Ionizable lipid and lipid nanoparticle compositions for delivery of nucleic acids

Abstract

Novel ionizable lipids are provided. Novel lipid nanoparticle compositions are also provided for delivering nucleic acid materials to cells in vitro and in vivo, with different and improved pharmacokinetic profiles compared to those typically observed in the art. Methods for using the compositions in research and as therapeutic agents are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 425,969, filed November 16, 2022, and U.S. Provisional Application No. 63 / 455,243, filed March 28, 2023, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] 1. Introduction There are many instances in which delivery of nucleic acids is desirable, including research, diagnostic, and therapeutic applications. One example of such a therapeutic application is gene therapy, which can be used to treat genetic disorders and other conditions. Genetic disorders, while individually rare, collectively represent a significant disease burden, particularly for children, resulting in significant disability and mortality.

[0003] In the field of gene therapy, viral vectors, such as AAV-based vectors, are commonly used to deliver genes into cells. However, AAV vectors are limited in the size of the gene cargo they can package. Therefore, any gene cargo larger than 4.7 kB is not suitable for delivery using AAV vectors, limiting the usefulness of such vectors for many indications. In addition, viral vectors such as AAV induce antibody responses, limiting re-administration, which is not suitable for some indications. Furthermore, in indications where target cells are dividing, such as the liver, expression from successfully transduced cells may decrease or be lost with cell division and turnover, requiring re-administration, but due to immunological memory, re-administration may not be possible or effective. Furthermore, many subjects have pre-existing immunity to commonly used viral vectors such as AAV, which can limit even initial treatment with AAV gene therapy. Furthermore, viral vectors such as AAV may be toxic at the doses required to achieve therapeutic benefit in some indications.

[0004] Lipid nanoparticles (LNPs) offer an alternative to viral gene therapy. Lipid nanoparticles have been developed and used to deliver many RNA therapeutics, but RNA delivered by lipid nanoparticles has a limited therapeutic lifespan. DNA delivered by lipid nanoparticles designed for RNA delivery suffers from low efficiency and significant activation of innate immune responses in treated subjects. New delivery vehicles for delivering nucleic acids, such as DNA, to cells have the potential to significantly advance numerous scientific endeavors, particularly in vivo for patients requiring gene therapy and in vitro for research applications. Summary of the Invention

[0005] 2. Overview Provided herein is a novel ionizable lipid, which has an ionizable head group connected to lipid tail via linear alkyl core.The linear alkyl core can have n carbon atoms, and n-1 carbon atoms in the linear alkyl core are connected to lipid tail.Also provided herein is a novel lipid nanoparticle (LNP) composition for delivering nucleic acid material to cells in vitro and in vivo, which has different and improved pharmacokinetic profile compared with that typically observed in the art.Also provided is a method for using the composition of the present invention in research and as a therapeutic agent. [Brief explanation of the drawings]

[0006] 3. Brief description of the drawings [Figure 1] Figures 1A-C describe studies conducted to evaluate how the structure of ionizable lipids affects the efficacy and toxicity of DNA-LNPs. (Figure 1A) Formulation details for the test articles. The ionizable lipids were varied in all formulations. The ionizable cationic lipids tested were ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and two ionizable cationic lipids, L-2 and L-3, of the present disclosure. The phospholipid in all formulations was DSPC. The nucleic acid cargo used in all formulations was nanoplasmid DNA (npDNA) containing the hAAT promoter driving expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (Figure 1B) EPO serum levels were measured in wild-type BALB / c mice 4 hours after intravenous administration of the test substance at a dose of 1 mg / kg. (Figure 1C) Serum IL-6 cytokine levels were measured 4 hours after intravenous administration of the test substance at a dose of 1 mg / kg to wild-type BALB / c mice.

[0007] [Figure 2] Figures 2A-O describe further studies conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs. (Figures 2A-2B) Formulation details for the test articles. In these formulations, the ionizable lipids were varied. Two benchmark ionizable lipids, ALC-0315 and MC3, are described above. Other benchmark ionizable lipids tested included LP01 (see Finn et al. Cell Reports, 2018, 22:2227), SM102 (see Sabnis et al. Molecular Therapy, 2018, 26:1509), and ARCT (see Rajappan et al. Organic Process R&D, 2021, 25:1383). Three ionizable lipids of the present disclosure, L-5, L-15, and L-9, were also tested. The phospholipids in these formulations were either DSPC (Figure 2B) or DOPE (Figure 2C). The nucleic acid cargo used in all formulations was nanoplasmid DNA (npDNA) containing the hAAT promoter driving expression of the EPO transgene. Good encapsulation efficiency and small size were observed for all test substances (Figure 2B). (Figure 2C) Seven days after intravenous administration of the test substances in Figures 2A and 2B at doses of 1 or 0.3 mg / kg, EPO protein levels were measured in the serum of wild-type BALB / c mice. (Figures 2D-I) Four hours after intravenous administration of the test substance in Figure 2A (formulated with DSPC phospholipid) at doses of 1 or 0.3 mg / kg, IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC levels were measured in the serum of wild-type BALB / c mice. (Figures 2J-O) Four hours after intravenous administration of the test substance (compounded with DOPE phospholipid) in Figure 2B, the levels of IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC were measured in the serum of wild-type BALB / c mice.

[0008] [Figure 3]Figures 3A-F describe further studies conducted to evaluate how additional ionizable lipid structures affect the efficacy and toxicity of DNA-LNPs. (Figures 3A-3B) Formulation details for the test articles. In these formulations, the ionizable lipid was varied. The benchmark ionizable lipid, ALC-0315, is described above. Other benchmark ionizable lipids were tested, including A9 (see Han et al. Nature Communications, 2021, 12:7233) and ssOP (see Tanaka et al. Pharmaceuticals, 2021, 13:544). Three ionizable lipids of the present disclosure, L-12, L-13, and L-14, were also tested. The phospholipids in these formulations were either DSPC (Figure 3A) or DOPE (Figure 3B). The nucleic acid cargo used in all formulations was nanoplasmid DNA (npDNA) containing the hAAT promoter driving expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test substances. (Figure 3C) Seven days after intravenous administration of the test substances at doses of 1 or 0.3 mg / kg, EPO protein levels were measured in the serum of wild-type BALB / c mice. (Figures 3D-I) Four hours after intravenous administration of the test substances (formulated with DSPC phospholipids) in Figure 3A at doses of 1 or 0.3 mg / kg, IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC levels were measured in the serum of wild-type BALB / c mice. (Figures 3J-O) Four hours after intravenous administration of the test substances (formulated with DOPE phospholipids) in Figure 3B, IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC levels were measured in the serum of wild-type BALB / c mice.

[0009] [Figure 4]Figures 4A-C describe further studies conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs. (Figure 4A) Formulation details for the test articles. In these formulations, the ionizable lipids were varied. The benchmark ionizable lipid, ALC-0315, is described above. Five ionizable lipids of the present disclosure, L-9, L-10, and L-11, L-15, and L-16, were also tested. The phospholipid in these formulations was DSPC. The nucleic acid cargo used in all formulations was nanoplasmid DNA (npDNA) containing the hAAT promoter driving expression of the EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (Figure 4B) EPO protein levels were measured in the serum of wild-type BALB / c mice 3 days after intravenous administration of the test articles at doses of 1 or 0.3 mg / kg. (FIG. 4C) IL-6 cytokine levels were measured in the serum of wild-type BALB / c mice 4 hours after intravenous administration of the test substance at a dose of 1 or 0.3 mg / kg.

[0010] [Figure 5]Figures 5A-C describe further studies conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs. (Figure 5A) Formulation details for the test articles. In these formulations, the ionizable lipids were varied. Two benchmark ionizable lipids, ALC-0315 and ARCT, are described above. One additional benchmark ionizable lipid, CL1 (see Lam et al. Advanced Materials, 2023, 35:2209-624), was also tested. Four ionizable lipids of the present disclosure, L-17, L-21, L-19, and L-20, were also tested. The phospholipid in these formulations is DSPC. The nucleic acid cargo used in all formulations is nanoplasmid DNA (npDNA) containing the hAAT promoter driving expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (Figure 5B) Three days after intravenous administration of the test substance at a dose of 1 or 0.3 mg / kg, serum EPO protein levels were measured in wild-type BALB / c mice. (Figure 5C) Four hours after intravenous administration of the test substance at a dose of 1 or 0.3 mg / kg, serum IL-6 cytokine levels were measured in wild-type BALB / c mice.

[0011] [Figure 6]Figures 6A-K describe studies conducted to evaluate how the structure of ionizable lipids affects the efficacy and toxicity of LNPs co-formulated with both DNA and mRNA. (Figure 6A) Formulation details for the test articles. In these formulations, the ionizable lipid was varied. The benchmark ionizable lipid, CL1, is described above. Three ionizable lipids of the present disclosure, L-15, L-17, and L-18, were also tested. The phospholipid in these formulations was DOPE. The nucleic acid cargo used in all formulations included nanoplasmid DNA (npDNA) and mRNA mixed at a 1:3 (w / w) ratio of DNA:mRNA, where the npDNA contains a TTR promoter driving expression of a human factor IX (FIX) transgene. Good encapsulation efficiency and small size were observed for all test articles. (Figure 6B) Human FIX protein levels in plasma of wild-type BALB / c mice 21 days after intravenous administration of the test substance at a dose of 0.5 mg / kg DNA (1.5 mg / kg mRNA). (Figures 6C-K) Cytokine levels in serum of wild-type BALB / c mice 4 hours after intravenous administration of the test substance at a dose of 0.5 mg / kg DNA (1.5 mg / kg mRNA). DETAILED DESCRIPTION OF THE INVENTION

[0012] 4. Detailed Description 4.1 Lipid Nanoparticle Composition Novel lipid nanoparticle compositions are provided for delivering nucleic acids to cells in vitro and in vivo, which have different and improved pharmacokinetic profiles compared to those typically observed in the art. Methods for using the lipid nanoparticle compositions of the present disclosure in research and as therapeutic agents are also provided.

[0013] "Lipid nanoparticle" refers to a lipid composition that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., DNA and / or RNA), proteins, small molecules, etc., to a desired target site. In lipid nanoparticles, nucleic acid agents can be encapsulated in lipids, thereby protecting the agents from enzymatic degradation.

[0014] Generally, lipid nanoparticles comprise several lipid components, including, for example, an ionizable lipid, one or more helper lipids (e.g., non-cationic lipids), and a lipid that prevents aggregation of the nanoparticles (also called a coating lipid or a conjugated lipid, e.g., a PEG-lipid). In some embodiments, the present disclosure provides lipid nanoparticle (LNP) compositions described herein that comprise a nucleic acid, wherein the nucleic acid is substantially encapsulated by the lipid component of the LNP.

[0015] 4.2 Ionizable lipids The lipid nanoparticles (LNPs) of the present disclosure can include ionizable lipids. As summarized above, novel ionizable lipids are provided herein. Ionizable lipids are typically used in lipid nanoparticles (LNPs) to condense their nucleic acid cargo, such as DNA or RNA, at low pH and to promote membrane association and fusogenicity. The term "ionizable lipid" refers to a lipid containing an ionizable group that has a net charge at a selected pH (e.g., pH 6.5 or less) but can remain neutral at higher pHs, such as physiological pH. The pH sensitivity of such ionizable lipids may be desirable for providing intracellular delivery of nucleic acid cargo. Ionizable lipids have fewer interactions with cell membranes when neutral, and can then become charged upon uptake into the interior of endosomes in target cells, where the pH is lower than the extracellular environment. Protonated, and therefore positively charged, ionizable lipids can promote membrane destabilization and facilitate endosomal escape of nanoparticles.

[0016] In some embodiments, the ionizable lipid is a cationic lipid. The term "cationic lipid" refers to a lipid that has a net positive charge at a selected pH (for example, a pH of 6.5 or less). In some embodiments, the ionizable lipid is a cationic lipid that is positively charged or contains at least one ionizable amino group that is protonated at a selected pH, for example, a pH of 6.5 or less. In some embodiments, the cationic lipid contains one or more tertiary amino groups, for example, a trialkylamino group.

[0017] As disclosed herein, ionizable lipids comprise an ionizable head group (e.g., an ionizable amino group) connected to a lipid tail via a linear alkyl core. The linear alkyl core can have n carbon atoms, with n-1 carbon atoms in the linear alkyl core being linked to a lipid tail. In some embodiments, the linear alkyl core has 3 carbon atoms and 2 lipid tails. In some embodiments, the linear alkyl core has 4 carbon atoms and 3 lipid tails. In some embodiments, the linear alkyl core has 5 carbon atoms and 4 lipid tails. In some embodiments, the linear alkyl core has 6 carbon atoms and 5 lipid tails.

[0018] In some embodiments, the cationic lipids comprise a protonatable tertiary amine (e.g., pH-titratable) head group, a linear alkyl core, and a hydrocarbon chain (e.g., C 18 C8 to C alkyl chains 20In some embodiments, the cationic lipid comprises a protonatable tertiary amine head group, a linear alkyl core, a hydrocarbon chain (e.g., as described herein), and an ester linkage between the linear alkyl core and the hydrocarbon chain. ...

[0019] An embodiment of the present disclosure provides an ionizable lipid compound of formula (I): (ZLY)-W n -(XR) (n-1) (I) Including, During the ceremony, Z is an ionizable head group; L is an optionally substituted (C1 to C 12 ) alkylene, Y is a linking group, W n is a linear alkyl core of n carbon atoms, where n is 3 to 6; X is an optional linking group; Each R is independently a lipid tail.

[0020] In some embodiments of Formula (I), n is 4 to 6 such that the linear alkyl core has 4 to 6 carbon atoms. In some cases, n is 4 such that the linear alkyl core has 4 carbon atoms. In some cases, n is 5 such that the linear alkyl core has 5 carbon atoms. In some cases, n is 6 such that the linear alkyl core has 6 carbon atoms. In some embodiments, n is 3 such that the linear alkyl core has 3 carbon atoms.

[0021] In some embodiments of Formula (I), the linear alkyl core W n teeth, [ka] and where * represents the point of attachment to Y and each ** represents the point of attachment to X.

[0022] In some embodiments of Formula (I), the linear alkyl core W n teeth, [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and each G 2 is independently H or —CHOH. In certain instances, at least one G 2 is H. In certain cases, both G 2 The group is H. In certain cases, at least one G 2 is -CH2OH. In certain cases, both G 2 In certain cases, one of the G 2 is H and the other is —CH2OH.

[0023] In some embodiments of Formula (I), the linear alkyl core W n teeth, [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and G 2 is H or -CHOH. In certain instances, G 2 is H. In a particular case, G 2 is -CHOH.

[0024] In some embodiments of Formula (I), W n teeth, [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and G 2 is H or -CHOH. In certain instances, G 2 is H. In a particular case, G 2 is -CHOH.

[0025] In some embodiments of Formula (I), the linear alky core W n teeth: [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and G 1 is H or G 1 and W to which Y is bonded n is a group cyclically linked to Y which, together with the carbon atoms of G, provides a heterocyclic ring. 1 is cyclically linked to Y to form a 5-membered heterocycle. 1 is cyclically linked to Y to form a 6-membered heterocycle. 1 is H.

[0026] In some embodiments of Formula (I), the linear alkyl core W n teeth, [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and G 1 is H or G 1 and W to which Y is bonded n is a group cyclically linked to Y which, together with the carbon atoms of G, provides a heterocyclic ring; 2 is H or -CHOH. In certain instances, G 1 is cyclically linked to Y to form a 5-membered heterocycle. 1 is cyclically linked to Y to form a 6-membered heterocycle. 1 is H. In a particular case, G 2 is H. In a particular case, G 2 is -CHOH. In certain cases, G 1 and G 2 At least one of G is H. In certain cases, 1 and G 2 are both H. In certain cases, G 1 is H and G 2 is -CH2OH. In certain cases, G 1 is cyclically linked to Y to provide a heterocycle, and G 2 is H. In a particular case, G 1 is cyclically linked to Y to provide a heterocycle, and G 2 is -CHOH.

[0027] In some embodiments of Formula (I), the linear alkyl core W n teeth, [ka] and where * represents the point of attachment to Y, each ** represents the point of attachment to X, and G 1 is H or G 1 and W to which Y is bonded n is a group cyclically linked to Y which, together with the carbon atoms of G, provides a heterocyclic ring; 2 is H or -CHOH. In certain instances, G 1 is cyclically linked to Y to form a 5-membered heterocycle. 1 is cyclically linked to Y to form a 6-membered heterocycle. 1 is H. In a particular case, G 2 is H. In a particular case, G 2 is -CHOH. In certain cases, G 1 and G 2 At least one of G is H. In certain cases, 1 and G 2 are both H. In certain cases, G 1 is H and G 2 is -CH2OH. In certain cases, G 1 is cyclically linked to Y to provide a heterocycle, and G 2 is H. In a particular case, G 1 is cyclically linked to Y to provide a heterocycle, and G 2 is -CHOH.

[0028] As described herein above, in formula (I), the alkyl linear core W nis linked to the ionizable head group Z through a linking group Y. By "linking group" is meant a linking moiety that connects two groups via a covalent bond. The linking group Y can be linear, branched, cyclic, a single atom, or a covalent bond. Examples of such linking groups include, but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amide, ureylene, imide, ether, thioether, carbonate, alkyldioxy, oximino, amino, carbonyl, heterocycle (e.g., cyclic acetal), and the like.

[0029] In some embodiments of Formula (I), Y is —O—, —C(R 10 )2-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S—, wherein R 10 teeth 、 H and C 1~6 In some cases, Y is selected from -O-, -OC(O)-, -C(O)O-, and -OC(O)NR 10 In some cases, Y is -O-. In some cases, Y is -OC(O)-. In some cases, Y is -OC(O)NR 10 - and R 10 is H. In some cases, Y is -C(R 10 )2-, and each R 10 is H. In some cases, Y is -C(O)O-. In some cases, Y is -OC(O)O-. In some cases, Y is -SC(O)NR 10 - and each R 10 is H. In some cases, Y is —C(O)NR 10 - and each R 10is H. In some cases, Y is -NR 10 C(O)-, and each R 10 is H. In some cases, Y is -S-. In some cases, Y is -NR 2 In some instances, Y is -NR 10 C(O)O-, and each R 10 is H. In some instances, Y is -NR 10 C(O)S-, and each R 10 is H.

[0030] In some embodiments, W n is the group G adjacent to the point of attachment to the linking group Y. 1 In some embodiments, G 1 is cyclically linked to the linking group Y to provide a heterocycle. 1 is cyclically linked to Y to provide a 5-membered heterocycle. In some embodiments, the 5-membered heterocycle is a cyclic acetal. In some embodiments, G 1 is cyclically linked to Y to provide a 6-membered heterocycle. In some embodiments, the 6-membered heterocycle is a cyclic acetal.

[0031] As described herein above, in formula (I), the linking group Y is an optionally substituted (C1-C 12) alkylene L is linked to the ionizable head group Z. In some embodiments of Formula (I), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In some embodiments, L is (C2-C4) alkylene or substituted (C2-C4) alkylene. In certain cases, L is C2-alkylene or substituted C2-alkylene. In certain cases, L is C3-alkylene or substituted C3-alkylene. In certain cases, L is C4-alkylene or substituted C4-alkylene. In certain cases, L is C5-alkylene or substituted C5-alkylene. In certain cases, L is C6-alkylene or substituted C6-alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3. In certain cases, L is -(CH2)4-. In certain cases, L is -(CH2)5-. In certain cases, L is -(CH2)6-.

[0032] In some embodiments, -YLZ has the formula -O(CH) r Z, where r is 2 to 6. In some embodiments, -YLZ is of the formula -OC(O)(CH) r Z, where r is 2 to 6. In some embodiments, -YLZ is of the formula -OC(O)NH(CH) r Z, where r is 2 to 6. In some embodiments, -YLZ is of the formula -CH2(CH2) r Z, where r is 2 to 6. In some cases, r is 2 to 4. In some cases, r is 2. In some cases, r is 3. In some cases, r is 4.

[0033] As described herein, the ionizable lipid of formula (I) comprises an ionizable head group. In some embodiments, the ionizable head group comprises a primary, secondary, or tertiary amine that can be protonated at physiological pH. In some embodiments, the ionizable head group comprises a tertiary amino group. In certain embodiments, the ionizable head group comprises a group of formula -NR 11 R 12 R 11 and R 12 are each independently alkyl or substituted alkyl. In some embodiments, R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 In some embodiments, R 11 and R 12 are each independently 1~3 Alkyl or substituted C 1~3 In some embodiments, R 11 and R 12 are C 1~3 In some embodiments, R 11 and R 12 are each methyl. In some embodiments, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0034] As described herein, formula (I) can be linked to a core W, optionally via an additional linking group X. n 2-5 lipid tails R (e.g., -(XR) (n-1) ) is included.

[0035] In some embodiments, the ionizable lipid of formula (I) comprises a linking group X. The linking group X can be linear, branched, cyclic, or a single atom. Examples of such linking groups include, but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, amide, ureylene, imide, ether, thioether, thiocarbamate, carbonate, alkyldioxy, oximino, amino, carbonyl, etc. In some embodiments of formula (I), each X is independently -(CH2) s OC(O)-,-(CH2) s C(O)O-,-(CH2) s OC(O)O-,-(CH2) s OC(O)NR10 -,-(CH2) s O-,-(CH2) s SC(O)NR 10 -,-(CH2) s C(O)NR 10 -,-(CH2) s NR 10 C(O)-,-(CH2) s S-,-(CH2) s NR 10 -,-(CH2) s NR 10 C(O)O- and -(CH2) s NR 10 C(O)S—, wherein R 10 is H and C 1~6 alkyl, and s is 0 to 6. In some embodiments, each X is independently —(CH) s OC(O)-, -(CH2) s C(O)O- and -(CH2) s In some embodiments, each X is selected from -OC(O)O-. s OC(O)-, where s is 0, 1, or 2. In some embodiments, each X is -(CH) s C(O)O—, where s is 0, 1, or 2. In some embodiments, each X is —(CH) s OC(O)O-, where s is 0, 1, or 2. In some embodiments, at least one X group is -(CH) s O- and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH) s OC(O)NR 10 - and R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s SC(O)NR 10 - and R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s C(O)NR 10 - and R10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s NR 10 C(O)- and R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s S-, where s is 0, 1, or 2. In some embodiments, at least one X group is -(CH) s NR 10 - and R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s NR 10 C(O)O- and R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is —(CH) s NR 10 C(O)S- and R 10 is H and s is 0, 1 or 2.

[0036] In some embodiments of Formula (I), each X is independently -OC(O)-, -C(O)O-, -OC(O)O-, -O-, or -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, and R 10 teeth 、 H and C 1~6In some embodiments, each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In some embodiments, each X is -OC(O)-. In some embodiments, each X is -C(O)O-. In some embodiments, each X is -OC(O)O-. In some embodiments, at least one X group is -O-. In some embodiments, at least one X group is -OC(O)NR 10 - and R 10 is H. In some embodiments, at least one X group is —SC(O)NR 10 - and R 10 is H. In some embodiments, at least one X group is —C(O)NR 10 - and R 10 is H. In some embodiments, at least one X group is —NR 10 C(O)- and R 10 is H. In some embodiments, at least one X group is -S-. In some embodiments, at least one X group is -NR 10 - and R 10 is H. In some embodiments, at least one X group is —NR 10 C(O)O- and R 10 is H. In some embodiments, at least one X group is —NR 10 C(O)S- and R 10 is H.

[0037] In some embodiments of Formula (I), each -XR is independently -(CH) s OC(O)R, -(CH2) s C(O)OR, -(CH2) s OC(O)OR, -(CH2) s OR, -(CH2) s OC(O)NR 10 R, -(CH2) s SC(O)NR 10 R, -(CH2) sC(O)NR 10 R, -(CH2) s NR 10 C(O)R, -(CH2) s SR, -(CH2) s NR 10 R, -(CH2) s NR 10 C(O)OR and -(CH2) s NR 10 C(O)SR, R 10 is selected from H and C alkyl, s is 0 to 6, and each R is independently a lipid tail. In some embodiments, each -XR is -(CH) s In some embodiments, each -XR is -(CH) s In some embodiments, each -XR is -(CH2) s In some embodiments, each -XR is -(CH) s In some embodiments, each -XR is -(CH) s OC(O)NR 10 In some embodiments, each -XR is -(CH) s SC(O)NR 10 In some embodiments, each -XR is -(CH) s C(O)NR 10 In some embodiments, each -XR is -(CH) s NR 10 In some embodiments, each -XR is -(CH2) s In some embodiments, each -XR is -(CH) s NR 10 In some embodiments, each -XR is -(CH) s NR 10 In some embodiments, each -XR is -(CH2) s NR 10 It is C(O)SR.

[0038] In some embodiments of Formula (I), each -XR is -OC(O)R, C(O)OR, OC(O)OR, OR, OC(O)NR 10 R, SC(O)NR 10 R, C(O)NR 10 R, NR 10 C(O)R, SR, NR 10 R, NR 10 C(O)OR and NR 10 C(O)SR independently selected from R 10 is H and C 1~6 alkyl, and each R is independently a lipid tail. In some embodiments, each -XR is OC(O)R. In some embodiments, each -XR is -C(O)OR. In some embodiments, each -XR is OC(O)OR. In some embodiments, each -XR is OR. In some embodiments, each -XR is OC(O)NR 10 In some embodiments, each -XR is S-C(O)NR 10 In some embodiments, each -XR is -C(O)NR 10 In some embodiments, each -XR is -NR 10 In some embodiments, each -XR is -SR. In some embodiments, each -XR is -NR 10 In some embodiments, each -XR is -NR 10 In some embodiments, each -XR is -NR 10 C(O)SR. In some embodiments of Formula (I), each lipid tail is independently an aliphatic hydrocarbon group that is linear or branched, saturated or unsaturated, and / or optionally contains a cyclic group.

[0039] In some embodiments of Formula (I), each R is a straight chain hydrocarbon group optionally containing one or more cyclic groups. In some embodiments, each R is a C5-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20In some embodiments, each R is a straight chain hydrocarbon group independently selected from C6 to C8. 12 Alkyl and C6-C 12 In some embodiments, at least one R is a straight-chain hydrocarbon group independently selected from alkenyl. In some embodiments, at least one R is a straight-chain hydrocarbon group comprising a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle and heteroaryl, and any of the monocyclic or bicyclic groups is optionally substituted.

[0040] In some embodiments of Formula (I), at least one R is a branched hydrocarbon group, optionally comprising one or more cyclic groups. In some embodiments, each R is a branched hydrocarbon group, optionally comprising one or more cyclic groups. In some embodiments, the branched hydrocarbon group contains 8 to 20 carbon atoms. In some embodiments, the branched hydrocarbon group contains 8 carbon atoms. In some embodiments, the branched hydrocarbon group contains 9 carbon atoms. In some embodiments, the branched hydrocarbon group contains 10 carbon atoms. In some embodiments, the branched hydrocarbon group contains 11 carbon atoms. In some embodiments, the branched hydrocarbon group contains 12 carbon atoms. In some embodiments, the branched hydrocarbon group contains 13 carbon atoms. In some embodiments, the branched hydrocarbon group contains 14 carbon atoms. In some embodiments, the branched hydrocarbon group contains 15 carbon atoms. In some embodiments, the branched hydrocarbon group contains 16 carbon atoms. In some embodiments, the branched hydrocarbon group contains 17 carbon atoms. In some embodiments, the branched hydrocarbon group contains 18 carbon atoms. In some embodiments, the branched hydrocarbon group contains 19 carbon atoms. In some embodiments, the branched hydrocarbon group contains 20 carbon atoms. In some embodiments, the branched hydrocarbon group is saturated. In some embodiments, the branched hydrocarbon group is unsaturated. In some embodiments, R is a group of the formula -CH(R 7 )2, and each R 7are independently C5~C 12 Alkyl or C5-C 12 In some embodiments, each R is an alkenyl. 7 is C5-alkyl or C5-alkenyl. In some embodiments, each R 7 is C-alkyl or C-alkenyl. In some embodiments, each R 7 is a C7-alkyl or C7-alkenyl. 7 is C8-alkyl or C8-alkenyl. In some embodiments, each R 7 is a C9-alkyl or C9-alkenyl. In some embodiments, each R 7 is C 10 -Alkyl or C 10 -alkenyl. In some embodiments, each R 7 is C 11 -Alkyl or C 11 -alkenyl. In some embodiments, each R 7 is C 12 -Alkyl or C 12 In some embodiments, at least one R is a branched hydrocarbon group containing a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, and any of the monocyclic or bicyclic groups is optionally substituted.

[0041] In some embodiments, R is a straight-chain or branched hydrocarbon group containing one or more cyclic groups. In some embodiments, the cyclic group is an optionally substituted monocyclic cycloalkyl. In some embodiments, the cyclic group is an optionally substituted bicyclic cycloalkyl. In some cases, the cyclic group is an optionally substituted monocyclic aryl group. In some cases, the cyclic group is an optionally substituted bicyclic aryl group. In some cases, the cyclic group is an optionally substituted monocyclic or bicyclic heterocyclic group. In some cases, the cyclic group is an optionally substituted monocyclic or bicyclic heteroaryl group.

