Lipid compounds and uses thereof

Abstract

The present disclosure relates to lipid compounds of formula (I): A-(CH2)n-CX-BZ-R1(I), where R1 represents a lipophilic or hydrophobic tail group and R1 is an optionally substituted, branched, saturated or unsaturated C 10 ~C 55 is a hydrocarbon radical, optionally interrupted by one or several atoms of oxygen or nitrogen and / or one or several moieties -(C=O)-, -O-(C=O)- or -(C=O)-O-, and one nitrogen atom, if present in the backbone, may be linked directly or indirectly to the Z radical, Z is a spacer arm, B represents O or NH, X is O or S, n is 0, 1, 2, 3, 4, 5 or 6, A is (i) R2R3N-, (ii) Y is O or N, Alk is an alkylene moiety in the range C2 to C6, R2 and R3 are each independently linear or branched. or (iii) a 4-8 membered saturated heterocyclic radical containing 3-7 carbon atoms and 1 or 2 nitrogen atoms, which 4-8 membered saturated heterocyclic radical is linked to the remainder of the molecule by a carbon or nitrogen atom and is optionally substituted by 1-4 substituents selected, independently of one another, from linear or branched (C1-C6) alkyl groups,

Description

[Technical Field]

[0001] The present disclosure relates to the field of novel lipid compounds. The novel lipid compounds are ionizable cationic lipids. The lipid compounds can be used in combination with other lipid components, such as neutral lipids, structured lipids, and polymer-conjugated lipids, to form lipid nanoparticles. The lipid nanoparticles can be used to deliver therapeutic agents, such as nucleic acids. [Background technology]

[0002] The field of polynucleotide therapeutics has made significant progress in recent years. Polynucleotides include various nucleic acid-based compounds, such as messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, or immunostimulatory nucleic acids. Some nucleic acids, such as mRNA, plasmids, and ssDNA, can be used, for example, to induce the expression of specific cellular products useful for treating diseases associated with protein or enzyme deficiencies or to express vaccine antigens to induce specific immune responses. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether native to the system or not. The expression product of a nucleic acid can enhance existing protein levels, replace defective or non-functional versions of proteins, or introduce new proteins and associated functions in cells or organisms exposed to foreign proteins to induce specific immune responses.

[0003] However, there are many challenges associated with delivering polynucleotides to affect desired responses in biological systems, and effective delivery of polynucleotides to their intracellular sites of action remains a major challenge. To be efficiently delivered to their sites of action, polynucleotides must (i) be protected from enzymatic and non-enzymatic degradation, (ii) be properly distributed to the desired biological compartment, (iii) be effectively and efficiently internalized by the target cell, and then (iv) be delivered to the intracellular compartment where the relevant translation machinery resides.

[0004] Lipid nanoparticles (LNPs) have proven to be efficient for delivering various types of therapeutically active agents into cells (Thi et al., Vaccines, 2021, 9(4), 359). For example, LNPs containing nucleic acids, such as LNP-mRNA, have attracted great interest and have recently demonstrated their efficacy and safety in the vaccine field, which may prove dramatically important in managing the Covid-19 pandemic (Reichmuth et al., Therapeutic delivery, 2016, 7(5), 319-334; Khurana et al., Nano today, 2021, 38, 101142).

[0005] For example, lipid nanoparticles formed from cationic lipids combined with other lipid components, such as neutral lipids, cholesterol, and PEGylated lipids, have been used to protect polynucleotides from degradation and promote their cellular uptake.

[0006] Although lipid nanoparticle-based vehicles containing cationic lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a great need for improved lipid nanoparticle-based delivery systems.

[0007] There remains a need for improved cationic and ionizable lipids that exhibit improved pharmacokinetic properties and are capable of delivering various types of polynucleotides with enhanced efficiency to a wide variety of cell types and tissues.

[0008] There is also a need for novel cationic ionizable lipids that have reduced toxicity and that can efficiently deliver encapsulated polynucleotides to target cells, tissues, and organs.

[0009] There is a need for new cationic ionizable lipids that can be easily cleared in vivo after administration and that have reduced toxicity.

[0010] There is a need for biodegradable cationic ionizable lipids.

[0011] The improved cationic lipids and lipid nanoparticles for polynucleotide delivery also provide optimal polynucleotide / lipid ratios, protect the polynucleotide from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of polynucleotides.

