161] A10: colorless oil, 30% yield. 'H NMR (500 MHz, CDCI3) 8 8.04 (s, 2H), 4.55 - 4.39 (m, 4H), 4.31 (dd, J= 7.5, 3.8 Hz, 2H), 3.24 - 2.99 (m, 4H), 2.93 - 2.60 (m, 12H), 1.69 - 1.51 (m, 8H), 1.34 - 1.12 (m, 56H), 0.85 (t, J= 6.9 Hz, 12H);13C NMR (125 MHz, CDCI3) 8 170.89, 164.87, 65.68, 54.15, 52.99, 52.78, 48.66, 31.87, 29.56, 29.52, 29.50, 29.33, 29.30, 29.28, 27.15, 22.67, 14.11; HRMS (ESI) m / z calculated for C52H100N406 [M+H]+= 877.77156, found = 877.77167.

[0162] A12: colorless oil, 40% yield.XH NMR (500 MHz, CDCI3) 8 8.14 - 7.93 (m, 2H), 4.51 - 4.41 (m, 4H), 4.37 - 4.29 (m, 2H), 3.40 - 3.13 (m, 4H), 3.09 - 2.70 (m, 12H), 1.67 (dq, J = 14.0, 8.3, 6.8 Hz, 8H), 1.40 - 1.08 (m, 72H), 0.86 (t, J = 6.9 Hz, 12H);13C NMR (125 MHz, CDCI3) 8 170.50, 164.95, 65.71, 54.13, 52.66, 52.13, 48.67, 31.92, 29.64, 29.61, 29.57, 29.54, 29.52, 29.49, 29.45, 29.36, 29.34, 29.26, 29.24, 26.98, 23.43, 22.70, 14.13; HRMS (ESI) m / z calcd for C60H116N4O6 [M+H]+= 989.89676, found = 989.89686.

[0163] B10: colorless oil, 37% yield. 'H NMR (500 MHz, CDCI3) 8 7.57 (s, 2H), 4.55 - 4.34 (m, 4H), 4.30 (dd, J= 6.9, 3.3 Hz, 2H), 2.90 - 2.57 (m, 12H), 2.54 - 2.37 (m, 4H), 1.98 - 1.84(m, 4H), 1.64 - 1.47 (m, 8H), 1.32 - 1.16 (m, 56H), 0.86 (t, J= 6.9 Hz, 12H);13C NMR (125 MHz, CDCh) 8 172.51, 164.93, 65.48, 54.36, 52.68, 52.17, 31.88, 31.24, 29.57, 29.53, 29.36, 29.30, 27.22, 24.66, 22.67, 20.46, 14.12; HRMS (ESI) m / z calculated for C54H104N4O6 [M+H]+= 905.80286, found = 905.80334.

[0164] B12: colorless oil, 44% yield. 'H NMR (500 MHz, CDCh) 8 7.64 (s, 2H), 4.53 - 4.37 (m, 4H), 4.31 (dd, .7= 6.7, 3.4 Hz, 2H), 2.91 - 2.68 (m, 12H), 2.58 - 2.38 (m, 4H), 1.97 (s, 4H), 1.67 - 1.49 (m, 8H), 1.32 - 1.17 (m, 72H), 0.86 (t, J = 6.9 Hz, 12H);13C NMR (125 MHz, CDCh) 8 172.37, 164.92, 65.53, 54.37, 52.53, 52.09, 31.92, 31.15, 29.65, 29.63, 29.57, 29.56, 29.47, 29.35, 29.32, 27.15, 24.31, 22.69, 20.17, 14.13; HRMS (ESI) m / z calculated for C62H120N4O6 [M+H]+= 1017.92806, found = 1017.92822.Example 2 - Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids

[0165] FIGS. 1A-1E illustrate the formulation of the inventive stereopure lipids into stable LNPs. As shown in FIG. IB, stereopure lipids were formulated with the inventive lipids, cholesterol, two PEG-lipid variants (C14PEG2K or CISPEG2K), and either DOPE or DDAB as helper lipids. The four components were mixed at four molar ratios, as shown in FIG. 1C. This created a library of 64 chemically distinct LNPs (FIG. ID). Forty-seven met inclusion criteria (which was a monodisperse dynamic light scattering (DLS) spectrum with a hydrodynamic diameter between 20 and 200 nm) and were therefore pooled and intravenously injected. FIG. IE shows hydrodynamic diameters of the LNPs as a function of lipid composition. FIG. IF provides hydrodynamic diameter of the pooled LNP control (green). The LNP pool diameter was within the range of diameters of LNPs comprising the pool, suggesting the pool did not come out of solution. FIG. 1G, provides a schematic of the SANDS assay; each LNP carries the same mRNA encoding aVHH as well as a unique identifying barcode. FIG. 1H provides functional mRNA delivery measured by the percent of cells expressing aVHH protein in the liver, spleen, and bone marrow. FIG. II depicts normalized delivery for LNPs as well as an the unencapsulated barcode (green), which is a negative control, n = 9 - 13 LNPs per group in FIG. IE; n = 3 mice / group in FIG. 1H. In FIGS. IE and 1H, data are plotted as mean + / - standard deviation.

[0166] Nanoparticle Formulation. LNPs were formulated with aVHH or Cre as previously described. Nucleic acids were diluted in 10 mM citrate buffer, while the remaining components were diluted in 100% ethanol. The phases were then microfluidically mixed.

[0167] Nanoparticle Characterization. LNP hydrodynamic diameter was measured using high throughput dynamic light scattering (DynaPro Plate Reader II, Wyatt). LNPs were diluted in sterile IX PBS and analyzed. In the case of the barcoding assays, LNPs were only included if, as noted, they had a hydrodynamic diameter between 20 and 200 nm as well as a correlation function with 1 inflection point. In all cases, particles were dialyzed into IX PBS.

[0168] All four test lipids generated stable LNPs with a similar hydrodynamic diameter (FIG. IE). Hydrodynamic diameter of the pooled LNPs was measured as a control for particles coming out of solution when mixed. The results were within the range of the LNPs comprising the pool, which suggested that the LNPs did not come out of solution (FIG. IF). The 47 LNPs were intravenously administered at a total nucleic acid dose of 1.0 mg / kg (i.e., 0.021 mg / kg / LNP, on average, FIG. 1G). Twenty -four hours later, the liver, spleen, and bone marrow were isolated, and these tissues were digested into single cell suspensions. The percentages of aVHH+cells for 15 cell types were quantified using FACS (FIG. 1H). Compared to PBS-treated mice, which were used as a gating control, higher percentages of aVHH+liver and spleen cells were observed relative to bone marrow. However, also observed were a small number of aVHH+cells that expressed CD34. Since multipotent progenitors (MPPs) and shorter-term HSCs express CD34, additional investigation concerning this signal was conducted (one important caveat is that long lived mouse HSCs do not express CD34).Example 3 - Delivery of mRNA From Lipid Nanoparticle Formed With Inventive Lipids

[0169] FIGS. 2A-2I illustrate how an inventive nanoparticle, designated LNPSCdelivers mRNA to CD34 cells in vivo. FIG. 2A depicts the chemical and biophysical traits of LNP 1, LNP15, and LNPSC, three LNPs identified by DNA barcoding to have distinct tropism. FIG. 2B illustrates the workflow to identify LNPSC: nanoparticles identified in the in vivo screen were tested individually, then analyzed in more detail. FIG. 2C provides the normalized delivery of LNP1, LNP15, or LNPSCacross all cell types in the liver and spleen, (n = 3 mice / group, average + / - standard error of the mean. Two- way ANOVA, ****p < 0.0001, ***p = 0.0001, ns = 0.4910). Functional delivery of LNP1, LNP15, or LNPSCafter the LNPs were tested individually, in the liver, spleen, and bone marrow (FIG. 2D) (n = 3 mice / group, average + / - standard error of the mean). FIG. 2E provides the percent of cells expressing tdTomato in the bone marrow three days after LNPSCcarrying Cre mRNA were injected, (n = 3 mice / group,average + / - standard deviation. One-way ANOVA, ****p < 0.0001). FIG. 2F provides the percent of CD34+cells expressing tdTomato in the bone marrow decreases with the LNPSCCre mRNA dose, n = 2 / 3 mice / group, average + / - standard deviation. One-way ANOVA, ***P = 0.0004, **P < 0.0093, *P = 0.0126, ns > 0.0691). FIG. 2G provides the percent of liver cells expressing tdTomato after mice were treated with 0.2 mg / kg LNPSCCre mRNA. (n = 3 mice / group, average + / - standard deviation). FIG. 2H illustrates how LNPSCformulated with Cas9 mRNA and sgLox was injected into mice engineered to report Cas9-mediated gene editing via LoxP editing. FIG. 21 provides the percent of CD34+cells expressing tdTomato after a 2.0 mg / kg injection of LNPSCcarrying Cas9 mRNA and sgLox. (n = 3 mice / group, average + / - standard deviation).

[0170] One advantage of barcoding is that delivery mediated by many LNPs can be quantified in a single experiment. The normalized delivery of each LNP in the liver and spleen was calculated. Normalized delivery is a validated approach to quantify the amount of LNP that reaches a given cell, relative to other LNPs. This led to three chemically distinct LNPs (FIG. 2A) predicted by the screen to exhibit differential liver tropism (FIG. 2B). Specifically, normalized delivery predicted LNP1 to have higher liver tropism; LNP15 to have intermediate liver tropism; and LNP30, which was subsequently designated LNP stem cell (LNPSC), to have lower liver tropism (FIG. 2C). One limitation of this analysis was the inability to sequence barcodes from CD34+cells, since the number of transfected cells was low. To overcome this, all three LNPs were analyzed one by one in a further experiment. Specifically, LNP1, LNP15, and LNPSCwere formulated and intravenously injected with 1 mg / kg aVHH mRNA, and aVHFF cells were measured in the liver, spleen, and bone marrow.

[0171] Consistent with the screening data, a higher percentage of aVHH+cells was observed in the liver with LNP1 and a higher percentage of aVHFF cells was observed in the spleen and marrow using LNPSC(FIG. 2D). From these two lines of evidence it was considered that liver detargeting LNPs could be identified using SANDS.