[0042] In some embodiments of Formula (I), R is [ka] where: Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted, straight-chain or branched, saturated or partially unsaturated C1-C 20 is an aliphatic group, r, p, and q each independently represent an integer of 0 to 20.

[0043] In some embodiments, R is [ka] where: Cy A and Cy Bare each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted, straight-chain or branched, saturated or partially unsaturated C1-C 10 is an aliphatic group, r, p, and q each independently represent an integer of 0 to 10.

[0044] In some embodiments, R is [ka] where: Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted linear or branched, saturated or partially unsaturated C1-C6 aliphatic group; r, p, and q each independently represent an integer of 0 to 6. In some embodiments, R is [ka] [ka] and [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] where each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. In some embodiments, R is [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] and each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain. [ka] where each # represents the point of attachment to X or the point of attachment of R to the straight or branched hydrocarbon chain.

[0045] In some embodiments, the compound of formula (I) has formula (IIA): [ka] wherein X, R, Y, L and Z are as defined herein above.

[0046] In certain embodiments of Formula (IIA), Y is —O—, —OC(O)—, and —OC(O)NR 10 - selected from R 10 is H and C 1~6 In certain embodiments of Formula (IIA), Y is -O-. In certain embodiments of Formula (IIA), Y is -OC(O)-. In certain embodiments of Formula (IIA), Y is -OC(O)NR 10 - and R 10 is H.

[0047] In certain embodiments of Formula (IIA), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In certain cases, L is (C2-C6) alkylene. In certain cases, L is (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0048] In certain embodiments of Formula (IIA), Z is a tertiary amine. In certain instances, Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 In certain instances, R 11and R 12 are C 1~3 In certain instances, R 11 and R 12 are each methyl. In certain cases, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0049] In certain embodiments of Formula (IIA), each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In certain embodiments of Formula (IIA), at least one X is -OC(O)-. In some cases, each X is -OC(O)-. In certain embodiments of Formula (IIA), at least one X is -C(O)O-. In certain cases, each X is -C(O)O-. In certain embodiments of Formula (IIA), at least one X is -OC(O)O-. In certain cases, each X is -OC(O)O-.

[0050] In certain embodiments of Formula (IIA), each -XR is of the formula -OC(O)R. In certain embodiments, each -XR is of the formula -C(O)OR. In certain embodiments, each -XR is of the formula -OC(O)OR.

[0051] In certain embodiments of Formula (IIA), each R is a C-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 In certain instances, each R is selected from C to C alkynyl. 12 Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is a C-C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0052] In certain embodiments of Formula (IIA), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of Formula (IIA), R is -CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 It is alkenyl.

[0053] In certain embodiments of Formula (IIA), at least one R is a linear or branched hydrocarbon group containing one or more cyclic groups. In certain embodiments of Formula (IIA), R is -(CH) t J(CH2) u wherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0054] In certain embodiments of Formula (IIA), the compound has Formula (IIIA): [ka] It is of During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is -O-, -OC(O)- and -OC(O)NR 10 - selected from Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

[0055] In some embodiments of Formula (IIA), the compound is of Formula (IIIA): During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is -O-, -OC(O)- and -OC(O)NR 10 - selected from Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl and —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 It is alkenyl.

[0056] In certain embodiments of Formula (IIIA), Y is —O—, —OC(O)—, and —OC(O)NR 10 - selected from R 10 is H and C 1~6 In certain embodiments of Formula (IIIA), Y is -O-. In certain embodiments of Formula (IIIA), Y is -OC(O)-. In certain embodiments of Formula (IIA), Y is -OC(O)NR 10- and R 10 is H.

[0057] In certain embodiments of Formula (IIIA), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.

[0058] In certain embodiments of Formula (IIIA), R 11 and R 12 In certain cases, R 11 and R 12 At least one of R is methyl. 11 and R 12 are the same. In a particular case, R 11 and R 12 and R are both methyl. 11 and R 12 At least one of R is ethyl. 11 and R 12 and R are both ethyl. 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0059] In certain embodiments of Formula (IIIA), each R is a C5-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 In certain instances, each R is selected from C to C alkynyl. 12 Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is selected from C to C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0060] In certain embodiments of Formula (IIIA), at least one R is —CH(R 7 )2, and each R 7 are independently C5~C 12Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 It is alkenyl.

[0061] In certain embodiments of Formula (IIIA), R is —(CH) t J(CH2) u wherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 5. In some instances, t is 2 and u is 3.

[0062] In certain embodiments of the compound of Formula (I), the compound has the formula (IIB): [ka] wherein X, R, Y, L and Z are as defined herein.

[0063] In certain embodiments of Formula (IIB), Y is —O—, —OC(O)—, —OC(O)NR 10 -, -NR 10 C(O)-, -NR 10 C(O)O- and -NR 10 C(O)S-, and R 10 is H and C 1~6In certain embodiments of Formula (IIB), Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-. In certain cases, Y is -NHC(O)-. In certain cases, Y is -NHC(O)O-. In certain cases, Y is -NHC(O)S-.

[0064] In certain embodiments of Formula (IIB), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In certain cases, L is (C2-C6) alkylene. In certain cases, L is (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0065] In certain embodiments of Formula (IIB), Z is a tertiary amine. In certain instances, Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 In certain instances, R 11 and R 12 are C 1~3 In certain instances, R 11 and R 12 are each methyl. In certain cases, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0066] In certain embodiments of Formula (IIB), each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In certain embodiments of Formula (IIB), at least one X is -OC(O)-. In some cases, each X is -OC(O)-. In certain embodiments of Formula (IIB), at least one X is -C(O)O-. In certain cases, each X is -C(O)O-. In certain embodiments of Formula (IIB), at least one X is -OC(O)O-. In certain cases, each X is -OC(O)O-.

[0067] In certain embodiments of Formula (IIB), each -XR is of the formula -OC(O)R. In certain embodiments of Formula (IIB), each -XR is of the formula -C(O)OR. In certain embodiments of Formula (IIB), each -XR is of the formula -OC(O)OR.

[0068] In certain embodiments of Formula (IIB), each R is a C-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 In certain instances, each R is selected from C to C alkynyl. 12 Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is a C-C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0069] In certain embodiments of Formula (IIB), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of Formula (IIB), R is -CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 It is alkenyl.

[0070] In certain embodiments of Formula (IIB), at least one R is a linear or branched hydrocarbon group containing one or more cyclic groups. In certain embodiments of Formula (IIA), R is -(CH) t J(CH2) u wherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0071] In certain embodiments of Formula (IIB), the compound has Formula (IIIB): [ka] It is of During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is selected from -NHC(O)-, -NHC(O)O- and -NHC(O)S-; Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl and —CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

[0072] In some embodiments of Formula (IIB), the compound is of Formula (IIIB): During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-; Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl and —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 It is alkenyl.

[0073] In certain embodiments of Formula (IIIB), Y is -NHC(O)-. In certain cases, Y is -NHC(O)O-. In certain cases, Y is -NHC(O)S-.

[0074] In certain embodiments of Formula (IIIB), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.

[0075] In certain embodiments of Formula (IIIB), R 11 and R 12 In certain cases, R 11 and R 12 At least one of R is methyl. 11 and R 12 are the same. In a particular case, R 11 and R 12 and R are both methyl. 11 and R 12At least one of R is ethyl. 11 and R 12 and R are both ethyl. 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0076] In certain embodiments of Formula (IIIB), each R is a C-C 20 Alkyl, C5-C 20 Alkenyl and C5-C20 In certain instances, each R is selected from C to C alkynyl. 12 Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is selected from C to C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0077] In certain embodiments of Formula (IIIB), at least one R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 It is alkenyl.

[0078] In certain embodiments of Formula (IIIB), R is —(CH) t J(CH2) u wherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 3. In some instances, t is 2 and u is 3.

[0079] In some embodiments, the compound of formula (I) has the formula (IIC): [ka] wherein X, R, Y, L and Z are as defined herein above.

[0080] In certain embodiments of Formula (IIC), Y is —C(R 10 )2- and -O-; R 10 is H and C 1~6 In certain embodiments of Formula (IIC), Y is -O-. In certain embodiments of Formula (IIC), Y is -C(R 10 )2- and R 10 is H.

[0081] In certain embodiments of Formula (IIC), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In certain cases, L is (C2-C6) alkylene. In certain cases, L is (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0082] In certain embodiments of formula (IIC), Z is a tertiary amine. In certain instances, Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 In certain instances, R 11 and R 12 are C 1~3 In certain instances, R 11 and R 12are each methyl. In certain cases, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0083] In certain embodiments of Formula (IIC), each X is independently —(CH) s OC(O)-, -(CH2) sC(O)O-, -(CH2) s OC(O)O-, where s is 0 to 6. In some cases, at least one X is selected from -(CH) s OC(O)-, where s is 0, 1, or 2. In some cases, each X is -(CH) s OC(O)-, and s is 0, 1, or 2. In certain embodiments of formula (IIC), at least one X is -(CH) s C(O)O—, where s is 0, 1, or 2. In certain instances, each X is —(CH) s In certain embodiments of Formula (IIC), s is 1 and each X is -CHC(O)O-. In certain embodiments of Formula (IIC), s is 2 and X is -(CH)C(O)O-. In certain embodiments of Formula (IIC), at least one X is -(CH) s OC(O)O-, where s is 0, 1, or 2. In certain instances, each X is -(CH) s and s is 0, 1, or 2. In certain embodiments of formula (IIC), s is 0 and X is -OC(O)O-.

[0084] In certain embodiments of Formula (IIC), each -XR has the formula -(CH) s OC(O)R, where s is 0 to 6. In certain embodiments, each -XR has the formula -(CH) s C(O)OR, where s is 0 to 6. In certain embodiments, each -XR has the formula -(CH) s OC(O)OR, where s is 0 to 6.

[0085] In certain embodiments of Formula (IIC), each R is a C-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 In certain instances, each R is selected from C to C alkynyl. 12Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is a C-C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0086] In certain embodiments of Formula (IIC), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of Formula (IIC), R is -CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C6-C9 alkenyl.

[0087] In certain embodiments of Formula (IIC), at least one R is a linear or branched hydrocarbon group containing one or more cyclic groups. In certain embodiments of Formula (IIA), R is -(CH) t J(CH2) uwherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0088] In certain embodiments of Formula (IIC), the compound has Formula (IIIC): [ka] It is of During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is -O- and -C(R 10 )2- is selected from each s is independently 0, 1, or 2; W is —O— or —CH—; Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and each of t and u is 1-10.

[0089] In some embodiments of Formula (IIC), the compound is of Formula (IIIC), wherein: R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is -O- and -C(R 10 )2- is selected from s is 0 to 2, W is O or CH2; Each R is -CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 It is alkenyl.

[0090] In certain embodiments of Formula (IIIC), Y is —C(R 10 )2- and -O-; R 10 is H and C 1~6 In certain embodiments of Formula (IIIC), Y is -O-. In certain embodiments of Formula (IIIC), Y is -C(R 10 )2- and R 10 is H.

[0091] In certain embodiments of Formula (IIIC), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.

[0092] In certain embodiments of Formula (IIIC), R 11 and R 12 In certain cases, R 11 and R 12 At least one of R is methyl. 11 and R 12 are the same. In a particular case, R 11 and R 12 and R are both methyl. 11 and R 12 At least one of R is ethyl. 11 and R 12 and R are both ethyl. 11 and R 12and R are both propyl. 11 and R 12 and R are both n-propyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are both isopropyl. 11 and R 12 and R are independently optionally substituted butyl. 11 and R 12 and R are independently optionally substituted n-butyl. 11 and R 12 and R are independently optionally substituted sec-butyl. 11 and R 12 and R are independently optionally substituted butyl. In certain instances, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, —CHCHOH, —CH(CH)CHOH, —CHCH(OH)CH, and —CHCHCHOH. In certain instances, each R 11 and R 12 are independently optionally substituted C 1~4 Alkyl, C 1~3 Alkyl, C 1~4 Heteroalkyl and C 1~3 heteroalkyl.

[0093] In certain embodiments of Formula (IIIC), W is -CH2-. In certain embodiments of Formula (IIIC), W is -O-.

[0094] In certain embodiments of Formula (IIIC), s is 0. In certain embodiments, s is 1. In certain embodiments, s is 2.

[0095] In certain embodiments of Formula (IIIC), W is -CH- and s is 0. In certain cases, W is -CH- and s is 1. In certain cases, W is -O- and s is 0.

[0096] In certain embodiments of Formula (IIIC), each R is C5-C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 In certain instances, each R is selected from C to C alkynyl. 12 Alkyl, C5-C 12 Alkenyl and C5-C 12 In certain instances, each R is selected from C to C alkynyl. 12 In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. In certain cases, each R is a C alkyl. 10 In certain instances, each R is C 11 In certain instances, each R is C 12 It is alkyl.

[0097] In certain embodiments of Formula (IIIC), at least one R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 In certain instances, each R is -CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is —CH(R 7 )2, and each R 7 is C5~C 12 In certain instances, each R is -CH(R7 )2, and each R 7 is C6-C9 alkyl. In certain instances, each R is -CH(R 7 )2, and each R 7 is C6-C9 alkenyl.

[0098] In certain embodiments of Formula (IIIC), R is —(CH) t J(CH2) u wherein J is a cyclic group, and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 3. In some instances, t is 2 and u is 3.

[0099] In some embodiments of Formula (I), (IIA), (IIIA), (IIB), (IIIB), (IIC), and (IIIC), each R is independently [ka] where: Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted, straight-chain or branched, saturated or partially unsaturated C1-C 20 is an aliphatic group, r, p, and q each independently represent an integer of 0 to 20.

[0100] In some embodiments, R is [ka] where: Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted, straight-chain or branched, saturated or partially unsaturated C1-C 10 is an aliphatic group, r, p, and q each independently represent an integer of 0 to 10.

[0101] In some embodiments, R is [ka] where: Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted linear or branched, saturated or partially unsaturated C1-C6 aliphatic group; r, p, and q each independently represent an integer of 0 to 6.

[0102] In certain embodiments, the lipid is selected from the compounds in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0103] 4.3 Further ionizable lipids In some embodiments, the lipid nanoparticle composition can include one or more additional ionizable lipid components in addition to the ionizable lipid of Formula (I) (e.g., as described above). Any convenient lipid that has a net positive charge at or near physiological pH can be used as the additional ionizable lipid in the compositions described herein.

[0104] Non-limiting examples of cationic lipid are described in detail herein.The cationic lipid and related analogues that are useful in lipid nanoparticles of the present disclosure include but are not limited to the lipids described in United States Patent Application Publication No. 20060083780 and United States Patent Application Publication No. 20060240554; United States Patent No. 5,208,036; United States Patent No. 5,264,618; United States Patent No. 5,279,833; United States Patent No. 5,283,185; United States Patent No. 5,753,613; and United States Patent No. 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes.Further cationic lipids of interest include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), and heptatriaconta-6,9,28, 31-Tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N, These include, but are not limited to, N-trimethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.Cl"); 3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA"), dioctadecylamidoglycylcarboxyspermine ("DOGS"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). Additionally, numerous commercially available preparations of cationic lipids can be used, such as, for example, Lipofectin (including DOTMA and DOPE available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE available from GIBCO / BRL). In certain embodiments, the cationic lipid is an amino lipid containing one or two fatty acyl or fatty alkyl chains.

[0105] Further exemplary ionizable lipids that can be adapted for use in the lipid nanoparticles of the present disclosure are those described in International PCT Patent Publication Nos. WO 2015 / 095340, WO 2015 / 199952, WO 2018 / 011633, WO 2017 / 049245, WO 2015 / 061467, WO 2012 / 040184, WO 2012 / 000104, WO 2015 / 074085, WO 2016 / 081029, WO 2017 / 004143, WO 2017 / 075531, WO 2017 / 117528, WO 2011 / 022460, WO 2013 / 148541, WO 2013 / 116126, WO 2011 / 153120, WO 2012 / 044638, WO 2012 / 054365, WO 2011 / 090965, WO 2013 / 016058, WO 2012 / 162210, WO 2008 / 042973, WO 2010 / 129709, WO 2010 / 144740, WO 2 012 / 099755, WO 2013 / 049328, WO 2013 / 086322, WO 2013 / 086373, WO 2011 / 071860, WO 2009 / 132131, WO 2010 / 048536, WO 2010 / 088537, WO 2010 / 054401, WO 2010 / 054406, WO 2010 / 054405, WO 2010 / 054384, WO 2012 / 016184, WO 2009 / 086558, WO International Publication No. 2010 / 042877, International Publication No. 2011 / 000106, International Publication No. 2011 / 000107, International Publication No. 2005 / 120152, International Publication No. 2011 / 141705, International Publication No. 2013 / 126803, International Publication No. 2006 / 007712, International Publication No. 2011 / 038160, International Publication No. 2005 / 121348, International Publication No. 2011 / 066651, International Publication No. 2009 / 127060, International Publication No. 2011 / 141704, International Publication No. 2006 / 069782, International Publication No. 2012 / 031043,WO 2013 / 006825, WO 2013 / 033563, WO 2013 / 089151, WO 2017 / 099823, WO 2015 / 095346, and WO 2013 / 086354, as well as U.S. Patent Application Publication Nos. 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178 541, U.S. Patent Application Publication No. 2013 / 0303587, U.S. Patent Application Publication No. 2015 / 0141678, U.S. Patent Application Publication No. 2015 / 0239926, U.S. Patent Application Publication No. 2016 / 0376224, U.S. Patent Application Publication No. 2017 / 0119904, U.S. Patent Application Publication No. 2012 / 0149894, U.S. Patent Application Publication No. 2015 / 0057373, U.S. Patent Application Publication No. 2013 / 0090372, U.S. Patent Application Publication No. 2013 / 0274523, U.S. Patent Application Publication No. 2013 / 0274504, U.S. Patent Application Publication No. 201 3 / 0274504, U.S. Patent Application Publication No. 2009 / 0023673, U.S. Patent Application Publication No. 2012 / 0128760, U.S. Patent Application Publication No. 2010 / 0324120, U.S. Patent Application Publication No. 2014 / 0200257, U.S. Patent Application Publication No. 2015 / 0203446, U.S. Patent Application Publication No. 2018 / 0005363, U.S. Patent Application Publication No. 2014 / 0308304, U.S. Patent Application Publication No. 2013 / 0338210, U.S. Patent Application Publication No. 2012 / 0101148, U.S. Patent Application Publication No. 2012 / 0027796, U.S. Patent Application Publication No. 2012 / 0058144, U.S. Patent Application Publication No. 2013 / 0323269, U.S. Patent Application Publication No. 2011 / 0117125, U.S. Patent Application Publication No. 2011 / 0256175, U.S. Patent Application Publication No. 2012 / 0202871, U.S. Patent Application Publication No. 2011 / 0076335, U.S. Patent Application Publication No. 2006 / 0083780, U.S. Patent Application Publication No. 2013 / 0123338, U.S. Patent Application Publication No. 2015 / 0064242, U.S. Patent Application Publication No. 2006 / 0051405, U.S. Patent Application Publication No. 2013 / 0065939,Nos. 2006 / 0008910, 2003 / 0022649, 2010 / 0130588, 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920.

[0106] 4.4 Helper lipids In addition to the ionizable lipid components described herein, the LNPs of the present disclosure can also include one or more helper lipids. In some embodiments, the helper lipid is a neutral lipid. In some embodiments, the neutral lipid is zwitterionic, e.g., has an overall net charge of zero.

[0107] Neural lipids include, for example, phospholipids, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the compositions described herein is generally guided by considerations such as LNP size and the stability of the LNP in the bloodstream. Generally, the LNPs of the present disclosure include a helper lipid component comprising a neutral lipid that is a phospholipid. Non-limiting examples of phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. In some embodiments, the neutral lipid component is a lipid having two acyl groups (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or can be isolated or synthesized by well-known techniques. In one embodiment, the neutral lipid is C 10 ~C 30 In one embodiment, the saturated fatty acids include fatty acids having carbon chain lengths in the range of C 10 ~C 30 Neutral lipids having mono- or diunsaturated fatty acids with carbon chain lengths in the range of 0.1 to 1.0 are used. Furthermore, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. The neutral lipids can also be composed of sphingomyelin or dihydrosphingomyelin.

[0108] In some embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG).

[0109] In some embodiments, the phospholipid has a hydrocarbon chain or "tail" having 12-24 carbons, e.g., 16-20 carbons, 18-22 carbons, or 12-18 carbons. In some embodiments, the phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. In some embodiments, the phospholipid tail does not contain any double bonds, i.e., the bonds are saturated. In some embodiments, the phospholipid tail is unsaturated, i.e., contains one or more double bonds, e.g., 1, 2, 3, 4, or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, i.e., contains one or more triple bonds, e.g., 1, 2, 3, 4, or 5 triple bonds. In some embodiments, the phospholipid tail contains one or more ring structures. In some embodiments, one or more ring structures are selected from the group consisting of: a 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; a 5- to 6-membered aryl; a 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and each ring structure is selected from the group consisting of 0-7 R A and each R is selected from a 7- to 10-membered bicyclic aryl independently substituted with a aryl group. A are independently halogen or C 1~12 The ring structure is selected from an optionally substituted group selected from aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl. In some such examples, the ring structure is cholesterol or a cholesterol derivative. In some embodiments, the phospholipid is symmetrical, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetrical, i.e., the phospholipid contains two different hydrocarbon chains.

[0110] In some embodiments, the helper lipids are each independently an optionally substituted, branched or straight chain, partially unsaturated or saturated C9-C 24 It is or comprises an aliphatic symmetric or asymmetric aliphatic phospholipid moiety.

[0111] In some embodiments, the helper lipid comprises one or more optionally substituted and / or optionally bridged ring structures in the hydrophobic tail. Exemplary helper lipids of this class include: [ka] and [ka] Includes:

[0112] In some embodiments, the helper lipid comprises phosphatidylethanolamine (PE). Phosphatidylethanolamine (PE) is a class of phospholipids that incorporate ethanolamine as a head group. In some embodiments, the phosphatidylethanolamine is selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2-dioleyl-sn-glycero-3-phosphoethanolamine) (Δ9-cisPE, or DOPE), palmitoyl-oleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE), dimyristoylphosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) (DPPE), and the like. amine) (DMPE), (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE), monomethyl-phosphatidyl-ethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), dielaidoyl-phosphatidylethanolamine (DEPE), lysophosphatidylethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE). In certain embodiments, the phosphatidylethanolamine is dioleoylphosphatidylethanolamine (also called 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or (Δ9-cis)PE, or DOPE), which has an 18 carbon and one saturated bond tail ("18-1"), as shown below: [ka]

[0113] In some embodiments, the helper lipid comprises phosphatidylcholine (PC). Phosphatidylcholine (PC) is a class of phospholipids that incorporate choline as a head group. In some embodiments, the phosphatidylcholine is selected from the group consisting of phosphatidylcholine, distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine) (DSPC), dioleoylphosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine) (Δ9-cisPC, or DOPC), dipalmitoylphosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (DPPC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("20-1PC" or "20:1PC"), egg yolk phosphatidylcholine, and the like. The phosphatidylcholine is selected from the group consisting of phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dierucoylphosphatidylcholine (DEPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), and 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC). In certain embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also called 1,2-distearoyl-sn-glycero-3-phosphocholine) with an 18-carbon, unsaturated tail ("18-0"), as shown below. [ka]

[0114] In certain embodiments, the phosphatidylcholine is dioleoylphosphatidycholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, also known as (Δ9-cis)PC or DOPC), which has 18 carbons and one saturated bond tail ("18-1"), as shown below. [ka]

[0115] In certain embodiments, the phosphatidylcholine is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (delta9-cisPC), which has a tail of 16 carbons and one saturated bond ("16-1"), as shown below. [ka]

[0116] In certain embodiments, the phosphatidylcholine is an asymmetric lipid having one 16-carbon tail and a second 18-carbon tail. In some such cases, the 18-carbon phosphatidylcholine tail has one saturated bond, such as 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (also referred to as "16-0 / 18-1PC," "16:0 / 18:1PC," or POPC), as shown below. [ka]

[0117] In certain embodiments, the phosphatidylcholine is 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below. [ka]

[0118] In certain embodiments, the phosphatidylcholine is 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below. [ka]

[0119] In certain embodiments, the phosphatidylcholine is 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below. [ka]

[0120] In certain embodiments, the phosphatidylcholine is 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below. [ka]

[0121] In some embodiments, the helper lipid comprises a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyl oleyol phosphatidylglycerol (POPG).

[0122] In some embodiments, the helper lipid comprises a phosphatidylserine, such as phosphatidylserine or dioleoylphosphatidylserine (DOPS).

[0123] In some embodiments, the helper lipid comprises a lecithin, such as lecithin or lysolecithin.

[0124] In some embodiments, the helper lipid comprises a sphingomyelin (SM), such as egg sphingomyelin (ESM).

[0125] In some embodiments, the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebroside, or dicetyl phosphate.

[0126] In some embodiments, LNPs can further comprise components such as sterols to provide membrane integrity. One exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Exemplary cholesterol derivatives are described in International Application Publication No. WO 2009 / 127060 and U.S. Patent Application Publication No. 2010 / 0130588. Components that provide membrane integrity, such as sterols, can account for 0-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, such components comprise 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0127] Thus, the neutral lipid component of the LNP can further comprise cholesterol or a derivative or analog thereof. A variety of cholesterol analogs and derivatives can be adapted for use in the LNPs of the present disclosure. In some embodiments, the helper lipid component comprises cholesterol.

[0128] In some embodiments, the LNPs comprise a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or a derivative or analog thereof.

[0129] In some embodiments, the LNP comprises a neutral lipid component comprising a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.

[0130] In some embodiments, the LNPs comprise a neutral lipid component comprising a DOPE phospholipid and cholesterol. In some embodiments, the LNPs comprise a neutral lipid component comprising a DSPC phospholipid and cholesterol. In some embodiments, the LNPs comprise a neutral lipid component comprising a DOPC phospholipid and cholesterol.

[0131] 4.5 Other ingredients The LNPs of the present disclosure can also include one or more additional lipid components. Such lipids can be selected to provide a desired profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability, and biodistribution.

[0132] In some embodiments, the LNP can further comprise a non-cationic lipid. Non-ionic lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be a neutral uncharged lipid, a zwitterionic lipid, or an anionic lipid. Non-cationic lipids are typically used to enhance membrane fusion. Exemplary non-cationic lipids contemplated for use in these methods and compositions are described in International Application PCT / US2018 / 050042, published as International Publication No. WO 2019051289A1. Exemplary non-cationic lipids are described in International Application Publication No. WO 2017 / 099823 and US Patent Application Publication No. 2018 / 0028664.

[0133] Non-limiting examples of non-cationic lipids include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like.

[0134] In some embodiments, the LNPs comprise one or more lipids capable of reducing aggregation. Generally, lipids capable of reducing aggregation can be attached to the surface of the LNPs and comprise at least one hydrocarbon tail or chain linked to a hydrophilic group that provides reduced LNP aggregation. Thus, lipids capable of reducing aggregation may be referred to as conjugated lipids or coated lipids.

[0135] Lipids that can reduce particle aggregation can include conjugated lipid molecules, such as polyethylene glycol (PEG). Generally, these are used to inhibit the aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic polymer-lipid conjugates (CPL), or mixtures thereof. In one embodiment, LNPs contain either PEG-lipid conjugates or ATTA-lipid conjugates. In certain embodiments, PEG-lipid conjugates or ATTA-lipid conjugates are used together with CPL.

[0136] In some embodiments, the lipid that can reduce aggregation is a PEG-lipid, which refers to a lipid having one or more hydrocarbon tails linked to one or more polyethylene glycol (PEG) moieties via an optional linker.

[0137] It is understood that the PEG moiety may include terminal modifications, for example, to provide conjugation to a lipid tail via an optional linker. The PEG moiety may be terminated as a hydroxyl group or an alkyl ether (e.g., a methoxy terminal group). In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol)-conjugated lipid. Interesting PEG-lipids include, but are not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or a mixture thereof.

[0138] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Further exemplary PEG-lipid conjugates are For example, U.S. Patent Nos. 5,885,613, 6,287,591, U.S. Patent Application Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, and 2017 / 0119904. In some embodiments, PEG-lipids are prepared using methods described in U.S. Patent Application Publication Nos. The PEG-lipid is a compound disclosed in U.S. Patent Application Publication No. 2018 / 0028664. In some embodiments, the PEG-lipid is disclosed in U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.PEG-lipids include PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-omegal-methyl-poly(ethylene glycol), PEG-D MB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] may be one or more. In some examples, the PEG-lipid may be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.