[0012] In addition, the lipid-polynucleotide particles must be well tolerated and must provide an adequate therapeutic index so that patient treatment with an effective dose of polynucleotide is not associated with unacceptable toxicity and / or risk to the patient.

[0013] Furthermore, the lipid-nucleic acid particles must be stable as a liquid formulation when stored for extended periods at 4-8° C. in a pharmaceutically acceptable buffer.

[0014] The present disclosure aims to meet these needs in whole or in part. Summary of the Invention [Means for solving the problem]

[0015] According to one of its objects, the present disclosure provides a lipid compound of formula (I): A-(CH2) n -CX-BZ-R1(I) (In the formula, - R1, C 10 ~C 57 , or C10 ~C 55 is a lipophilic or hydrophobic tail group of - Z may be a spacer arm having 2 to 24, such as 2 to 18, such as 4 to 12 carbon atoms in a branched or unbranched, linear, saturated or unsaturated hydrocarbon chain, interrupted by one or several atoms of oxygen and / or a moiety selected from among -SS-;-(OC)-;-(CO)-O-;-O-(OC)-;-S-;-NH-, -NH-(OC)-;-(OC)-NH- and -NH-(CO)-O-, such as -(CO)-O-;-O-(OC)- and -NH-(CO)-O-, wherein the chain optionally having an oxygen atom or a moiety selected from among -NH-(OC)-*-O-(OC)--*;-(CO)-O-*; and -(OC)- is linked at its end to a hydrophobic tail group, * indicating a single bond linking the moiety to the hydrophobic tail group, B is an oxygen atom or an —NH— group, X may be an oxygen atom or a sulfur atom; n may be 0, 1, 2, 3, 4, 5 or 6; - A is - the group R2R3N-, where R2 and R3 are independently a linear or branched (C1-C6) alkyl group; the NR2R3-Alk-Y- group, in which Y is an oxygen atom or a nitrogen atom, Alk can be a C2-C6 alkylene moiety, and R2 and R3, independently of each other, are a linear or branched (C1-C6) alkyl group; - a 4-8 membered saturated heterocyclic radical containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms, which 4-8 membered saturated heterocyclic radical is linked to the rest of the molecule by a carbon or nitrogen atom and is optionally substituted by 1 to 4 substituents selected, independently of one another, from linear or branched (C1-C6) alkyl groups; a lipid compound of formula (I) which is a group selected from the group consisting of or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0016] According to another object, the present disclosure provides a lipid compound of formula (I): A-(CH2) n -CX-BZ-R1(I) (In the formula, - R1, C 10 ~C 57 , or C 10 ~C 55 where R1 is an optionally substituted, branched or unbranched, straight chain, saturated or unsaturated, C 10 ~C 57 , or C 10 ~C 55 with a hydrocarbon backbone optionally interrupted by one or several atoms of oxygen or nitrogen and / or one or several moieties -(C=O)-, -O-(C=O)- or -(C=O)-O-, and one nitrogen atom, if present in the backbone, which may be linked directly or indirectly to a Z radical, - Z may be a spacer arm having 2 to 24, such as 2 to 18, such as 4 to 12 carbon atoms in an unbranched, linear, saturated or unsaturated hydrocarbon chain, interrupted by one or several atoms of oxygen and / or a moiety selected from among -SS-;-(OC)-;-(CO)-O-;-O-(OC)-;-S-;-NH-, -NH-(OC)-;-(OC)-NH- and -NH-(CO)-O-, such as -(CO)-O-;-O-(OC)- and -NH-(CO)-O-, and optionally having an oxygen atom or a moiety selected from among -NH-(OC)-*-O-(OC)--*;-(CO)-O-*; and -(OC)-, the chain being linked at its end to the hydrophobic tail group, * indicating a single bond linking the moiety to the hydrophobic tail group, B is an oxygen atom or an —NH— group, X may be an oxygen atom or a sulfur atom; n may be 0, 1, 2, 3, 4, 5 or 6; - A is - the group R2R3N-, where R2 and R3 are independently a linear or branched (C1-C6) alkyl group; the NR2R3-Alk-Y- group, in which Y is an oxygen atom or a nitrogen atom, Alk can be a C2-C6 alkylene moiety, and R2 and R3, independently of each other, are a linear or branched (C1-C6) alkyl group; - a 4-8 membered saturated heterocyclic radical containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms, which 4-8 membered saturated heterocyclic radical is linked to the rest of the molecule by a carbon or nitrogen atom and is optionally substituted by 1 to 4 substituents selected, independently of one another, from linear or branched (C1-C6) alkyl groups; a lipid compound of formula (I) which is a group selected from the group consisting of or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0017] Surprisingly, as detailed in the Examples section, the inventors have observed that the novel lipid compounds disclosed herein enable the formulation of improved compositions, e.g., lipid nanoparticles, for in vitro and in vivo delivery of mRNA and / or other oligonucleotides or oligonucleotides.