[0172] It was then investigated whether LNPSCcould target CD34+cells. To control for the chance that delivery was specific to the aVHH reporter, the mRNA payload was changed. LNPSCwas formulated to carry mRNA encoding Cre, and it was injected into Ail4 mice. Ail4 mice have a Lox-Stop-Lox-tdTomato construct driven by CAG50; if Cre mRNA is translated into Cre protein, which translocates into the nucleus and edits the Stop, cells express tdTomato51. Threedays after administration, cells from bone marrow were isolated, and dose-dependent tdTomato expression was observed in CD34+cells (FIGS. 2E and 2F). At the 2.0 mg / kg dose, off-target delivery in the liver was low. Less than 10% tdTomato+cells was observed in hepatocytes (FIG. 2G), six-fold less delivery compared to CD34+cells at the same dose. LNPSCwas then evaluated with a third payload, namely, mRNA encoding SpCas9 as well as sgRNA targeting Lox sites (sgLox). Seven days after injecting the LNPs into a reporter mouse with cells that become tdTomato+after sgLox gene editing (FIG. 2H), tdTomato+CD34+cells were observed (FIG. 21). Example 4 - Delivery of mRNA From Lipid Nanoparticle to Cells Expressing LSK, LSK / SLAM, and ESLAM

[0173] While CD34 is expressed on multipotent progenitors in mice, short-term HSCs in mice, and long- term HSCs in NHPs and humans, it is not expressed on long term HSCs in mice. As a result, biologists that study mouse HSCs often use one of three gene combinations to define stem- and stem-like cells. One definition, named LSK, requires the co-expression of c-Kit and Seal cell surface proteins and the absence of lineage (Lin) markers (c-Kit+Sca+Lin‘). A second definition, named LSK / SLAM, quantifies CD 150 and CD48 in addition to the LSK markers (c- Kit+Sca+Lin-CD150+CD48-). Finally, the ESLAM phenotype (CD45+EPCR+CD150+CD48 ) is also used to purify mice HSPCs. The rarity of these cells makes quantifying in vivo delivery using those flow cytometry challenging. It also does not easily allow scientists to account for potential heterogeneity within an LSK, LSK / SLAM, or ESLAM population. The present study therefore executed a multiomic single-cell RNA sequencing (scRNA-seq)-based pipeline to (i) define cell subtypes (via their transcriptome) and (ii) quantify LNPsc-mediated aVHH delivery. Specifically, LNPSCwas formulated to carry aVHH mRNA, then injected into mice. Twenty-four hours later, cells were isolated from the bone marrow, scRNA-seq was performed, and the data was processed using STARsolo and analyzed using BBrowser2. This generated cell subtype transcriptome data. This dataset was complemented with DNA-tagged antibodies targeting XYZ, so protein levels could also be measured. Finally, anti-aVHH DNA-tagged antibodies were used to quantify aVHH protein and these reads were defined as a pseudogene, which allowed quantification of functional delivery of aVHH mRNA in the same cells. Unsupervised clustering divided the 8563 cells into 20 clusters, which were visualized using t-distributed Stochastic Neighbor Embedding (t-SNE) (FIG. 2A). We then ran several controls. First, each mouse sample was compared the others and it was confirmed that they were evenly distributedwithin each cluster (FIG. 5 A). Next, a comparison was made of number of RNA features, counts and mitochondrial percentages to previous scRNA-seq datasets (FIG. 5B); the present results were similar.

[0174] To quantify delivery mediated by LNPSCin the cell subtypes, we overlaid aVHH protein expression was overlaid onto the t-SNE: aVHEF cells were observed in all clusters, with some clusters showing higher delivery percentages than others (FIG. 3B). The analysis was then focused on determining the clusters that contained HSCs. An analysis of the protein expression of canonical HSC surface markers was undertaken, and it was found that cluster 16 contained the highest HSC marker expression levels using all three previously described strategies (FIG. 3C). Approximately 60% of the cells in cluster 16 contained aVHH protein, providing evidence of line of evidence of LNP-mediated mRNA delivery in a transcriptionally defined HSC; it was also noted that this percentage was higher than all other clusters (FIG. 3D). As a control for cell type definitions, gene expression in each cluster was compared to the single cell Mouse Cell Atlas (scMCA). Consistent with the present analysis, cluster 16 was identified as HSC progenitor cells (FIG. 4A). The scMCA also suggested that cluster 14 likely contained a significant number of HSCs (FIG. 4A); also observed was delivery in cluster 14, albeit in lower percentages than in cluster 16 (FIG. 3D).

[0175] Even within these transcriptionally defined populations, there can be heterogeneity. Specifically, HSCs do not always clearly group in one distinct cluster, and clusters that contain true stem cells can contain other cell types. The present study found two lines of evidence consistent with this, including the expression of CD48+ in some cells within cluster 16 as well as low expression of canonical cell surface markers in cluster 14. To ensure delivery was measured in individual cells that were true HSCs, a single cell heatmap of clusters 14 (FIG. 4B) and 16 (FIG. 4C) was generated. Individual stem cells are highlighted. Cells that expressed higher values of CD48 were removed, since this marker can be expressed on shorter term HSC-like cells in mice. Each cell that met this new rigorous definition was denoted, and it was found that 58% of the cells from cluster 16 and 9% from cluster 14 cells were aVHH+, respectively.

[0176] There is now enough clinical data using RNA-, LNP- or gene therapy-based drugs that the FDA has published guidance documents for future clinical trials. These documents strongly suggest that drugs will benefit from (i) chemical simplicity, which will make consistent manufacturing at human scales more likely and (ii) a clear way to predict how a specific geneticintervention will affect the corresponding disease. In the case of HSCs, there is an abundance of (ii) in preclinical models as well as patients treated with ex vivo therapies. As a result, there now exists clinically de-risked gene targets, clinical trial sites familiar with these diseases, and patients who have been willing undergo ex vivo procedures despite their high rates of morbidity. These conditions support efforts to achieve in vivo HSC targeting mediated by a chemically simple LNP.Example 5 - Delivery of mRNA to Non-Human Primate Cells

[0177] FIGS. 6A-B provides Luciferase images (FIG. 6A) and quantification (FIG. 6B) in NHPs bone marrow CD34-, bone marrow CD34+, and liver cells after LNP67 (i.e., CSS-A10: cholesterol: C14PEG2OOO: DOTAP at molar ratios of 35: 47.5: 2.5: 15) carrying mRNA encoding luciferase was administered systemically administered. The data demonstrate that LNP67 functionally delivers mRNA to the bone marrow at the clinically relevant doses of 0.25 and 0.40 mg / kg, without the use of any pretreatment. Notably, the LNPs worked after they were frozen, shipped across country, thawed, and administered.Example 6 - Delivery of mRNA to CD34+ Cells

[0178] FIG. 7 illustrates delivery of mRNA by LNP67 to CD34+ non-human primate cells at doses as low as 0.25 mg / kg. In addition, in all three animals treated with 0.4 mg / kg, LNP67 delivers mRNA to CD34+ cells as much or more than the liver.Example 7 - Synthesis and Characterization of Exemplary Ionizable LipidsCompound 382-(didecylamino)ethan-l -ol

[0179] To a round bottom flask containing a solution of ethanolamine (98.8 uL, 1.64 mmol) in 1,2-dichloroethane (16.4 mL) was added decanal (1.23 mL, 6.55 mmol), followed by sodium triacetoxyborohydride (1.04g, 4.91 mmol), and the resulting suspension was stirred overnight at room temperature. Upon reaction completion, the reaction was diluted with 30 mL DCM, washed with 10 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSCL, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (10g column, 0-10% MeOH / DCM) yielded product as a colorless oil that solidified upon standing. Yield - 541 mg (97% yield) Mass Spec - 342 (m+1) 'H NMR (400 MHz, CDC13) 5 3.79 (t, J = 5.0 Hz, 2H), 2.89 (t, J= 5.0 Hz, 2H), 2.79 (t, J= 8.1 Hz, 4H), 1.66 (s, 2H), 1.36 - 1.26 (m, 32H), 0.90 (t, J= 6.7 Hz, 6H). bis(2-(didecylamino)ethyl) (((2S,5S)-3,6-dioxopiperazine-2,5-diyl)bis(methylene)) bis(carbonate) - Compound 038

[0180] To a vial containing a 0 °C solution of triphosgene (26.0 mg, 0.098 mmol) in DCM (0.5 mL) was added a solution of 2-(didecylamino)ethan-l-ol (100 mg, 0.293 mmol) in DCM (0.5 mL). Pyridine (0.0236 mL, 0.293 mmol) was then added, and the resulting solution was stirred for 2 hours, slowly warming to rt. Upon full conversion of the starting material, the reaction was concentrated in vacuo to an orange solid. A suspension of cyclo(ser,ser) (25.5 mg, 0.146 mmol) in dimethylacetamide (1 mL) was added to the solid, and reaction was stirred at 45 °C for 5 days. Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water x 3, and brine, dried over MgSO4,and concentrated in vacuo to a brown oil. Purification via Flash chromatography (5 g HC column, 0-20% MeOH / DCM, 0.1% NH3) yielded product. Yield: 14.9 mg (11.2% yield) 'H NMR (400 MHz, CDC13) 8 12.07 (s, 2H), 9.62 (s, 3H), 8.34 (t, J= 7.9 Hz, 2H), 7.92 (t, J= 7.0 Hz, 3H), 5.17 (s, 3H), 3.03 (q, J= 7.3 Hz, 8H), 2.47 (s, 6H), 1.41 - 0.89 (m, 66H), 0.81 (t, J = 6.7 Hz, 10H).Compound 51 & Compound 53didecylglycine

[0181] To a solution of glycine (100 mg, 1.33 mmol) in methanol (1.5 mL) and water (0.9 mL) was added sodium cyanoborohydride (209.3 mg, 3.33 mmol), and the resulting solution was stirred at rt for 5 minutes. Decanal (0.752 mL, 4.00 mmol) was then added, and the resulting solution was stirred at rt for overnight. Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over calcium chloride and concentrated in vacuo to a white foam. Purification via Flash chromatography (5g HC column, 0-20% MeOH / DCM) yielded product as a white foam. Yield: 346.1 mg (73% yield)1H NMR (400 MHz, CDC13) 5 3.61 (s, 2H), 3.21 - 3.01 (m, 4H), 1.71 (q,7.9 Hz, 4H), 1.30 (d, J = 17.5 Hz, 28H), 0.90 (t, J = 6.7 Hz, 6H).((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(ethane-2,l-diyl) bis(2-(didecylamino)acetate) - Compound 51

[0182] To a solution of didecylglycine (126.7 mg, 0.371 mmol) in DCM (1.23 mL) was added EDCI (71.1 mg, 0.371 mmol) and DMAP (1.5 mg, 0.012 mmol), and the resulting solution was stirred at rt for 45 min. Cyclo(homoserine, homoserine) (25 mg, 0.124 mmol) was added, and the resulting solution was stirred overnight at rt. Upon reaction completion, the reaction wasconcentrated in vacuo to a colorless gum. Purification via Flash chromatography (5g HC column, 0-30% MeOH / DCM) yielded product is a colorless, sticky gum. Yield: 103.9 mg (97% purity, 93% yield) Mass Spec - 877.6 (M+l) 'H NMR (400 MHz, CDC13) 5 6.76 (s, 1H), 4.55 (dd, J= 12.0, 5.4 Hz, 1H), 4.17 (dt, J= 12.1, 6.3 Hz, 1H), 4.02 (d, J= 9.2 Hz, 1H), 3.34 (s, 3H), 2.66 - 2.37 (m, 8H), 1.99 (d, J = 14.5 Hz, 1H), 1.59 (s, 8H), 1.45 (d, J = 7.8 Hz, 7H), 1.28 (s, 56H), 0.90 (t, J= 6.7 Hz, 13H).((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(2-(didecylamino)acetate) - Compound 53

[0183] To a solution of didecylglycine (153.1 mg, 0.431 mmol) in DMF (1.4 mL) was added EDCI (82.6 mg, 0.431 mmol) and DMAP (5.3 mg, 0.043 mmol), and the resulting solution was stirred at rt for 45 minutes. Cyclo(serine, serine) (25 mg, 0.144 mmol) was the added, and the reaction was stirred overnight at rt. After 24 hours, the reaction temperature was increased to 40 °C and the reaction was stirred for an additional 3 days. Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless gum. Purification via Flash chromatography (5 g HC column, 0-30% MeOH / DCM) yielded product. Yield: 68.8 mg (56.4% yield) Mass Spec - m / z = 849.8 (M+l) ’H NMR (400 MHz, CDC13) 8 4.34 (s, 1H), 3.39 (s, 3H), 2.94 (s, 4H), 2.66 - 2.45 (m, 2H), 1.66 (s, 5H), 1.58 - 1.11 (m, 64H), 0.91 (t, J = 6.7 Hz, 13H).Compound 57(5)-(3,6-dioxopiperazin-2-yl)methyl didecylglycinate - Compound 57