[0139] As mentioned above, instead of PEG-lipid, lipids conjugated with molecules other than PEG can also be used.For example, instead of PEG-lipid or in addition to PEG-lipid, polyoxazoline (POZ)-lipid conjugate, polyamide-lipid conjugate (such as ATTA-lipid conjugate) and cationic-polymer lipid (CPL) conjugate can be used.Exemplary conjugated lipids, that is, PEG-lipid, (POZ)-lipid conjugate, ATTA-lipid conjugate and cationic polymer-lipid, are described in International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, 2006 / 010392, 2007 / 010392, 2008 / 010392, 2009 / 010392, 2010 / 010392, 2010 / 010392, 2011 / 010392, 2012 / 010392, 2013 / 010392, 2014 / 010392, 2015 / 010392, 2016 / 010392, 2017 / 010392, 2018 / 010392, 2019 / 010392, 2014 / 010392, 2015 / 010392, 2016 ...6 / 010392 Nos. 008 / 147438, 2009 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104 and and U.S. Patent Application Publication Nos. 2010 / 006282, U.S. Patent Application Publication Nos. 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 03 76224, 2016 / 0317458, 2013 / 0303587, 2013 / 0303587, and 20110123453, as well as U.S. Patents U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559.

[0140] 4.6 Targeting Ligands In some embodiments, it may be desirable to limit the transfection of nucleic acids to certain cells or tissues. For example, the liver may be an interesting target organ, in part, due to its central role in protein metabolism and production, and therefore for diseases caused by defects in liver-specific gene products (e.g., urea cycle disorders), in which specific targeting of cells (e.g., hepatocytes) may be beneficial.

[0141] In some embodiments, the LNP further comprises a component comprising a targeting ligand. The targeting ligand can be selected as desired based on the target cell or tissue to which the LNP of the present disclosure is desired to be directed. In some embodiments, the targeting ligand is a ligand for a cell surface receptor. In some embodiments, the cell surface receptor is the asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells.

[0142] In some embodiments, the targeting ligand is a ligand for ASGPR, such as an N-acetylgalactosamine (GalNAc)-containing ligand. A variety of GalNAc-containing ligands and ligands, including multivalent GalNAc ligands, are available for use in the LNPs of the present disclosure, including, for example, those disclosed in International Publication No. 2021178725, the entire disclosure of which is incorporated herein by reference.

[0143] In some embodiments, PEG-lipid is linked to a targeting ligand. In some embodiments, an interesting targeting ligand (for example, as described herein) is linked to the end of PEG moiety. For example, the trisGalNac ligand conjugated to PEG-lipid can provide LNP binding to ASGPR receptor of target cell, leading to endocytosis of LNP.

[0144] 4.7 Lipid nanoparticles containing lipids of formula (I) In some embodiments, the LNP comprises an ionizable lipid of formula (I) (e.g., as described herein); a nucleic acid cargo (e.g., as described herein); an additional ionizable lipid (e.g., as described herein); a phospholipid (e.g., as described herein), cholesterol (e.g., as described herein); and a lipid capable of reducing aggregation (e.g., as described herein).

[0145] In some embodiments of LNPs, the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of DNA. In some embodiments of LNPs, the nucleic acid cargo comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of RNA. In certain embodiments of LNPs, the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof, and further comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, etc., or a chemically modified derivative thereof.

[0146] In some embodiments of LNPs, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), and derivatives thereof. In certain cases, the phospholipid is phosphatidylethanolamine (PE). In certain cases, the phospholipid is phosphatidylcholine (PC). In certain embodiments of LNPs, the phospholipids each independently comprise hydrocarbon chains having 12 to 24 carbons. In some cases, the hydrocarbon chains each independently comprise 16 to 20 carbons. In certain cases, the hydrocarbon chains are saturated. In certain cases, the hydrocarbon chains are unsaturated. In certain cases, the hydrocarbon chains each independently comprise 1 to 4 double bonds. In certain cases, the phospholipid comprises two different hydrocarbon chains. In certain embodiments of LNPs, the phospholipid comprises dioleoylphosphatidylethanolamine (DOPE, 18:1PE). In certain cases, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain cases, the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In certain cases, the phospholipid comprises 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (delta9-cisPC). In certain cases, the phospholipid comprises 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). In certain cases, the phospholipid comprises a mixture of dioleoylphosphatidylethanolamine (DOPE, 18-1) and dioleoylphosphatidylcholine (DOPC, 18-1).

[0147] In certain embodiments of LNPs, the lipid capable of reducing aggregation is a PEG-lipid. In certain instances, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0148] In certain embodiments, the LNP further comprises a targeting ligand (e.g., as described herein). In certain cases, the targeting ligand comprises GalNac. In certain embodiments, the targeting ligand is linked to a ligand capable of reducing aggregation. In certain cases, the lipid capable of reducing aggregation linked to the targeting ligand is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0149] In some embodiments, the LNPs comprise an ionizable lipid of formula (I) (e.g., as described herein); a phospholipid that is DOPE, cholesterol, and a lipid that can reduce aggregation that is PEG-DMG.

[0150] In some embodiments, the LNPs comprise an ionizable lipid, which is a cationic lipid containing a tertiary amino ionizable group; a phospholipid, which is a phosphatidylethanolamine (e.g., DOPE), cholesterol, and an aggregation-reducing lipid, which is PEG-DMG, and / or PEG-DSG-GalNAc or PEG-DSPE-GalNAc.

[0151] In some embodiments, the LNP comprises an ionizable lipid, which is a cationic lipid containing a tertiary amino ionizable group, a phospholipid, which is a phosphatidylcholine (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and a coating lipid (polyethylene glycol-dimyristylglycerol, PEG-DMG), e.g., as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0152] Typically, lipid particles are prepared containing a total lipid-to-DNA (mass or weight) ratio of about 5:1 to 50:1. This is also referred to as the ratio of positively chargeable polymeric amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups, or N / P ratio. In some embodiments, the N / P ratio (mass / mass ratio; w / w ratio) can be within the range of about 1:1 to about 50:1, about 7:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and DNA can be adjusted to provide a desired N / P ratio, e.g., 3:1 ("3"), 4:1 ("4"), 5:1 ("5"), 6:1 ("6"), 7:1 ("7"), 8:1 ("8"), 9:1 ("9"), 10:1 ("10"), 11:1 ("11"), 12:1 ("12"), 13:1 ("13"), 14:1 ("14"), or higher. Generally, the total lipid content of the lipid particle formulation can range from about 5 mg / mL to about 30 mg / mL.

[0153] In some embodiments, the N / P ratio is 5 to 30. In certain cases, the N / P ratio is 7. In certain cases, the N / P ratio is 14. In certain cases, the N / P ratio is 28.

[0154] In some embodiments, the lipid nanoparticles have an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 10 to about 300 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 25 to about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution with an average size (e.g., diameter) of about 70 nm to about 200 nm, more typically with an average size of about 100 nm or less.

[0155] In some embodiments, the LNPs have an average diameter of 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm, 25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.

[0156] In some embodiments, the LNPs have an average diameter of 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm.

[0157] In some embodiments, the LNPs have an average diameter of 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.

[0158] In some embodiments, the structural characteristics of target tissue can be utilized to direct the distribution of LNPs to such target tissue.For example, to target hepatocytes, LNPs can be sized so that their dimensions are smaller than the fenestrations of the endothelial layer lining the hepatic sinusoids in the liver, and thus the LNPs can easily penetrate these endothelial fenestrations to reach the target hepatocytes.In some embodiments, LNPs can be sized so that the particle dimensions are large enough to limit or clearly avoid distribution into certain cells or tissues.For example, LNPs can be sized so that their dimensions are larger than the fenestrations of the endothelial layer lining the hepatic sinusoids, thereby limiting the distribution of LNPs to hepatocytes.In such embodiments, large LNPs do not easily penetrate the endothelial fenestrations, and instead are removed by the macrophage Kupffer cells lining the hepatic sinusoids. In some embodiments, the size of the LNPs is in the range of about 25-250 nm or 25 nm-100 nm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.

[0159] Without limitation, the ionizable lipids can account for 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the molar content of the ionizable lipids can be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the ionizable lipids account for about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles. In some embodiments, the ionizable lipids account for about 50 mol% to about 85 mol%, about 50 mol% to about 80 mol%, about 50 mol% to about 75 mol%, about 50 mol% to about 70 mol%, about 50 mol% to about 65 mol%, about 50 mol% to about 60 mol%, about 55 mol% to about 65 mol%, or about 55 mol% to about 70 mol% (or any fraction or range therein) of the total lipids present in the particles. In certain embodiments, the cationic lipid comprises 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol% or 60 mol% (or any fraction thereof) of the total lipid present in the particle.

[0160] The neutral lipid component can account for 10-60% (mol) of the total lipids present in the lipid nanoparticle. For example, the non-cationic lipid content is 10-50% (mol) or 20-55% (mol) of the total lipids present in the lipid nanoparticle. In some embodiments, the non-cationic lipids comprise about 10 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 45 mol%, or about 37 mol% to about 42 mol% (or any fraction or range therein) of the total lipids present in the particle. In certain embodiments, the non-cationic lipid comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46%, 47%, 48%, 49%, or 50% (or any fraction or range therein) of the total lipid present in the particle.

[0161] In embodiments in which the lipid particle contains a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may comprise up to about 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle. In certain embodiments, the mixture of phospholipids and cholesterol or a cholesterol derivative comprises up to 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46%, 47%, 48%, 49%, or 50% (or any fraction or range therein) of the total lipid present in the particle.

[0162] In some embodiments, the LNP comprises a phospholipid component in the mixture in an amount of about 2 mol% to about 20 mol%, about 2 mol% to about 15 mol%, about 2 mol% to about 12 mol%, about 4 mol% to about 15 mol%, or about 4 mol% to about 10 mol% (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture comprises about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, about 5 mol% to about 8 mol%, about 6 mol% to about 9 mol%, about 6 mol% to about 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction thereof or range therein) of the total lipid present in the particle.

[0163] In some embodiments, the LNP comprises the cholesterol component in the mixture in an amount of about 25 mol% to about 45 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 27 mol% to about 37 mol%, about 25 mol% to about 30 mol%, or about 35 mol% to about 40 mol% (or any fraction or range therein) of the total lipid present in the particle. In some embodiments, the cholesterol component in the mixture comprises about 25 mol% to about 35 mol%, about 27 mol% to about 35 mol%, about 29 mol% to about 35 mol%, about 30 mol% to about 35 mol%, about 30 mol% to about 34 mol%, about 31 mol% to about 33 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36%, 37%, 38%, or 39% (or any fraction or range therein) of the total lipid present in the particle.

[0164] It is understood that the molar percentages of the components described herein in the LNPs are target amounts, and that the actual amount of each lipid component present in the formulation may vary, for example, by ±5 mol%.

[0165] In some embodiments, the LNP comprises a lipid (e.g., a PEG-lipid conjugate) capable of reducing aggregation in an amount of about 1.5% to about 4%, e.g., about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25% (or any fraction or range therein) of the total lipid present in the particle. According to some embodiments, the lipid capable of reducing aggregation is present at 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% (or any fraction or range therein) of the total lipid present in the particle.

[0166] In various embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2: 1 to about 8: 1. In some embodiments, the lipid nanoparticles do not include any phospholipids.

[0167] In certain embodiments, the LNP is a) 40-60 mol% of ionizable lipids of the total lipids present; b) phospholipids that are 6-20 mol% of the total lipids present; c) cholesterol at 35-45 mol% of the total lipids present, and d) Lipids capable of reducing aggregation by 1.5 to 2.5 mol% of the total lipids present Includes: In certain embodiments, the LNP is a) 40-60 mol% of ionizable lipids of the total lipids present; b) phospholipids that are 10-20 mol% of the total lipids present; c) cholesterol at 35-45 mol% of the total lipids present, and d) Lipids capable of reducing aggregation by 1.5 to 2.5 mol% of the total lipids present Includes: In certain embodiments, the LNP is a) 40-49 mol% of ionizable lipids of the total lipids present; b) phospholipids that are 10-20 mol% of the total lipids present; c) cholesterol at 35-45 mol% of the total lipids present, and d) Lipids capable of reducing aggregation by 1.5 to 2.5 mol% of the total lipids present Includes:

[0168] In some embodiments, the ratio of ionizable lipid:phospholipid:cholesterol:PEG (as a percentage of total lipid content) is A:B:C:D; aA=40%~60%, B=5%~20%, C=25%~50% and D=1.5%~3.0%, and A+B+C+D=100% bA=40%~60%, B=6%~20%, C=35%~45% and D=1.5%~2.5%, and A+B+C+D=100% bA = 40%-60%, B = 10%-20%, C = 35%-45% and D = 1.5%-2.5%, and A+B+C+D = 100%; cA = 40%-49%, B = 10%-20%, C = 35%-45% and D = 1.5%-2.5%, and A+B+C+D = 100%; dA=40%~49%, B=10%~20%, C=35%~45% and D=1.5%~2.5%, and A+B+C+D=100%; dA=39%~60%, B=10%~25%, C=20%~30% and D=0%~3%, and A+B+C+D=100%; eA=40%~60%, B=10%~25%, C=20%~30% and D=0%~3%, and A+B+C+D=100%; fA=45%~50%, B=20%~25%, C=25%~30% and D=0%~1%, and A+B+C+D=100%; gA=40%~60%, B=10%~30%, C=20%~45% and D=0%~3%, and A+B+C+D=100%; hA=40%~60%, B=10%~30%, C=25%~45% and D=0%~3%, and A+B+C+D=100%; iA=45%~55%, B=10%~20%, C=30%~40% and D=1%~2%, and A+B+C+D=100%; jA=45%~50%, B=10%~15%, C=35%~40% and D=1%~2%, and A+B+C+D=100%; kA=45%~65%, B=5%~20%, C=20%~45% and D=0%~3%, and A+B+C+D=100%; mA=45%, B=15%, C=37.5% and D=2.5%; nA=57%, B=12%, C=28.5% and D=2.5% lA=50%~60%, B=5%~15%, C=30%~45% and D=0%~3%, and A+B+C+D=100%; mA = 55% to 60%, B = 5% to 15%, C = 30% to 40% and D = 1% to 2%, and A + B + C + D = 100%; or nA=55%-60%, B=5%-10%, C=30%-35% and D=1%-2%, and A+B+C+D=100%.

[0169] 4.8 Nucleic Acid Cargo In many embodiments, a given lipid nanoparticle can contain a cargo or payload to be delivered to a cell. Of particular interest in some embodiments are cargoes containing polynucleotides. In some embodiments, the polynucleotide is DNA. DNA nucleic acid compositions of any structure can be included in the LNPs of the present disclosure. For example, the DNA can be circular, such as a plasmid, nanoplasmid, minicircle, covalently closed circular DNA, or a circular viral genome. As another example, the DNA can be linear, such as doggybone or other closed-end DNA, or a linear viral genome. As another example, the DNA can be multivalent, such as tri-DNA. The DNA can be single-stranded or double-stranded, or a hybrid of single and double strands. The DNA can be chemically modified. In some embodiments, the polynucleotide is RNA. RNA nucleic acid compositions of any structure can be included in the LNPs of the present disclosure. For example, the RNA can be linear or circular. The RNA can be mRNA, siRNA, shRNA, guide RNA (gRNA), microRNA (miRNA), or circular RNA (circRNA). The RNA can be chemically modified.

[0170] One or more additional compounds can be therapeutic agents.The therapeutic agent can be selected from any class suitable for therapeutic purposes.In other words, the therapeutic agent can be selected from any class suitable for therapeutic purposes.In other words, the therapeutic agent can be selected according to the desired treatment purpose and biological effect. For example, if the DNA in the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate)). In another example, if the DNA-containing LNP is useful for treating an infectious disease, the additional compound can be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In yet another example, if the DNA-containing LNP is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as DNA encoding different proteins or different compounds such as therapeutic agents, can be used in the compositions and methods of the present invention. In some embodiments, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is an immunostimulatory agent.

[0171] 4.9 Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising a nucleic acid (e.g., DNA) encapsulated in lipid nanoparticles and a pharmaceutically acceptable carrier or excipient.In some aspects, the present disclosure provides a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients.In some embodiments, the lipid nanoparticle formulation further comprises sucrose, Tris, trehalose and / or glycine.

[0172] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0173] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substance used in pharmaceutical formulations.

[0174] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts. Pharmaceutically acceptable salts include, for example, salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as salts of acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycosaminoglycan, and the like. These include acid addition salts (formed with free amino groups of the protein) formed with organic acids such as, but not limited to, benzoic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts.Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0175] 4.10 Preparation method Any convenient method can be used to prepare the LNP of the present disclosure.The LNP composition can be prepared by high-energy mixing of ethanolic lipids with aqueous DNA at low pH, which protonates ionizable lipids and provides favorable energetics for DNA / lipid association and particle nucleation.The particles can be further stabilized through aqueous dilution and removal of organic solvent.The particles can be concentrated to a desired level.

[0176] 4.11 How to use As shown in the examples and figures herein, the LNPs and LNP pharmaceutical compositions of the present disclosure, when formulated with nucleic acids, are less toxic in vivo, e.g., at least 2-fold less toxic, e.g., 3-, 4-, or 5-fold less toxic, in some cases 10-, 20-, or 50-fold less toxic, and in certain cases 100-fold less toxic, than industry-standard LNPs (comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid) administered at the same dose. "Less toxic" means, for example, that they induce a reduced immune response, characterized by reduced amounts of one or more cytokines, upon administration to an organism.

[0177] At the same time, the LNPs and LNP pharmaceutical compositions of the present disclosure have been observed to be effective in delivering their nucleic acid cargo to target cells of interest, including being as effective or more effective in delivering their nucleic acid cargo to target cells of interest than the same industry-standard LNP administered at the same dose, e.g., having two-fold or more efficacy, e.g., three-fold, four-fold, or five-fold or more efficacy, and in some cases, 10-fold, 20-fold, or 50-fold efficacy, and in certain cases, 100-fold or more efficacy. By "more effective," we mean being able to deliver more nucleic acid cargo to cells, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, e.g., a two-fold or more increase, e.g., a three-fold, four-fold, or five-fold increase, e.g., a 10-fold, 20-fold, or 50-fold increase, and in some cases, a 100-fold or more increase.

[0178] In other words, the LNPs of the present disclosure demonstrate an improved pharmacokinetic (PK) profile that broadens the therapeutic index of the composition. The therapeutic index, or therapeutic ratio, refers to the range of doses at which a drug is effective without unacceptable adverse events, and is calculated as the ratio comparing the blood concentration at which the drug becomes toxic to the concentration at which the drug is effective. This improvement over the prior art makes the LNPs of the present disclosure more suitable for delivering nucleic acids, including DNA, to cells in vitro and in vivo, and therefore finds many uses in many applications, including the delivery of nucleic acids, including DNA, to cells for research and therapeutic applications.

[0179] In carrying out such a method, the cell is typically contacted with a composition, such as an LNP or a pharmaceutical composition thereof, in an amount effective to deliver the agent into the cytoplasm of the cell. In some embodiments, the contacting is in vitro. In other embodiments, the contacting is in vivo. In some embodiments, the method further comprises measuring the amount of protein produced.

[0180] The present disclosure further provides a method for treating or preventing a disease in a subject in need thereof, wherein an effective amount of a therapeutic composition described herein is administered to the subject. The route of administration will, of course, vary depending on the location and nature of the disease being treated, and may include, for example, intradermal, transdermal, subdermal, parenteral, intranasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The encapsulated polynucleotide compositions described herein are useful in the treatment of any indication in which it is beneficial to deliver a therapeutic cargo into target cells.

[0181] The present disclosure further provides a method of immunizing a subject against a disease, wherein an effective amount of a therapeutic composition described herein is administered to the subject. The route of administration will, of course, vary depending on the location and nature of the immunizing agent, and may include, for example, intradermal, transdermal, subdermal, parenteral, intranasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumor, perfusion, lavage, direct injection, and oral administration.

[0182] The present disclosure further provides a particle of the present disclosure, a vector of the present disclosure, a recombinant DNA of the present disclosure, or a composition thereof for use as a pharmaceutical. In some embodiments, the pharmaceutical is for expressing a protein in a cell. In some embodiments, the expression of the protein is for treating a disease in which a cell is deficient in the protein. In some embodiments, the expression of the protein is for treating a disease in which another cell is deficient in the protein. In some embodiments, the pharmaceutical is for treating cancer. In some embodiments, the pharmaceutical is for immunization against a disease.

[0183] 4.12 Usefulness The subject methods and compositions can be used in any application where delivery of cargo nucleic acids is desired, for example, as described above.Interesting applications include both research and therapeutic applications.Interesting applications include, but are not limited to, research, diagnostic, and therapeutic applications.In some examples, cargo nucleic acids that can be introduced into cells and subsequently into the nucleus via the methods of the present invention include those encoding research proteins, diagnostic proteins, and therapeutic proteins.

[0184] A research protein is a protein whose activity has utility in research protocols. Thus, a research protein is a protein used in experimental procedures. A research protein can be any protein with such utility, and in some examples, a research protein is a protein domain that is also provided in research protocols by being expressed in cells from an encoding vector. Specific examples of research proteins include, but are not limited to, transcriptional regulators of inducible expression systems, members of signal production systems, such as enzymes and their substrates, hormones, prohormones, proteases, enzyme activity regulators, perturbers and peptide aptamers, antibodies, regulators of protein-protein interactions, genome-modifying proteins such as CRE recombinases, meganucleases, zinc finger nucleases, CRISPR / Cas-9 nucleases, TAL effector nucleases, and cell reprogramming proteins such as Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin-28.

[0185] A diagnostic protein is a protein whose activity has utility in diagnostic protocols. Thus, a diagnostic protein is a protein used in diagnostic procedures. A diagnostic protein can be any protein with such utility. Examples of specific types of diagnostic proteins include, but are not limited to, members of signal-producing systems, such as enzymes and their substrates, labeled binding members, such as labeled antibodies and their binding fragments, peptide aptamers, and the like.

[0186] Proteins of interest further include therapeutic proteins, including, but not limited to, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors and enzymes.

[0187] Target cells to which nucleic acids can be delivered according to embodiments of the present disclosure can vary widely. Interesting target cells include, but are not limited to, cell lines such as HeLa, HEK, CHO, and 293, mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples, and the like. Some non-limiting examples of mammalian cells include, but are not limited to, mouse cells, rat cells, hamster cells, rodent cells, and non-human primate cells. In some embodiments, the target cells are human cells. It should also be understood that the target cells can be of any cell type. For example, the target cells can be stem cells, which can include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, umbilical cord blood stem cells, or adult stem cells (i.e., tissue-specific stem cells). In other cases, the target cells can be any differentiated cell type found in a subject. Cells of interest include both dividing and non-dividing cells. Examples of specific target cells of interest include, but are not limited to, hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiac muscle cells, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, and the like.

[0188] The targeted cells can include cells at a targeted location, such as the liver, or cells near or adjacent to hepatocytes, such as hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), hepatic sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.

[0189] In some instances, interesting applications are therapeutic applications, such as in the treatment of disease.For example, the compositions and methods of the present application can be used to deliver nucleic acid sequences to cells to complement genetic defects.As a non-limiting example, the compositions of the present application can be used in the treatment of genetic defects that affect the function of liver cells, or in the treatment of genetic defects elsewhere in the body that can be treated by utilizing liver cells as biofactories to secrete defective proteins.

[0190] 4.13 Definition In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "includes" should be interpreted as "including, but not limited to"). Where a specific number of introduced claim recitations is intended, such intent will be explicitly set forth in the claim, and it will be further understood by those skilled in the art that, absent such recitation, no such intent exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as suggesting that introducing a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such introduced claim recitation to embodiments containing only one such recitation, even when that same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same is true for the use of definite articles used to introduce claim recitation. Moreover, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the recitation "two recitations," without other modifiers, means at least two recitations, or more than two recitations).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0191] Additionally, when features or aspects of the disclosure are described in the form of a Markush group, those skilled in the art will recognize that the disclosure is also described with respect to any individual member or subgroup of members of that Markush group.

[0192] As will be understood by those skilled in the art, for any and all purposes, e.g., with respect to providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be divided into at least 2, 3, 4, 5, or 10 equal parts. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," and "less than," is inclusive of the recited numbers, and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 entities refers to groups having 1, 2, 3, 4 or 5 entities, etc.

[0193] The terms "individual," "subject," and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a patient / animal patient. In some embodiments, the subject is a human patient. In some embodiments, the subject may have or is suspected of having a disorder or health condition associated with a gene of interest (GOI). In some embodiments, the subject is a human who has been diagnosed at or after the time of diagnosis as being at risk for a disorder or health condition associated with the GOI. In some cases, a diagnosis of having a risk for a disorder or health condition associated with the GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequences near the GOI in the genome that can affect expression of the GOI.

[0194] The term "treatment" when used in reference to a disease or condition means that at least an improvement in the symptoms associated with the condition afflicting an individual is achieved, and improvement is used broadly to refer to at least a decrease in the parameters associated with the condition being treated (e.g., hemophilia A), such as the magnitude of the symptoms. Thus, treatment also includes situations in which a pathological condition or at least its associated symptoms are completely inhibited, e.g., prevented from occurring or completely eliminated, such that the host is no longer afflicted with the condition or at least the symptoms that characterize the condition. Thus, treatment includes (i) prevention, i.e., preventing clinical symptoms from developing, e.g., reducing the risk of clinical symptoms occurring, including preventing disease progression; (ii) inhibition, i.e., halting the onset or further development of clinical symptoms, e.g., alleviating or completely inhibiting active disease.

[0195] As used herein, the terms "effective amount," "pharmaceutically effective amount," or "therapeutically effective amount" refer to a sufficient amount of a composition to provide a desired benefit when administered to a subject with a particular condition. Thus, the term "therapeutically effective amount" refers to an amount of therapeutic cells or a composition having therapeutic cells sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount also includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slow the progression of disease symptoms), or reverse disease symptoms. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.

[0196] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering a compound(s) of interest to a subject. A "pharmaceutically acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0197] As used herein, a "pharmaceutical composition" is intended to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that can induce an undesirable response in a subject (e.g., the compound(s) in the pharmaceutical composition are of pharmaceutical grade). Pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, etc.

[0198] As used herein, the phrases "having the formula" or "having the structure" are not intended to be limiting and are used in the same manner as the term "comprising" is commonly used. The term "independently selected from" is used herein to indicate that the listed elements, such as R groups, can be the same or different.

[0199] As used herein, the terms "may," "optional," "optionally," or "may optionally" mean that the subsequently described circumstance may or may not occur, such that the statement includes when the subsequently described circumstance occurs or does not occur. For example, the phrase "optionally substituted" means that non-hydrogen substituents may or may not be present on a given atom, and thus the statement includes structures in which non-hydrogen substituents are present and structures in which non-hydrogen substituents are absent.

[0200] "Acyl" refers to the groups HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heptyl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted aryl-C(O)-, substituted hetero ... Heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CHC(O)-.

[0201] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoradical) typically, but not necessarily, containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl and cyclohexyl. Generally, but not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" contemplates alkyl groups of 1 to 6 carbon atoms. "Substituted alkyl" refers to an alkyl substituted with one or more substituents, including when two hydrogen atoms are replaced from the same carbon atom in an alkyl substituent, as in a carbonyl group (i.e., the substituted alkyl group may contain a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl substituents in which at least one carbon atom has been replaced with a heteroatom, as described in more detail below. Unless otherwise indicated, the terms "alkyl" and "lower alkyl" include straight-chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkyl or lower alkyl, respectively.

[0202] The term "substituted alkyl" refers to a group in which one or more carbon atoms in the alkyl chain are substituted with -O-, -N-, -S-, -S(O) n- (wherein n is 0-2), -NR- (wherein R is hydrogen or alkyl), and optionally substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hetero and -NRaRb, where R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic.

[0203] The term "alkenyl" refers to a straight-chain, branched, or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, etc. Again, generally, but not necessarily, an alkenyl group herein can contain 2 to about 18 carbon atoms, e.g., 2 to 12 carbon atoms. The term "lower alkenyl" contemplates an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted with one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl in which at least one carbon atom has been replaced with a heteroatom. Unless otherwise indicated, the terms "alkenyl" and "lower alkenyl" include straight-chain, branched, cyclic, unsubstituted, substituted and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0204] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, etc. Again, generally, but not necessarily, alkynyl groups herein can contain from 2 to about 18 carbon atoms, and such groups can further contain from 2 to 12 carbon atoms. The term "lower alkynyl" contemplates an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl in which at least one carbon atom has been replaced with a heteroatom. Unless otherwise indicated, the terms "alkynyl" and "lower alkynyl" include straight-chain, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0205] The term "aryl," unless otherwise specified, generally, but not necessarily, refers to an aromatic substituent containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings fused together, directly linked, or indirectly linked (such as when different aromatic rings are bonded to a common group such as a methylene or ethylene moiety). Aryl groups can contain, for example, 5 to 20 carbon atoms; as a further example, aryl groups can contain 5 to 12 carbon atoms. For example, aryl groups can contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. "Substituted aryl" refers to an aryl moiety substituted with one or more substituents, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom has been replaced with a heteroatom, as described in more detail below. Aryl is a stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C6 ring aryl, exemplified by, but not limited to, phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazolyl, which may be further substituted with 1 to 5 members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl. 14 (See, e.g., Katritzky, Handbook of Heterocyclic Chemistry.) Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0206] The term "alkylene" refers to a diradical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may contain heteroatoms. "Lower alkylene" refers to an alkylene linkage containing 1 to 6 carbon atoms. Examples include methylene (-CH-), ethylene (-CHCH-), propylene (-CHCHCH-), 2-methylpropylene (-CH-CH(CH)-CH-), hexylene (-(CH)-), and the like.