[0018] The LNPs obtained using the lipid compounds of the present disclosure were shown to be small and uniform in size and were able to induce strong protein expression.

[0019] The improved lipid nanoparticles are useful for the expression of proteins encoded by mRNA. The lipid nanoparticles disclosed herein can be used to regulate, up-regulate, or down-regulate protein expression by delivering either miRNA or miRNA inhibitors to regulate the expression of endogenous proteins, or mRNA or plasmids for the expression of transgenes. The lipid nanoparticles disclosed herein can also be used to induce pharmacological effects resulting from protein expression or protection against infection through the delivery of mRNA encoding appropriate antigens or antibodies, such as influenza antigens.

[0020] The lipid nanoparticles disclosed herein can also be used to induce pharmacological effects resulting from the expression of proteins such as erythropoietin (EPO), which are useful in the treatment of metabolic diseases or diseases resulting from protein deficiency.

[0021] The compounds disclosed herein can be in cationic form.

[0022] In the compounds disclosed herein, R1 is an optionally substituted, branched or unbranched, straight-chain, saturated or unsaturated, C10-C55 hydrocarbon radical, with a hydrocarbon backbone optionally interrupted by one or several atoms of oxygen or nitrogen and / or one or several moieties -CO-, -O-CO- or -CO-O-, and one nitrogen atom, if present in the backbone, may be directly or indirectly linked to a Z radical as defined herein.

[0023] In the compounds disclosed herein, R1 can be a group selected from the group consisting of:

[0024] [Table 1]

[0025] [Table 2]

[0026] [Table 3]

[0027] [Table 4]

[0028] [Table 5]

[0029] [Table 6]

[0030] [Table 7]

[0031] In the compounds disclosed herein, Z is a spacer arm having 2 to 24, e.g., 2 to 18, e.g., 4 to 12 carbon atoms in a branched or unbranched, linear, saturated or unsaturated hydrocarbon chain, the chain being interrupted by one or several oxygen atoms, e.g., Z represents a radical of the formula: -((CH2)2-O) m -(CH2) r (T) q -*(Z); where: - * denotes a single bond connecting the radical to the hydrophobic tail group; m is an integer from 1 to 12, for example from 2 to 4, for example 4; - r is 0 or an integer from 1 to 4, - q is zero or one, - T is selected from the group consisting of -(OC)-; -(CO)-O-**; -O-(OC)-**; and -NH-(CO)-O-**, where ** represents a single bond connecting the group to the hydrophobic tail group.

[0032] In the compounds disclosed herein, B can be an oxygen atom.

[0033] In the compounds disclosed herein, B can be an —NH— group.

[0034] In the compounds disclosed herein, X can be an oxygen atom.

[0035] In the compounds disclosed herein, n can be 0, 1, 2, 3, or 4.

[0036] In the compounds disclosed herein, A is selected from the group consisting of -N(CH3)2, -N(CH2-CH2-CH3)2, O-(CH2)2N(CH3)2, N-(CH2)2N(CH3)2 and NR2R3-Alk-Y- groups, where Y can be an oxygen atom or a nitrogen atom, Alk is a C2-C6 alkylene, and R2 and R3 are, independently of each other, a straight-chain or branched (C1-C6) alkyl group.

[0037] In the compounds disclosed herein, A is a 4-8 membered saturated heterocyclic radical containing 3-7 carbon atoms and 1 or 2 nitrogen atoms, wherein the 4-8 membered saturated heterocyclic radical is linked to the remainder of the molecule by a carbon or nitrogen atom and is optionally substituted by 1 to 4 substituents, each independently selected from a straight-chain or branched (C1-C6) alkyl group.

[0038] The compounds disclosed herein can have an apparent pKa of less than 7 or in the range of 4.5-7.