[0184] To a solution of di decylglycine (11 1.0 mg, 0.312 mmol) in DMF (2.1 mL) was added EDCI (60.0 mg, 0.312 mmol) and DMAP (5.1 mg, 0.042 mmol), and the resulting solution was stirred at rt for 45 minutes. Cyclo(glycine, serine) (30 mg, 0.208 mmol) was then added, and the reaction was stirred overnight at rt. After one day, the reaction temperature was increased to 40 °C, and the reaction was stirred over the for 3 days. Upon reaction completion, the reaction wasdiluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless gum. Purification via Flash chromatography (5g HC column, 0-30% MeOH / DCM w / 0.1% NH3) yielded product.Yield: 9.6 mg (98% purity, 10% yield) MASS SPEC: 482.4 (M+l) Ti NMR (400 MHz, CDC13) 8 6.42 (s, 1H), 6.36 (s, 1H), 4.51 (dd, J= 11.4, 3.2 Hz, 1H), 4.28 (dd, J = 11.4, 5.6 Hz, 1H), 4.20 (d J= 4.3 Hz, 1H), 4.10 - 3.86 (m, 1H), 3.28 (s, 1H), 2.55 - 2.25 (m, 4H), 1.35 (q, J = 7.4 Hz, 5H), 1.19 (s, 30H), 0.81 (t, J= 6.6 Hz, 6H).Compound 64(5)-N-decyl-N-(oxiran-2-ylmethyl)decan-l-amine

[0185] To a vial containing didecylamine (1.00 g, 3.36 mmol) was added water (9 uL) and s- epichlorohydrin (0.316 mL, 4.03 mmol). The reaction was then heated to 40 °C and the reaction was stirred overnight. Upon full consumption of didecylamine starting material, 2 mL 36% NaOH was added and vigorously stirred at rt overnight. Upon reaction completion, the reaction was diluted with 10 mL water, extracted with 3 x 10 mL DCM, washed combined organic phases with 10 mL brine, dried over MgSO4, and concentrated in vacuo to a yellow oil. Purification via Flash chromatography (0-10% EtOAc / DCM) yielded product as a yellow liquid. Yield: 490.5 mg (98% purity, 40% yield) Mass Spec - 354.4 (M+l) 'H NMR (400 MHz, CDC13) 8 3.07 (dq, J = 6.9, 3.7 Hz, 1H), 2.82 - 2.70 (m, 2H), 2.52 (dtt, J= 14.9, 12.4, 6.6 Hz, 6H), 1.47 (t, J= 7.1 Hz, 5H), 1.30 (d, J= 5.6 Hz, 29H), 0.90 (t, J= 6.6 Hz, 6H).(5)-3-(((A)-3-(didecylaniino)-2-hydroxypropoxy)niethyl)piperazine-2, 5-dione - Compound 64

[0186] To a suspension of cyclo(Gly,Ser) (50.0 mg, 0.346 mmol) in isopropanol (1.7 mL) was added (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-l -amine (147 mg, 0.416 mmol), and the resulting suspension was stirred at 80 °C overnight. Upon reaction completion, 0.1 mL DBU wasadded, then the reaction was concentrated in vacuo to a yellow solid. Purification via Flash chromatography (5g HC column, 5-12% MeOH / DCM) yielded product. Yield: 24.2 mg (14% yield, 98% purity) Mass Spec - 498.6 (M+l) 'HNMR (400 MHz, CDC13) 5 6.82 (1H, d, br., 7=25.9 Hz), 6.41 (1H, d, br., 7=25.9 Hz) 4.32-3.46 (6H, m), 3.36-2.82 (2H, m), 2.82-2.26 (6H, m), 1.60-1.39 (4H, m), 1.28 (28H, s), 0.90 (6H, t, J= 6.3 Hz).Compound 65((25,55)-5-benzyl-3,6-dioxopiperazin-2-yl)methyl didecylglycinate - Compound 65

[0187] To a suspension of didecyllglycine (91.1 mg, 0.256 mmol) in DMF (2.1 mL) was added EDCI (49.1 mg, 0.256 mmol) and DMAP (2.61 mg, 0.021 mmol), and the resulting suspension was stirred at rt for 45 minutes. Cyclo(Phe,Ser) (50.0 mg, 0.213 mmol) was then added, and the reaction was stirred over the weekend at rt. Upon reaction completion, the reaction was diluted with 20 mL DCM, washed with 5 mL each saturated sodium bicarbonate solution, water x 3, and brine, dried over MgSCU, and concentrated in vacuo. Purification via Flash chromatography (5g HC column, 0-20% MeOH / DCM) yielded product. Yield: 28.8 mg (23.7% yield) Mass Spec - 572.4 (M+l) >H NMR (400 MHz, CDC13) 8 7.43 - 7.28 (m, 3H), 7.25 - 7.18 (m, 2H), 6.31 (s, 1H), 6.01 (s, 1H), 4.41 (dd, J= 11.3, 3.3 Hz, 1H), 4.29 - 4.14 (m, 2H), 3.60 (dd, 7= 11.4, 7.4 Hz, 1H), 3.38 (d, 7= 3.7 Hz, OH), 3.02 (dd, J= 13.9, 8.7 Hz, 1H), 2.53 (dd, J= 8.8, 6.3 Hz, 4H), 1.42 (t, J = 7.5 Hz, 5H), 1.26 (d, J = 3.4 Hz, 30H), 0.88 (t, J = 6.7 Hz, 6H).Compound 85terf-butyl (2-(didecylamino)ethyl)carbamate

[0188] To a flask containing rV-Boc-ethylenediamine (600 mg, 3.74 mmol) in DCM (37.4 mL) was added decanal (2.8 mL, 14.98 mmol), followed by sodium triacetoxyborohydride (2.38 g, 11.24 mmol), and the resulting suspension was stirred over the a few days at room temperature. Upon reaction completion, the reaction was diluted with 75 mL DCM, washed with 25 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSCL, and concentrated in vacuo to a colorless oil. Purification via flash chromatography (25g column, 0- 20% MeOH / DCM) yielded product as a colorless oil. Yield: 1.383 g (100% purity, 88% yield) Mass Spec - 441.6 (m+1) 'H NMR (400 MHz, CDC13) 8 3.50 (d, J = 8.6 Hz, 2H), 2.91 (d, J = 52.8 Hz, 4H), 1.64 (d, J = 43.8 Hz, 4H), 1.46 (s, 9H), 1.41 - 1.08 (m, 30H), 0.90 (t, J = 6.7 Hz, 6H).A'-didecylethaiie-1.2-diainine

[0189] To a vial containing tert-butyl (2-(didecylamino)ethyl)carbamate (1.38 g, 3.14 mmol) was added HCI in dioxane (15.69 mL, 62.76 mmol, 4M), and the resulting solution was stirred at rt for 4 hours. Upon reaction completion, the reaction was concentrated in vacuo, dissolved in 60 mL DCM, washed with 10 mL each IM NaOH, water, and brine, dried over MgSCU, and concentrated in vacuo to a dark yellow oil. Yield: 1.006g (94% yield) Mass Spec - 341.1 (m+1) ’HNMR (400 MHz, CDC13) 8 3.22 (dt, J = 30.4, 6.3 Hz, 4H), 2.90 - 2.77 (m, 4H), 1.76 - 1.52 (m, 4H), 1.30 (d, J = 13.0 Hz, 28H), 0.90 (t, J = 6.7 Hz, 6H).(5)-(3,6-dioxopiperazin-2-yl)methyl (2-(didecylamino)ethyl)carbamate - Compound 85

[0190] To a solution of cyclo(gly,ser) (25 mg, 0.173 mmol) in acetonitrile (1.7 mL) was added 4-nitrophenylchloroformate (52.4 mg, 0.260 mmol), DMAP (4.2 mg, 34.7 umol), and pyridine (27.9 uL, 0.347 mmol), and the resulting solution was stirred at rt overnight. Upon full consumption of starting material, N^NMidecylethane-l^-diamine (118.2 mg, 0.347 mmol) was added, and the resulting solution was stirred at rt overnight. Upon reaction completion, the reaction was concentrated in vacuo. Purification via flash chromatography (5 g HC column, 0- 10% MeOH / DCM, 20 CV gradient) yielded product as a colorless oil. Yield: 17 mg (19% yield, 98% purity) Mass Spec: 511.4 (M+l) ’H NMR (400 MHz, CDC13) 8 8.39 (dt, 11.4, 5.9 Hz, 1H), 4.74 - 4.57 (m, 5H), 4.50 - 4.40 (m, OH), 4.19 (dd, J= 17.1, 7.0 Hz, OH), 3.91 (dd, J = 17.2, 5.3 Hz, OH), 3.83 - 3.55 (m, 1H), 3.39 - 2.92 (m, 5H), 2.77 - 2.62 (m, 2H), 1.72 (d, J= 9.7 Hz, 2H), 1.56 (t, J= 7.9 Hz, 2H), 1.29 (d, J= 7.8 Hz, 28H), 0.90 (t, J= 6.7 Hz, 6H).Compound 16((2A,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-((frr / - butoxycarbonyl)amino)propanoate)

[0191] To a lOOmL flask was added 500 mg of cyclo (serine, serine) (2.87 mmol), 1.625 g of 7V-boc-P-alanine(8.61 mmol), 1.64 g of l-Ethyl-3-(3-diniethylaminopropyl)carbodiimide (EDCI) (8.61 mmol), 1.30 g of Hydroxybenzotriazole (HOBt), (8.61 mmol), 2.1 ml of N,N- diisopropylethylamine (DIPEA) (22 mol), 20 mg of 4-Dimethylaminopyridine (DMAP)(cat.), 30 ml of DMF. The mixture was stirred at RT (room temperature) overnight. Upon reaction completion, the solution was concentrated in vacuo, the residue was suspended in 150 ml of DCM, washed with water, saturated sodium bicarbonate solution, and brine, the combinedaqueous phases were extracted with 80 ml of DCM twice, and the combined organic phases were concentrated in vacuo. Flash chromatography purification (0 to 10% MeOH / DCM) afforded 1.38 g of the desired product with 46.5% yield with some DMF. 1H NMR (400 MHz, CDC13) 8 7.51 - 7.41 (s, 2H), 7.01 (s, 2H), 4.59 (m, 2H), 4.36 (m, 4H), 3.42 (m, 4H), 2.56 (m, 4H), 1.43 (s,18H).((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(didecylamino)propanoate - Compound 16

[0192] To a 100 ml flask with ((2\5,S')-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3 - (( / e?7-butoxycarbonyl)amino)propanoate) (1.38 g, 2.67 mmol) was added 25 ml 4N HC1 in dioxane. The mixture was stirred at room temperature for 1.5 hrs., after the solvents were removed in vacuo. The residue was then dissolved in DCM (100 mL), and to the resulting solution was added aldehyde (2.67g. 16.2 mmol), and NaBH(OAc)3(2.83g;13.85 mmol). The reaction mixture was stirred at room temperature for overnight. Upon reaction completion, the reaction was quenched with saturated NaHCCh solution, extracted with DCM, dried over Na2SCU and concentrated. Purification via reverse phase flash chromatography (0 to 100% acetonitrile in water (0.1% TFA)) followed by desalting with 3.5% K2CO3 solution / chloroform afforded 417 mg of clean product. (47.5% yield) (CSSA-10). 1H NMR (400 MHz, CDC13) 8 7.45 (s, 2H), 4.50 (m, 4H), 4.37 - 4.17 (m, 2H), 2.80(m, 4H), 2.65 - 2.43 (m, 12H), 1.42 (m, 8H), 1.26 (m, 56H), 0.88 (m,12H).Compound 61(Z)-dec-5-enal:

[0193] To a 250 ml flask was added (l-Pentyl)triphenylphosphonium bromide(24.8g, 60 mmol) and 100ml of THF. The reaction was cooled to 0°C, then 98 ml of 0.7 M KTMDS in toluene was added. The resulting mixture was stirred at 0°C, for one hour, after which the reaction was cooled -78°C and tetrahydro-27 / -pyran-2-ol (4.08g.40 mmol) in 20 ml of THF was added slowly. The mixture was slowly warmed up to RT for over weekend, added saturated NH4CI solution (100 ml), extracted with EtOAc (100 ml, three times), dried over MgSCh , filtered, concentrated. Flash chromatography purification on a 50g column afforded 3.31g of the desired (Z)-dec-5-en-l-ol (53% yield). 1H NMR (400 MHz, CDC13) 5 5.36 (m, 2H), 4.95 (s, 1H), 3.64 (m, 2H), 2.14 - 1.97 (m, 2H), 1.95 - 1.69 (m, 2H), 1.58 (m,4H), 1.40 - 1.20 (m, 4H), 1.01 - 0.69 (m, 3H).