[0207] Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to the diradical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0208] The term "amino" refers to the group -NRR', where R and R' are independently hydrogen or non-hydrogen substituents, including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0209] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0210] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents or from 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0211] "Heteroaryl" refers to an aromatic group of 1 to 15 carbon atoms, e.g., 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Such heteroaryl groups can have a single ring (such as pyridinyl, imidazolyl, or furyl) or multiple condensed rings in the ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), provided that at least one ring in the ring system is aromatic, provided that the point of attachment is through an aromatic ring atom. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of a heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. The term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of a heteroaryl substituent, such heteroaryl groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0212] The terms "heterocycle," "heterocyclic," and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridged and spiro ring systems, and having 3 to 15 ring atoms, including 1 to 4 heteroatoms. These ring heteroatoms are selected from nitrogen, sulfur, and oxygen; in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of a heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0213] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, These include, but are not limited to, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0214] Unless otherwise constrained by the definition of a heterocyclic substituent, such heterocyclic groups may be optionally substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0215] "Substituted," as in "substituted alkyl," "substituted aryl," etc., as implicit in some of the preceding definitions, means that in an alkyl, aryl, or other moiety, at least one hydrogen atom bonded to a carbon (or other) atom has been replaced with one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups, and hydrocarbyl moieties C1-C6. 24 Alkyl (C1-C 18 Contains alkyl, C1-C 12 alkyl, further including C1-C6 alkyl), C2-C 24 Alkenyl (C2-C 18 Contains alkenyl, C2-C 12 alkenyl, further including C2-C6 alkenyl), C2-C 24 Alkynyl (C2-C 18 C2-C, including alkynyl 12 alkynyl, further including C2-C6 alkynyl), C5-C30 Aryl (C5-C 20 C5-C, including aryl 12 (further including aryl), and C6-C 30 Aralkyl (C6~C 20 C6-C, including aralkyl 12 (Further including aralkyl). The above hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically listed. Unless otherwise indicated, any of the groups described herein should be interpreted as including substituted and / or heteroatom-containing moieties in addition to unsubstituted groups.

[0216] "Linkage" or "linker," as in "linking group," "linker moiety," etc., refers to a linking moiety that connects two groups via a covalent bond. A linker can be linear, branched, cyclic, or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, but not limited to, amide (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), epithio (-S-), epidioxy (-OO-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)nO-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), and the like. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone can be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between backbone atoms can be saturated or unsaturated, and typically there are no more than one, two, or three unsaturated bonds in the linker backbone. The linker can include one or more substituents, for example, with alkyl, aryl, or alkenyl groups. Linkers include, but are not limited to, poly(ethylene glycol) units (e.g., —(CH—CH—O)—); ethers, thioethers, amines, alkyls (e.g., (C1-C 12) alkyl), such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, where two or more atoms of the cyclic group, such as 2, 3, or 4 atoms, are included in the backbone. The linker may be cleavable or non-cleavable. Any convenient orientation and / or connection of the linker to the linked groups may be used.

[0217] When the term "substituted" appears before or after a list of possible substituted groups, it is intended that the term apply to all members of that group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl."

[0218] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituents, as defined below.

[0219] In addition to the groups disclosed for each individual term herein, one or more hydrogens on a saturated carbon atom in the specified group or radical (any two hydrogens on a single carbon can be substituted, e.g., ═O, ═NR) can also be substituted. 70 , =N-OR 70 , =N2 or =S), unless otherwise specified, is a -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R70 、 -OSO2O - M + 、 -OSO2OR 70 、 -P(O)(O - )2(M + )2、 -P(O)(OR 70 )O - M + 、 -P(O)(OR 70 )2、 -C(O)R 70 、 -C(S)R 70 、 -C(NR 70 )R 70 、 -C(O)O - M + 、 -C(O)OR 70 、 -C(S)OR 70 、 -C(O)NR 80 R 80 、 -C(NR<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 are independently 70 Or, two R 80 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl which may optionally contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have —H or C1-C3 alkyl substitution; each M + is a counterion with a net positive charge. + are independently, e.g., K + , Na + , Li + Alkaline ions such as; + N(R 60 ) 4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 or [Ba 2+ ] 0.5 (The "subscript 0.5" means that one of the counterions to such divalent alkaline earth ions may be the ionized form of a compound of the invention and the other may be a typical counterion such as chloride, or that a two-ionized compound disclosed herein may serve as a counterion to such divalent alkaline earth ions, or that a doubly ionized compound of the invention may serve as a counterion to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl and N-morpholinyl.

[0220] Further to the disclosure herein, substituents for hydrogens on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are designated by -R unless otherwise specified. 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as defined above, except that in the case of a substituted alkene or alkyne, the substituent may be -O - M + , -OR 70 , -SR 70 or -S - M + isn't it.

[0221] In addition to the groups disclosed for each individual term herein, substituents for the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as defined above.

[0222] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0223] Unless otherwise indicated, the naming of substituents not explicitly defined herein is arrived at by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0224] For any of the groups disclosed herein that contain one or more substituents, it is understood that such groups do not contain any sterically impractical and / or synthetically impractical substitutions or substitution patterns. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0225] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of the compound.Salts, solvates, hydrates, and prodrug forms of the compound are also of interest.All such forms are encompassed by the present disclosure.Therefore, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomeric forms, including their pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers.In certain embodiments, the compounds can be metabolized into pharmaceutically active derivatives.

[0226] Those skilled in the art will appreciate that, as used herein, in small molecule structures [ka] It will be understood that a bond designated as refers to a bond that, in some embodiments, is a single (e.g., saturated) bond and, in some embodiments, is a double (e.g., unsaturated) bond. For example, the following structure: [ka] teeth, [ka] and [ka] is intended to encompass both.

[0227] Unless otherwise specified, a reference to an atom includes isotopes of that atom. For example, a reference to H includes isotopes of that atom. 1 H, 2 H (i.e., D) and 3 includes H (i.e., T), and any reference to C 12 C and all isotopes of carbon ( 13 C, etc.)

[0228] Definitions of other terms and concepts appear throughout the detailed description.

[0229] The following examples are offered by way of illustration and not by way of limitation. 5. Enumerated Embodiments Embodiment 1. An ionizable lipid compound of formula (I): (ZLY)-W n -(XR) (n-1) (I) During the ceremony, aZ is an ionizable head group, bL is optionally substituted (C1 to C 12 ) alkylene, cY is a linking group, dW n is a linear alkyl core of n carbon atoms, where n is 3 to 6; eX is an optional linking group; f. Each R is independently a lipid tail. Embodiment 2. The compound of embodiment 1, wherein n is 4 to 6. Embodiment 3. The compound of embodiment 2, wherein n is 4. Embodiment 4. The compound of embodiment 2, wherein n is 5. Embodiment 5. The compound of embodiment 2, wherein n is 6. Embodiment 6. The compound of embodiment 1, wherein n is 3. Embodiment 7. W n but, [ka] and [ka] is selected from During the ceremony, * represents the point of attachment to Y; Each ** represents a point of attachment to X, G 1 is H or G 1 and W to which Y is bonded n is a group cyclically linked to Y which together with the carbon atom of G 2 is H or -CH2OH A compound according to any one of embodiments 1 to 6. Embodiment 8. Y is —O—, —C(R 10 )2-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S—, wherein R 10 teeth 、 H and C 1~6 The compound of any one of embodiments 1-6, wherein the alkyl is selected from: Embodiment 9. Y is -O-, -OC(O)- and -OC(O)NR 10 The compound of embodiment 8, selected from: Embodiment 10. The compound of embodiment 8, wherein Y is —CH—. Embodiment 11. G 1is cyclically linked to Y and the W to which they are bonded n which together with the carbon atom of provides a heterocyclic ring. Embodiment 12. The compound of any one of embodiments 1 to 11, wherein L is (C2-C6)alkylene or substituted (C2-C6)alkylene. Embodiment 13. The compound of embodiment 12, wherein L is —(CH 2 ) 2 —. Embodiment 14. The compound of embodiment 12, wherein L is —(CH 2 ) 3 —. Embodiment 15. The compound of any one of embodiments 1-14, wherein Z comprises a tertiary amino group. Embodiment 16. Z is -NR 11 R 12 and R 11 and R 12 and n is independently 0 or 1. The compound of embodiment 15, wherein each is independently alkyl or substituted alkyl. Embodiment 17. R 11 and R 12 are respectively, C 1~6 The compound of embodiment 16, wherein the aryl is alkyl. Embodiment 18. R 11 and R 12 are respectively, C 1~3 The compound of embodiment 17, wherein the aryl is alkyl. Embodiment 19. R 11 and R 12 and each is methyl. Embodiment 20. R 11 and R 12 and each is ethyl. Embodiment 21. Each X is independently -(CH) s OC(O)-,-(CH2) s C(O)O-,-(CH2) s OC(O)O-,-(CH2) s OC(O)NR 10 -,-(CH2) s O-,-(CH2) s SC(O)NR 10-,-(CH2) s C(O)NR 10 -,-(CH2) s NR 10 C(O)-,-(CH2) s S-,-(CH2) s NR 10 -,-(CH2) s NR 10 C(O)O- and -(CH2) s NR 10 C(O)S—, wherein R 10 is H and C 1~6 The compound of any one of embodiments 1-20, wherein s is selected from alkyl, and s is 0-6. Embodiment 22. Each X is independently -OC(O)-, -C(O)O-, -OC(O)O-, -O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S—, wherein R 10 teeth 、 H and C 1~6 The compound of any one of embodiments 1-21, wherein the alkyl is selected from: Embodiment 23. The compound of embodiment 22, wherein each X is independently selected from —OC(O)—, —C(O)O—, and —OC(O)O—. Embodiment 24. The compound of embodiment 23, wherein each -XR is -OC(O)R. Embodiment 25. The compound of any one of embodiments 1-24, wherein each R is independently an aliphatic hydrocarbon group that is linear or branched, saturated or unsaturated, and / or optionally contains a cyclic group. Embodiment 26. The compound of any one of embodiments 1-25, wherein each R is a straight chain hydrocarbon group optionally containing one or more cyclic groups. Embodiment 27. Each R is C5 to C 20 Alkyl, C5-C 20 Alkenyl and C5-C20 The compound of any one of embodiments 1-26, wherein the compound is selected from alkynyl. Embodiment 28. Each R is C to C 12 Alkyl and C6-C 12 The compound of embodiment 27, selected from alkenyl. Embodiment 29. The compound of embodiment 26, wherein at least one R is a straight chain hydrocarbon group, including a cyclic group. Embodiment 30. The compound of embodiment 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, any of the monocyclic or bicyclic groups being optionally substituted. Embodiment 31. The compound of any one of embodiments 1-25, wherein at least one R is a branched hydrocarbon group, optionally including a cyclic group. Embodiment 32. The compound of embodiment 31, wherein each R is a branched hydrocarbon group. Embodiment 33. The compound of embodiment 32, wherein the branched hydrocarbon group contains 8 to 20 carbon atoms. Embodiment 34. The compound of any one of embodiments 31-33, wherein the branched hydrocarbon group is saturated. Embodiment 35. The compound of any one of embodiments 31-33, wherein the branched hydrocarbon group is unsaturated. Embodiment 36. R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 The compound of any one of embodiments 31-35, wherein the compound is alkenyl. Embodiment 37. The compound of embodiment 31, wherein at least one R is a branched hydrocarbon group that includes a cyclic group. Embodiment 38. The compound of embodiment 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, any of the monocyclic or bicyclic groups being optionally substituted. Embodiment 39. The compound has formula (IIA): [ka] 2. The compound of embodiment 1, wherein Embodiment 40. Y is -O-, -OC(O)- and -OC(O)NR 10 - selected from R 10 is H and C 1~6 The compound of embodiment 39, wherein the aryl group is selected from alkyl. Embodiment 41. The compound of embodiment 40, wherein Y is —O—. Embodiment 42. The compound of embodiment 40, wherein Y is —OC(O)—. Embodiment 43. Y is —OC(O)NR 10 41. The compound of embodiment 40, wherein Embodiment 44. A compound according to any one of embodiments 39 to 43, wherein L is (C2-C6)alkylene or substituted (C2-C6)alkylene. Embodiment 45. The compound of embodiment 44, wherein L is —(CH 2 ) 2 —. Embodiment 46. The compound of embodiment 44, wherein L is —(CH 2 ) 3 —. Embodiment 47. The compound of embodiment 44, wherein L is —(CH 2 ) 4 —. Embodiment 48. Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 The compound of any one of embodiments 39-47, wherein is alkyl. Embodiment 49. R 11 and R 12 are respectively, C 1~3 The compound of embodiment 48, wherein R is alkyl. Embodiment 50. R 11 and R 12 and R are each methyl. Embodiment 51. R 11 and R 12and each is ethyl. Embodiment 52. The compound of any one of embodiments 39-51, wherein each X is independently selected from —OC(O)—, —C(O)O—, and —OC(O)O—. Embodiment 53. Each R is C5 to C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 The compound of any one of embodiments 39-52, wherein the alkynyl is selected from: Embodiment 54. The compound of any one of embodiments 39-52, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms, optionally further containing one or more cyclic groups. Embodiment 55. R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 The compound of embodiment 54, wherein the compound is alkenyl. Embodiment 56. The compound has Formula (IIIA): [ka] During the ceremony, R 11 and R 12 are each independently 1~3 is alkyl, q is 1 to 4; Y is -O-, -OC(O)- and -OC(O)NR 10 - selected from Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10. 40. The compound of embodiment 39, wherein: Embodiment 57. The compound has Formula (IIB): [ka] 2. The compound of embodiment 1, wherein Embodiment 58. Y is -O-, -OC(O)-, -OC(O)NR 10 -, -NR 10 C(O)-, -NR 10 C(O)O- and -NR 10 C(O)S-, and R 10 is H and C 1~6 The compound of embodiment 57, wherein the aryl group is selected from alkyl. Embodiment 59. The compound of embodiment 58, wherein Y is selected from —NHC(O)—, —NHC(O)O—, and —NHC(O)S—. Embodiment 60. A compound according to any one of embodiments 57-59, wherein L is (C2-C6)alkylene or substituted (C2-C6)alkylene. Embodiment 61. The compound of embodiment 60, wherein L is —(CH 2 ) 2 —. Embodiment 62. The compound of embodiment 60, wherein L is —(CH 2 ) 3 —. Embodiment 63. The compound of embodiment 60, wherein L is —(CH 2 ) 4 —. Embodiment 64. Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 The compound of any one of embodiments 57-59, wherein is alkyl. Embodiment 65. R 11 and R 12 are respectively, C 1~3 The compound of embodiment 64, wherein the aryl is alkyl. Embodiment 66. R 11 and R 12 and R are each methyl. Embodiment 67. The compound of any one of embodiments 57-66, wherein each X is independently selected from —OC(O)—, —C(O)O—, and —OC(O)O—. Embodiment 68. Each R is C5 to C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 The compound of any one of embodiments 57-67, wherein the compound is selected from alkynyl. Embodiment 69. The compound of any one of embodiments 57-67, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms, optionally further containing one or more cyclic groups. Embodiment 70. R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 The compound of embodiment 69, wherein the compound is alkenyl. Embodiment 71. The compound has Formula (IIIB): [ka] During the ceremony, R 11 and R 12 are each independently 1~3 is alkyl, q is 1 to 4; Y is selected from -NHC(O)-, -NHC(O)O- and -NHC(O)S-; Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10. 58. The compound of embodiment 57, wherein: Embodiment 72. The compound has Formula (IIC): [ka] 2. The compound of embodiment 1, wherein Embodiment 73. Y is -O-, -OC(O)-, -OC(O)NR 10 - and -C(R 10 )2-, and R 10 is H and C 1~6 The compound of embodiment 72, wherein the aryl group is selected from alkyl. Embodiment 74. The compound of embodiment 73, wherein Y is —O—. Embodiment 75. Y is -C(R 10 )2-. Embodiment 76. A compound according to any one of embodiments 72 to 75, wherein L is (C2-C6)alkylene or substituted (C2-C6)alkylene. Embodiment 77. A compound according to any one of embodiments 76, wherein L is —(CH 2 ) 2 —. Embodiment 78. A compound of any one of embodiments 76, wherein L is —(CH 2 ) 3 —. Embodiment 79. A compound of any one of embodiments 76, wherein L is —(CH 2 ) 4 —. Embodiment 80. Z is -NR 11 R 12 and R 11 and R 12 are each independently 1~6 Alkyl or substituted C 1~6 The compound of any one of embodiments 72-79, wherein is alkyl. Embodiment 81. R 11 and R 12 are respectively, C 1~3 The compound of embodiment 80, wherein the aryl is alkyl. Embodiment 82. R 11 and R 12 and R are each methyl. Embodiment 83. Each X is independently —(CH)s OC(O)-, -(CH2) s C(O)O-, -(CH2) s The compound according to any one of embodiments 72-82, wherein s is selected from OC(O)O—, and s is 0-6. Embodiment 84. The compound of any one of embodiments 72-82, wherein each s is 0. Embodiment 85. The compound of any one of embodiments 72-82, wherein each s is 1. Embodiment 86. The compound of any one of embodiments 72-82, wherein each s is 3. Embodiment 87. Each R is C5 to C 20 Alkyl, C5-C 20 Alkenyl and C5-C 20 The compound of any one of embodiments 72-86, wherein the compound is selected from alkynyl. Embodiment 88. The compound of any one of embodiments 72-86, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms, optionally further containing one or more cyclic groups. Embodiment 89. R is —CH(R 7 )2, and each R 7 are independently C5~C 12 Alkyl or C5-C 12 The compound of embodiment 88, wherein the compound is alkenyl. Embodiment 90. The compound has Formula (IIIC): [ka] During the ceremony, R 11 and R 12 are each independently 1~3 Alkyl and C 1~4 heteroalkyl; q is 1 to 4; Y is -O- and -C(R 10 )2- is selected from each s is independently 0, 1, or 2; W is -O- and -C(R 10 )2- and Each R is independently C5 to C 20 Alkyl, C5-C 20 Alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u Each R 7 are independently C5~C 12 Alkyl or C5-C 12 alkenyl, J is a cyclic group, and each of t and u is 1 to 10. 73. The compound of embodiment 72, wherein Embodiment 91. Each R is independently: [ka] Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R x and R y are each independently a bond or an optionally substituted, straight-chain or branched, saturated or partially unsaturated C1-C 20 is an aliphatic group, r, p, and q are each independently an integer of 0 to 20. 91. The compound of any one of embodiments 1-90, wherein Embodiment 92. At least one R is [ka] [ka] and [ka] , where each # represents the point of attachment to X or the point of attachment of R to the linear or branched hydrocarbon chain. The compound of embodiment 91, comprising a moiety selected from: Embodiment 93. A lipid nanoparticle comprising an ionizable lipid compound according to any one of embodiments 1 to 92. Embodiment 94. The lipid nanoparticle of embodiment 93, further comprising a neutral lipid and a lipid capable of reducing aggregation. Embodiment 95. The lipid nanoparticle of embodiment 94, wherein the neutral lipid comprises a phospholipid. Embodiment 96. The lipid nanoparticle of embodiment 94 or 95, wherein the neutral lipid comprises cholesterol. Embodiment 97. a. Nucleic acid, b. ionizable lipids, C. phospholipids, d. cholesterol, and e. Lipids that can reduce aggregation 97. The lipid nanoparticle of embodiment 96, comprising: Embodiment 98. The lipid nanoparticle of embodiment 97, wherein the nucleic acid comprises DNA. Embodiment 99. The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises RNA. Embodiment 100. The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises DNA and RNA. Embodiment 101. The lipid nanoparticle of embodiment 100, wherein the RNA is selected from mRNA, gRNA, and siRNA. Embodiment 102. The lipid nanoparticle of any one of embodiments 97 to 101, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG) and derivatives thereof. Embodiment 103. The lipid nanoparticle of embodiment 102, wherein the phospholipid is phosphatidylethanolamine (PE). Embodiment 104. The lipid nanoparticle of embodiment 103, wherein the phospholipid is phosphatidylcholine (PC). Embodiment 105. The lipid nanoparticle of any one of embodiments 97-104, wherein the phospholipids each independently comprise a hydrocarbon chain having from 12 to 24 carbons. Embodiment 106. The lipid nanoparticle of embodiment 105, wherein the phospholipids each independently comprise a hydrocarbon chain having from 16 to 20 carbons. Embodiment 107. The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chain is saturated. Embodiment 108. The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chain is unsaturated and / or further comprises a carbocyclyl. Embodiment 109. The lipid nanoparticle of embodiment 108, wherein the hydrocarbon chains each independently comprise 1 to 4 double bonds. Embodiment 110. The lipid nanoparticle of any one of embodiments 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains. Embodiment 111. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). Embodiment 112. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). Embodiment 113. The lipid nanoparticle of embodiment 104, wherein the phospholipid comprises 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (Δ9Δ9-CisPC). Embodiment 114. The lipid nanoparticle of embodiment 106, wherein the lipid nanoparticle comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Embodiment 115. The lipid nanoparticle of embodiment 104, wherein the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Embodiment 116. The lipid nanoparticle of any one of embodiments 103 to 115, wherein the lipid capable of reducing aggregation is a PEG-lipid. Embodiment 117. The lipid nanoparticle of embodiment 116, wherein the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]). Embodiment 118. The lipid nanoparticle of any one of embodiments 94 to 117, further comprising a targeting ligand. Embodiment 119. The lipid nanoparticle of embodiment 118, wherein the targeting ligand comprises GalNAc. Embodiment 120. The lipid nanoparticle of embodiment 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation. Embodiment 121. The lipid nanoparticle of embodiment 120, wherein the lipid capable of reducing aggregation is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]). Embodiment 122. The lipid nanoparticle according to any one of embodiments 94 to 121, wherein the N / P ratio (ratio of moles of amine groups of the cationic lipid to moles of phosphate groups of the DNA) is 5 to 30. Embodiment 123. The lipid nanoparticle of embodiment 122, wherein the N / P ratio is 7. Embodiment 124. The lipid nanoparticle of embodiment 122, wherein the N / P ratio is 14. Embodiment 125. The lipid nanoparticle of embodiment 122, wherein the N / P ratio is 28. Embodiment 126. a. 40-60 mol% of ionizable lipids of the total lipids present; b. phospholipids that make up 6-20 mol% of the total lipids present; c. Cholesterol at 35-45 mol% of the total lipids present, and d. Lipids capable of reducing aggregation by 1.5-2.5 mol% of the total lipids present 126. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: Embodiment 127. a. 40-60 mol% of ionizable lipids of the total lipids present; b. phospholipids that make up 10-20 mol% of the total lipids present; c. Cholesterol at 35-45 mol% of the total lipids present, and d. Lipids capable of reducing aggregation by 1.5-2.5 mol% of the total lipids present 126. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: Embodiment 128. ae) 40-49 mol% of ionizable lipids of the total lipids present; bf) phospholipids that are 10-20 mol% of the total lipids present; cg) cholesterol, 35-45 mol% of the total lipids present, and dh) Lipids that can reduce aggregation of 1.5 to 2.5 mol% of the total lipids present 126. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: Embodiment 129. A pharmaceutical composition comprising the lipid nanoparticles of any one of embodiments 94 to 128 and a pharmaceutically acceptable excipient, carrier or diluent. Embodiment 130. A method for delivering a nucleic acid into a cell, comprising contacting said cell with a lipid nanoparticle of any one of embodiments 94 to 128. Embodiment 131. The method of embodiment 130, wherein the cell is in vitro. Embodiment 132. The method of embodiment 130, wherein the cell is in vivo. Embodiment 133. A method for delivering a nucleic acid for the in vivo production of a target protein, comprising: 130. A method comprising systemically administering to a subject in need thereof the pharmaceutical composition of embodiment 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and wherein the administering of the pharmaceutical composition results in long-term, stable expression of the target protein. [Example]

[0230] 6. Example The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0231] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells and kits for the methods referred to in or related to this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc. and repositories such as, for example, Addgene, Inc., American Type Culture Collection (ATCC).

[0232] material and method LNP formulation. LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or a DNA / mRNA mixture) as described by Prud'homme et al. (J Pharm Sci 2018). Briefly, a lipid excipient mixture (ionizable lipids, helper lipids, cholesterol, PEG-lipids, and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of nucleic acid is prepared in a low pH buffer ranging from 3.0 to 4.0. Then, using a commercially available mixer device, the lipid mixture is mixed with the aqueous nucleic acid solution at a flow rate ratio of 1:3 (V / V). The resulting solution is immediately diluted with a buffer pH range of 5.0 to 6.5. The diluted LNPs are subjected to dialysis purification against a second buffer with a pH range of 7.0 to 8.0. The LNP solution was concentrated using a 100,000 MWCO Amicon Ultra centrifuge tube (Millipore Sigma) followed by filtration through a 0.2 μm PES sterile-grade filter. Particle size was determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency was calculated using the Quant-it RiboGreen assay kit.

[0233] EPO and cytokine detection in serum. Blood was collected via retro-orbital bleeding into serum separator tubes and processed into serum. Serum samples could be stored at -80°C from collection until analysis. The U-PLEX Human EPO Assay from MSD was used according to the manufacturer's instructions to quantify serum levels of human EPO protein driven by expression from the DNA payload. The Mouse ProInflammatory 7-Plex Tissue Culture Kit from MSD was used according to the manufacturer's instructions to quantify serum levels of mouse cytokines resulting from exposure to DNA-LNPs.

[0234] FIX detection in plasma. Blood was collected via retro-orbital bleeding into K2EDTA tubes and processed to plasma. Plasma samples could be stored at -80°C from collection until analysis. Plasma levels of human FIX after administration of LNP were quantified using the U-Plex assay on the MSD platform. Briefly, a monoclonal mouse anti-human FIX antibody (Prolytix, clone AHIX-5041) was conjugated to biotin and used as a capture reagent on streptavidin-coated plates. A polyclonal goat anti-human FIX antibody (Cedarlane, clone CL20040AP) was conjugated to Sulfo-TAG and used as a detection reagent in a standard configuration for quantification of electrochemiluminescence (ECL) signal using a QuickPlex SQ 120MM instrument from MSD. Pooled normal human plasma (Affinity Biologicals, FRNCP0125), a pool of normal citrated human plasma collected from a minimum of 20 donors, was used to generate a standard curve and calculate % normal human FIX levels. The assay was confirmed to be specific for human FIX, did not cross-react with mouse FIX, and showed very low background levels in untreated mouse plasma samples. Example 1. Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (L-1) [ka]

[0235] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-(dimethylamino)butanoate [ka]

[0236] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1 equivalent) and diisopropylethylamine (3 equivalents) in DMF (4 volumes) is added 2,5-dioxopyrrolidin-1-yl 4-(dimethylamino)butanoate (2 equivalents). The reaction mixture is heated at 90°C for 16 hours, then cooled to room temperature and quenched by the addition of water and MTBE. The organic layer is collected and the aqueous layer is further extracted with MTBE (3 x 3 volumes). The combined organic extracts are washed with 10% CuSO4 (2 volumes), then brine (2 volumes), dried over MgSO4, then filtered, concentrated, and purified by column chromatography. The recovered starting material is resubjected to these reaction conditions.

[0237] Synthesis of 1,2,4,5-tetrahydroxypentan-3-yl 4-(dimethylamino)butanoate [ka]

[0238] To a solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-(dimethylamino)butanoate in methanol (5 V) is added aqueous HCl (1 M). The reaction mixture is monitored for completion by LCMS. Once complete, the reaction mixture is concentrated under reduced pressure with some toluene azeotrope to complete dryness.