[0039] The compounds disclosed herein have the formula (II): [ka] wherein Z, n, and R1 may be as defined herein; R4 can be a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; R12 can be a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; p may be equal to 0 or 1, for example, p may be equal to 0; R5, R6, R7, R8 and R9 may be, independently of one another, a moiety selected from among -CH2-; -CHR12- and -NH-, and one of R5, R6, R7, R8 and R9 involved in the bond to the remainder of the molecule, a moiety selected from among -CH-; -CR12- and -N-, with the proviso that only one of R5, R6, R7, R8 and R9 is -NH- or -N-; B is an oxygen atom or an —NH— group, for example an oxygen atom, formula (II), or one of its pharmaceutically acceptable salts; the compound may be in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0040] The compounds disclosed herein have the formula (IIa): [ka] wherein R1 may be as defined herein; n can be 0, 1, 2, 3, 4, 5 or 6, for example 0-4, such as 0, 1 or 2; r can be 0, 1, 2, 3 or 4, for example, 0, 1 or 2; R4-R9, R12 and p can be as defined herein, for example, p is equal to 0; Formula (IIa), wherein m can be an integer from 1 to 12, for example an integer from 2 to 6, for example 4; or one of its pharmaceutically acceptable salts; the compound may be in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0041] The compounds disclosed herein have the formula (III): [ka] wherein R1 may be as defined herein; n can be 0, 1, 2, 3, 4, 5 or 6, for example 0-4, such as 0, 1 or 2; m can be an integer from 1 to 12, for example an integer from 2 to 6, for example 4; r can be 0, 1, 2, 3 or 4, for example, 0, 1 or 2; R4 to R8, R12 and p may be as defined herein, for example, p is equal to 0, Formula (III): or one of its pharmaceutically acceptable salts; the compound may be in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0042] The compounds disclosed herein have the formula (IV): [ka] During the ceremony, R1 may be as defined herein; -Q can be a moiety selected from the group consisting of -O(C=O)-*; -(C=O)-O*; -O(C=O)O*; -N(C=O)O-* and -O(C=O)N-*, wherein * is the moiety (CH2CHO) m indicates the connection to n may be 0, 1, 2, 3, 4, 5 or 6, for example 1 to 5, for example 2, 3 or 4; r can be 0, 1, 2, 3 or 4, for example, 0, 1 or 2; R10 and R11 are each independently a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group or a propyl group; m may be an integer from 1 to 12, for example an integer from 2 to 6, for example 4, Formula (IV), or one of its pharmaceutically acceptable salts; the compound may be in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0043] In some embodiments, Q can be a moiety selected from the group consisting of -(C=O)-O*; -O(C=O)O*; -N(C=O)O-* and -O(C=O)N-*, where * is the moiety (CH2CHO) m Indicates a connection to

[0044] The compounds disclosed herein have the formula (V): [ka] wherein Formula (V), wherein R1, R10, R11, n, m, and r may be as defined herein. or one of its pharmaceutically acceptable salts; the compound may be in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0045] The compounds disclosed herein are selected from the group consisting of:

[0046] [Table 8]

[0047] [Table 9]

[0048] [Table 10]

[0049] [Table 11]

[0050] [Table 12]

[0051] [Table 13]

[0052] [Table 14]

[0053] [Table 15]

[0054] [Table 16]

[0055] or one of its pharmaceutically acceptable salts; compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms, in particular compounds (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), or the group consisting of compounds (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), or the group consisting of compounds VII, XII, XIV, XV, XVI, XIX, XX and XXI.

[0056] In one of its aims, the present disclosure relates to compositions or lipid nanoparticles (LNPs) comprising a lipid component that includes at least one lipid compound disclosed herein.

[0057] The lipid component may further comprise at least one lipid selected from neutral lipids, structured lipids, and optionally PEG-lipids.

[0058] The neutral lipid may be selected from the group consisting of phosphatidylcholines, such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines, such as DOPE, DPPE, DMPE, DSPE, DLPE; DEPE; DPPS; DOPG; sphingomyelin; and ceramide; and mixtures thereof.

[0059] The structured lipid may be selected from the group consisting of sterols or their esters, tomatine, alpha-tocopherol, and corticosteroids, and mixtures thereof.