[0194] To a 250 ml flask was added a solution of DMSO (2.85 ml, 3.12g, 40 mmol) in DCM (40 ml) to a solution of oxalyl chloride (3 ml, 3.35 mmol) in DCM (40 ml) at -78°C over a period of 10 minutes, stirred the mixture for 30 minutes, added a solution of (Z)-dec-5-en-l-ol (3.31g, 21 mmol) in 30 ml of DCM, stirred the mixture at -78°C. for two hours, added EtsN in 20 ml of DCM, kept stirred at -78°C for one hour, slowly warmed up to RT for overnight, added DCM, saturated sodium bicarbonate solution, extracted with DCM twice, washed with brine, dried and concentrated, to afford 2.5g of crude (Z)-dec-5-enal (71% yield). 1H NMR (400 MHz, CDC13) 8 9.77 (s 1H), 5.49 - 5.22 (m, 4H), 2.43 (m, 2H), 2.18 - 2.01 (m, 4H), 1.69 (m, 2H), 1.52 - 1.39 (m,4H), 0.90 (m, 3H).((21?,51?)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(di((Z)-dec-5-en-l- yl)amino)propanoate)

[0195] The compound was synthesized according to the same procedure as Compound 16. Isolated product with 9% yield. 1H NMR (400 MHz, CDC13) 8 7.34 (s, 2H), 5.35 (m, 8H), 4.50 (m, 4H), 4.29 (m, 2H), 2.80 (s, 4H), 2.66 - 2.40 (m, 12H), 2.02 (m, 16H), 1.45- 1.19 (m, 32H), 1.01 - 0.71 (m, 12H).Compound 62((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(di((Z)-dec-4-en-l- yl)amino)propanoate)

[0196] This compound was synthesized according to the same procedure as Compound 16. Isolated product with 19.2% yield. 1H NMR (400 MHz, CDC13) 8 7.33 (s, 2H), 5.35(m,8H), 4.60 - 4.40 (m, 4H), 4.29 (m, 2H), 2.81 (m, 4H), 2.53 (m, 12H), 2.01 (m, 16H), 1.45- 1.18 (m, 32H), 0.89 (m, 12H).Compound 659-methyldecanal

[0197] To a 250 ml flask was added 7-bromoheptan-l-ol (4.875g, 25 mmol) and 40 ml of THF. The resulting solution was cooled to -78°C, and then isobutylmagnesium bromide in ether (50 ml, 2M. 100 mmol) and Li2CuCl4solution (0.1 mmol / L in THF, 5 ml. 0.5 mmol) was added. The resulting mixture was slowly warmed up to RT for overnight. Upon reaction completion, the reaction was quenched with 200 ml of saturated NH4CI solution, extracted with EtOAc twice, washed with saturated NaHCO.i solution and brine, dried over Na2SC>4 and concentrated to afford crude product. Purification via flash chromatography on a 25g column with 0 to 50% EtOAc / Hexanes afforded 2.01g of 9-methyldecan-l-ol. (47% yield). 1H NMR (400 MHz, CDC13) 8 3.73 - 3.53 (m, 2H), 1.64 - 1.42 (m, 5H), 1.42 - 1.22 (m, 7H), 0.89 (m ,3H).

[0198] Into 250 ml flask was added a solution of DMSO (2.2 ml, 2.34g, 30 mmol) in DCM (20 ml) to a solution of oxalyl chloride (25 mmol, 2.12 ml) in DCM (30 ml) at -78°C over a period of 10 minutes. The reaction was stirred for 30 minutes, then a solution of 9-methyldecan- l-ol(2.01g, 11.68 mmol) in 30 ml of DCM was added and the reaction was stirred at -78°C for two hours. EtaN in 20 ml of DCM was then added, the reaction was stirred at -78°C for one hour, then it slowly warmed up to RT overnight. Upon reaction completion, the reaction was quenched with water, the aqueous phase was extracted with DCM, and the combined organic phases were washed with brine, dried and concentrated. Purification via flash chromatography (3 to 30% EtOAc / Hexanes) afforded 900 mg of 9-methyldecanal (45% yield). 1H NMR (400 MHz, CDC13) 8 9.75 (s, 1H), 2.41 (m, 2H), 1.76 - 1.52 (m, 4H), 1.43 - 1.17 (m, 9H), 0.85 (m, 6H).((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(bis(9- methyldecyl)amino)propanoate)

[0199] ((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(bis(9- methyldecyl)amino)propanoate) was synthesized according to the same procedure as NAVA- 016. Isolated product with 3% yield. INMR 1H NMR (400 MHz, CDC13) 8 7.52 (s, 2H), 4.50 (m, 4H), 4.37 - 4.17 (m, 2H), 2.80(m, 4H), 2.65 - 2.43 (m, 12H), 1.5(m, 4H), 1.42-1.20 (m, 56H), 0.88 (m,24H).Compound 68((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(bis(3- (pentyldisulfaneyl)propyl)amino)propanoate)

[0200] This compound was synthesized according to the same procedure as Compound 16. Isolated product with 11.8% yield. 1H NMR (500 MHz, CDC13) 8 6.96 (s, 2H), 4.52 (m. 2H), 4.45 (m, 2H), 4.34 (m 2H), 2.80 (m,4H), 2.68-2.50 (m, 28H),1.80 (m, 8H), 1.67 (m 8H), 1.41 - 1.30 (m, 8H), 1.03 - 0.79 (m,12H)Compound 69((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(bis(2- methyldecyl)amino)propanoate)

[0201] This compound was synthesized according to the same procedure as Compound 16. Isolated product with 4.3% yield. 1H NMR (500 MHz, CDC13) 5 6.45 (s, 2H), 4.60 (s, 2H), 4.29 (m, 4H), 2.72-2.05 (m, 16), 1.42 - 1.20 (m, 56H), 1.00 (m,4H), 0.94 - 0.75 (m, 24H).Compound 703-((terf-butoxycarbonyl)(decyl)amino)propanoic acid

[0202] A solution of methyl acrylate (4.4g, 562 mmol) in 40 ml of THF was added to a solution of decan-l-amine (12g, 40 mmol) in 40 ml of THF at 0°C. The mixture was stirred at 30°C for three days. Upon reaction completion, the solvent was removed in vacuo, and theresulting residue was dissolved in a solution of 8g of Eta N (80 mmol) in 60 ml of DCM. The resulting solution was added dropwise to a solution of BOC2O (11.4g, 52 mmol) in 30 ml of DCM. The mixture was stirred at 30°C for overnight. Upon reaction completion, the reaction was quenched with water, the aqueous phase was extracted with DCM twice, and the combined organic phases were dried over MgSCU and concentrated. Purification via flash chromatography (EtOAc / Hexanes) afforded 11.82 g of methyl 3-((ter / -butoxycarbonyl)(decyl)amino)propanoate (86% Yield). 1H NMR (500 MHz, CDC13) 8 3.67 (s, 3H), 3.45 (m, 2H), 3.16 (m, 2H), 2.56 (m, 2H), 1.44 (s, 9H), 1.25 (m, 16H), 0.96 - 0.73 (m, 3H).

[0203] To a solution of methyl 3-((ter / -butoxycarbonyl)(decyl)amino)propanoate (11.82g, 34 mmol) in 80 ml of EtOH and 40 ml of water at 0°C was added 2.3 g of LIOH.(96 mmol). The mixture was stirred at 30°C for overnight. Upon reaction completion, the reaction was concentrated in vacuo to remove the EtOH. The resulting aqueous solution was acidified with IN HC1 to pH=5, extracted with EtOAc three times, and the combined organic phases were dried over MgSO4 and concentrated to afford 11.4 g of 3 -(('Zc / 7-butoxy carbonyl) (decyl)amino)propanoic acid with 100% yield. 1H NMR (500 MHz, CDC13) 8 3.48 (m, 2H), 3.20 (m, 1H), 3.10 (m, 2H), 2.56 (m, 2H), 1.45 (s, 9H), 1.25 (s, 16H), 0.91(m, 3H).3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-((terC butoxycarbonyl)(decyl)amino)propanoate)

[0204] To a solution of (3S, 68’)-3,6-bis(hydroxymethyl)piperazine-2, 5-dione (1.044g, 6 mmol) in DMF (20 mL) was added 3-(( / e / 7-butoxycarbonyl)(decyl)amino)propanoic acid (5.93g, 18 mmol), EDCI (3.438g, 18 mmol), HOBt (2.718g, 18 mmol), and Hunig’s base (6 ml), and the resulting solution was stirred at RT for overnight. Upon reaction completion, the solvent was removed in vacuo. The resulting residue was dissolved DCM, washed with water and brine, dried over Na2SC>4 ,and concentrated in vacuo. Purification via flash chromatography (0 to 10% MeOH / DCM) afforded 3.5g of (3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(( / c / 7- butoxycarbonyl)(decyl)amino)propanoate)with 62% yield. 1H NMR (500 MHz, CDC13) 8 7.92 - 7.71 (m, 2H), 7.51 - 7.36 (m, 2H), 4.60 (m, 2H), 4.32 (m, 4H), 3.46 (s, 4H), 3.29 - 3.10 (m, 4H), 2.57 (m, 4H), 1.60 - 1.39 (m, 18H), 1.25 (m, 32H), 0.87 (m, 6H).((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(((Z)-dec-4-en-l- yl)(decyl)amino)propanoate)

[0205] This compound was synthesized according to the same procedure as NAVA-016. Isolated product with 23% yield. 1H NMR (500 MHz, CDC13) 8 7.44 (s, 2H), 5.55 - 5.25 (m, 4H), 4.65 - 4.41 (m, 4H), 4.28 (m, 2H), 2.79 (m, 4H), 2.65 - 2.43 (m, 12H), 2.00 (8H), 1.46 (m,4H), 1.40-1.25(m, 44H), 0.88(m, 12H).Compound 71(3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(2-methyldecyl)amino)propanoate)

[0206] The compound was synthesized according to the same procedure as NAVA-070.Isolated product with 81% yield. 1H NMR (500 MHz, CDC13) 8 6.54 (m, 2H), 4.60 (m 2H), 4.32 (m 4H), 3.51 (m, 4H), 3.41 (m, 4H), 2.92 - 2.64 (m, 8H), 2.54 - 2.31 (m, 8H), 2.16 (m, 8H), 1.72 - 1.51 (m, 4H), 1.48 - 1.35(m, 44H), 0.89 (m, 12H).Compound 723-(pentyldisulfaneyl)propanal

[0207] To a mixture of pentane- 1 -thiol (1.55g, 15 mmol, 1.89 ml) and methyl 3- mercaptopropanoate (15 mmol, 1.6 ml) was added KMnO4 over 5 minutes, The mixture was stirred at RT overnight. Upon reaction completion, the solution was diluted with ether, filteredthrough a short celite pad. The celite pad was washed with ether, and the filtrate was concentrated in vacuo. Purification via flash chromatography on a 25g column with 0 to 5% EtOAc / Hexanes yielded 950 mg of the desired product (43% yield). 1H NMR (500 MHz, CDC13) 6 3.70 (s,3H), 2.96 - 2.83 (m, 2H), 2.83 - 2.72 (m, 2H), 2.67 (m, 2H), 1.75 - 1.56 (m, 2H), 1.45 - 1.28 (m, 4H), 1.00 - 0.76 (m, 3H).