[0239] Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) [ka]

[0240] Mix 1,2,4,5-tetrahydroxypentan-3-yl 4-(dimethylamino)butanoate (1 equivalent), decanoic acid (5.5 equivalents), EDCI (6 equivalents), DMAP (2 equivalents), DIEA (8 equivalents), and ACN (10 mL) at 0°C and then at room temperature. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 x 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. Example 2. Synthesis of 1,4,5-tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yldecanoate (L-2) [ka]

[0241] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol [ka]

[0242] A solution of 1,2,3,4,5-pentahydroxypentane (25 g, 1 equiv.), p-toluenesulfonic acid (2.83 g, 0.1 equiv.), and 2,2-dimethoxypropane (37.65 g, 2.2 equiv.) in methanol (250 mL) was stirred overnight at room temperature under a nitrogen atmosphere. K2CO3 (5 g) was added to the reaction mixture and stirred for 1 h. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 20 mL). The filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (200 mL), and 50 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. The fraction was concentrated to zero under vacuum while maintaining the temperature below 35 °C. 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 wt / wt.) was loaded onto the column, followed by the final step of preparing dried silica gel onto which the reaction mixture was absorbed. The product was purified using CombiFlash. Elution was performed with PE / EA (100:0 to 50:50 gradient, collecting every 200 ± 10 mL). Samples were taken for TLC analysis (EA:PE = 1:1), and the acceptable products were combined. This gave 12 g (31.4%) of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as a colorless oil.

[0243] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate [ka]

[0244] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (10 g, 1 equiv.) in THF (100 mL) was added 4-nitrophenyl carbonochloridate (9.55 g, 1.1 equiv.) in portions. TEA (13.07 g, 3 equiv.) was added dropwise at 0° C. under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 60 minutes. The resulting mixture was used directly in the next step without further purification.

[0245] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate [ka]

[0246] Dimethylaminopropylamine (6.6 g, 2.0 equiv.) was added dropwise to a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-nitrophenyl carbonate (110 mL THF solution) under a nitrogen atmosphere at 0°C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 60 minutes. The resulting mixture was quenched with 50 mL of water and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (2 × 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 15 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. The fraction was concentrated to zero under vacuum while maintaining the temperature below 35°C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 30 w / w) was loaded onto the column, followed by the final step of absorbing the reaction mixture onto dry silica gel. The product was purified using CombiFlash. It was eluted with CHCl / MeOH (gradient from 100:0 to 10:1, collecting every 100 ± 10 mL). Samples were taken for TLC analysis (CHCl / MeOH = 5:1), and the acceptable products were combined. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate (7 g, 45.1% over two steps) was obtained as a yellow oil.

[0247] Synthesis of 1,2,4,5-tetrahydroxypentan-3-yl N-[3-(dimethylamino)propyl]carbamate [ka]

[0248] A solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate (7 g, 1 equivalent) in HCl (6 M, 70 mL) was stirred overnight at 50° C. under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure to give 1,2,4,5-tetrahydroxypentan-3-yl N-[3-(dimethylamino)propyl]carbamate (4 g, 73.48%) as a pale yellow oil.

[0249] Synthesis of 1,4,5-tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yldecanoate [ka]

[0250] To a stirred solution of 1,2,4,5-tetrahydroxypentan-3-yl N-[3-(dimethylamino)propyl]carbamate (4 g, 1 equiv.) and decanoyl chloride (21.77 g, 114.152 mmol, 8 equiv.) in DCM (100 mL) was added TEA (14.44 g, 142.690 mmol, 10 equiv.) dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography using the following conditions: column, XSelect CSH Prep C18 5 μm; mobile phase, B: MeCN: i-PrOH = 1:1; A: water (0.1% TFA), 50% to 95% gradient over 15 min; flow rate: 50 mL / min; detector, ELSD. The organic solvent was removed under reduced pressure, and the aqueous phase was adjusted to pH 8 with NaHCO3 (5% aqueous solution) and extracted with heptane (50 mL * 2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give 1,4,5-tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yldecanoate (961.2 mg, 7.51%) as a pale yellow oil. LCMS: (ES, m / z): 898 [M+H] + ; 1 H-NMR:(400 MHz, CDCl3, ppm):δ 5.914-5.626 (m, 1H), 5.367-5.211 (m, 3H), 4.281-4.293 (m, 2H), 4.145-4.068 (s, 2H), 3.275-3.197 (m, 2H), 2.369-2.213 (m, 16H), 1.680 (s, 10H), 1.335 (s, 48H), 0.982-0.826 (m, 12H). Example 3. Synthesis of 3-(4-(dimethylamino)butoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (L-3) [ka]

[0251] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate [ka] To a 100 mL three-necked round-bottom flask, bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (4 g, 17.221 mmol, 1 equiv.), DCM (40 mL), and TEA (3.49 g, 34.442 mmol, 2 equiv.) were added at room temperature. To the above mixture, MsCl (2.96 g, 25.831 mmol, 1.5 equiv.) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature for an additional 1 h. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with CHCl (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. This gave bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (8.1 g, crude) as a brown oil. LCMS: (ES, m / z): 311 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 4.851 (t, J = 4.5 Hz, 1H), 4.307-4.247 (m, 1H), 4.153-4.068 (m, 2H), 4.045-3.967 (m, 2H), 3.145-3.070 (m, 3H), 1.457-1.330 (m, 12H).

[0252] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine [ka]

[0253] To a 100 mL three-necked round-bottom flask, NaH (870.29 mg, 21.759 mmol, 1.5 equiv, 60%) and THF (25 mL) were added at room temperature. To the above mixture, 4-(dimethylamino)butan-1-ol (1.7 g, 14.506 mmol, 1 equiv) and THF (25 mL) were added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for an additional 30 minutes. To the above mixture, bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (4.95 g, 15.957 mmol, 1.1 equiv) and THF (25 mL) were added dropwise at 0 °C. The resulting mixture was stirred at 60 °C overnight. The reaction was quenched with saturated NH4Cl(aq) at 0 °C. The mixture was basified to pH 8 with saturated NaHCO3(aq). The resulting mixture was extracted with EtOAc (5×100 mL) and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with CHCl / MeOH (5:1) to give {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine (4.6 g, 80.6% yield over two steps) as a brown solid. LCMS: (ES, m / z): 332 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 4.155-4.095 (m, 2H), 4.060-4.011 (m, 2H), 3.929-3.873 (m, 2H), 3.723 (t, J = 6.1 Hz, 2H), 3.563 (d, J = 4.6 Hz, 1H), 2.742-2.689 (m, 2H), 2.545 (s, 6H), 1.809-1.707 (m, 2H), 1.653-1.586 (m, 2H), 1.429 (s, 6H), 1.339 (s, 6H).

[0254] Synthesis of 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetrol [ka]

[0255] To a 50 mL three-necked round-bottom flask was added {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine (2.6 g, 7.844 mmol, 1 equiv.), HO (5 mL), and acetic acid (21 mL) at room temperature. The resulting mixture was stirred at 80 °C for 6 h. The mixture was allowed to cool to room temperature. The resulting mixture was concentrated in vacuo. This afforded 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetrol (4.8 g, crude) as a brown oil. LCMS: (ES, m / z): 252 [M+1] + . 1 H NMR (300 MHz, DMSO-d6) δ 3.634-3.582 (m, 2H), 3.547-3.483 (m, 5H), 3.393-3.334 (m, 3H), 3.238-3.196 (m, 1H), 2.219-2.161 (m, 2H), 2.106 (s, 6H), 1.442-1.383 (m, 4H).

[0256] Synthesis of 1,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yldecanoate [ka]

[0257] To a 250 mL three-necked round-bottom flask, 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetrol (3.2 g, 12.733 mmol, 1 equiv.), DCM (160 mL), and TEA (20.62 g, 203.728 mmol, 16 equiv.) were added at room temperature. To the above mixture, decanoyl chloride (24.28 g, 127.330 mmol, 10 equiv.) was added dropwise at 0 °C. The resulting mixture was stirred at 40 °C for an additional 2 h. The mixture was allowed to cool to room temperature. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with CHCl (2 × 100 mL), and the organic phase was collected and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with CH2Cl2 / MeOH (5:1) to give 4 g (crude). The residue was purified by reversed-phase HP-flash chromatography using the following conditions: Column, XSelect CSH Prep C 18 5 μm; mobile phase, B: MeCN: i-PrOH = 1:1; A: water (0.1% TFA), 50% to 95% gradient in 15 min; flow rate: 50 mL / min; detector, ELSD. The residue was dissolved in hexane (100 mL). The combined organic layers were washed with saturated NaHCO3 solution (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This afforded 1,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yldecanoate (699.5 mg, 11.07% yield over two steps) as a pale yellow oil. LCMS: (ES, m / z): 869 [M+1] + . 1 H NMR (300 MHz, Chloroform-d, ppm):δ 5.229-5.179 (m, 2H), 4.429-4.378 (m, 2H), 4.163-4.101 (m, 2H), 3.639 (t, J= 5.3 Hz, 1H), 3.602-3.564 (m, 2H), 2.355-2.251 (m, 16H), 1.647-1.539 (m, 12H), 1.325-1.253 (m, 48H), 0.901-0.856 (m, 12H). Example 4. Synthesis of 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyl tridodecanoate (L-4) [ka]

[0258] Synthesis of pent-4-ene-1,2,3-triol [ka]

[0259] To a mixture of 1-(2,2-dimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (1 eq.) in MeOH (1V) was added HCl (6M) (0.13 g, 3.544 mmol, 0.2 eq.). The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was concentrated under reduced pressure, and the material was thoroughly dried under vacuum for further reaction.

[0260] Synthesis of pent-4-ene-1,2,3-triyl tridodecanoate [ka]

[0261] To a mixture of pent-4-ene-1,2,3-triol (1 eq.), dodecanoic acid (3.4 eq.), and DMAP (1 eq.) in DCM (10 V) was added EDCI (1.44 g, 7.525 mmol, 1 eq.). The reaction mixture was stirred at 20 °C for 6 h. The resulting mixture was diluted with DCM (5 V). The resulting mixture was washed with water (3 × 1 V) and brine (1 V). The resulting solution was dried over anhydrous Na2SO4. After filtration, the resulting mixture was concentrated under reduced pressure. The residue was dissolved in DCM (1 V) and silica gel (type: ZCX-2, 100-200 mesh, 5.00 w. / w.) was added. TLC analysis (PE / EA = 5:1).

[0262] Synthesis of 4-oxobutane-1,2,3-triyltridodecanoate [ka]

[0263] To a mixture of pent-4-ene-1,2,3-triyltridodecanoate (1 eq.) in THF (40 V) and HO (20 V), add KOsO·2HO (0.12 eq.). The reaction mixture is stirred at 20 °C for 10 min, and then NaIO (5.0 eq.) and 2,6-lutidine (5.0 eq.) are added at 20 °C. The reaction mixture is stirred at 20 °C for 18 h. The resulting mixture is diluted with EA (20 V). The resulting mixture is washed 3 × 20 V with water and brine (30 V). The resulting solution is dried over anhydrous NaSO. The resulting mixture is concentrated under reduced pressure. TLC analysis (PE / EA = 10:1).

[0264] Synthesis of 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyl tridodecanoate [ka] A mixture of 4-oxobutane-1,2,3-triyltridodecanoate (1 equiv.), 4-(dimethylamino)butane-1,2-diol (1.5 equiv.), DMAc dimethyl acetal (10 equiv.), and camphorsulfonic acid (2 equiv.) is heated at reflux overnight in DCE (40 V). The reaction mixture is cooled to 0 °C and then poured into a rapidly stirring mixture of EA (30 V) and saturated aqueous NaHCO (30 V). When the pH exceeds 7, the organic layer is collected, and the product is further extracted from the aqueous layer with EA (3 × 20 V). The organic extracts are combined, washed with brine (30 V), dried over MgSO, filtered, concentrated, and then purified by silica column chromatography to give 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyltridodecanoate. Example 5. Synthesis of 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate (L-5) [ka]

[0265] Synthesis of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol [ka]

[0266] To a stirred solution of 2,3,4-trihydroxybutanal (10 g, 1 equiv.) and 1-(2,4-dimethoxyphenyl)methanamine (13.92 g, 1 equiv.), H2SO4 (1.63 g, 0.2 equiv.), and Raney Ni (3.57 g, 0.5 equiv.) in EtOH (100 mL) was introduced H2 (3 atm) at room temperature. The resulting mixture was stirred at 45 °C for 6 h. The resulting mixture was filtered, and the filter cake was washed with ethanol (2 × 20 mL). The filtrate was concentrated under reduced pressure. The crude product, 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol (16 g), was used directly in the next step without further purification.

[0267] Synthesis of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane [ka]

[0268] To a stirred solution of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol (16 g, 1 equiv., crude) and TBSCl (28 g, 3.1 equiv.) in DMF (160 mL) was added imidazole (16 g, 4.0 equiv.) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The mixture was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN (0.1% TFA) in water, 40% to 90% gradient in 20 min; detector, UV 220 nm. This gave 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 22.3%) as a brown oil.

[0269] Synthesis of N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide [ka]

[0270] A solution of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 1 equiv.) and 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 1 equiv.), HATU (6.02 g, 1.2 equiv.), and DIEA (5.12 g, 3 equiv.) in DMF (50 mL) was stirred at room temperature under a nitrogen atmosphere for 1 hour. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN (0.1% TFA) in water, 30% to 90% gradient in 20 min; detector, UV 220 nm. This afforded N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide (4.2 g, 51.89%) as a brown oil.

[0271] Synthesis of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide [ka]

[0272] A solution of N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide (4.2 g, 6.852 mmol, 1 equiv.) and TBAF (1.79 g, 6.852 mmol, 1 equiv.) in THF (40 mL) was stirred at room temperature under a nitrogen atmosphere for 3 hours. The resulting mixture was concentrated in vacuo. The crude product, N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide (5 g, crude), was used directly in the next step without further purification.

[0273] Synthesis of 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate [ka]

[0274] A solution of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide (5 g, 13.005 mmol, 1 equiv.) and lauric acid (9.12 g, 45.518 mmol, 3.5 equiv.), EDC.HCl (8.08 g, 52.020 mmol, 4 equiv.), and DMAP (1.58 g, 13.0 mmol, 1 equiv.) in DCM (50 mL) was stirred at room temperature under a nitrogen atmosphere for 4 hours. The resulting mixture was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN (0.1% TFA) in water, 30% to 90% gradient in 20 min; detector, UV 220 nm. This afforded 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate (3 g, 24.77%) as a white semisolid.

[0275] Synthesis of 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate [ka]

[0276] A solution of 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate (2.5 g, 2.684 mmol, 1 equiv.) in HCl (gas) in 1,4-dioxane (25 mL) was stirred at room temperature under a nitrogen atmosphere for 6 hours. The resulting mixture was concentrated in vacuo. The residue was purified by reverse-phase HP-flash chromatography using the following conditions: column, XSelect CSH Prep C18 5 μm; mobile phase, B:MeCN:i-PrOH = 1:1; A:water (0.1% TFA), 45% to 85% gradient in 15 min; flow rate: 50 mL / min; detector, ELSD. This gave 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yldodecanoate TFA salt (683.0 mg, 32.57%) as a white semi-solid. LCMS: (ES, m / z): 782 [M+H] + ; 1 H-NMR:(400 MHz, CDCl3, ppm):δ 12.182 (s, 1H),7.283-7.193 (m, 1H),5.261-5.187 (m, 2H), 4.365-4.336 (m, 1H), 4.156-4.110 (m, 1H), 3.635-3.607 (m, 1H), 3.402-3.367 (m, 1H), 3.367-2.893 (m, 2H), 2.864 (s, 1H), 2.693-2.298 (m, 12H), 2.112 (s, 2H), 1.613 (s, 6H), 1.272 (s, 48H), 0.992-0.910 (m, 9H). Example 6. Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayltetranonanoate (L-6) [ka]

[0277] Synthesize 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayltetranonanoate in the same manner as (L-1), using nonanoic acid instead of decanoic acid. Example 7. Synthesis of 3-(((3-(dimethylamino)propyl)carbamoyl)oxy)pentane-1,2,4,5-tetrayltetranonanoate (L-7) [ka]

[0278] Synthesize 3-(((3-(dimethylamino)propyl)carbamoyl)oxy)pentane-1,2,4,5-tetrayltetranonanoate in the same manner as (L-2), using nonanoyl chloride instead of decanoyl chloride. Example 8. Synthesis of 3-(4-(dimethylamino)butoxy)pentane-1,2,4,5-tetrayltetranonanoate (L-8) [ka]

[0279] Synthesize 3-(4-(dimethylamino)butoxy)pentane-1,2,4,5-tetrayltetranonanoate in the same manner as (L-3), using nonanoyl chloride instead of decanoyl chloride. Example 9. Synthesis of 3-(3-(dimethylamino)propoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (L-9) [ka]

[0280] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol [ka]

[0281] A solution of 1,2,3,4,5-pentahydroxypentane (20 g, 131.5 mmol, 1 equiv.), p-toluenesulfonic acid (2.26 g, 13.15 mmol, 0.1 equiv.), and 2,2-dimethoxypropane (30.1 g, 289.3 mmol, 2.2 equiv.) in methanol (200 mL) was stirred at room temperature under a nitrogen atmosphere for 16 h. K2CO3 (5 g) was added to the reaction mixture, which was then stirred at room temperature for 1 h. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 20 mL). The filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (200 mL), and 40 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. The mixture was concentrated to zero under vacuum, maintaining the temperature below 35 °C. 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 wt / wt.) was loaded onto the column, and then, in the final step, the reaction mixture was absorbed onto the dried silica gel. The product was purified using CombiFlash and then eluted with PE / EA (100:0 to 50:50 gradient, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (EA:PE = 1:1). The acceptable products were combined. This gave 15 g (49.1%) of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as a colorless oil.

[0282] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate [ka]

[0283] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (5 g, 21.526 mmol, 1 equiv.) and TEA (4.36 g, 43.052 mmol, 2 equiv.) in CHCl (50 mL) under a nitrogen atmosphere at 0 °C, MsCl (3.70 g, 32.289 mmol, 1.5 equiv.) was added dropwise. The resulting mixture was stirred at room temperature for an additional 3 h. The resulting mixture was washed with water (2 × 50 mL). The aqueous layer was extracted with CHCl (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (6 g, 88.01%) as a yellow oil.

[0284] Synthesis of 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-dimethylpropan-1-amine [ka]

[0285] To a stirred solution of NaH (1.74 g, 43.620 mmol, 3 equiv) in THF (15 mL) was added 1-propanol, 3-(dimethylamino)- (1.5 g, 14.540 mmol, 1.00 equiv) in portions at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C for an additional 0.5 h. To the above mixture was added bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (6.77 g, 21.810 mmol, 1.5 equiv) in portions at 0 °C. The resulting mixture was stirred at 60 °C for an additional 6 h. The reaction was quenched by the addition of NH4Cl(aq) (15 mL) at room temperature. The aqueous layer was extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (50 mL) and 15 g of silica gel (type: ZCX-2, 100-200 mesh, 3 wt. / wt.) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 150 g of silica gel (type: ZCX-2, 100-200 mesh, 10 wt. / wt.) was loaded onto a column, after which the reaction mixture was absorbed onto dry silica gel in the final step. The product was purified using CombiFlash and then eluted with CHCl / MeOH (9:1) (gradient from 100:0 to 90:10, collecting every 200 ± 10 mL). Samples were analyzed by TLC (CHCl / MeOH = 9:1), and compatible products were combined. This gave {3-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}dimethylamine (1.07 g, 16.46%).

[0286] Synthesis of 3-(3-(dimethylamino)propoxy)pentane-1,2,4,5-tetraol [ka]

[0287] To a 100 mL three-necked round-bottom flask was added {3-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}dimethylamine (1 g, 3.150 mmol, 1 equivalent) and hydrogen chloride (6 M, 10 mL) at room temperature. The resulting mixture was stirred at 60 °C for 6 hours. The resulting mixture was concentrated under reduced pressure. The crude product mixture was used directly in the next step without further purification.

[0288] Synthesis of 1,4,5-tris(decanoyloxy)-3-[3-(dimethylamino)propoxy]pentan-2-yldecanoate (L-9) [ka]

[0289] To a 100 mL round-bottom flask, 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetrol (1 g, 4.214 mmol, 1 equiv.), DCM (10 mL), and capric acid (3.99 g, 23.177 mmol, 5.5 equiv.) were added at room temperature. EDCI (4.85 g, 25.284 mmol, 6 equiv.) and DMAP (1.03 g, 8.428 mmol, 2 equiv.) were added to the above mixture at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was quenched by the addition of water (50 mL) at room temperature. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, CHCN / IPA (1:1) (0.1% TFA) in water, 40% to 90% gradient in 20 min; detector, ELSD. CHCN was removed from the fractions under reduced pressure and basified to pH 8 with saturated NaHCO3 (aq). The aqueous layer was extracted with n-heptane (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This afforded 1,4,5-tris(decanoyloxy)-3-[3-(dimethylamino)propoxy]pentan-2-yldecanoate (0.561 g, 8.47%) as a yellow oil. LCMS: (ES, m / z): 855 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ:5.412-5.135 (m, 2H), 4.491-4.305 (m, 2H), 4.295-4.032 (m, J = 12.1, 6.4 Hz, 2H), 3.753-3.373 (m, 3H), 2.591-2.061 (m, 16H), 1.853-1.496 (m, 10H), 1.274 (d, J = 6.1 Hz, 48H), 0.878 (t, J = 6.7 Hz, 12H). Example 10. Synthesis of 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (L-10) [ka]

[0290] Synthesis of (4-bromobut-2-yn-1-yl)diethylamine HBr salt [ka]

[0291] To a 250 mL round-bottom flask was added 4-(diethylamino)but-2-yn-1-ol (11 g, 78.014 mmol, 1 eq.) and DCM (110 mL) at room temperature under a nitrogen atmosphere. Then, a solution of PBr3 (63.26 g, 233.688 mmol, 3 eq.) in DCM (100 mL) was added with stirring at 0 °C for 15 min. The mixture was stirred at room temperature for 4 h. LCMS showed the reaction was complete. The reaction was then quenched by the addition of 300 mL of Na2CO3 (saturated aqueous solution). The resulting solution was extracted with 3 × 100 mL of DCM. The organic layers were combined. The organic phase was washed with 1 × 150 mL of Na2CO3 (saturated aqueous solution) and 1 × 150 mL of brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with ethyl acetate / hexane (83 / 17) to give the product (4-bromobut-2-yn-1-yl)diethylamine HBr salt (7.2 g, 32.61%) as a colorless solid.

[0292] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine [ka]

[0293] To a 250 mL three-necked round-bottom flask, under a nitrogen atmosphere, bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol HBr salt (6.9 g, 50.707 mmol, 1.5 equiv.) and toluene (80 mL) were added at room temperature. To this was added NaH (8.11 g, 338.050 mmol, 10 equiv., 60%) at room temperature. The mixture was stirred at room temperature for 1 hour. To another 100 mL round-bottom flask, (4-bromobut-2-yn-1-yl)diethylamine (6.9 g, 33.805 mmol, 1 equiv.), Na2CO3 (10.75 g, 101.415 mmol, 3 equiv.), and toluene (100 mL) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was added to the above reaction mixture. The resulting mixture was stirred at 80 °C overnight. LCMS indicated that the reaction was complete. The reaction was then quenched by the addition of 100 mL of 5% citric acid (aqueous solution). The resulting solution was extracted with 3 × 100 mL of ethyl acetate. The organic layers were combined. The organic phase was washed with 1 × 200 mL of Na2CO3 (saturated aqueous solution) and 1 × 200 mL of brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with ethyl acetate / hexane (3 / 2) to give the product {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine (2.66 g, 30.73%) as a pale red oily liquid.

[0294] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}diethylamine [ka]

[0295] To a 250 mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was added {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine (2.65 g, 11.253 mmol, 1 equiv.), MeOH (40 mL), and Pd / C (10%, 1.2 g). The flask was evacuated and flushed with nitrogen three times, then flushed with hydrogen. The mixture was hydrogenated under a hydrogen atmosphere (30 psi) at room temperature for 18 hours. LCMS indicated the reaction was complete. The mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure to afford {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}diethylamine (1 g, 37.31%) as a yellow oil.

[0296] Synthesis of 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride [ka]

[0297] To a 250 mL three-necked round-bottom flask was added (4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyldiethylamine 950 mg, 9.736 mmol, 1 equiv) and THF (5 mL) at room temperature under a nitrogen atmosphere. To this was added HCl (6 M, 40 mL) dropwise over 15 minutes at 0°C. The mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated to give 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride (700 mg, 88.64%).

[0298] Synthesis of 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) [ka]

[0299] To a 150 mL three-necked round-bottom flask were added 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride (700 mg, 3.579 mmol, 1 equiv.), capric acid (3.39 g, 19.684 mmol, 5.5 equiv.), DMAP (0.87 g, 7.158 mmol, 2 equiv.), EDCI (4.12 g, 21.474 mmol, 6 equiv.), and ACN (21 mL) at room temperature. The mixture was stirred at room temperature for 16 hours. The reaction solution was depressurized, concentrated in vacuo, and 200 mL of DCM was added. The mixture was washed with 5% citric acid solution (3 × 100 mL), then three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by Flash-Prep-HPLC under the following conditions: C18 silica gel column; mobile phase, A: 0.05% TFA in water / B: CH3CN (0% CH3CN increased to 95% within 15 min), eluent was collected (gradient: A: 0.05% TFA B: CH3CN = 15 / 85); detector, ELSD. The organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CO3 (aq). The aqueous layer was extracted with heptane (3 x 100 mL). The resulting mixture was concentrated under reduced pressure. This gave 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (0.5145 g, 25.84%). LCMS: (ES, m / z): 896.7 [M+1] + H-NMR (300 MHz, CDCl3) δ:5.360-5.189 (m, 2H), 4.434-4.357 (m, 2H), 4.163-4.101 (m, 2H), 3.658-3.502 (m, 3H), 2.562-2.491 (m, 4H), 2.452-2.409(m, 2H), 2.352-2.245 (m, 8H), 1.606-1.463 (m, 12H), 1.266 (s,48H), 1.044-0.977 (m, 6H), 0.900-0.856 (m, 12H). Example 11. Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl 3-(4-propylphenyl)propanoate (L-11) [ka]

[0300] Synthesis of 3-(4-propylphenyl)acrylic acid [ka]

[0301] To a 40 mL vial, benzaldehyde, 4-propyl- (10 g, 67.474 mmol, 1 equiv.), pyridine (2.67 mL, 33.737 mmol, 0.5 equiv.), and malonic acid (7.72 g, 74.221 mmol, 1.1 equiv.) were added at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The mixture was allowed to cool to room temperature. The precipitated solid was collected by filtration and washed with 3 × 60 mL of water and 3 × 60 mL of methyl t-butyl ether / heptane (2:1). After filtration, the resulting solid was dried under infrared light. This afforded (2E)-3-(4-propylphenyl)prop-2-enoic acid (9.5 g, 72.90%) as a white solid.

[0302] Synthesis of 3-(4-propylphenyl)propanoic acid [ka]

[0303] To a 250 mL round-bottom flask was added (2E)-3-(4-propylphenyl)prop-2-enoic acid (9.5 g, 49.936 mmol, 1 equiv.), EA (95 mL), MeOH (95 mL), and Pd / C (4.75 g, 46.984 mmol) at room temperature. The resulting mixture was stirred under a hydrogen atmosphere at room temperature for 12 hours. After filtration, the filtrate was concentrated under reduced pressure. This afforded 3-(4-propylphenyl)propanoic acid (8.58 g, 88.48%) as a white solid. LCMS: (ES, m / z): 193 [M+1] + .

[0304] Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl 3-(4-propylphenyl)propanoate [ka]

[0305] To a 40 mL vial, 3-(4-propylphenyl)propanoic acid (4.46 g, 23.177 mmol, 5.5 equiv.), 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetrol (1 g, 4.214 mmol, 1.00 equiv.), DMAP (1.03 g, 8.428 mmol, 2 equiv.), EDCI (6.47 g, 33.712 mmol, 8 equiv.), and DCM (10 mL) were added at room temperature. The resulting mixture was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase HP-flash chromatography using the following conditions: column, XSelect CSH Prep C18 5 μm; mobile phase, B: CH3CN, A: water (0.1% TFA), gradient 50% to 95% in 15 min; flow rate: 50 mL / min; detector, ELSD. The organic solvent was removed under reduced pressure and the mixture was basified to pH 8 with saturated Na2CO3 (aq). The aqueous layer was extracted with heptane (3 × 100 mL). The resulting mixture was concentrated under reduced pressure. This afforded 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl 3-(4-propylphenyl)propanoate (0.5574 g, 13.33%) as a yellow oil. LCMS: (ES, m / z): 934.6 [M+1] + . 1H NMR (300 MHz, chloroform-d) δ 7.070 (s, 16H), 5.438 (t, J = 5.4 Hz, 1H), 5.281 (d, J= 5.4 Hz, 1H), 5.075 (d, J = 5.9 Hz, 1H), 4.304-4.172 (m, 1H), 3.775 (s, 1H), 3.469-3.308 (m, 2H), 3.299-3.165 (m, 2H), 2.923-2.807 (m, 8H), 2.706-2.148 (m, 24H), 1.827 (m, 2H), 1.657-1.537 (m, 8H), 0.974-0.873 (m, 12H). Example 12. Synthesis of 1,11-bis(pentadecan-8-yl) 6-[3-(dimethylamino)propoxy]undecandioate (L-12) [ka]

[0306] Synthesis of trideca-1,12-dien-7-ol [ka]

[0307] To a 500 mL three-necked round-bottom flask was added Mg (16.40 g, 674.955 mmol, 5 equiv) and THF (50 mL) at room temperature. 6-Bromohex-1-ene (55.03 g, 337.478 mmol, 2.5 equiv) was added dropwise over minutes at 55 °C. The resulting mixture was stirred at 55 °C for an additional 1 h. Ethyl formate (10 g, 134.991 mmol, 1 equiv) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for an additional 2 h. The reaction was quenched with saturated NH4Cl (aq) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with heptane / EA (10:1) to give trideca-1,12-dien-7-ol (23.5 g, 88.67%) as a pale yellow oil.