[0060] The sterol or ester thereof may be selected from the group consisting of cholesterol and its derivatives, ergosterol, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, diosgenin, sitosterol, sitostanol, campesterol, fecosterol, brassicasterol, tomatidine, ursolic acid, 24-methylenecholesterol, cholesteryl margallate, cholesteryl oleate and cholesteryl stearate, and mixtures thereof.

[0061] The PEG-lipid may be selected from the group consisting of PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, DSPC-PEG, DSPE-PEG, PEG-cer, mPEG-N,N-ditetradecylacetamide, PEG-dialkyloxypropylcarbamate, and mixtures thereof.

[0062] The composition of the LNP may comprise, relative to the total molar amount of lipid components, about 30% to about 70% molar amount of a lipid compound disclosed herein, about 0% to about 50% molar amount of a neutral lipid, about 20% to about 50% molar amount of a structured lipid, and about 1% to about 15% molar amount of a PEG-lipid.

[0063] The LNP compositions disclosed herein can further comprise at least one biologically active agent.

[0064] The biologically active agent can be a nucleic acid.

[0065] The nucleic acid can encode at least one antigen.

[0066] In one of its aims, the present disclosure relates to pharmaceutical compositions comprising at least one composition or LNP disclosed herein and a pharmaceutically acceptable excipient.

[0067] In one of its aims, the present disclosure relates to immunogenic compositions comprising at least one composition or LNP disclosed herein, for example, immunogenic compositions comprising nucleic acid encoding at least one antigen.

[0068] In one of its aims, the present disclosure relates to a composition or LNP disclosed herein for use as a medicament.

[0069] In one of its objects, the disclosure relates to a composition or LNP disclosed herein for use in a method for preventing and / or treating a disease selected from the group consisting of an infectious disease, an allergy, an autoimmune disease, a blood disorder, a metabolic disease, a neurological disease, and a cancer disease. [Brief explanation of the drawings]