[0208] To a solution of methyl 3-(pentyldisulfaneyl)propanoatein(950 mg, 4.27 mmol) in 30 ml of Z-BuOMe at -78°C was added 9 ml of 1 M DIBAL-H (9 mmol) in hexanes. The mixture was stirred at -78°C for 2 hours, after which was added 5 ml of MeOH and 40 ml of a saturated Rochelle salt solution, and the resulting mixture was stirred at RT for one hour. Ether was then added, the aqueous phase was extracted with EtOAc twice, and the combined organic phases were dried and concentrated to afford 877 mg of crude 3-(pentyldisulfaneyl)propanal (100%). 1H NMR (500 MHz, CDC13) 8 9.82 (s, 1H), 3.05 - 2.77 (m, 4H), 2.74 - 2.54 (m, 2H), 1.72 - 1.56 (m, 2H), 1.46 - 1.27 (m, 4H), 0.97 - 0.76 (m, 3H).(3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(3-(pentyldisulfaneyl)propyl)amino)propanoate)

[0209] This compound was synthesized according to the same procedure as NAVA-070. Isolated product with 14% yield. 1H NMR (500 MHz, CDC13) 8 7.23 - 7.15 (m, 2H), 4.62 - 4.38 (m, 4H), 4.36 - 4.14 (m, 1H), 2.79 (m, 4H), 2.74 - 2.63 (m, 8H), 2.63 - 2.44 (m, 12H), 1.87- 1.75 (m, 4H), 1.67 (m, 8H), 1.54 - 1.29 (m, 36H), 0.89 (m, 12H).Compound 73((2A,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(nonyl)amino)propanoate)

[0210] This compound was synthesized according to the same procedure as NAVA-070.Isolated product with 15% yield. 1H NMR (500 MHz, CDC13) 8 7.50 (s, 1H), 7.26 (m, 2H), 4.67 - 4.42 (m, 4H), 4.28 (m, 2H), 2.95 - 2.71 (m, 4H), 2.64 - 2.45 (m, 8H), 1.41 (m, 8H), 1.37 - 1.17 (m, 52H), 0.88 (t, J = 6.9 Hz, 12H).(3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(9-methyldecyl)amino)propanoate)

[0211] This compound was synthesized according to the same procedure as Compound 70. Isolated product with 16.9% yield. 1H NMR (400 MHz, CDC13) 6 7.47 (s, 2H), 4.65 - 4.41 (m, 4H), 4.41 - 4.14 (m, 4H), 2.62 - 2.39 (m, 12H), 1.52 (m, 2H), 1.43 (m, 4H), 1.26 (m, 60H), 0.98 - 0.68 (m, 18H).Compound 81((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(2- (hexyldisulfaneyl)ethyl)amino)propanoate)

[0212] This compound was synthesized according to the same procedure as Compound 70. Isolated product with 8.3% yield. 1H NMR (400 MHz, CDC13) 5 7.40 (m, 2H), 4.82 - 4.54 (m, 2H), 4.44 (m, 4H), 4.32 - 4.03 (m, 2H), 3.27 (m, 4H), 3.03 - 2.55 (m, 20H), 1.82 (m, 8H), 1.60 - 1.49 (m, 40H), 1.15(m, 12H).Compound 84((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(decyl(ethyl)amino)propanoate)

[0213] This compound was synthesized according to the same procedure as NAVA-070. Isolated product with 28% yield. 1H NMR (400 MHz, CDC13) 3 7.69 (m, 2H), 4.67 - 4.38 (m, 4H), 4.29 (m, 2H), 2.78 (m, 8H), 2.70 - 2.35 (m, 12H), 1.42 (m, 6H), 1.14- 0.96 (m, 32H), 0.88 (m, 6H).Compound 89((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(4-nitrophenyl) bis(carbonate)

[0214] To a suspension of (35, 65)-3,6-bis(hydroxymethyl)piperazine-2, 5-dione (348mg, 2 mmol) and pyridine (395 mg, 5.0 mmol) in 20 ml of THF at 0°C was added 4-nitrophenyl chloroformate (956.41 mg, 4.40 mmol). The resulting mixture was stirred at RT for overnight.Upon reaction completion, the solution was diluted with EtOAc, washed 0.5N HC1, and filtered to yield the product (550 mg) (54% yield) 1H NMR (400 MHz, DMSO) 8 8.63 (s, 2H), 8.33 -8.12 (m, 4H), 7.57 - 7.42 (m, 4H), 4.72 - 4.43 (m, 4H), 4.39 (m,2H), 3.68 - 3.52.((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis((2-(didecylamino)ethyl)carbamate)

[0215] To a 50 ml flask with 512 mg of / c / 7-butyl (2-(didecylamino)ethyl)carbamate(1.16 mmol, 3 eq.) was added 10 ml of 20% TFA in DCM, and the resulting solution was stirred for 1 hour. Upon reaction completion, the removed solvents were removed in vacuo. To the residue was added a solution of 150mg ((25,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(4- nitrophenyl) bis(carbonate)(0.38 mmol), and 1 ml of DIPEA (5.6 mmol) in 20 ml of DMF. The mixture was stirred at RT for overnight. Upon reaction completion, the solution was concentrated in vacuo, the residue was suspended in EtOAc, washed with water, sodium bicarbonate solution, brine, dried over Na2SO4 and concentrated. Flash chromatography purification (0-20% MeOH / DCM) yielded product. 54% yield. 1H NMR (400 MHz, CDC13) 8 7.22 (m, 2H), 7.09 (m 2H), 4.70 - 4.54 (m, 2H), 4.31 (m, 2H), 4.18 m2H), 4.05 (m,2H), 3.64 (m, 2H), 3.53 - 3.40 (m, 4H), 3.18 -3.06 (m, 6H), 2.69 - 2.42 (m, 4H), 1.28 m, 64H), 0.90 (m, 12H).Compound 92bis(2-(di((Z)-dec-4-en-l-yl)amino)ethyl) (((25,55)-3,6-dioxopiperazine-2,5- diyl)bis(methylene)) bis(carbonate)

[0216] This compound was synthesized according to the same procedure as NAVA-016. Isolated product with 19% yield. 1H NMR (400 MHz, CDC13) 8 7.37 (m, 2H), 5.57 - 5.13 (m, 8H), 4.65 (m, 2H), 4.48 - 4.20 (m, 4H), 3.56 - 3.06 (m, 4H), 2.68 (m, 4H), 2.64 - 2.44 (m, 8H), 2.01 (m, 16H), 1.50(m, 8H), 1.46 - 1.21 (m, 24H), 0.97(m, 12H).Compound 98((2A,55)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis((2-(bis(3- (pentyldisulfaneyl)propyl)amino)ethyl)carbamate)

[0217] Synthesized according to the same procedure as NAVA-016. Isolated product with 18% yield. 1H NMR (400 MHz, CDC13) 8 6.98 (m, 2H),4.68 (m, 2H), 4.53 - 4.30 (m, 4H), 3.34 (m, 2H), 3.22 (m, 4H), 2.70 (m, 14H), 2.56 (m, 12H), 1.82 (m, 8H), 1.67 (m, 8H), 1.47 - 1.19 (m, 16H), 0.91 (m, 12H).Compound 56((27?,51?)-3,6-dioxopiperazine-2,5-diyl)bis(methylene) bis(3-(didecylamino)propanoate)

[0218] This compound was synthesized according to the same procedure as NAVA-016. Isolated product with 18% yield. 1H NMR (400 MHz, CDC13) 5 7.45 (s, 2H), 4.50 (m, 4H),4.29 (m, 2H), 2.80 (m, 4H), 2.60 - 2.38 (m, 12H), 1.42 (m, 8H), 1.26 (m, 56H), 0.88 (m, 12H).Example 8 - Delivery of mRNA to Mouse Using Lipid Nanoparticle Formulated With Exemplary Diketopiperazine LipidsFormulation of Lipid Nanoparticles Using Diketopiperazine Lipids

[0219] Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 7. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids, cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2.Experimental Protocols for Mouse

[0220] Dose Preparation: LNPs were prepared using the NanoAssemblr Ignite system or the NanoAssemblr Spark system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol. After mixing, resulting LNPs were dialyzed in IX PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 pm pore size PES filter. Where the nanoparticles contained a DNA barcode in addition to mRNA, LNPs were not until a second dialysis step. Nanoparticles were then analyzed in the Unchained Stunner to determine whether they meet the quality control criteria: diameter (20 nm - 200 nm), poly dispersity index (< 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 < 3.0 and A330 < 1.25). The nanoparticles contained a DNA barcode in addition to the mRNA, and passed the quality control criteria were pooled and dialyzed again in IX PBS for 1 hour in a 100 kDa dialysis cassette.

[0221] Dose Administration: Doses were calculated based on animal weight the day of administration. LNPs were administered as a bolus via a tail vein using an insulin syringe with a 28 gauge. Maximum volume for administrations was set to 20 pL / gram based on approved IACUC protocols. Body weight was monitored after administration until sacrifice.

[0222] Tissue Collection: Animals injected with LNPs containing reporter mRNA were sacrificed in a carbon dioxide chamber 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely on top of Petri dishes into ~1 cm3cubes (with the exception of bone marrow) and placed into individual aliquots containing specific digestive enzyme cocktails depending on the organ.-n-

[0223] Tissue Digestion: Samples were digested as discussed below: i. Liver Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (200 U / mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 °C for 30 minutes at 750 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (0.5% v / v) FcX block and transferred to microcentrifuge tubes to stain. ii. Lung Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (200 U / mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 °C for 20 minutes at 750 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (0.5% v / v) FcX block and transferred to microcentrifuge tubes to stain. iii. Kidney Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (200 U / mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 °C for 30 minutes at 750 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (0.5% v / v) FcX block and transferred to microcentrifuge tubes to stain. iv. Heart Digestion: Digestive enzyme cocktail containing a mix of Collagenase II (1 mg / mL), Dispase (0.6 U / mL), DNAse I (200 U / mL), FBS (2% v / v), and RPMI-1640 solvent was used. Tissues were incubated at 37 °C for 1 hour at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (0.5% v / v) FcX block and transferred to microcentrifuge tubes to stain. v. Spleen Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (200 U / mL) in RPMI-1640 solvent. Tissues were incubated at 37 °C for 10 minutes at750 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (5% v / v) FcX block and transferred to microcentrifuge tubes to stain. vi. Bone Marrow Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (200 U / mL) in RPMI-1640 solvent. Tissues were incubated at 37 °C for 10 minutes at 750 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Mouse Monoclonal (5% v / v) FcX block and transferred to microcentrifuge tubes to stain.