[0308] Synthesis of N,N-dimethyl-3-(trideca-1,12-dien-7-yloxy)propan-1-amine [ka] To a 500 mL three-necked round-bottom flask was added trideca-1,12-dien-7-ol (10 g, 50.934 mmol, 1 equiv.) and toluene (200 mL) at room temperature. To the mixture was added NaH (4.00 g, 166.554 mmol, 3.27 equiv.) in portions over 10 minutes at 0° C. The resulting mixture was further stirred at 85° C. overnight. To the mixture was added (3-chloropropyl)dimethylamine hydrochloride (15.28 g, 101.868 mmol, 2 equiv.) in portions at 80° C. The resulting mixture was further stirred at 80° C. for 8 hours. The mixture was allowed to cool to room temperature. The resulting mixture was diluted with water (300 mL). The resulting mixture was extracted with EtOAc (2×200 mL). The combined organic layers were washed with water (2×200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (10:1) to give N,N-dimethyl-3-(trideca-1,12-dien-7-yloxy)propan-1-amine (12.5 g, 87.19%) as a pale yellow oil.

[0309] Synthesis of 6-(3-(dimethylamino)propoxy)undecane dihydrochloride [ka]

[0310] To a 1000 mL four-neck round-bottom flask, N,N-dimethyl-3-(trideca-1,12-dien-7-yloxy)propan-1-amine (7.6 g, 27.000 mmol, 1 equiv.) and AcOH (140 mL) were added at room temperature. To the above mixture, KMnO (17 g, 107.573 mmol, 3.98 equiv., in 700 mL of HO) was added dropwise at room temperature. The resulting mixture was stirred at 15 °C for an additional 3 h. The reaction was quenched by the addition of NaSO (17 g) and NaHSO (3 g) at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, AQ-C18 silica gel; mobile phase, MeCN (0.1% HCl) in water, 10% to 35% gradient in 16 min; detector, ELSD. This gave 6-(3-(dimethylamino)propoxy)undecane dihydrochloride (2.9 g, 30.35%) as a pale yellow oil.

[0311] Synthesis of 1,11-bis(pentadecan-8-yl)6-[3-(dimethylamino)propoxy]undecane dioate [ka]

[0312] To a 100 mL round-bottom flask, 6-(3-(dimethylamino)propoxy)undecane dihydrochloride (850 mg, 2.402 mmol, 1 equiv.), pentadecan-8-ol (1.4 g, 6.129 mmol, 2.55 equiv.), ACN (17 mL), TEA (728 mg, 7.206 mmol, 3.0 equiv.), and DMAP (100 mg, 0.819 mmol, 0.34 equiv.) were added at room temperature. EDCI (1.4 g, 7.303 mmol, 3.04 equiv.) was added in portions at room temperature. The resulting mixture was stirred at room temperature for an additional 16 hours. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with saturated Na2CO3 (2 × 20 mL), MeOH / HO (4:1, 5 × 20 mL), HO (3 × 20 mL), and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN (0.1% TFA) in water, 35% to 70% gradient in 10 min; detector, MS. The product fractions were concentrated under vacuum to remove ACN, and saturated Na2CO3 (30 mL) was added. The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with MeOH / HO (4:1, 5 × 20 mL), HO (3 × 20 mL), and brine (20 mL), and dried over anhydrous Na2SO4. 4で The mixture was dried. After filtration, the filtrate was concentrated under reduced pressure to give 1,11-bis(pentadecan-8-yl) 6-[3-(dimethylamino)propoxy]undecandioate (680 mg, HPLC: 95.1%, yield: 34.24%) as a pale yellow oil. LCMS: (ES, m / z): 738.7 [M+H] + ; 1H NMR:(400 MHz, chloroform-d) δ 4.892-4.830 (m, 2H), 3.435 (t, J = 6.4 Hz, 2H), 3.208-3.183 (m, 1H), 2.363 (br, 2H), 2.301-2.245 (m, 10H), 1.761-1.726 (m, 2H), 1.709-1.693 (m, 4H), 1.658-1.586 (m, 14H), 1.511-1.412 (m, 42H), 0.877 (t, J = 6.8 Hz, 12H). Example 13. Synthesis of di(heptadecan-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate (L-13) [ka]

[0313] Synthesis of nona-1,8-dien-5-ol [ka]

[0314] To a stirred solution of Mg (32.81 g, 1349.910 mmol, 5.0 equiv.) and I2 (200 mg, 0.788 mmol) in THF (100 mL) was added 4-bromo-1-butene (91.12 g, 674.955 mmol, 2.5 equiv.) dropwise at room temperature under a nitrogen atmosphere. The mixture was stirred at 55 °C for 1 h under a nitrogen atmosphere. Ethyl formate (20 g, 269.982 mmol, 1 equiv.) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for an additional 2 h. The reaction was quenched with saturated NH4Cl(aq) (400 mL, 20 V) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 200 mL, 20 V). The combined organic layers were washed with brine (200 mL, 10 V) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (volume ratio) (gradient from 100:0 to 90:10, and the product eluent PE / EA = 92 / 8 was collected). A sample was analyzed by TLC (PE / EA = 10:1) to give nona-1,8-dien-5-ol (26.0 g, 64.01%) as a pale yellow oil.

[0315] Synthesis of N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine [ka]

[0316] To a stirred solution of nona-1,8-dien-5-ol (26 g, 185.415 mmol, 1 equiv) in toluene (500 mL, 20 V) was added NaH (22.25 g, 556.245 mmol, 3.0 equiv, 60%) in portions at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at 80 °C for 8 h under a nitrogen atmosphere. To the above mixture was added (3-chloropropyl)dimethylamine hydrochloride (58.68 g, 371.428 mmol, 2.0 equiv) at 80 °C. The resulting mixture was stirred at 80 °C for an additional 16 h. The reaction was quenched with saturated NH4Cl(aq) (500 mL, 20 V) at room temperature. The resulting mixture was extracted with EtOAc (2 × 250 mL, 20 V). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with CHCl / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the product eluent CHCl / MeOH = 90:10 was collected). A sample was taken for TLC analysis (CHCl / MeOH = 10:1) to give N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine (35 g, 74.96%) as a pale yellow oil.

[0317] Synthesis of 4-(3-(dimethylamino)propoxy)heptanedioic acid [ka]

[0318] To a stirred solution of N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine (12 g, 53.244 mmol, 1 equiv.) in AcOH (240 mL, 20 V) was added KMnO (33.66 g, 212.976 mmol, 4.0 equiv.) dissolved in water (1.2 L, 100 V) at room temperature. The resulting mixture was stirred at room temperature for 1 h. The reaction was quenched at room temperature with solid NaSO (33.66 g, 212.976 mmol, 4.0 equiv.) and stirred at room temperature for 30 min. The resulting mixture was concentrated under reduced pressure. The residue was purified by slurrying with DMF (1 L, 80 V). The crude product was used directly in the next step without further purification.

[0319] Synthesis of di(heptadecan-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate [ka]

[0320] To a stirred solution of 4-(3-(dimethylamino)propoxy)heptanedioic acid (6.00 g, 22.961 mmol, 1.0 equiv), DMAP (5.61 g, 45.922 mmol, 2.00 equiv), and 9-heptadecanol (14.72 g, 57.402 mmol, 2.50 equiv), EDCI (13.20 g, 68.883 mmol, 3.00 equiv) was added at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched by the addition of water (1.0 L, 80V) at room temperature. The resulting mixture was extracted with EtOAc (2 x 300 mL, 60V). The combined organic layers were washed with brine (300 mL, 30V) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio). (Gradient: 100:0 to 90:10, and product eluent: DCM / MeOH = 95:5 was collected.) A sample was analyzed by TLC (DCM:MeOH = 10:1) to give di(heptadecan-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate (601.1 mg, 3.55%) as a yellow oil. LCMS: (ES, m / z): 738.7 [M+H] + ; 1 H NMR (400 MHz, chloroform-d) δ 4.874-4.843 (m, 2H), 3.486-3.455 (m, 2H), 3.314-3.285 (m, 1H), 2.480 (br, 2H), 2.387-2.293 (m, 10H), 1.818-1.752 (m, 6H), 1.512-1.499 (m, 8H), 1.296-1.258 (m, 48H), 0.894-0.878 (m, 12H). Example 14. Synthesis of (3-{[1,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2-yl]oxy}propyl)dimethylamine (L-14) [ka]

[0321] Synthesis of 2,2-dimethyl-1,3-dioxan-5-ol [ka]

[0322] A 500 mL round-bottom flask was charged with 2,2-dimethyl-1,3-dioxan-5-one (15 g, 115.258 mmol, 1 equiv.) and THF (150 mL) at room temperature under a N atmosphere. To this was added LiAlH (4.37 g, 115.258 mmol, 1.0 equiv.) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction was quenched by the addition of water (4.5 mL), 4.5 mL (wt. % 15% NaOH), and 13.5 mL of water at 0 °C. The resulting solution was diluted with 300 mL of THF, and to this was added NaSO (30 g). The mixture was warmed to room temperature and stirred for 15 min. The resulting mixture was filtered, and the filter cake was washed with EA (2 × 100 mL). The filtrate was concentrated under reduced pressure to give 2,2-dimethyl-1,3-dioxan-5-ol (12 g, 78.78%) as a colorless oil.

[0323] Synthesis of {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine [ka]

[0324] A 100 mL round-bottom flask was charged with 2,2-dimethyl-1,3-dioxan-5-ol (5 g, 37.833 mmol, 1 eq) and DMF (100 mL) at room temperature under a N atmosphere. To this was added NaH (2.27 g, 94.582 mmol, 2.5 eq) at 0° C. The mixture was stirred at room temperature for 1 hour. To this was added (3-chloropropyl)dimethylamine (5.52 g, 45.400 mmol, 1.2 eq, in 10 mL of DMF) at room temperature. The mixture was stirred at 50° C. for 16 hours. The reaction was quenched by the addition of NH4Cl (sat., 50 mL) at 5° C. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography, washed and eluted with (MeOH / DCM=1 / 1) to give {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine (2.1 g, 25.54%) as a yellow solid.

[0325] Synthesis of 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride [ka]

[0326] A 40 mL vial was charged with {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine (1.0 g, 4.602 mmol, 1 equiv.) and DCM (20 mL) at room temperature under a N atmosphere. To this was added HCl (gas) in 1,4-dioxane (4 M, 5 mL) at 0 °C. The mixture was stirred at 0 °C for 1 hour. The resulting mixture was concentrated in vacuo to give 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride (780 mg, crude) as a yellow oil. The crude product was used directly in the next step without further purification.

[0327] Synthesis of heptadecan-9-yl 4-nitrophenyl carbonate [ka]

[0328] A 250 mL round-bottom flask was charged with 9-heptadecanol (5 g, 19.495 mmol, 1 equiv.), DMAP (0.95 g, 7.798 mmol, 0.4 equiv.), TEA (3.95 g, 38.990 mmol, 2 equiv.), and THF (100 mL) at room temperature under a N atmosphere. 4-Nitrophenyl carbonochloridate (4.32 g, 21.445 mmol, 1.1 equiv. in 20 mL of THF) was added at 0 °C. The mixture was stirred at 70 °C for 2 hours. The resulting mixture was diluted with THF (100 mL). The resulting mixture was filtered, and the filter cake was washed with THF (2 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with Hep / EA (volume ratio). (Gradient from 100:0 to 90:10, and product eluent Hep / EA = 97 / 3 was collected.) A sample was analyzed by TLC (Hep:EA = 10:1) to give heptadecan-9-yl 4-nitrophenyl carbonate (3.5 g, 41.52%) as a pale yellow oil.

[0329] Synthesis of (3-{[1,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2-yl]oxy}propyl)dimethylamine [ka]

[0330] A 250 mL round-bottom flask was charged with 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride (0.79 g, 3.736 mmol, 0.45 equiv.), DMAP (0.23 g, 1.868 mmol, 0.5 equiv.), TEA (1.51 g, 14.944 mmol, 4 equiv.), and DMF (16 mL) at room temperature under a N atmosphere. To this was added heptadecan-9-yl 4-nitrophenyl carbonate (3.5 g, 8.302 mmol, 1 equiv. in 30 mL of DMF) at room temperature. The mixture was stirred at 80 °C for 16 h. The resulting mixture was diluted with EA (300 mL) and washed with 5 wt% citric acid (1 × 50 mL), NaHCO (1 × 50 mL), and water (2 × 150 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio) (gradient: 100:0 to 90:10, and the product eluent DCM / MeOH = 97:3 was collected). A sample was analyzed by TLC. (DCM:MeOH = 10:10.2) gave (3-{[1,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2-yl]oxy}propyl)dimethylamine (800 mg) as a yellow oil. The product was dissolved in n-heptane (100 mL). The n-heptane phase was washed with MeOH / HO (4:1) (2 × 10 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give (3-{[1,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2-yl]oxy}propyl)dimethylamine (584.1 mg, 9.07%) as a yellow oil. LCMS: (ES, m / z): 742.18 [M+1] + ; 1H NMR (400 MHz, chloroform-d) δ 4.732-4.671 (m, 2H), 4.272-4.177 (m, 4H), 3.781-3.768 (m, 1H), 3.677-3.645 (m, 2H), 2.406 (s, 2H), 2.273 (s, 6H), 1.799-1.766 (m, 2H), 1.623-1.517 (m, 8H), 1.338-1.285 (m, 48H), 0.918-0.855 (m, 12H). Example 15. Synthesis of 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propyl]nonanedioate (L-15) [ka]

[0331] Synthesis of methyl 3-(2,6-dioxocyclohexyl)propanoate [ka]

[0332] A 250 mL round-bottom flask was charged with 1,3 cyclohexanedione (25 g, 222.959 mmol, 1 equiv.), DMF (50 mL), CsCO (43.59 g, 133.775 mmol, 0.6 equiv.), and methyl acrylate (23.03 g, 267.551 mmol, 1.2 equiv.). The reaction mixture was stirred at 80 °C for 12 h. The reaction was then quenched by adding 200 mL of water / ice. The pH of the solution was adjusted to 6 with HCl (1 mol / L). The resulting solution was extracted with 3 × 200 mL of ethyl acetate, and the organic layers were combined. The resulting mixture was washed with 200 mL of NaCl. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This afforded methyl 3-(2,6-dioxocyclohexyl)propanoate (35 g, crude) as a yellow oil.

[0333] Synthesis of 5-oxononanedioic acid [ka]

[0334] A 250 mL round-bottom flask was charged with methyl 3-(2,6-dioxocyclohexyl)propanoate (35 g, 176.573 mmol, 1 equiv.) and HCl (1 M) (70 mL). The reaction mixture was stirred at 110° C. for 12 h. The resulting mixture was concentrated in vacuo. The residue was dissolved in 200 mL of MTBE and stirred for 1 h. The solid was collected by filtration. This afforded 5-oxononanedioic acid (15 g, 42.01%) as a brown solid.

[0335] Synthesis of pentadecan-8-ol [ka]

[0336] A 1.0 L round-bottom flask was charged with 8-pentadecanone (15 g, 66.253 mmol, 1 equiv.), THF (450 mL), MeOH (150 mL), and NaBH (7.52 g, 198.759 mmol, 3.0 equiv.). The reaction mixture was stirred at 20 °C for 3 h. The reaction mixture was poured into 500 mL of ice water. The resulting mixture was extracted with EA (3 × 500 mL). The combined organic layers were washed with water (3 × 100 mL) and NaCl (100 mL, aq.) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give pentadecan-8-ol (10 g, crude) as a white solid.

[0337] Synthesis of 1,9-bis(pentadecan-8-yl)5-oxononanedioate [ka]

[0338] A 250 mL round-bottom flask was charged with 5-oxononanedioic acid (5.0 g, 24.727 mmol, 1 equiv.), pentadecan-8-ol (10.17 g, 44.509 mmol, 1.8 equiv.), DCM (100 mL), DMAP (3.02 g, 24.727 mmol, 1 equiv.), and EDCI (10.43 g, 54.399 mmol, 2.2 equiv.). The reaction mixture was stirred at room temperature for 12 h. The resulting mixture was diluted with DCM (100 mL). The reaction was quenched at 5 °C by the addition of citric acid (5% aqueous solution) (60 mL) and washed with 2 × 30 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with Hep / EA (volume ratio). (A gradient from 100:0 to 90:10 was run, and the product eluent Hep / EA = 95:5 was collected.) The sample was analyzed by TLC (Hep:EA = 10:10.2). The resulting mixture was concentrated under reduced pressure to give 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (10.2 g, 66.21%) as a pale yellow oil.

[0339] Synthesis of 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate [ka]

[0340] A 250 mL round-bottom flask was charged with 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (5.5 g, 8.828 mmol, 1 equiv.) and THF (44 mL) at room temperature under a N atmosphere. To this was added [3-(chloromagnesio)propyl]dimethylamine (88.28 mL, 88.280 mmol, 10 equiv.) dropwise at −60° C. The mixture was stirred at −60° C. for 2 h. The reaction was quenched by the addition of NH4Cl(aq) (80 mL) at 5° C. and washed with 2 × 40 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (volume ratio) (gradient: 100:0 to 70:30, and the product eluent: DCM / MeOH = 82 / 18 was collected). The sample was analyzed by TLC (DCM:MeOH=2:10.1). The resulting mixture was concentrated under reduced pressure to give 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.7 g, 43.07%) as a pale yellow oil.

[0341] Synthesis of 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propylidene]nonanedioate [ka] Et3SiH (10 equivalents), BF 3. Et2O (10 equivalents), DCM (1V), 45℃, 2 hours, 62.51%

[0342] A 40 mL vial was charged with 1,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.5 g, 3.520 mmol, 1 equiv.) and DCM (5 mL) at room temperature under a N atmosphere. To this was added EtSiH (4.09 g, 35.200 mmol, 10 equiv.) and BF·EtO (5.00 g, 35.200 mmol, 10 equiv.) at room temperature. The mixture was stirred at 45 °C for 2 h. The resulting mixture was diluted with DCM (100 mL). The reaction was quenched by the addition of NaHCO (aq.) (25 mL) at 5 °C and washed with 2 × 25 mL of water. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (vol. / vol.). (Gradient from 100:0 to 90:10, and product eluent DCM / MeOH = 94 / 6 was collected.) A sample was taken for TLC analysis (DCM:MeOH = 10:10.15). The resulting mixture was concentrated under reduced pressure to give 1,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propylidene]nonanedioate (1.6 g, 62.51%) as a pale yellow oil.

[0343] Synthesis of 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propyl]nonanedioate (L-15) [ka]

[0344] A 100 mL vial was charged with 1,9-bis(pentadecan-8-yl)5-[3-(dimethylamino)propylidene]nonanedioate (1.6 g, 2.312 mmol, 1 equiv.) and EtOH (32 mL). Pd / C (0.49 g, 0.462 mmol, 0.2 equiv., 10%) was added at room temperature. The mixture was stirred at room temperature under an H atmosphere (30 psi) for 2 hours. The resulting mixture was filtered, and the filter cake was washed with EA (2 × 32 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (volume ratio) (gradient: 100:0 to 90:10, and the product eluate DCM / MeOH = 95:5 was collected). A sample was analyzed by TLC (DCM:MeOH = 10:10.2). The filtrate was concentrated under reduced pressure. The residue was dissolved in n-heptane (160 mL, 100 V). The n-heptane phase was then washed with MeOH / HO (4:1) (2 × 32 mL, 10 V), MeCN / HO (4:1) (2 × 32 mL, 10 V), water (32 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give 1,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]nonanedioate (1.0640 g, 66.31%) as a pale yellow oil. LCMS: (ES, m / z): 694.9 [M+H] + . 1 H NMR (400 MHz, chloroform-d) δ4.934-4.832 (m, 2H), 2.333-2.172 (m, 12H), 1.681-1.564 (m, 4H), 1.558-1.477 (m, 8H), 1.471-1.399 (m, 2H), 1.389-1.203 (m, 47H), 0.896 (t, J = 6.8 Hz, 12H). Example 16. Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris(dodecanoyloxy)pentan-2-yldodecanoate (L-16) [ka]

[0345] To a 40 mL vial, 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetrol (1 g, 4.214 mmol, 1 equiv.), lauric acid (4.64 g, 23.177 mmol, 5.5 equiv.), EDCI (4.85 g, 25.284 mmol, 6 equiv.), DMAP (1.03 g, 8.428 mmol, 2 equiv.), DIEA (4.36 g, 33.712 mmol, 8 equiv.), and ACN (10 mL) were added at room temperature. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography using the following conditions: column, XSelect CSH Prep C 18 5 μm; mobile phase, B: CH3CN, A: water (0.1% TFA), 50% to 95% gradient in 15 min; flow rate: 50 mL / min; detector, ELSD. CH3CN was removed from the fractions under reduced pressure and basified to pH 8 with saturated NaHCO3 (aq). The aqueous layer was extracted with n-heptane (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This afforded 3-[3-(dimethylamino)propoxy]-1,4,5-tris(dodecanoyloxy)pentan-2-yldodecanoate (0.6852 g, 20.24%) as a yellow oil. LCMS: (ES, m / z): 967 [M+1] + . 1H NMR (300 MHz, chloroform-d) δ 5.469 (t, J= 5.5 Hz, 1H), 5.381-5.255 (m, 1H), 5.225-5.110 (m, 1H), 4.373-4.261 (m, 1H), 4.073-3.925 (m, 1H), 3.557-3.353 (m, 4H), 2.456-2.265 (m, 10H), 2.258-2.104 (m, 6H),1.735-1.675 (m, 2H),1.650-1.532 (m, 8H), 1.267 (d, J = 4.9 Hz, 64H), 0.880 (t, J = 6.6 Hz, 12H). Example 17. Synthesis of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (L-17) [ka]

[0346] Synthesis of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid [ka]

[0347] A solution of 5-oxononanedioic acid (4 g, 19.782 mmol, 1 equiv.) in DCM (60 mL) was treated with DMAP (0.48 g, 3.956 mmol, 0.2 equiv.) and pentadecan-8-ol (2.94 g, 12.858 mmol, 0.65 equiv.) under a nitrogen atmosphere at 25° C., followed by the addition of EDCI (4.17 g, 21.760 mmol, 1.1 equiv.) in portions at 25° C. The resulting mixture was stirred at 25° C. under a nitrogen atmosphere for 18 hours. The mixture was acidified to pH 5 with 0.05 M HCl. The resulting mixture was washed with 1×50 mL of 0.05 M HCl and 1×100 mL of brine. The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 15 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 160 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was added to the column, and then the reaction mixture was absorbed onto the dry silica gel in the final step. The product was purified using CombiFlash. It was eluted with PE / EA (2:1) (gradient from 10:1 to 2:1, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (PE / EA = 1:1), and the acceptable product was combined. This gave 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (4 g, 49.01%) as a pale yellow oil. LCMS: (ES, m / z): 413.2 [M+H] + . 1 H NMR:(400 MHz, chloroform-d) δ4.939-4.830 (m, 2H), 2.560-2.464 (m, 4H), 2.409 (t, J = 7.213 Hz, 2H), 2.332 (t, J= 7.242 Hz, 2H), 1.978-1.862 (m, 4H), 1.577-1.462 (m, 4H), 1.347-1.211 (m, 20H), 0.934-0.863 (m, 6H).

[0348] Synthesis of 2-(bromomethyl)-octahydro-1H-indene [ka]

[0349] A solution of octahydro-1H-inden-2-ylmethanol (16 g, 103.726 mmol, 1 equiv.) in DCM (300 mL) was treated with triphenylphosphine (40.81 g, 155.589 mmol, 1.5 equiv.) under a nitrogen atmosphere at 0 °C, followed by the addition of carbon tetrabromide (51.60 g, 155.589 mmol, 1.5 equiv.) in portions at 0 °C. The resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 18 h. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL), and 15 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w. / w.) was added. The mixture was concentrated to zero under vacuum, maintaining the temperature below 35 °C. 160 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was loaded onto the column, followed by the final step of preparing dried silica gel to absorb the reaction mixture. The product was purified using CombiFlash. It was eluted with PE / EA (100:1) (gradient from 1:0 to 30:1, collecting every 200 ± 10 mL). Samples were taken for TLC analysis (PE / EA = 20:1), and the acceptable products were combined. This afforded 2-(bromomethyl)-octahydro-1H-indene (22 g, 97.67%) as a colorless oil. 1 H NMR: (400 MHz, chloroform-d) δ 3.480-3.378 (m, 2H), 2.663-2.368 (m, 1H), 2.072-1.691 (m, 4H), 1.644-0.887 (m, 10H).

[0350] Synthesis of 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene [ka]

[0351] A mixture of NaH (5.80 g, 144.898 mmol, 2.3 equiv, 60%) in DMSO (200 mL) was stirred at 25° C. for 1 hour under a nitrogen atmosphere. To the above mixture, toluenesulfonylmethyl isocyanide (TosMIC) (12.3 g, 62.999 mmol, 1.00 equiv) and TBAI (2327 mg, 6.300 mmol, 0.1 equiv) were added portionwise at 25° C. The resulting mixture was stirred at 25° C. for an additional 3 hours. To the above mixture, 2-(bromomethyl)-octahydro-1H-indene (21.89 g, 100.798 mmol, 1.6 equiv) in DMSO (100 mL) was added dropwise over 20 minutes at 25° C. The resulting mixture was stirred at 25° C. for an additional 18 hours. The reaction was quenched with saturated NH4Cl (aq) at 25° C. The resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (3 × 300 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 50 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 500 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was loaded onto a column, after which the reaction mixture was absorbed onto the dry silica gel in the final step. The product was purified using CombiFlash. Elution was performed with PE / EA (20:1) (gradient from 50:0 to 10:1, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (PE / EA=10:1) and the acceptable products were combined to give 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene (9 g, 30.54%) as a yellow oil. LCMS: (ES, m / z): 468.4 [M+H] + . 1H NMR: (400 MHz, chloroform-d) δ 7.932-7.853 (m, 2H), 7.480-7.392 (m, 2H), 2.512 (s, 3H), 2.273-1.769 (m, 14H), 1.567-1.407 (m, 8H), 1.390-1.068 (m, 12H).

[0352] Synthesis of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one [ka]

[0353] A solution of 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene (9 g, 19.243 mmol, 1 equiv.) in DCM (60 mL) was stirred at 25 °C. To the above mixture, HCl (5 mL, 4 M in MeOH) was added dropwise at 25 °C. The resulting mixture was stirred at 25 °C for an additional 3 h. Brine (100 mL) was added. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine (2 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 30 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w. / w.) was added. The mixture was concentrated under vacuum to zero, maintaining the temperature below 35°C. 300 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was loaded onto the column, and then, in the final step, the dry silica gel was prepared by absorbing the reaction mixture. The product was purified using CombiFlash. It was eluted with PE / EA (30:1) (gradient from 50:0 to 10:1, collecting every 200 ± 10 mL). Samples were taken for TLC analysis (PE / EA = 10:1), and the acceptable products were combined. The residue was purified by silica gel column chromatography eluting with PE / EA (30:1) to give 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one (5 g, 85.90%) as a pale yellow solid. LCMS-: (ES, m / z): 303.4 [M+H] + . 1 H NMR: (400 MHz, chloroform-d) δ 2.668-2.308 (m, 6H), 2.039-1.690 (m, 8H), 1.633-0.949 (m, 20H).

[0354] Synthesis of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol [ka]

[0355] A solution of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one (5 g, 16.529 mmol, 1 equiv.) in tetrahydrofuran (50 mL) was treated with NaBH (1.38 g, 36.364 mmol, 2.2 equiv.) at 0 °C, followed by dropwise addition of methanol (50 mL) at 0 °C. The resulting mixture was stirred at 25 °C for 3 h. The reaction was quenched by adding saturated NH Cl (aq.) (20 mL) at 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with brine (2 × 200 mL) and dried over anhydrous Na SO . After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL), and 10 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w. / w.) was added. The fractions were concentrated under vacuum while maintaining the temperature below 35°C. 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was loaded onto the column, and then, in the final step, the dry silica gel was prepared by absorbing the reaction mixture. The product was purified using CombiFlash. It was eluted with PE / EA (30:1) (gradient from 50:0 to 20:1, collecting every 200 ± 10 mL). Samples were taken for TLC analysis (PE / EA = 20:1), and the acceptable products were combined. This gave 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol (4.6 g, 91.39%) as a colorless oil. LCMS: (ES, m / z): 287.3 [M-18+H] + . 1 H NMR: (400 MHz, chloroform-d) δ 3.698-3.574 (m, 1H), 2.394-2.044 (m, 2H), 1.970-1.701 (m, 8H), 1.605-1.090 (m, 25H).