[0070] [Figure 1] FIG. 1 is a schematic diagram of the synthesis of compound VI (Example 1). [Figure 2] FIG. 1 is a schematic diagram of the synthesis of compound VII (Example 2). [Figure 3] FIG. 1 is a schematic diagram of the synthesis of compound VIII (Example 3). [Figure 4] FIG. 1 is a schematic diagram of the synthesis of compound X (Example 5). [Figure 5] FIG. 1 is a schematic diagram of the synthesis of compound XI (Example 6). [Figure 6] FIG. 1 is a schematic diagram of the synthesis of compound XII (Example 7). [Figure 7] FIG. 1 is a schematic diagram of the synthesis of compound XIII (Example 8). [Figure 8] FIG. 1 is a schematic diagram of the synthesis of compound XIV (Example 9). [Figure 9] FIG. 1 is a schematic diagram of the synthesis of compound XV (Example 10). [Figure 10] FIG. 1 is a schematic diagram of the synthesis of compound XVI (Example 11). [Figure 11] FIG. 1 is a schematic diagram of the synthesis of compound XVII (Example 12). [Figure 12] FIG. 1 is a schematic diagram of the synthesis of compound XIX (Example 14). [Figure 13] FIG. 1 is a schematic diagram of the synthesis of compound XX (Example 15). [Figure 14] FIG. 1 is a schematic diagram of the synthesis of compound XXI (Example 16). [Figure 15] FIG. 1 is a schematic diagram of the synthesis of compound XXIII (Example 18). [Figure 16] FIG. 1 is a schematic diagram of the synthesis of compound XXVI (Example 41). [Figure 17] FIG. 1 is a schematic diagram of the synthesis of compound XXVII (Example 19). [Figure 18] FIG. 1 is a schematic diagram of the synthesis of compound XXVIII (Example 20). [Figure 19] FIG. 1 is a schematic diagram of the synthesis of compound XXIX (Example 21). [Figure 20] FIG. 1 is a schematic diagram of the synthesis of compound XXX (Example 22). [Figure 21] FIG. 1 is a schematic diagram of the synthesis of compound XXXII (Example 24). [Figure 22] FIG. 1 is a schematic diagram of the synthesis of compound XXXIII (Example 25). [Figure 23A] FIG. 1 is a schematic diagram of the synthesis of compound XXXIV (Example 26). [Figure 23B] FIG. 1 is a schematic diagram of the synthesis of compound XXXIV (Example 26). [Figure 24] FIG. 1 is a schematic diagram of the synthesis of compound XXXV (Example 27). [Figure 25] FIG. 1 is a schematic diagram of the synthesis of compound XXXVI (Example 28). [Figure 26A] FIG. 1 is a schematic diagram of the synthesis of compound XXXVII (Example 29). [Figure 26B] FIG. 1 is a schematic diagram of the synthesis of compound XXXVII (Example 29). [Figure 27] FIG. 1 is a schematic diagram of the synthesis of compound XXXVIII (Example 30). [Figure 28] FIG. 1 is a schematic diagram of the synthesis of compound XXXIX (Example 31). [Figure 29] FIG. 1 is a schematic diagram of the synthesis of compound XLVII (Example 32). [Figure 30]FIG. 1 is a schematic diagram of the synthesis of compound XLI (Example 33). [Figure 31] FIG. 1 is a schematic diagram of the synthesis of compound XLII (Example 34). [Figure 32] FIG. 1 is a schematic diagram of the synthesis of compound XLIII (Example 35). [Figure 33] FIG. 1 is a schematic diagram of the synthesis of compound XLIV (Example 36). [Figure 34A] FIG. 1 is a schematic diagram of the synthesis of compound XLV (Example 37). [Figure 34B] FIG. 1 is a schematic diagram of the synthesis of compound XLV (Example 37). [Figure 35] FIG. 1 is a schematic diagram of the synthesis of compound XLVI (Example 38). [Figure 36] FIG. 1 is a schematic diagram of the synthesis of compound XXIV (Example 39). [Figure 37] FIG. 1 is a schematic diagram of the synthesis of compound XXV (Example 40). [Figure 38] FIG. 1 is a schematic diagram of the synthesis of compound XXXI (Example 23). [Figure 39] Figure 39A depicts the luminescence (Figure 39A) or cell viability (Figure 39B) obtained at 24 hours in Huh7 cells transfected with LNP Lip(VII) and LNP SS-OP containing mRNA-luc and formulated in two different molar ratios: 50 / 39.5 / 10 / 0.5(-1) and 40 / 44.5 / 15 / 0.5(-2). [Figure 40] FIG. 10 depicts whole-body bioluminescence imaging (BLI) obtained in mice treated with LNP SS-OP (filled squares) or LNP Lip.(VII) (filled circles) containing luciferase encoding mRNA or PBS (filled pentagons). [Figure 41] Figure 41 shows the mRNA-luc in vivo distribution of luciferase protein expression in mice treated with LNP SS-OP (Figure 41A), LNP Lip.(VII) (Figure 41B), or PBS (Figure 41C) at 6 hours (black bars), 24 hours (white bars), or 48 hours (gray bars) post-injection. [Figure 42]FIG. 42B depicts mRNA-luc ex vivo tissue distribution of luciferase protein expression in mice treated with LNP SS-OP (FIG. 42A) or LNP Lip.(VII) (FIG. 42B). [Figure 43] FIG. 1 shows plasma secretion of EPO (ng / mL) in mice measured 6 hours after injection of LNP Lip.(XIV), LNP Lip.(XVII), LNP Lip.(XVIII), LNP Lip.(XIX), LNP Lip.(XXX), LNP Lip.(XXXI), LNP Lip.(XXXII), LNP Lip.(XXXIII), LNP Lip.(XXXIV), LNP Lip.(XXXV), LNP Lip.(XXXVI), LNP Lip.(XXXVII), LNP Lip.(XXXVIII), and LNP Lip.MC3, which contain non-replicating, highly purified mRNA encoding human erythropoietin. [Figure 44] This figure shows the HI titers of mice measured 3 weeks after the second immunization with LNP Lip.(VII), LNP Lip.(VIII), or LNP Lip.(XII), which contain natural non-replicating mRNA encoding the full-length hemagglutinin (HA) of influenza virus A / California / 07 / 09 strain (H1N1). DETAILED DESCRIPTION OF THE INVENTION

[0071] definition The terms used herein generally have their ordinary meanings in the art, within the context of this disclosure and within the specific context in which each term is used. Particular terms are discussed below or elsewhere herein to provide further guidance in describing the compositions and methods of the present disclosure and how to make and use them. The following definitions are provided herein, including in the claims.

[0072] The term "pharmaceutically acceptable salts" includes addition salts of compounds disclosed herein derived from the combination of such compounds with, for example, non-toxic acid addition salts.