[0224] Cell Population Staining: After an incubation period of 15 minutes in FcX block reagent at 4 °C, each tissue was stained with a cocktail containing a combination of the following antibodies for 45 minutes at 4 °C or 30 minutes at room temperature. i. Liver Panel: anti-mouse CD3 (17A2), anti-mouse CD45.2 (104), anti-mouse CD1 lb (MI / 70), anti-mouse CD68 (FA-11), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), anti-mouse NK1.1 (PK136), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii. Lung Panel: anti-mouse CD8a (53-6.7), anti-mouse CD326 (G8.8), anti-mouse / human CD49f (GoH3), anti-mouse CD4 (RM4-5), anti-mouse CD3 (17A2), anti-mouse CD45.2 (104), anti-mouse GDI lb (MI / 70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iii. Kidney Panel: anti-mouse CD45.2 (104), anti-mouse CDl lb (MI / 70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), anti-mouse CD309 (89B3A5), anti-mouse Podocalyxin (10B9), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.iv. Heart Panel: anti-mouse CD45.2 (104), anti-mouse CDl lb (MI / 70), anti-mouse CD31 (390), anti-mouse TER-119 (TER- 119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v. Spleen Panel: anti-mouse CD8a (53-6.7), anti-mouse CD19 (6D5), anti-mouse CD1 lb (MI / 70), anti-mouse CD3 (17A2), anti-mouse TER-119 (TER-119), anti-mouse NK1.1 (PK136), anti-mouse CD4 (RM4-5), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi. Bone Marrow Panel: anti-mouse CD4 (RM4-5), anti-mouse CD117 (2B8), anti-mouse CD8a (53-6.7), anti-mouse CD45.2 (104), anti-mouse CD1 lb (MI / 70), anti-mouse Seal (D7), anti-mouse TER-119 (TER-119), anti-mouse CD3 (17A2), anti-mouse CD34 (SA376A4), antimouse CD11c (), Lineage Cocktail (CD3 / GR-1 / CD1 lb / CD45R / B220 / TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.

[0225] Flow Cytometry Readouts: Different cell populations were analyzed for reporter protein expression relative to an untreated primate. The results were expressed as the percentage of cells that were positive for the reporter protein. Where LNPs contained DNA barcodes in addition to the mRNA, populations expressing reporter protein were sorted into QuickExtract buffer to isolate intracellular DNA.

[0226] DNA Sequencing of Sorted Samples: DNA barcodes of sorted cells were amplified under two rounds of PCR and Illumina handles were attached for compatibility with Next Generation Sequencing. Amplified samples were pooled, cleaned, diluted to 1 nM, and loaded into an iSeq cartridge for sequencing. Samples were analyzed for relative DNA barcode counts for selection of LNP candidates to advance in the pipeline.

[0227] The results of LNP screening in mouse for LNP formulations containing various exemplary diketopiperazine lipids as disclosed herein are shown in FIGS.4-14. In These figures, certain LNP formulations were selected from high-throughput, barcoding based, screens. During high-throughput screening, each LNP formulation was numbered as LNP-1 through LNP-XYZ in database, referred to as the sample number for that LNP formulation. For instance, LNP-151 indicates the 151st LNP formulation for a screen.

[0228] FIGS. 8A-8F illustrate the flow cytometry results for five high-throughput, barcodingbased, screens in mouse using the LNP formulations containing Compound 16 in the liver (FIG.8 A), lung (FIG. 8B), spleen (FIG. 8C), bone marrow (FIG. 8D), kidney (FIG. 8E), and heart (FIG. 8F). For each screen, LNPs were formulated with Compound 16 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10: 1.

[0229] In Screen 1 , the PEG lipid included C 14PEG2K PE, C 18PEG2K PE, DMG-PEG2K, or DSG-PEG2K; and the helper lipid included DOPE, DOTMA, DOTAP, or DDAB. The ratios for the lipid components were:

[0230] In Screen 2, the PEG lipid included C14PEG2K PE, C18PEG2K PE, or DSG-PEG2K; and the helper lipid included DOTAP or DDAB. The molar ratios for the lipid components were:[00231J In Screen 3, the PEG lipid included C14PEG2K PE, C18PEG2K PE, or DSG-PEG2K; and the helper lipid included DOTAP or DDAB. The molar ratios for the lipid components were:

[0232] In Screen 4, the PEG lipid included C14PEG2K PE, C18PEG2K PE, or DSG-PEG2K; and the helper lipid included DDAB. The molar ratios for the lipid components were:

[0233] In Screen 5, the PEG lipid included Cl 8PEG2K PE or DSG-PEG2K; and the helper lipid included DDAB. The molar ratios for the lipid components were:

[0234] The results in FIGS. 8A-8F show that the LNP delivery was found in multiple cell populations in the liver of mouse, including endothelial cells; and in multiple cell populations in the lung of mouse, including endothelial, basal, and stem cells. As shown in FIG. 8D, LNP delivery to progenitor cells in the bone marrow was improved across screens. FIGS. 8E-8F show that the LNP delivery was found in the kidney and heart in mouse, predominantly in endothelial cells.

[0235] FIGS. 9A-9F show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 9A), lung (FIG. 9B), bone marrow (FIG. 9C), spleen (FIG. 9D), kidney (FIG. 9E), and heart (FIG. 9F). To conduct this experiment, a number of LNPs that performed well in Screen 1 (a high-throughput, barcoding based, screen disclosed in FIGS.8A-8F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 16) for FIGS. 9A-9F are characterized below:

[0236] As shown in FIG. 9A, among the LNP formulation assessed, the highest delivery came from LNP-108. FIG. 9B shows significant levels of delivery in endothelial cells in lung, and relatively lower levels of delivery in basal cells in lung with the most LNP formulations assessed. FIG. 9C shows that in the bone marrow, certain LNP formulation (e.g., LNP-108) had the highest representation across all progenitor populations, though the delivery across the board was low. FIG. 9E-9F show that LNP delivery was found in the kidney and heart in mouse, predominantly in endothelial cells.

[0237] FIGS. 10A-10F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 10A), lung (FIG. 10B), bone marrow (FIG. 10C), spleen (FIG. 10D), kidney (FIG. 10E), and heart (FIG. 10F). To conduct this experiment, a number of LNPs that performed well in Screen 2 (a high-throughput, barcoding based, screen disclosed in FIGS.8A-8F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 16) for FIGS. 10A-10F are characterized below:

[0238] The results in FIGS. 10A-10F show that LNP delivery was found the liver, lung, bone marrow, spleen, kidney, and heart in mouse, although the levels of delivery to spleen, kidney and heart were not significant. Certain LNP formulations (e.g., LNP-1025 and LNP-1029) had substantial delivery in most cell populations in the lung in mouse, with little to no off-target in most cell populations in the liver in mouse. Off-target was predominantly found in endothelial cells. LNP-997 formulation had a similar delivery profile, although with a higher delivery to liver endothelial cells. In addition, certain LNP formulations showed some delivery in progenitor populations in the bone marrow of mouse.

[0239] FIGS. 11A-11C show the flow cytometry results for individual LNP formulations selected from Screen 4 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 11 A), lung (FIG. 1 IB), and bone marrow (FIG. 11C). To conduct this experiment, a number of LNPs that performed well in Screen 4 (a high- throughput, barcoding based, screen disclosed in FIGS. 8A-8F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 16) for FIGS. 11 A-11C are characterized below:

[0240] The results in FIGS. 11A-11C show that LNP delivery was found the liver, lung, and bone marrow in mouse. FIG. 11 A shows that with most LNP formulations, delivery to the liver was largely constrained to endothelial cells. FIG. 1 IB shows that in the lung of mouse, high delivery was observed across most cell populations, whereas the delivery was at or near saturation in endothelial cells, basal cells, and stem cells with most LNP formulations assessed. FIG. 11C shows that in the bone marrow, LNP delivery has highest levels in LSK-defined HSCs and CD34+ progenitor cells.

[0241] FIGS. 12A-12C show the flow cytometry results for individual LNP formulations selected from Screen 5 (discussed in FIGs. 8A-8F) in mouse using the LNP formulations containing Compound 16 in the liver (FIG. 12A), lung (FIG. 12B), and bone marrow (FIG. 12C). To conduct this experiment, a number of LNPs that performed well in Screen 5 (a high- throughput, barcoding based, screen disclosed in FIGS. 8A-8F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 16) for FIGS. 12A-12C are characterized below:

[0242] The results in FIGS. 12A-12C show that LNP delivery was found the liver, lung, and bone marrow in mouse. FIG. 12A shows that with most LNP formulations, delivery to the liver was largely constrained to endothelial cells. FIG. 12B shows that in the lung of mouse, highdelivery was observed across most cell populations, whereas the delivery was at or near saturation in endothelial cells, basal cells, and stem cells with many LNP formulations assessed (e.g., LNP-1516, LNP-1562, LNP-1564, and LNP-1570). FIG. 12C shows that in the bone marrow, LNP delivery has relatively higher levels in LSK progenitor cells and CD34+ progenitor cells than in other cell populations in bone marrow in mouse.

[0243] FIGS. 13A-13F illustrate the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 61 in the liver (FIG. 13A), lung (FIG. 13B), spleen (FIG. 13C), bone marrow (FIG. 13D), kidney (FIG. 13E), and heart (FIG. 13F). For the screen, LNPs were formulated with Compound 61 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. The PEG lipid included C14-PEG2K PE or C18-PEG2K PE; and the helper lipid included DDAB or DSPC. The molar ratios for the lipid components were:

[0244] The results in FIGS. 13A-13F show that the LNP delivery was found in multiple cell populations in the liver of mouse, including endothelial cells; and in multiple cell populations in the lung of mouse, including endothelial cells and macrophages. As shown in FIGS. 13C-13F, the LNP delivery was also found in the spleen, bone marrow, kidney, and heart in mouse.

[0245] FIGS. 14A-14F show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 13A-13F) in mouse using the LNP formulations containing Compound 61 in the liver (FIG. 14A), lung (FIG. 14B), bone marrow (FIG. 14C), spleen (FIG. 14D), kidney (FIG. 14E), and heart (FIG. 14F). To conduct this experiment, a number of LNPs that performed well in Screen 1 (a high-throughput, barcoding based, screen disclosed in FIGS. 13A-13F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 61) for FIGS. 14A-14F are characterized below:

[0246] The results in FIGS. 14A-14F show that the LNP delivery was found for certain LNP formulation (e.g., LNP- 1753) in the liver of mouse, and limited delivery in certain cell populations in the spleen and kidney of mouse.