[0356] Synthesis of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-oxononanedioate [ka]

[0357] A solution of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (6 g, 14.542 mmol, 1 equiv.) in DCM (120 mL) was treated with 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol (5.31 g, 17.450 mmol, 1.2 equiv.) and DMAP (1.79 g, 14.620 mmol, 1 equiv.) under a nitrogen atmosphere at 25 °C, followed by the addition of EDCI (3.64 g, 18.913 mmol, 1.3 equiv.) in portions at 25 °C. The resulting mixture was stirred at 45 °C under a N atmosphere for 18 h. The resulting mixture was diluted with DCM (120 mL). The combined organic layers were washed with 0.05 M HCl (1 × 100 mL) and brine (1 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 30 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 300 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was added to the column, and then, in the final step, the dry silica gel was prepared by absorbing the reaction mixture. The product was purified using CombiFlash. It was eluted with PE / EA (5:1) (gradient from 20:1 to 4:1, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (PE / EA = 3:1), and the acceptable products were combined. This gave 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-oxononanedioate (3.7 g, 36.40%) as a pale yellow oil. LCMS: (ES, m / z): 721.5 [M+Na] + . 1H NMR:(400 MHz, Chloroform-d) δ5.015-4.907 (m, 1H), 4.913-4.831 (m, 1H), 2.483 (t, J = 7.2 Hz, 4H), 2.320 (t, J= 7.2 Hz, 4H), 2.145-2.005 (m, 1H), 1.984-1.741 (m, 12H), 1.747-1.607 (m, 3H), 1.607-1.414 (m, 3H), 1.408-1.194 (m, 28H), 1.199-1.048 (m, 3H), 0.941-0.839 (m, 6H).

[0358] Synthesis of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate [ka]

[0359] To a stirred solution of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-oxononanedioate (3.5 g, 5.006 mmol, 1 equiv.) in tetrahydrofuran (50 mL) was added dropwise [3-(chloromagnesio)propyl]dimethylamine (75.10 mL, 75.090 mmol, 15 equiv.) at −60° C. under a nitrogen atmosphere. The resulting mixture was stirred at −60° C. for 2 hours under a nitrogen atmosphere. The reaction was quenched by adding saturated NH4Cl(aq) (50 mL) at −60° C. The mixture was acidified to pH 6 with 0.05 M HCl. The resulting mixture was extracted with CHCl (2 × 100 mL). The combined organic layers were washed with brine (1 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 10 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was added to the column, and then, in the final step, the dry silica gel was prepared by absorbing the reaction mixture. The product was purified using CombiFlash. It was eluted with CHCl / MeOH (20:1) (gradient from 50:1 to 10:1, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (CHCl / MeOH = 10:1), and the acceptable products were combined. This gave 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.5 g, 63.51%) as a pale yellow oil. LCMS: (ES, m / z): 786.7 [M+1] + . 1H NMR: (400 MHz, chloroform-d) δ 5.046-4.780 (m, 2H), 3.087-2.944 (m, 2H), 2.758 (s, 6H), 2.439-2.222 (m, 4H), 2.205-2.015 (m, 1H), 1.999-1.750 (m, 10H), 1.744-1.568 (m, 9H), 1.574-1.405 (m, 18H), 1.405-1.043 (m, 33H), 0.946-0.835 (m, 6H).

[0360] Synthesis of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl(5Z)-5-[3-(dimethylamino)propylidene]nonanedioate [ka]

[0361] To a stirred solution of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.4 g, 3.052 mmol, 1 equiv.) and triethylsilane (3.55 g, 30.520 mmol, 10 equiv.) in DCM (50 mL) was added BF3.Et2O (4.33 g, 30.520 mmol, 10 equiv.) dropwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 40 °C under a nitrogen atmosphere for 2 h. The combined organic layers were washed with NaHCO3 (2 × 50 mL), brine (1 × 50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (10 mL) and 10 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) was added. The mixture was concentrated under vacuum to zero fractions while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w / w) was added to the column, and then, in the final step, the dry silica gel was prepared by absorbing the reaction mixture. The product was purified using CombiFlash. It was eluted with CHCl / MeOH (20:1) (gradient from 50:1 to 10:1, collecting every 200 ± 10 mL). A sample was taken for TLC analysis (CHCl / MeOH = 10:1), and the acceptable products were combined. This gave 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl(5Z)-5-[3-(dimethylamino)propylidene]nonanedioate (1.5 g, 63.97%) as a pale yellow oil. LCMS: (ES, m / z): 768.7 [M+H] + . 1 H NMR: (400 MHz, chloroform-d) δ 5.228-5.086 (m, 1H), 5.021-4.786 (m, 2H), 2.563-2.176 (m, 13H), 1.182-1.992 (m, 5H), 1.992-1.773 (m, 8H), 1.773-1.588 (m, 7H), 1.593-1.054 (m, 45H), 1.653-0.658 (m, 7H).

[0362] Synthesis of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (L-17) [ka]

[0363] To a stirred solution of 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl(5Z)-5-[3-(dimethylamino)propylidene]nonanedioate (1.3 g, 1.692 mmol, 1 equiv.) in EtOH (40 mL) was added Pd / C (0.26 g, 2.443 mmol, 1.44 equiv.) at 25° C. under a nitrogen atmosphere. The flask was evacuated and flushed with nitrogen three times, then with hydrogen. The mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The resulting mixture was filtered, and the filter cake was washed with EA (2×100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in heptane (150 mL). The resulting mixture was washed with 2 x 100 mL of ACN / HO (5:1, 50 mL) and 1 x 100 mL of ACN (50 mL). The heptane phase was concentrated under reduced pressure. This afforded 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (0.5205 g, 38.46%) as a pale yellow oil. LCMS: (ES, m / z): 770.7 [M+H] + . 1 H NMR: (400 MHz, chloroform-d) δ 5.008-4.822 (m, 2H), 2.333-2.082 (m, 12H), 1.961-1.734 (m, 8H), 1.726-1.397 (m, 23H), 1.392-1.197 (m, 36H), 1.193-1.005 (m, 4H), 0.935-0.800 (m, 6H). Example 18. Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (L-18) [ka]

[0364] Synthesis of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid [ka]

[0365] To a 500 mL round-bottom flask was added 5-oxononanedioic acid (11 g, 54.400 mmol, 1 equiv.), DCM (200 mL), pentadecan-8-ol (11.80 g, 51.680 mmol, 0.95 equiv.), DMAP (1.33 g, 10.880 mmol, 0.2 equiv.), and EDCI (11.47 g, 59.840 mmol, 1.1 equiv.) at room temperature. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (10:1) to give 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (10 g, 44.55% yield) as a yellow oil.

[0366] Synthesis of methyl 2-(1,3-dihydroinden-2-ylidene)acetate [ka]

[0367] To a 2000 mL three-necked round-bottom flask, methyl 2-(dimethoxyphosphoryl)acetate (103.35 g, 567.485 mmol, 2.5 equiv.) and THF (900 mL) were added at room temperature. To the above mixture, NaH (22.70 g, 567.485 mmol, 2.5 equiv., 60%) was added portionwise at 0° C. The resulting mixture was stirred at 0° C. for an additional 30 minutes. To the above mixture, 2-indanone (30 g, 226.994 mmol, 1 equiv. in 200 mL of THF) was added dropwise at 0° C. The resulting mixture was stirred at room temperature overnight. The reaction was quenched by the addition of saturated NH4Cl(aq) (300 mL) at 0° C. The resulting mixture was extracted with heptane (3×200 mL). The combined organic layers were washed with saturated aqueous Na2CO3 (2 × 50 mL), water (2 × 50 mL), MeOH / HO (4:1, 4 × 50 mL), water (2 × 50 mL), and brine (1 × 50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (98:2) to give methyl 2-(1,3-dihydroinden-2-ylidene)acetate (25.6 g, yield: 59.68%) as an orange oil.

[0368] Synthesis of methyl 2-(octahydro-1H-inden-2-yl)acetate [ka]

[0369] To a solution of methyl 2-(1,3-dihydroinden-2-ylidene)acetate (25 g, 132.819 mmol, 1 equiv.) in 750 mL of AcOH was added Pd / C (10%, 12.5 g) in a 1 L pressure tank reactor. The mixture was hydrogenated at 110 °C under 40 atm of hydrogen pressure for 24 h. The resulting mixture was cooled to room temperature. The reaction mixture was filtered, and the filtrate was concentrated. The resulting mixture was diluted with water (250 mL). The resulting mixture was extracted with heptane (3 × 200 mL). The combined organic layers were washed with water (2 × 500 mL), saturated NaHCO (2 × 300 mL), MeOH (4 × 200 mL), and brine (1 × 300 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This gave methyl 2-(octahydro-1H-inden-2-yl)acetate (21 g, yield: 80.55%) as a pale yellow solid.

[0370] Synthesis of 2-(octahydro-1H-inden-2-yl)ethanol [ka]

[0371] To a 500 mL four-necked round-bottom flask, methyl 2-(octahydro-1H-inden-2-yl)acetate (20 g, 101.890 mmol, 1 equiv.) and THF (200 mL) were added at room temperature. To the above mixture, LiAlH4 (40 mL, 80.000 mmol, 0.79 equiv.) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature overnight. The reaction was quenched by the addition of water (3 mL) at 0 °C. To the resulting mixture, aqueous NaOH (3 mL, 15% w / w) and water (9 mL) were added dropwise. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in heptane (500 mL). The resulting mixture was washed with 2 × 200 mL of water, 3 × 200 mL of water / MeOH (1:4), 2 × 200 mL of aqueous citric acid (5% w / w), 2 × 200 mL of saturated NaHCO and brine (200 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (10:1) to give 2-(octahydro-1H-inden-2-yl)ethanol (14.3 g, yield: 83.40%) as a pale yellow oil.

[0372] A synthesis giving 2-(2-bromoethyl)-octahydro-1H-indene [ka]

[0373] To a 500 mL three-necked round-bottom flask, 2-(octahydro-1H-inden-2-yl)ethanol (14 g, 83.195 mmol, 1 equiv.) and DCM (280 mL) were added at room temperature. To the above mixture, PPh3 (32.73 g, 124.792 mmol, 1.5 equiv.) was added portionwise at room temperature. The resulting mixture was stirred at room temperature for an additional 10 minutes. To the above mixture, CBr4 (41.38 g, 124.792 mmol, 1.5 equiv. in 200 mL of DCM) was added dropwise at 10 °C. The resulting mixture was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with heptane (500 mL). The solid was removed by filtration, and the filter cake was washed with heptane (2 × 50 mL). The filtrate was washed with 2 × 200 mL of water, 3 × 200 mL of water / MeOH (1:4), 2 × 200 mL of aqueous citric acid (5% w / w), 2 × 200 mL of saturated NaHCO and brine (200 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with heptane to give 2-(2-bromoethyl)-octahydro-1H-indene (15.1 g, yield: 77.96%) as a pale yellow oil.

[0374] Synthesis of 2-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene [ka]

[0375] To a 250 mL three-necked round-bottom flask, DMSO (30 mL) and NaH (1.13 g, 28.252 mmol, 2.31 equiv, 60%) were added at room temperature. The resulting mixture was stirred at room temperature for an additional 1 hour. To the above mixture, TosMIC (2.39 g, 12.241 mmol, 1 equiv) was added portionwise over 10 minutes at room temperature. The resulting mixture was stirred at room temperature for an additional 1 hour. To the above mixture, TBAI (0.45 g, 1.224 mmol, 0.1 equiv) and 2-(2-bromoethyl)-octahydro-1H-indene (6.00 g, 25.706 mmol, 2.1 equiv, 99.3%) were added dropwise over 1 hour at 20 °C. The resulting mixture was stirred at room temperature overnight. The resulting mixture was diluted with saturated NH4Cl (120 mL). The resulting mixture was extracted with hexane (3 × 60 mL). The combined organic layers were washed with water (2 × 150 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (95:5) to give 2-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene (4.8 g, yield: 49.62%) as a pale yellow oil.

[0376] Synthesis of 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one [ka]

[0377] To a 250 mL three-necked round-bottom flask was added 2-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene (4 g, 7.592 mmol, 1 equivalent, 94.1%) and HCl (gas) in 1,4-dioxane at 0 °C. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with hexane (3 × 30 mL). The combined organic layers were washed with saturated NaHCO (2 × 50 mL), water (2 × 50 mL), and brine (1 × 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (95:5) to give 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one (2.8 g, yield: 95.13%) as a pale yellow oil.

[0378] Synthesis of 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol [ka]

[0379] To a 100 mL three-necked round-bottom flask was added 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one (2.5 g, 7.351 mmol, 1 equiv., 97.2%) and MeOH (25 mL) at 0 °C. To the above mixture, NaBH (0.28 g, 7.351 mmol, 1 equiv.) was added portionwise over 2 minutes at 0 °C. The resulting mixture was stirred at 0 °C for an additional 2 hours. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with hexane (3 × 30 mL). The combined organic layers were washed with saturated NaHCO (2 × 50 mL), water (2 × 50 mL), and brine (1 × 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (95:5) to give 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol (2.3 g, yield: 91.72%) as an off-white solid.

[0380] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate [ka]

[0381] To a 100 mL three-necked round-bottom flask were added 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol (1.8 g, 5.277 mmol, 1 equiv., 97.5%), 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (2.61 g, 6.332 mmol, 1.2 equiv.), DCM (36 mL), DIEA (1.36 g, 10.554 mmol, 2 equiv.), and DMAP (128.94 mg, 1.055 mmol, 0.2 equiv.) at room temperature. To the above mixture was added EDCI (1.52 g, 7.915 mmol, 1.5 equiv.) in portions at room temperature. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (30 mL). The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with saturated Na2CO3 (30 mL), MeOH / HO (4:1, 2 × 50 mL), water (50 mL), and brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (9:1) to afford 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate (3.6 g, yield: 93.25%) as a pale yellow oil.

[0382] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate [ka]

[0383] To a 250 mL three-necked round-bottom flask, 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate (3 g, 4.101 mmol, 1 equiv., 99.4%) and THF (30 mL) were added at room temperature. To the above mixture, [3-(chloromagnesio)propyl]dimethylamine (41.01 mL, 41.010 mmol, 10 equiv.) was added dropwise at -50 °C. The resulting mixture was stirred at -40 °C for an additional 2 h. The reaction was quenched by the addition of saturated NH4Cl(aq) (20 mL) at -30 °C. The mixture was allowed to warm to room temperature. The resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (1 × 20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with CHCl / MeOH (13:87) to give 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.1 g, yield: 59.11%) as a pale yellow oil.

[0384] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate [ka]

[0385] To a 50 mL round-bottom flask, 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2 g, 2.309 mmol, 1 equiv., 94%) and DCM (20 mL) were added at room temperature. EtSiH (2.68 g, 23.090 mmol, 10 equiv.) and BFEtO (3.28 g, 23.090 mmol, 10 equiv.) were added dropwise at room temperature. The resulting mixture was stirred at 40 °C for an additional 2 h. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with CHCl (3 × 20 mL). The combined organic layers were washed with brine (1 × 20 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with CHCl / MeOH (13:87) to give 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (1.28 g, yield: 49.05%) as a pale yellow oil.

[0386] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate [ka]

[0387] To a 50 mL round-bottom flask, 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl(4Z)-5-[3-(dimethylamino)propyl]non-4-enedioate (650 mg, 0.789 mmol, 1 equivalent, 96.7%) and EtOH (13 mL) were added at room temperature. Pd / C (150 mg) was added to the above mixture at room temperature. The mixture was hydrogenated under 30 psi hydrogen pressure at room temperature for 3 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was diluted with heptane (80 mL). The solids were removed by filtration. The filtrate was washed with MeOH / HO (4:1, 2 × 50 mL), water (50 mL), and brine (1 × 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This gave 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (504.9 mg, yield: 45.01%) as a colorless oil. LCMS: (ES, m / z): 798.7 [M+H] + . 1 H NMR:(400 MHz, Chloroform-d, ppm) δ 5.4.894-4.818 (m, 2H), 2.275-2.013 (m, 12H), 1.906-1.556 (m, 13H), 1.512-1.468 (m, 16H), 1.386-1.166 (m, 40H), 1.115-1.050 (m, 3H), 0.903-0.869 (m, 8H). Example 19. 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (L-19) [ka]

[0388] Synthesis of 1-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene [ka]

[0389] To a 500 mL four-neck round-bottom flask, DMSO (120 mL) and NaH (1.74 g, 72.329 mmol, 2.4 equiv.) were added at room temperature. To the mixture, TosMIC (5.88 g, 30.137 mmol, 1 equiv.) was added portionwise over 10 minutes at room temperature. The resulting mixture was stirred at room temperature for an additional 0.5 hours. To the mixture, TBAI (1.11 g, 3.014 mmol, 0.1 equiv.) was added portionwise over 10 minutes at room temperature. The resulting mixture was stirred at room temperature for an additional 15 minutes. To the mixture, 1-(2-bromoethyl)-4-methylbenzene (6 g, 30.137 mmol, 1 equiv.) was added dropwise over 15 minutes at room temperature. The resulting mixture was stirred at room temperature for an additional 18 hours. The reaction was quenched with 300 mL of saturated NH4Cl(aq.) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with water (2 × 200 mL) and brine (1 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (4:1) to give 1-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene (5 g, 38.44%) as a pale yellow oil.

[0390] Synthesis of 1,5-bis(4-methylphenyl)pentan-3-one [ka]

[0391] To a 250 mL round-bottom flask, 1-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene (5 g, 11.585 mmol, 1 equiv.) in MeOH (100 mL, 4 M) and HCl (g) were added at room temperature. The resulting mixture was stirred at room temperature for 18 hours. LCMS showed that the reaction was complete. The resulting mixture was concentrated in vacuo. The resulting mixture was diluted with 60 mL of sodium carbonate (5.0%, aq.). The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (4:1) to give 1,5-bis(4-methylphenyl)pentan-3-one (2.5 g, 81.01%) as a pale yellow oil.

[0392] Synthesis of 1,5-bis(4-methylphenyl)pentan-3-ol [ka]

[0393] A 250 mL three-necked round-bottom flask was charged with 1,5-bis(4-methylphenyl)pentan-3-one (2.5 g, 9.385 mmol, 1 equiv.), MeOH (4 mL), and THF (8 mL) at 0 °C. Subsequently, NaBH (0.99 g, 26.041 mmol, 1.5 equiv.) was added in portions at 0 °C. The resulting solution was stirred at 0 °C for 10 min. LCMS showed that the reaction was complete. The resulting mixture was quenched by the addition of citric acid (5%, 50 mL) at 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (1 × 200 mL), brine (1 × 200 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with EA:heptane (1:8) to give 1,5-bis(4-methylphenyl)pentan-3-ol (2.3 g, 91.31%).

[0394] Synthesis of 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate [ka]

[0395] To a 250 mL three-necked round-bottom flask, 1,5-bis(4-methylphenyl)pentan-3-ol (2.3 g, 8.569 mmol, 1 equiv.), 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (3.54 g, 8.569 mmol, 1 equiv.), EDCI (2.46 g, 12.854 mmol, 1.5 equiv.), DMAP (1.05 g, 8.569 mmol, 1 equiv.), and DCM (23 mL) were added at room temperature. The mixture was stirred at room temperature for 18 hours. LCMS indicated the reaction was complete. The resulting solution was diluted with 250 mL of DCM. The resulting solution was washed with 1 × 200 mL of citric acid (5%, aq.), 2 × 200 mL of water, and 1 × 200 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with Hep / EA (volume ratio) (gradient from 100:0 to 90:10, and the product eluent Hep / EA = 95:5 was collected) to give 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate (5.1 g, 89.77%) as a colorless oil.

[0396] Synthesis of 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate [ka]

[0397] A 250 mL round-bottom flask was charged with 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-oxononanedioate (3.5 g, 5.279 mmol, 1 equiv.) and tetrahydrofuran (10.5 mL) at room temperature under a N atmosphere. The mixture was cooled to -60 °C. To this was added [3-(chloromagnesio)propyl]dimethylamine (52.79 mL, 52.790 mmol, 10 equiv.) dropwise at -60 °C. The mixture was stirred at -60 °C for 2 hours. LCMS showed the reaction was complete. The reaction was quenched by the addition of 80 mL of NH Cl (sat.) below 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 70:30, and the product eluent DCM / MeOH = 82 / 18 was collected) to afford 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (1.3 g, 32.83%) as a colorless oil.

[0398] Synthesis of inseparable 1-(1,5-di-p-tolylpentan-3-yl)9-(pentadecan-8-yl)(Z)-5-(3-(dimethylamino)propylidene)nonanedioate, 1-(1,5-di-p-tolylpentan-3-yl)9-(pentadecan-8-yl)(E)-5-(3-(dimethylamino)propyl)non-4-enedioate, and 9-(1,5-di-p-tolylpentan-3-yl)1-(pentadecan-8-yl)(E)-5-(3-(dimethylamino)propyl)non-4-enedioate [ka]

[0399] A 50 mL round-bottom flask was charged with 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (1.3 g, 1.733 mmol, 1 equiv.) and DCM (0.55 mL, 8.665 mmol, 5 equiv.) under a N atmosphere at room temperature. To this was added BF EtO (2.46 g, 17.330 mmol, 10 equiv.) and EtSiH (2.02 g, 17.330 mmol, 10 equiv.) at room temperature. The mixture was stirred at 45 °C for 2 h. LCMS indicated the reaction was complete. The reaction was quenched by the addition of 25 mL of NaHCO (sat.) at 0 °C. The resulting mixture was extracted with DCM (2 × 50 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL) and dried over anhydrous NaSO. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the product eluent DCM / MeOH = 94 / 6 was collected). An inseparable mixture of product isomers (1.1 g, 86.82%) was obtained as a colorless oil.

[0400] Synthesis of 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate [ka]

[0401] A 100 mL round-bottom flask was charged with (1.1 g, 1.505 mmol, 1 equiv) and EtOH (22 mL). To this was added Pd / C (0.22 g, 10%) at room temperature. The mixture was stirred under H2 (30 psi) atmosphere at room temperature for 4 hours. LCMS showed that the reaction was complete. The resulting mixture was filtered, and the filter cake was washed with EA (2 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in n-heptane (110 mL, 100 V). The n-heptane phase was then washed with 2 × 30 mL of MeOH / H2O (4:1), 2 × 30 mL of MeCN / H2O (4:1), 1 × 30 mL of water, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give 1-[1,5-bis(4-methylphenyl)pentan-3-yl]9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (539.5 mg, 48.84%) as a colorless oil. LCMS: (ES, m / z): 733.6 [M+H] + . 1 H NMR (400 MHz, Chloroform-d, ppm) δ 7.122-7.050 (m, 8H), 5.031-5.007 (m, 1H), 4.990-4.862 (m, 1H), 2.686-2.615 (m, 4H), 2.585-2.268 (m, 18H), 1.989-1.811 (m, 4H), 1.668-1.534 (m, 10H), 1.360-1.282 (m, 27H), 0.921-0.876 (m, 6H). Example 20. Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (L-20) [ka]

[0402] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol [ka]

[0403] To a 2 L three-necked round-bottom flask, xylitol (40 g, 262.905 mmol, 1 equiv.), MeOH (400 mL, 10 V), 2,2-dimethoxypropane (600 mL, 15 V), and TsOH.HO (10 g, 52.581 mmol, 0.2 equiv.) were added at room temperature. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. To the mixture, KCO (7.263 g, 0.2 equiv.) was added in portions at room temperature. The resulting mixture was stirred at room temperature for an additional 30 minutes. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. To the mixture, 150 g of silica gel (type: ZCX-2, 100-200 mesh, 1.5 w. / w.) was added. The mixture was then concentrated to zero fractions under vacuum while maintaining the temperature below 35 °C. 700 g of silica gel (type: ZCX-2, 100-200 mesh, 9.0 wt. / wt.) was loaded onto a column, followed by the dried silica gel prepared by absorbing the reaction mixture from the last step. The product was purified using combi-flash. It was eluted with n-heptane / EA (gradient from 100:0 to 90:10). Samples were taken for TLC analysis (EA:n-heptane = 1:4), and the acceptable products were combined. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (36.1 g, 59.12% yield) was obtained as a pale yellow oil.

[0404] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate [ka]

[0405] A solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (25.8 g, 111.075 mmol, 1 equiv.) in DCM (300 mL) was treated with TEA (33.72 g, 333.225 mmol, 3 equiv.) at room temperature under a nitrogen atmosphere for 10 minutes. Subsequently, MsCl (19.08 g, 166.613 mmol, 1.5 equiv.) was added dropwise at 0° C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by the addition of saturated NH4Cl solution at room temperature. The resulting mixture was extracted with DCM (3 × 300 mL). The combined organic layers were washed with brine (2 × 300 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (35.8 g crude) as a brown solid.

[0406] Synthesis of 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan-1-amine [ka]

[0407] To a 1 L three-necked round-bottom flask were added 3-(diethylamino)propan-1-ol (10.57 g, 80.552 mmol, 2.5 equiv), THF (300 mL), and NaH (1.93 g, 80.552 mmol, 2.5 equiv) at 0 °C. The resulting mixture was stirred at 0 °C for 30 minutes under a nitrogen atmosphere. To the above mixture was added bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (10 g, crude) in portions at 0 °C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. The reaction was quenched with saturated NH4Cl (aq) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 300 mL). The combined organic layers were concentrated under reduced pressure. To the mixture was added 20 g of silica gel (type: ZCX-2, 100-200 mesh, 1.5 w / w). The mixture was then concentrated to zero under vacuum, maintaining the temperature below 35 °C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 9.0 w / w) was loaded onto a column, followed by the dried silica gel that had absorbed the reaction mixture from the last step. The product was purified using combi-flash. Elution was with a gradient of DCM / MeOH (100:0 to 90:10). A sample was taken for TLC analysis (DCM / MeOH (10:1)), and the acceptable product was combined. 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan-1-amine (1.9 g) was obtained as a pale yellow oil.

[0408] Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetraol [ka]

[0409] To a 100 mL round-bottom flask was added 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan-1-amine (1.9 g, 5.500 mmol, 1 equiv.) and HCl (6 M) (20 mL) at room temperature. The resulting mixture was stirred at 60° C. under a nitrogen atmosphere for 3 hours. The resulting mixture was concentrated in vacuo. The crude product (1.8 g) was used directly in the next step without further purification.

[0410] Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) [ka]

[0411] To a 250 mL round-bottom flask was added 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetraol (1.8 g, 6.783 mmol, 1 equiv.), ACN (100 mL), decanoic acid (7.01 g, 40.698 mmol, 6 equiv.), DMAP (1.66 g, 13.566 mmol, 2 equiv.), and EDCI (7.80 g, 40.698 mmol, 6 equiv.) at room temperature. The resulting mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure. The residue was dissolved in water (200 mL). The resulting mixture was extracted with heptane (3 × 200 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, CHCN and water (0.05% TFA), gradient from 50% CHCN to 95% in 15 min; detector, UV 200 nm. The product eluate was collected and concentrated. The resulting mixture was diluted with n-heptane (300 mL) and basified to pH 8-9 with saturated NaCO (3%). The organic layer was washed with brine (2 × 100 mL) and H2O / MeOH = 1 / 5 (1 × 100 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated. This afforded 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayltetrakis(decanoate) (526.8 mg, 8.80% yield) as a pale yellow oil. LCMS: (ES, m / z): 883 [M+1] + . 1 H NMR (300 MHz, CDCl3, ppm) δ 5.484 (t, J = 5.4 Hz, 1H), 5.347-5.311 (m, 1H), 5.205-5.171 (m,1H), 4.352-4.299 (m, 1H), 4.027-3.966 (m, 1H), 3.507-3.394 (m, 4H), 2.550-2.452 (m, 6H), 2.344-2.262 (m, 8H), 1.741-1.586 (m, 10H), 1.390-1.210 (m, 48H), 1.011 (t, J = 7.2 Hz, 6H), 0.879 (t, J = 6.9 Hz, 12H). Example 21. Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl 3-(octahydro-1H-inden-2-yl)propanoate [ka]

[0412] Synthesis of octahydro-1H-indene-2-carboxylic acid [ka]

[0413] To a solution of 2,3-dihydro-1H-indene-2-carboxylic acid (25 g, 154.142 mmol, 1 equiv.) in 500 mL of AcOH was added Pd / C (25 g) in a pressure tank. The mixture was hydrogenated at 120° C. under 30 psi hydrogen pressure for 24 h. After filtration through a Celite pad, the filtrate was concentrated under reduced pressure. The resulting mixture was diluted with water (250 mL). The mixture was extracted with EtOAc (2×250 mL). The combined organic layers were washed with brine (1×250 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded octahydro-1H-indene-2-carboxylic acid (22 g, 75.08%) as a brown oil.