[0073] The term "acid addition salts" includes inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as acetic acid, citric acid, propionic acid, tartaric acid, glutamic acid, salicylic acid, oxalic acid, methanesulfonic acid, para-toluenesulfonic acid, succinic acid, and benzoic acid, and related inorganic and organic acids.

[0074] Pharmaceutically acceptable salts of the compounds disclosed herein may also exist as various solvates, including water, methanol, ethanol, dimethylformamide, ethyl acetate, and the like. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in, or adventitious to, the solvent of crystallization, preparation or crystallization. Such solvates are within the scope of the present disclosure.

[0075] In the context of the present disclosure, the following chemical terms have the following meanings: Halogen atoms: fluorine, chlorine, bromine or iodine; Ct-Cz: a carbon chain that can have t to z carbon atoms (wherein t and z can have values ​​of 1 to 7); for example, C1-C4 is a carbon chain that can have 1 to 4 carbon atoms; Alk means branched or linear alkylene C2-C6; As used herein, C1-C4 alkyl refers to a C1-C4 normal saturated hydrocarbon, a secondary saturated hydrocarbon, or a tertiary saturated hydrocarbon, respectively. Non-limiting examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tertbutyl; C1-C4 alkoxy is intended to mean an —O—(C1-C4) alkyl group, wherein C1-C4 alkyl groups are as defined above. Non-limiting examples are methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy; heteroatom is understood to mean nitrogen, oxygen or sulfur; heteroaromatic ring means a 5- or 6-membered aromatic ring containing one or two heteroatoms; Aromatic ring refers to a monocyclic or polycyclic, e.g., a monocyclic aromatic hydrocarbon radical of 6 to 20 atoms, e.g., 6 atoms, derived by removing one hydrogen from a carbon atom of a parent aromatic ring system. An example of an aromatic ring disclosed herein is a phenyl group.

[0076] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In the event of a conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with and in the techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. Reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0077] Units, prefixes, and symbols are shown in their Systeme International des Unites (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, amino acid sequences are written left to right in amino to carboxy orientation, and nucleic acid sequences are written left to right in 5' to 3' orientation. The headings provided herein are not limitations of the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0078] Throughout this specification and the embodiments, the terms "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. The words "have" and "comprise" or variations such as "has," "(having), "comprises," or "comprising" are understood to mean the inclusion of stated elements (e.g., compositions of matter or method steps), but not the exclusion of any other elements. The term "consisting of" means the inclusion of the stated elements, excluding any additional elements. The term "consisting essentially of" means the inclusion of the stated elements and optionally other elements that do not materially affect the basic characteristics of the disclosure.

[0079] Various embodiments of the present disclosure that use the term "comprising" or equivalents are understood to encompass embodiments in which this term is replaced with "comprising only," "consisting of," or "consisting essentially of."

[0080] Whenever an embodiment is described herein with the language "comprising," it is understood that similar embodiments otherwise described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0081] The term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0082] Furthermore, "and / or," when used herein, should be considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," as used in phrases such as "A and / or B," is intended herein to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," as used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0083] The terms "approximately" or "about" are used herein to mean approximately, in the region of, or in the range of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above or below the stated value by a difference of, for example, 10 percent, up or down (higher or lower). In some embodiments, the term indicates a deviation from the stated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the stated numerical value. In some embodiments, "about" indicates a ±4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.01% from the indicated numerical value.

[0084] Within this disclosure, the terms "significantly" or "substantially" when used to qualify a difference or change with respect to a feature or parameter are intended to mean that the observed change or difference is noticeable and / or has statistical significance. Conversely, the terms "significantly" or "substantially" when used to qualify a similarity or identity with respect to a feature or parameter are intended to mean that the change or difference is not noticeable, or that the observed change or difference is not statistically different, or that the properties and functions of the relevant parameter or feature are not substantially affected.

[0085] As used herein, "administration" or "administering" refers to delivering a composition, e.g., lipid nanoparticles, described herein, to a subject. The composition can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, for example, orally, sublingually, chin-wise, nasally, rectally, vaginally, or via a pulmonary route. In some embodiments, administration is subcutaneous. In some embodiments, administration is intramuscular. In some embodiments, administration is intravenous. In some embodiments, administration is self-administration.