[0247] FIGS. 15A-15F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 56 and Compound 62 in the liver (FIG. 15 A), lung (FIG. 15B), spleen (FIG. 15C), bone marrow (FIG. 15D), kidney (FIG. 15E), and heart (FIG. 15F). For the screen, LNPs were formulated with a Lipomer (Compound 56 or Compound 62), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. The PEG lipid included C14-PEG2K PE or Cl 8- PEG2K PE; and the helper lipid included DSPC or DDAB. The molar ratios for the lipid components were:

[0248] The results in FIGS. 15A-15F show that the LNP delivery was found in multiple cell populations in the liver of mouse, including endothelial cells; in multiple cell populations in the lung of mouse, including endothelial cells and macrophages; and in multiple cell populations in the spleen of mouse. FIGS. 15E-15F show that the LNP delivery was found in the kidney and heart in mouse, predominantly in the endothelial cells and macrophages.

[0249] FIGS. 16A-16C show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs. 15A-15F) in mouse using the LNP formulations containing Compound 56 and Compound 62 in the liver (FIG. 16A), lung (FIG. 16B), and spleen (FIG. 16C). To conduct this experiment, a number of LNPs that performed well in Screen 1 (a high-throughput, barcoding based, screen disclosed in FIGS. 15A-15F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 56 or 62) for FIGS. 16A-16C are characterized below:

[0250] FIG. 16A shows that the LNP delivery for certain LNP formulation (e g., LNP-1942) was found in the liver of mouse. The LNP delivery was also found for certain LNP formulations in the spleen, although the levels of delivery were not significant.

[0251] FIGS. 17A-17F show the flow cytometry results for a high-throughput, barcodingbased, screen in mouse using the LNP formulations containing Compound 68, Compound 69, and Compound 70 in the liver (FIG. 17A), lung (FIG. 17B), spleen (FIG. 17C), bone marrow (FIG. 17D), kidney (FIG. 17E), and heart (FIG. 17F). For the screen, LNPs were formulated with a Lipomer (Compound 68, Compound 69, or Compound 70), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10: 1. The PEG lipid included C14-PEG2K PE or C18-PEG2K PE; and the helper lipid included DSPC or DDAB. The molar ratios for the lipid components were:

[0252] The results in FIGS. 17A-17F show that the LNP delivery was found in the liver and lung of mouse, although the levels of delivery were not significant.Example 9 - Delivery of mRNA to non-human Primate (NHP) Using Lipid Nanoparticles Formulated With the Exemplary Diketopiperazine LipidsFormulation of Lipid Nanoparticles Using Diketopiperazine Lipids

[0253] Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 7. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids,cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2.Experimental Protocols for non-human Primate

[0254] Predose Biopsy Collections: Blood (8 to 10 mL) were collected via a femoral vein into tubes containing K2EDTA from each animal predose (Day -20 to -22 based upon each animal’s dosing date). Additionally, bone marrow and liver biopsy samples were collected from all animals on Day -20 to -22 based upon the animal’s date of dosing. Animals underwent laparoscopic liver biopsy procedures while bone marrow were collected by perfusion of 30 mL of phosphate buffered saline (PBS) through one femur bone.

[0255] Animal Pretreatment: Primates were pretreated with a dose of 1.0 mg / kg of dexamethasone administered intravenously, 0.5 mg / kg of famotidine administered intravenously, and 1.5 mg / kg diphenhydramine administered intramuscularly both 16-24 hours and 1 hour before LNP-mRNA administration.

[0256] Dose Preparation: LNPs were prepared using the NanoAssemblr Ignite system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol. After mixing, resulting LNPs were dialyzed in IX PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 pm pore size PES filter. Nanoparticles were then analyzed to determine whether they meet the quality control criteria: using DLS for diameter (20 nm - 200 nm), poly dispersity index (< 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 < 3.0 and A330 < 1.25). The nanoparticles that met these criteria were diluted to a solution of IX PBS and 10% w / v trehalose, frozen at -20 °C, transferred to -80 °C, and shipped in dry ice the next day.

[0257] At the administration site, frozen LNPs were allowed to thaw at room temperature for at least 45 minutes. Once thawed, they were filtered using 0.22 pm pore size, 33 mm diameter PES filters. Nanodrop RNA concentration measurements were taken before and after filtering to calculate dose retention from the original LNP concentration.

[0258] Dose Administration: Doses were calculated based on animal weight the day of administration. LNPs were administered as a 30-minute infusion via a saphenous vein using a catheter set.

[0259] Post-dose Procedures: Body weight and temperature were monitored after infusion until sacrifice. Additionally, hematology values and clinical chemistry were tracked several times after administration to monitor the response of the animal to the infused LNP.

[0260] Tissue Collection: Animals injected with LNPs containing reporter mRNA were sacrificed by exsanguination under anesthesia 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely into ~3 cm3cubes (with the exception of bone marrow, blood, and spleen) and placed into individual aliquots of a solution containing 90% FBS and 10% DMSO. All samples will be stored in Mr. Frosty for control temperature change up to 72 hours at -70 to -90°C and then frozen in liquid nitrogen or at approximately -80°C until shipped.

[0261] Tissue Digestion: All samples were allowed to thaw at room temperature prior to processing. Once thawed, tissues were transferred to a complete media solution containing FBS and centrifuged to remove any traces of DMSO. After centrifugation samples were resuspended in tissue digestion solutions and processed as indicated below: i. Liver Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (400 U / mL), and RPMI-1640 solvent. Tissues were incubated at 37 °C for 30 minutes at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain. ii. Lung Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (400 U / mL), and RPMI-1640 solvent. Tissues were incubated at 37 °C for 45 minutes at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain.iii. Kidney Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U / mL), Collagenase XI (125 U / mL), Hyaluronidase (60 U / mL), DNAse I (400 U / mL), and RPML1640 solvent. Tissues were incubated at 37 °C for 30 minutes at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain. iv. Heart Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase II (1 mg / mL), Dispase (0.6 U / mL), DNAse I (400 U / mL), FBS (2% v / v), and RPML1640 solvent. Tissues were incubated at 37 °C for 1 hour at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain. v. Spleen Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (400 U / mL) in RPML1640 solvent. Tissues were incubated at 37 °C for 10 minutes at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain. vi. Bone Marrow Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (400 U / mL) in RPML1640 solvent. Tissues were incubated at 37 °C for 10 minutes at 1,000 rpm. Then, they were transferred through a 70 pm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 pL of MojoSort buffer containing Human Monoclonal (5% v / v) and Polyclonal (15% v / v) FcX block and transferred to microcentrifuge tubes to stain.

[0262] Cell Population Staining: After an incubation period of 15 minutes in FcX blockreagent at 4 °C, each tissue was stained with a cocktail containing a combination of the following antibodies for 45 minutes at 4 °C or 30 minutes at room temperature. i. Liver Panel: anti-human CD56 (REA196), anti-human CD31 (WM59), anti-human CD4 (OKT4), anti-NHP CD45 (D058-1283), anti-human CD3 (SP34-2), anti-mouse CDl lb (MI / 70), anti-human CD68 (REA886), anti-human CDl lc (S-HCL-3), anti-human CD8 (BW 135 / 80), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii. Lung Panel: anti-human CD31 (WM59), anti-human CD326 (MH99), anti-mouse CDl lb (MI / 70), anti-mouse / human CD49f (GoH3), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iii. Kidney Panel: anti-human CD31 (WM59), anti-mouse CDl lb (MI / 70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iv. Heart Panel: anti-human CD31 (WM59), anti-mouse CDl lb (MI / 70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v. Spleen Panel: anti-human CD56 (REA196), anti-human CD4 (OKT4), anti -human CD3 (SP34-2), anti-mouse CDl lb (MI / 70), anti-human CD20 (2H7), anti-human CDl lc (S- HCL-3), anti-human CD8 (BW135 / 80), anti-human CD31 (WM59), anti-mouse CDl lb (MI / 70), anti-human CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi. Bone Marrow Panel: anti-human CD123 (7G3), and-human CD117 (104D2), antihuman CD4 (OKT4), anti -mouse CD1 lb (MI / 70), anti -human CD34 (561 and 563), anti -human CD45RA (5H9), anti-human CD90 (REA897), anti -human CD8 (BW135 / 80), anti -human CD45RO (UCHL1), anti-human CD20 (2H7), anti-human CD2 (RPA-2.10), anti-human CD14 (M5E2), anti-human CD16 (3G8), anti-human CD23 (M-L233), anti-human CD21 (B-ly4), antihuman CD56 (REA196), anti-human CD3 (SP34-2), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.

[0263] Flow Cytometry Readouts: Different cell populations were analyzed for reporterprotein expression relative to an untreated primate. The results were expressed as the percentage of cells that were positive for the reporter protein.

[0264] The results of LNP screening in primate for a LNP formulation containing an exemplary diketopiperazine lipid as disclosed herein are shown in FIGS.18-19. In These figures, certain LNP formulations were selected from a high-throughput, barcoding based, screen.

[0265] FIGS. 18A-18F show the flow cytometry results for a LNP formulation containing Compound 16 in primate at various doses (0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg), in the liver (FIG. 18 A), lung (FIG. 18B), spleen (FIG. 18C), bone marrow (FIG. 18D), kidney (FIG.18E), and heart (FIG. 18F). The LNP formulations (Lipomer: Compound 16) for FIGS. 18A- 18F are characterized below:

[0266] As shown in FIG. 18A, a higher delivery was observed in the liver of NHPs at doses of 2.0 and 1.0 mg / kg, with lower levels of delivery at the lowest dose of 0.1 mg / kg. FIG. 18B shows consistently high levels of delivery across the 0.5-2 mg / kg dosage level in the lung of NHPs, although delivery was largely maintained in basal, stem, and epithelial cells at the lower dose of 0.1 mg / kg. FIG. 18C shows high levels of delivery across the 1-2 mg / kg dosage level in the spleen of NHPs, with lower levels of delivery at 0.5 mg / kg and 0.1 mg / kg. FIG. 18D shows a high delivery at 2 mg / kg dosage in the bone marrow of NHPs and a significant drop in delivery from 2 to 0.5 and 0.1 mg / kg in the bone marrow of NHPs. FIG. 18E and 18F show that the delivery in the kidney and heart of NHPs were observed predominant in endothelial cells, with a significant drop in delivery efficiency from 1 mg / kg to 0.5 mg / kg and 0.1 mg / kg.

[0267] FIGS. 19A-19F show the flow cytometry results for a screen of LNP formulations using the top performing individual LNP formulations containing Compound 18 in primate injected intravenously at 1.5 mg / kg, in the liver (FIG. 19A), lung (FIG. 19B), spleen (FIG. 19C), bone marrow (FIG. 19D), kidney (FIG. 19E), and heart (FIG. 19F). For the screen, LNPs were formulated with Compound 18 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, thetotal lipid to nucleic acid mass ratio was 10:1. The PEG lipid included C14-PEG2K PE, DSG- PEG2K, or C18-PEG2K PE; and the helper lipid included DDAB. The molar ratios for the lipid components were:

[0268] The results in FIGS. 19A-19F show that the LNP delivery was found in most cell populations in the liver and lung of NHPs, with high delivery in endothelial cells, basal, and stem cells. The results also show that the LNP delivery was found in most cell populations in the spleen and bone marrow of NHPs, with substantial delivery to hematopoetic stem cell populations. FIGS. 19E-19F show that the LNP delivery in kidney and heart were observed predominantly in endothelial cells.