[0414] Synthesis of octahydro-1H-inden-2-ylmethanol [ka]

[0415] To a stirred mixture of octahydro-1H-indene-2-carboxylic acid (22 g, 115.730 mmol, 1 equiv., 88.5%) in THF was added LiAlH (6.59 g, 173.595 mmol, 1.5 equiv.) dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 4 hours. The reaction was quenched by the addition of hydrochloric acid (2 M) (100 mL) at 0 °C. The resulting mixture was extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine (1 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded octahydro-1H-inden-2-ylmethanol (16 g, 79.68%) as a yellow oil.

[0416] Synthesis of octahydro-1H-indene-2-carbaldehyde [ka]

[0417] To a 250 mL three-necked round-bottom flask, octahydro-1H-inden-2-ylmethanol (15 g, 97.243 mmol, 1 equiv.) and DCM (150 mL) were added at room temperature. To the above mixture, Dess-Martin (45.37 g, 106.967 mmol, 1.1 equiv.) was added in portions at 0 °C. The resulting mixture was stirred at room temperature for an additional 2 h. The reaction was quenched by the addition of NaSO (aq.) (100 mL) at room temperature. The resulting mixture was extracted with CHCl (2 × 200 mL). The combined organic layers were washed with NaHCO (aq.) (3 × 200 mL) and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded octahydro-1H-indene-2-carbaldehyde (5.95 g, 26.69%) as a yellow oil.

[0418] Synthesis of methyl (2E)-3-(octahydro-1H-inden-2-yl)prop-2-enoate [ka]

[0419] Octahydro-1H-indene-2-carbaldehyde (5.7 g, 37.442 mmol, 1 equiv.), 2-MeTHF (60 mL), and methyl 2-(triphenyl-λ5-phosphanylidene)acetate (15.02 g, 44.930 mmol, 1.2 equiv.) were added to a 250 mL three-necked round-bottom flask at room temperature. The resulting mixture was stirred at room temperature for 2 h. The workup was combined with the previous 200 mg reaction mixture. The resulting mixture was concentrated under reduced pressure. The mixture was dissolved in DCM (100 mL) and 24 g of silica gel (type: ZCX-2, 100-200 mesh, 4 wt. / wt.) was added. The fractions were concentrated under vacuum while maintaining the temperature below 35 °C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w / w) was loaded onto the column, followed by the final step of preparing dried silica gel to absorb the reaction mixture. The product was purified using combi-flash. It was eluted with PE / EA (gradient from 100:0 to 90:10, collecting every 200 ± 10 mL). Samples were taken for TLC analysis (EA:PE = 5:1), and the acceptable products were combined. This afforded methyl (2E)-3-(octahydro-1H-inden-2-yl)prop-2-enoate (5.51 g, 63.00%) as a colorless oil.

[0420] Synthesis of methyl 3-(octahydro-1H-inden-2-yl)propanoate [ka]

[0421] To a solution of methyl (2E)-3-(octahydro-1H-inden-2-yl)prop-2-enoate (5 g, 24.004 mmol, 1 equiv.) in 50 mL of MeOH was added Pd / C (2.5 g) in a pressure tank. The mixture was hydrogenated at room temperature under 30 psi of hydrogen pressure for 12 hours. The workup was combined with the previous 500 mg reaction mixture. It was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 20 mL). The filtrate was concentrated under reduced pressure. This afforded methyl 3-(octahydro-1H-inden-2-yl)propanoate (5.1 g, 72.19%) as a colorless oil.

[0422] Synthesis of 3-(octahydro-1H-inden-2-yl)propanoic acid [ka]

[0423] To a 250 mL three-necked round-bottom flask was added methyl 3-(octahydro-1H-inden-2-yl)propanoate (5 g, 23.774 mmol, 1 equiv.), THF (50 mL), MeOH (50 mL), HO (50 mL), and LiOH.HO (2.00 g, 47.548 mmol, 2 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The organic solvent was removed under reduced pressure. The resulting mixture was extracted with EtOAc (2 × 50 mL), and the aqueous phase was collected. The aqueous phase was acidified to pH = 2 with HCl (6 M). The resulting mixture was extracted with EtOAc (2 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded 3-(octahydro-1H-inden-2-yl)propanoic acid (3.941 g, 74.10%) as a white solid.

[0424] Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl 3-(octahydro-1H-inden-2-yl)propanoate [ka]

[0425] To an 80 mL vial, 3-(octahydro-1H-inden-2-yl)propanoic acid (0.85 g, 4.330 mmol, 1 equiv.), ACN (8.5 mL), 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetrol (5.14 g, 21.650 mmol, 5 equiv.), EDCI (4.98 g, 25.980 mmol, 6 equiv.), DMAP (1.06 g, 8.660 mmol, 2 equiv.), and DIEA (4.48 g, 34.640 mmol, 8 equiv.) were added at room temperature. The resulting mixture was stirred at room temperature for an additional 3 h. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with heptane (3 × 150 mL). The collected organic phase was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography using the following conditions: column, XSelect CSH Prep C 18 5 μm; mobile phase, B: CH3CN, A: water (0.1% TFA), 50% to 95% gradient in 15 min; flow rate: 50 mL / min; detector, ELSD. The resulting mixture was concentrated under reduced pressure and basified to pH 8 with saturated Na2CO3 (aq). The aqueous layer was extracted with heptane (3 × 100 mL). The resulting mixture was concentrated under reduced pressure. This afforded 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl 3-(octahydro-1H-inden-2-yl)propanoate (0.5272 g, 12.90%) as a yellow oil. LCMS: (ES, m / z): 950.9 [M+1] + . 1H NMR (300 MHz, chloroform-d) δ 5.456 (t, J = 5.5 Hz, 1H), 5.342 (t, J = 5.5 Hz, 1H), 5.145 (q, J = 5.0 Hz, 1H), 4.395-4.285 (m, 1H), 4.000-3.875 (m, 1H), 3.584-3.377 (m, 4H), 2.413-2.252 (m, 10H), 2.243-2.184 (m, 6H), 1.999-1.799 (m, 14H), 1.763-1.584 (m, 15H), 1.540-1.418 (m, 14H). 1.352-1.245 (m, 21H), 1.161-1.027 (m, 6H). Example 22. Synthesis of 5-(dimethylamino)-1-((2-heptylnonanoyl)oxy)pentane-2,3-diylbis(decanoate) (L-22) [ka]

[0426] Synthesis of 2-(5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)acetaldehyde [ka]

[0427] To a 500 mL three-necked round-bottom flask were added 3,4,5-trihydroxypentanal (20 g, 149.108 mmol, 1 equiv.), pTsOH (1.28 g, 7.455 mmol, 0.05 equiv.), and acetone (200 mL) at room temperature. 2,2-Dimethoxypropane (18.64 g, 178.930 mmol, 1.2 equiv.) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at 0 °C for an additional 30 minutes. The mixture was basified to pH 8 with saturated NaHCO (aq.). The resulting mixture was concentrated under reduced pressure to remove acetone. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This gave 2-(5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)acetaldehyde (23 g, crude, GCMS purity: 78.2%) as a pale yellow oil.

[0428] Synthesis of (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methanol [ka]

[0429] To a 250 mL three-necked round-bottom flask were added 2-((4S,5S)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)acetaldehyde (6 g, 34.444 mmol, 1 equiv.), THF (30 mL), MeOH (60 mL), and AcOH (2.6 g, 43.296 mmol, 1.26 equiv.) at room temperature. Dimethylamine (2 M in THF, 22.4 mL, 44.800 mmol, 1.30 equiv.) was added dropwise to the mixture at room temperature. The resulting mixture was stirred at room temperature for an additional 30 minutes. To the mixture was added NaBHCN (3.0 g, 47.740 mmol, 1.39 equiv.) in portions over 10 minutes at room temperature. The resulting mixture was stirred at room temperature for an additional 4 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN (0.1% NH₃·HO) in water, 5% hold for 3 min, gradient from 5% to 55% in 12 min; detector, UV 220 nm. This afforded (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methanol (3.2 g, 45.70%) as a pale yellow oil.

[0430] Synthesis of (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-heptylnonanoate [ka]

[0431] To a 250 mL three-necked round-bottom flask was added ((4S,5S)-5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methanol (3.2 g, 15.742 mmol, 1 equiv.), ACN (64 mL), 2-heptylnonanoic acid (4.04 g, 15.742 mmol, 1 equiv.), and DMAP (0.58 g, 4.723 mmol, 0.3 equiv.) at room temperature. To the above mixture was added EDCI (4.53 g, 23.613 mmol, 1.5 equiv.) at room temperature. The resulting mixture was stirred at room temperature overnight. The resulting mixture was diluted with water (150 mL). The resulting mixture was extracted with heptane (3 × 80 mL). The combined organic layers were washed with aqueous NaCO (2 × 80 mL), water (100 mL), and brine (100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with CHCl / MeOH (98:2) to afford (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-heptylnonanoate (4.7 g, 54.96%) as a pale yellow oil.

[0432] Synthesis of 5-(dimethylamino)-2,3-dihydroxypentyl 2-heptylnonanoate [ka]

[0433] To a 100 mL three-necked round-bottom flask was added ((4S,5S)-5-(2-(dimethylamino)ethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-heptylnonanoate (1.0 g, 2.264 mmol, 1 equiv.) and DCM (20 mL) at −10 °C. To the above mixture was added a mixture of TFA (5 mL, 67.315 mmol, 29.73 equiv.) and HO (0.5 mL, 27.755 mmol, 12.26 equiv.) dropwise at −10 °C. The resulting mixture was stirred for an additional 1 h, maintaining the temperature between 10 °C and −5 °C. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated Na2CO3 (2 × 50 mL), water (2 × 50 mL), and brine (1 × 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This afforded 5-(dimethylamino)-2,3-dihydroxypentyl 2-heptylnonanoate (920 mg, 72.6% purity, 73....

Claims

1. Ionizable lipid compounds of formula (I): (Z-L-Y)-W ｎ -(X-R) （ｎ－１） (I) During the ceremony, Z is an ionizable head group; L is an optionally substituted (C １ ~C １２ ) alkylene; Y is a linking group; W ｎ is a linear alkyl core of n carbon atoms, n being 3 to 6; X is an optional linking group; Each R is independently a lipid tail.

2. The compound of claim 1, wherein n is 4 to 6.

3. 3. The compound of claim 2, wherein n is 4.

4. The compound of claim 2 wherein n is 5.

5. 3. The compound of claim 2, wherein n is 6.

6. 2. The compound of claim 1, wherein n is 3.

7. W ｎ but, 【Chemistry 1】 and 【Chemistry 2】 is selected from During the ceremony, * represents the point of attachment to Y; Each ** represents a point of attachment to X; G １ is H or G １ and W to which Y is bonded. ｎ is a group cyclically linked to Y which together with the carbon atom of G ２ is H or -CH ２ It is OH The compound according to any one of claims 1 to 6.

8. Y is —O—, —C(R １０ ) ２ -, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR １０ -, -SC(O)NR １０ -, -C(O)NR １０ -, -NR １０ C(O)-, -S-, -NR １０ -, -NR １０ C(O)O- and -NR １０ C(O)S—, wherein R １０ teeth 、 H and C １～６ The compound according to any one of claims 1 to 6, wherein the aryl group is selected from alkyl.

9. Y is —O—, —OC(O)—, or —OC(O)NR １０ The compound of claim 8, wherein the compound is selected from:

10. Y is -CH ２ The compound according to claim 8, wherein

11. G １ is cyclically linked to Y and the W to which they are attached ｎ 8. The compound of claim 7, wherein R is a group which, taken together with said carbon atom, provides a heterocyclic ring.

12. L is (C ２ ~C ６ ) alkylene or substituted (C ２ ~C ６ 12. The compound according to claim 1, wherein:

13. L is -(CH ２ ) ２ The compound according to claim 12, wherein

14. L is -(CH ２ ) ３ The compound according to claim 12, wherein

15. The compound of any one of claims 1 to 14, wherein Z comprises a tertiary amino group.

16. Z is -NR １１ R １２ and R １１ and R １２ 16. The compound of claim 15, wherein each is independently alkyl or substituted alkyl.

17. R １１ and R １２ are respectively, C １～６ 17. The compound of claim 16, wherein the compound is alkyl.

18. R １１ and R １２ are respectively, C １～３ 18. The compound of claim 17, wherein the compound is alkyl.

19. R １１ and R １２ 19. The compound of claim 18, wherein each is methyl.

20. R １１ and R １２ and each is ethyl.

21. Each X is independently -(CH ２ ) ｓ OC(O)-,-(CH ２ ) ｓ C(O)O-,-(CH ２ ) ｓ OC(O)O-,-(CH ２ ) ｓ OC(O)NR １０ -, -(CH ２ ) ｓ O-, -(CH ２ ) ｓ SC(O)NR １０ -, -(CH ２ ) ｓ C(O)NR １０ -, -(CH ２ ) ｓ NR １０ C(O)-,-(CH ２ ) ｓ S-, -(CH ２ ) ｓ NR １０ -, -(CH ２ ) ｓ NR １０ C(O)O- and -(CH ２ ) ｓ NR １０ C(O)S—, wherein R １０ is H and C １～６ 21. The compound of any one of claims 1 to 20, wherein s is selected from alkyl and s is 0 to 6.

22. Each X is independently —OC(O)—, —C(O)O—, —OC(O)O—, —O—, or —OC(O)NR １０ -, -SC(O)NR １０ -, -C(O)NR １０ -, -NR １０ C(O)-, -S-, -NR １０ -, -NR １０ C(O)O- and -NR １０ C(O)S—, wherein R １０ teeth 、 H and C １～６ 22. The compound of any one of claims 1 to 21, wherein the compound is selected from alkyl.

23. 23. The compound of claim 22, wherein each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

24. 24. The compound of claim 23, wherein each -X-R is -OC(O)R.

25. 25. The compound of any one of claims 1 to 24, wherein each R is independently an aliphatic hydrocarbon group that is linear or branched, saturated or unsaturated, and / or optionally contains a cyclic group.

26. 26. The compound of any one of claims 1 to 25, wherein each R is a straight chain hydrocarbon group optionally containing one or more cyclic groups.

27. Each R is C ５ ~C ２０ Alkyl, C ５ ~C ２０ Alkenyl and C ５ ~C ２０ 27. The compound of any one of claims 1 to 26, selected from alkynyl.

28. Each R is C ６ ~C １２ Alkyl and C ６ ~C １２ 28. The compound of claim 27, selected from alkenyl.

29. 27. The compound of claim 26, wherein at least one R is a straight chain hydrocarbon group that includes a cyclic group.

30. 30. The compound of claim 29, wherein said cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle and heteroaryl, any of said monocyclic or bicyclic groups being optionally substituted.

31. 26. The compound of any one of claims 1 to 25, wherein at least one R is a branched hydrocarbon group, optionally including a cyclic group.

32. 32. The compound of claim 31, wherein each R is a branched hydrocarbon group.

33. 33. The compound of claim 32, wherein the branched hydrocarbon group contains from 8 to 20 carbon atoms.

34. The compound of any one of claims 31 to 33, wherein the branched hydrocarbon group is saturated.

35. The compound according to any one of claims 31 to 33, wherein the branched hydrocarbon group is unsaturated.

36. R is -CH(R ７ ) ２ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ The compound of any one of claims 31 to 35, which is alkenyl.

37. 32. The compound of claim 31, wherein at least one R is a branched hydrocarbon group that includes a cyclic group.

38. 38. The compound of claim 37, wherein said cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, any of said monocyclic or bicyclic groups being optionally substituted.

39. The compound has formula (IIA): 【Transformation 3】 2. The compound of claim 1, wherein

40. Y is —O—, —OC(O)—, or —OC(O)NR １０ -, and R １０ is H and C １～６ 40. The compound of claim 39, wherein the alkyl is selected from alkyl.

41. 41. The compound of claim 40, wherein Y is -O-.

42. 41. The compound of claim 40, wherein Y is -OC(O)-.

43. Y is -OC(O)NR １０ The compound of claim 40, wherein

44. L is (C ２ ~C ６ ) alkylene or substituted (C ２ ~C ６ 44. The compound according to any one of claims 39 to 43, wherein:

45. L is -(CH ２ ) ２ The compound of claim 44, wherein

46. L is -(CH ２ ) ３ The compound of claim 44, wherein

47. L is -(CH ２ ) ４ The compound of claim 44, wherein

48. Z is -NR １１ R １２ and R １１ and R １２ are each independently C １～６ Alkyl or substituted C １～６ The compound of any one of claims 39 to 47, which is alkyl.

49. R １１ and R １２ are respectively, C １～３ 49. The compound of claim 48, which is alkyl.

50. R １１ and R １２ 50. The compound of claim 49, wherein each is methyl.

51. R １１ and R １２ 50. The compound of claim 49, wherein each is ethyl.

52. 52. The compound of any one of claims 39-51, wherein each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

53. Each R is C ５ ~C ２０ Alkyl, C ５ ~C ２０ Alkenyl and C ５ ~C ２０ 53. The compound of any one of claims 39 to 52, selected from alkynyl.

54. 53. The compound of any one of claims 39 to 52, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms optionally further containing one or more cyclic groups.

55. R is -CH(R ７ ) ２ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ 55. The compound of claim 54, which is alkenyl.

56. The compound has the formula (IIIA): 【Chemistry 4】 During the ceremony, R １１ and R １２ are each independently C １～３ Alkyl and C １～４ heteroalkyl; q is 1 to 4; Y is —O—, —OC(O)—, or —OC(O)NR １０ - is selected from, Each R is independently C ５ ~C ２０ Alkyl, C ５ ~C ２０ alkenyl, —CH(R ７ ) ２ and -(CH ２ ) ｔ J (CH ２ ) ｕ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

40. The compound of claim 39, wherein

57. The compound has formula (IIB): 【Transformation 5】 2. The compound of claim 1, wherein

58. Y is -O-, -OC(O)-, -OC(O)NR １０ -, -NR １０ C(O)-, -NR １０ C(O)O- and -NR １０ C(O)S—, R １０ is H and C １～６ 58. The compound of claim 57, wherein the alkyl is selected from:

59. 59. The compound of claim 58, wherein Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-.

60. L is (C ２ ~C ６ ) alkylene or substituted (C ２ ~C ６ 60. The compound of any one of claims 57 to 59, wherein:

61. L is -(CH ２ ) ２ The compound of claim 60, wherein

62. L is -(CH ２ ) ３ The compound of claim 60, wherein

63. L is -(CH ２ ) ４ The compound of claim 60, wherein

64. Z is -NR １１ R １２ and R １１ and R １２ are each independently C １～６ Alkyl or substituted C １～６ 60. The compound of any one of claims 57 to 59, which is alkyl.

65. R １１ and R １２ are respectively, C １～３ 65. The compound of claim 64, which is alkyl.

66. R １１ and R １２ 65. The compound of claim 64, wherein each is methyl.

67. 67. The compound of any one of claims 57-66, wherein each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

68. Each R is C ５ ~C ２０ Alkyl, C ５ ~C ２０ Alkenyl and C ５ ~C ２０ 68. The compound of any one of claims 57 to 67, selected from alkynyl.

69. 68. The compound of any one of claims 57 to 67, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms, optionally further containing one or more cyclic groups.

70. R is -CH(R ７ ) ２ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ 70. The compound of claim 69, which is alkenyl.

71. The compound has the formula (IIIB): 【Transformation 6】 During the ceremony, R １１ and R １２ are each independently C １～３ Alkyl or C １～４ heteroalkyl; q is 1 to 4; Y is selected from —NHC(O)—, —NHC(O)O—, and —NHC(O)S—; Each R is independently C ５ ~C ２０ Alkyl, C ５ ~C ２０ alkenyl, —CH(R ７ ) ２ and -(CH ２ ) ｔ J (CH ２ ) ｕ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

58. The compound of claim 57, wherein:

72. The compound has formula (IIC): 【Transformation 7】 2. The compound of claim 1, wherein

73. Y is -O-, -OC(O)-, -OC(O)NR １０ - and -C(R １０ ) ２ -, and R １０ is H and C １～６ 73. The compound of claim 72, wherein the alkyl is selected from alkyl.

74. 74. The compound of claim 73, wherein Y is -O-.

75. Y is -C(R １０ ) ２ The compound of claim 73, wherein

76. L is (C ２ ~C ６ ) alkylene or substituted (C ２ ~C ６ 76. The compound of any one of claims 72 to 75, wherein:

77. L is -(CH ２ ) ２ 77. The compound of any one of claims 76, wherein:

78. L is -(CH ２ ) ３ 77. The compound of any one of claims 76, wherein:

79. L is -(CH ２ ) ４ 77. The compound of any one of claims 76, wherein:

80. Z is -NR １１ R １２ and R １１ and R １２ are each independently C １～６ Alkyl or substituted C １～６ 80. The compound of any one of claims 72 to 79, which is alkyl.

81. R １１ and R １２ are respectively, C １～３ Alkyl and C １～４ 81. The compound of claim 80, wherein the alkyl is selected from heteroalkyl.

82. R １１ and R １２ 82. The compound of claim 81, wherein each is methyl.

83. Each X is independently -(CH ２ ) ｓ OC(O)-,-(CH ２ ) ｓ C(O)O-,-(CH ２ ) ｓ 83. The compound of any one of claims 72 to 82, selected from OC(O)O-, wherein s is 0 to 6.

84. 83. The compound of any one of claims 72 to 82, wherein each s is 0.

85. 83. The compound of any one of claims 72 to 82, wherein each s is 1.

86. 83. The compound of any one of claims 72 to 82, wherein each s is 3.

87. Each R is C ５ ~C ２０ Alkyl, C ５ ~C ２０ Alkenyl and C ５ ~C ２０ 87. The compound of any one of claims 72 to 86, selected from alkynyl.

88. 87. The compound of any one of claims 72 to 86, wherein at least one R is a branched hydrocarbon group containing from 8 to 20 carbon atoms optionally further containing one or more cyclic groups.

89. R is -CH(R ７ ) ２ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ 89. The compound of claim 88, which is alkenyl.

90. The compound has formula (IIIC): 【Transformation 8】 During the ceremony, R １１ and R １２ are each independently C １～３ Alkyl and C １～４ heteroalkyl; q is 1 to 4; Y is —O— and —C(R １０ ) ２ - is selected from, each s is independently 0, 1, or 2; W is —O— and —C(R １０ ) ２ - and Each R is independently C ５ ~C ２０ Alkyl, C ５ ~C ２０ alkenyl, —CH(R ７ ) ２ and -(CH ２ ) ｔ J (CH ２ ) ｕ and each R ７ are independently ５ ~C １２ Alkyl or C ５ ~C １２ alkenyl, J is a cyclic group, and each of t and u is 1 to 10.

73. The compound of claim 72, wherein

91. Each R is independently 【Chemistry 9】 Cy Ａ and Cy Ｂ are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5-12 membered monocyclic, bicyclic, bridged polycyclic and spirocyclic; R ｘ and R ｙ are each independently a bond or an optionally substituted linear or branched, saturated or partially unsaturated C １ ~C ２０ is an aliphatic group, r, p, and q are each independently an integer of 0 to 20. The compound according to any one of claims 1 to 90,

92. At least one R is 【Chemistry 10-1】 【Chemistry 10-2】 and 【Chemistry 11】 where each # represents the point of attachment to X or the point of attachment of R to the linear or branched hydrocarbon chain.

92. The compound of claim 91, comprising a moiety selected from:

93. A lipid nanoparticle comprising an ionizable lipid compound according to any one of claims 1 to 92.

94. 94. The lipid nanoparticle of claim 93, further comprising a neutral lipid and a lipid capable of reducing aggregation.

95. The lipid nanoparticle of claim 94, wherein the neutral lipid comprises a phospholipid.

96. The lipid nanoparticle of claim 94 or 95, wherein the neutral lipid comprises cholesterol.

97. a) Nucleic acid; b) ionizable lipids; c) phospholipids, d) cholesterol, and e) Lipids that can reduce aggregation The lipid nanoparticle of claim 96, comprising:

98. The lipid nanoparticle of claim 97, wherein the nucleic acid comprises DNA.

99. The lipid nanoparticle of claim 98, wherein the nucleic acid comprises RNA.

100. The lipid nanoparticle of claim 98, wherein the nucleic acid comprises DNA and RNA.

101. The lipid nanoparticle of claim 100, wherein the RNA is selected from mRNA, gRNA, and siRNA.

102. The lipid nanoparticle of any one of claims 97 to 101, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG) and derivatives thereof.

103. The lipid nanoparticle of claim 102, wherein the phospholipid is phosphatidylethanolamine (PE).

104. The lipid nanoparticle of claim 103, wherein the phospholipid is phosphatidylcholine (PC).

105. The lipid nanoparticle of any one of claims 97 to 104, wherein the phospholipids each independently comprise a hydrocarbon chain having 12 to 24 carbons.

106. The lipid nanoparticle of claim 105, wherein the phospholipids each independently comprise a hydrocarbon chain having 16 to 20 carbons.

107. The lipid nanoparticle of claim 105 or 106, wherein the hydrocarbon chain is saturated.

108. The lipid nanoparticle of claim 105 or 106, wherein the hydrocarbon chain is unsaturated and / or further comprises a carbocyclyl.

109. The lipid nanoparticle of claim 108, wherein each of the hydrocarbon chains independently contains 1 to 4 double bonds.

110. The lipid nanoparticle of any one of claims 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.

111. The lipid nanoparticle of claim 103, wherein the phospholipid comprises 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE).

112. The lipid nanoparticle of claim 103, wherein the phospholipid comprises 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

113. The lipid nanoparticle of claim 104, wherein the phospholipid comprises 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (Δ9Δ9-CisPC).

114. The lipid nanoparticle of claim 106, wherein the lipid nanoparticle comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

115. The lipid nanoparticle of claim 104, wherein the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

116. The lipid nanoparticle of any one of claims 103 to 115, wherein the lipid capable of reducing aggregation is a PEG-lipid.

117. The lipid nanoparticle of claim 116, wherein the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

118. The lipid nanoparticle of any one of claims 94 to 117, further comprising a targeting ligand.

119. The lipid nanoparticle of claim 118, wherein the targeting ligand comprises GalNAc.

120. 120. The lipid nanoparticle of claim 118 or 119, wherein the targeting ligand is linked to the lipid, which can reduce aggregation.

121. The lipid nanoparticle of claim 120, wherein the lipid capable of reducing aggregation is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

122. A lipid nanoparticle according to any one of claims 94 to 121, wherein the N / P ratio (ratio of moles of amine groups of the cationic lipid to moles of phosphate groups of the DNA) is 5 to 30.

123. The lipid nanoparticle of claim 122, wherein the N / P ratio is 7.

124. The lipid nanoparticle of claim 122, wherein the N / P ratio is 14.

125. The lipid nanoparticle of claim 122, wherein the N / P ratio is 28.

126. a) 40-60 mol % of ionizable lipids of the total lipids present; b) 6-20 mol % of phospholipids of the total lipids present; c) 35-45 mol% cholesterol of the total lipids present, and d) Lipids capable of reducing aggregation by 1.5-2.5 mol% of the total lipids present The lipid nanoparticle of any one of claims 94 to 125, comprising:

127. a) 40-60 mol % of ionizable lipids of the total lipids present; b) 10-20 mol % of phospholipids of the total lipids present; c) 35-45 mol% cholesterol of the total lipids present, and d) Lipids capable of reducing aggregation by 1.5-2.5 mol% of the total lipids present The lipid nanoparticle of any one of claims 94 to 125, comprising:

128. e) 40-49 mol % of ionizable lipids of the total lipids present; f) 10-20 mol% of phospholipids of the total lipids present; g) 35-45 mol% cholesterol of the total lipids present, and h) Lipids capable of reducing aggregation by 1.5-2.5 mol% of the total lipids present The lipid nanoparticle of any one of claims 94 to 125, comprising:

129. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 94 to 128 and a pharmaceutically acceptable excipient, carrier or diluent.

130. 129. A method for delivering a nucleic acid into a cell, comprising contacting said cell with a lipid nanoparticle according to any one of claims 94 to 128.

131. 131. The method of claim 130, wherein the cell is in vitro.

132. 131. The method of claim 130, wherein the cell is in vivo.

133. 1. A method for delivering a nucleic acid for the in vivo production of a target protein, comprising:

130. A method comprising systemically administering to a subject in need thereof the pharmaceutical composition of claim 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticle, and wherein the administration of the pharmaceutical composition results in long-term, stable expression of the target protein.

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