Claims

What is claimed:

1. A compound of F ormul a (I) :wherein:Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl;wherein: each t is independently 1, 2, 3, or 4; each Z is absent, -C(O)-, -C(O)O-, -C(O)N(R")-, or -S(O)(O)-;X is -O-, -S-, or -N(R")-; each L is independently a Ci-Cg alkylene optionally substituted by OH;R, R', and R'' each are independently H, OR14, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl;Ra, and Rbeach are independently H or branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci - C20 monovalent hydrocarbon chain, or represented by -(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic,each R11, R12, and R14are independently H, branched or unbranched alkyl or alkenyl, and each R13is independently H, alkyl, or OR14; provided that when Z is -C(O)-, Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-.

2. The compound of claim 1, having the formula of:

3. The compound of claim 1 or 2, wherein:Y is H; aryl; branched or unbranched Ci-Ce alkyl; Ci-Ce alkyl interrupted with a N, O, oreach R, R', Ra, and Rbare H; each t is independently 1 or 2; each Z is independently absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-;X is -O-, -S-, -NH-, or -N(OH)-; each L is independently a C1-C4 alkylene optionally substituted by OH; andRi and R2 are each independently represented by -(CH2)m-Q-(CH2)nH, wherein Q is absent, -CH=CH-, -C=C-, -S-S-, -N(OH)-, -C(O)NH-, -C(O)N(OH)-, -NHC(O)-, -N(OH)C(O)-,4. The compound of claim 1, having the formula of:

5. The compound of claim 4, having the formula of:

6. The compound according to any one of claims 1-5, wherein Z is -C(O)-.

7. The compound according to any one of claims 1-5, wherein at least one Z is absent, -C(O)O-, -C(O)NH-, or -S(O)(O)-.

8. The compound according to claim 3, wherein each L is independently a C1-C4 alkylene, or -CH2-CH(OH)-CH2- when the Z variable next to the L variable is absent.

9. The compound according to any one of claims 1-8, wherein Y is10. The compound of claim 9, having the formula of:

11. The compound of claim 10, having the formula of:

12. The compound according to any one of claims 1-8, wherein Y is H.

13. The compound according to any one of claims 1-8, wherein Y is branched or unbranched C1-C4 alkyl, a C1-C3 alkyl interrupted with an S atom, or benzyl.

14. The compound according to any one of claims 1-8, wherein15. The compound according to any one of claims 1-14, wherein each Ri and R2 are the same.

16. The compound according to any one of claims 1-14, wherein at least one, two, or three of the Ri and R2 variables are different than the other(s).

17. The compound according to any one of claims 1-14, wherein each Ri and R2 are independently branched or unbranched C1-C20 alkyl or branched or unbranched C2 -C20 alkenyl.

18. The compound according to claim 17, wherein each Ri and R2 are independently C9-C16 alkyl or C9-C16 alkenyl.

19. The compound according to claim 17, wherein each Ri and R2 are independently C9-C12 alkyl or C9-C12 alkenyl.

20. The compound according to claim 17, wherein Ri is C1-C4 alkyl and R2 is C9-C12 alkyl or C9-C12 alkenyl.

21. The compound according to any one of claims 1-14, wherein Q in each Ri and R2 variable is independently -CH=CH-, -C=C~, or -S-S-.

22. The compound according to any one of claims 1-14, wherein Q in each Ri and R2 variable is independently -C(O)O- or -OC(O)-.

23. The compound according to any one of claims 1-14, wherein Q in each Ri and R2 variable is independently -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or-NHC(O)-.

24. The compound according to any one of claims 1-14, wherein one of Ri and R2 is Ci -Ci6 branched or unbranched alkyl; and in the other Ri or R2, Q is -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)NH-,-C(O)N(OH)-, -N(OH)C(O)-, or -NHC(O)-.

25. A method of making a compound of formulating(i) reactingin the presence of a coupling agent, an organic base, and a solvent to form an intermediate compound, and(ii) reacting the intermediate compound formed in (i) with an acid, and then withform the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(C(Rn)(R12))m-Q- (C(Ru)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

26. The method of claim 25, wherein the coupling agent is EDCI, the organic base is DIPEA, and the solvent is DMF, DCM, or a mixture thereof.

27. The method of claim 26, wherein: the reactant iform correspondingrespectively, and the reagents in step (i) further comprises a benzotriazole or its derivative.

28. The method of claim 27, wherein the benzotriazole or its derivative is HOBt, HAT'U, or a mixture thereof.

30. The method of claim 25, wherein: the acid in step (ii) is HC1 in dioxane, or TFA; andO the reagents in step (ii) that reacts withfurther comprise sodium triacetoxy hy drob orate (N aB H(O Ac)? ) .

31. A method of making a compound of formulareactingO R1(a)in the presence of an organic base,R1I(b)in the presence of an organic carbonate, orR1(c)in the presence of an organic coupling agent to form a carbamate, wherein: each t is independently 1, 2, 3, or 4; each L is independently a Ci-Ce alkylene optionally substituted by OH;R, R', Ra, and Rbeach are independently H, branched or unbranched Ci-Ce alkyl, or C3-C7 cycloalkyl; andR1and R2each are independently branched or unbranched, saturated or unsaturated Ci -C20 monovalent hydrocarbon chain, or represented by -(C(Rn)(R12))m-Q- (C(Rn)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C=C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(Rn)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14are independently H, branched or unbranched alkyl or alkenyl.

32. The method of claim 31, wherein the reaction reacts with (a)in the presence of EDCI and DMAP.R1HO^33. The method of claim 31, wherein the reaction reacts with (b) L R2in the presence of triphosgene (bi s(trichlorom ethyl carbonate)) and pyridine.R1I34. The method of claim 31, wherein the reaction reacts with (c)in the presence of 4-nitrophenyl chloroformate, pyridine, and DMAP.

35. A lipid nanoparticle comprising the compound according to any one of claims 1-24.

36. The lipid nanoparticle according to claim 35, further comprising a helper lipid.

37. The lipid nanoparticle according to claim 36, wherein the helper lipid is a phospholipid.

38. The lipid nanoparticle according to claim 36, wherein the helper lipid is 1,2-dioeoyl-sn- glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dimethyldioctadecylammonium (DDAB), didodecyldimethylammonium bromide (DDAB), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), 18: 1 PA, N- methyldioctadecylamine (MDOA), N,N-dicotadecylaniline, sn-(3-myristoyl-2-hydroxy)- glycerol-l-phospho-sn-3'-(T,2'-dimyristoyl)-glycerol (14:0 Hemi BMP), 1,2-dimyristoyl-sn- glycero-3 -phosphate (DMPA; 14:0 PA), l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA; 18: 1 PA), l,2-distearoyl-sn-glycero-3-phosphate (DSP A; 18:0 PA), or a combination thereof.

39. The lipid nanoparticle according to claim 38, wherein the helper lipid is 1,2-dioeoyl-sn- glycero-3-phosphoethanolamine (DOPE), dimethyl dioctadecylammonium (DDAB), 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 2-distearoyl-sn-glycero-3-phosphocholine(DSPC), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), or a combination thereof.

40. The lipid nanoparticle according to any one of claims 35-39, further comprising a sterol.

41. The lipid nanoparticle according to claim 40, wherein the sterol is cholesterol, a cholesterol derivative, or a combination thereof.

42. The lipid nanoparticle according to any one of claims 35-41, further comprising a PEG- modified lipid.

43. The lipid nanoparticle according to claim 42, wherein the PEG-modified lipid is PEG- DMG or PEG-PE.

44. The lipid nanoparticle according to claim 42, wherein the PEG-modified lipid is 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyl eneglycol)-2000 (DMPE-PEG2K; 14:0PEG2K PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000 (DSPE-PEG2K; 18:0 PEG2K PE), 1,2-dimyristoyl-sn- glycero-3-methoxypolyethylene glycol (DMG-PEG 2K), 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159), distearoyl-rac-glycerol-PEG2K (DSG-PEG 2K), or a combination thereof.

45. The lipid nanoparticle according to claim 35, further comprising a helper lipid, a cholesterol, and a PEG-modified lipid, wherein the molar ratio of the compound: the helper lipid: the cholesterol : the PEG lipid is about 20-60 : 7-50 : 5-70 : 0.5-3, or about 30-45 : 12-40 : 18-47 : 1-2.5.

46. The lipid nanoparticle according to claim 45, wherein the molar ratio of the compound: the helper lipid : the cholesterol : the PEG lipid is about 25 : 33 : 40 : 2, about 30 : 22.5 : 45 : 2.5, about 30:39:30: 1, about 30 : 50 : 18 : 2, about 35 : 35 : 27.5 : 2.5, about 35 : 43 : 20 : 2, about35:44.5: 18:2.5, about 35: 16:46.5:2.5, about 45: 13:39.5:2.5, about 50 : 40 : 7.5 : 2.5, about 50 :17.5 : 30 : 2.5, about 50 : 12.5 : 35 : 2.5, about 50 : 10 : 38.5 : 1.5, or about 35: 15:47.5:2.5.

47. The lipid nanoparticle according to claim 35, further comprising a helper lipid and a PEG-modified lipid, wherein the molar ratio of the compound: the helper lipid: the PEG lipid is about 20-60 : 10-60 : 0.5-3, about 30-55 : 30-55 : 1-3, or about 32-50 : 40-55 : 1.5-2.5.

48. The lipid nanoparticle according to any one of claims 35-47, having a diameter of about 20-200 nm.

49. The lipid nanoparticle according to any one of claims 35-48, further comprising a therapeutic agent.

50. The lipid nanoparticle according to claim 49, wherein the therapeutic agent is a nucleic acid molecule, protein or peptide, carbohydrate or glycoprotein, small molecule, or any combination thereof.

51. The lipid nanoparticle according to claim 49, wherein the therapeutic agent is DNA or RNA.

52. The lipid nanoparticle according to claim 51, wherein the therapeutic agent is an mRNA.

53. The lipid nanoparticle according to any one of claims 35-52, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to lung, heart, kidney, liver, splenic, or marrow cells of a subject.

54. The lipid nanoparticle according to claim 53, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung natural killer (NK) cells, or other immune cells within the lung.

55. The lipid nanoparticle according to any one of claims 35-52, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to stem-like cells and / or hematopoietic stem cells (HSCs).

56. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 35-55 and a pharmaceutically acceptable carrier.

57. A method for delivering a therapeutic agent to a subject, comprising administering to the subject a lipid nanoparticle according to any one of claims 35-55.

58. The method according to claim 57, wherein the therapeutic agent is delivered to lung, heart, kidney, liver, splenic, or marrow cells of the subject.

59. The method according to claim 57, wherein the therapeutic agent is delivered to one or more of lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung macrophages, lung natural killer (NK) cells, or other immune cells within the lung; heart endothelial cells, heart epithelial cells, heart dendritic cells, heart B cells, heart T cells, heart natural killer (NK) cells, heart macrophages, or other immune cells within the heart; kidney endothelial cells, kidney epithelial cells, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, kidney macrophages, or other immune cells within the kidney; liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, or other immune cells within the liver; spleen dendritic cells, spleen neutrophils, spleen macrophages, spleen B cells, spleen T cells, spleen natural killer (NK) cells, or other immune cells within the spleen; lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, or lymphatic natural killer (NK) cells of the subject.

60. The method according to claim 57, wherein the therapeutic agent is delivered to bone marrow stem-like cells, and / or bone marrow hematopoietic stem cells (HSCs).

Images