[0086] The term "antigen" includes any molecule, e.g., a peptide or protein, that elicits an immune response and / or contains at least one epitope against which an immune response is directed. For example, an antigen is a molecule that induces an immune response, e.g., specific for the antigen or a cell expressing the antigen, optionally after processing. After processing, the antigen can be expressed by an MHC molecule and specifically react with T lymphocytes (T cells). Thus, the antigen or a fragment thereof should be recognizable by a T cell receptor and, in the presence of an appropriate costimulatory signal, be capable of inducing clonal expansion of T cells bearing T cell receptors that specifically recognize the antigen or a fragment, resulting in an immune response against the antigen or a cell expressing the antigen. Any suitable antigen that is a candidate for an immune response can be envisioned. The antigen can correspond to or be derived from a naturally occurring antigen. Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents; the pathogen or antigen can also be a tumor antigen. The antigen can be provided as a nucleic acid encoding the antigen. After administration to an individual, the nucleic acid can be translated into a peptide or protein that is capable of eliciting an immune response.

[0087] The term "charged lipid" is intended to refer to any of several lipid species that exist in positively or negatively charged form within a useful physiological range, e.g., from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DODAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Choi).

[0088] As used herein, the term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is human.

[0089] The expression "ionizable cationic lipid" refers to a lipid that contains one or more groups that can be protonated at physiological pH but deprotonated at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines that can form cationic groups at physiological pH. Cationic ionizable lipid compounds may also further include C6-C 24 They may contain one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0090] The expression "lipid component" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, which are generally characterized by being poorly soluble in water but soluble in many organic solvents. Lipid is a general term that includes fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids (lipochromes), and fatty acids. Within this disclosure, "lipid" includes neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids.

[0091] The expression "lipid nanoparticle" (LNP) refers to a particle having at least one dimension on the nanometer scale (e.g., 1-1000 nm, or e.g., 10-800 nm, e.g., about 80 to about 200 nm, as measured by nanoparticle tracking analysis (NTA)). LNPs can comprise at least one lipid compound disclosed herein. LNPs can further comprise neutral lipids, structured lipids, and / or PEG-lipids. LNPs can be included in formulations that can be used to deliver biologically active agents, such as prophylactic, therapeutic, or diagnostic agents, to a desired target site (e.g., cells, tissues, organs, tumors, etc.).

[0092] The expression "neutral lipid" refers to any lipid component that is either non-ionizable or a neutral zwitterionic compound at a selected pH, e.g., physiological pH. Such lipids include, but are not limited to, neutral sphingolipids such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), or ceramide. Neutral lipids may be synthetic or naturally occurring. Neutral lipids are sometimes referred to as "helper" lipids.

[0093] Depending on the context, the term "nucleotide" or "polynucleotide" can encompass a single nucleic acid as well as multiple nucleic acids. Within this disclosure, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids can have any three-dimensional structure and can perform any function, known or unknown. In some embodiments, a polynucleotide is an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In some embodiments, a polynucleotide contains conventional phosphodiester bonds. In some embodiments, a polynucleotide contains non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids can contain regulatory elements such as promoters, enhancers, ribosomal binding sites, or transcription termination signals. "Nucleic acids," "polynucleotides," and "oligonucleotides" can be linear or circular.The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (locuses) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-end DNA (ceDNA), self-amplifying RNA (saRNA), stranded DNA (ssDNA), small interfering RNA (siRNA) and microRNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acids can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of a nucleic acid can be interrupted by non-nucleotide components. Nucleic acids can be further modified after polymerization, such as by conjugation with a labeling component. The term "complement of a nucleic acid" means a nucleic acid molecule that has a complementary base sequence and reverse orientation compared to a reference sequence, such that it can hybridize with the reference sequence with complete fidelity. "Recombinant" as applied to nucleic acids means that the nucleic acid is the product of a combination of various in vitro cloning, restriction and / or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.

[0094] The phrases "PEG-lipid" or "PEGylated lipid" are used interchangeably and are intended to refer to a molecule containing both a lipid moiety and a polyethylene glycol moiety. PEG-lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), etc.

[0095] As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from natural biological sources or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be produced by any method, including chemical synthesis. An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be readily removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0096] The expressions "sterol" or "steroid alcohol" are used interchangeably and are intended to refer to a group of lipids consisting of a sterane core with a hydroxyl moiety. Examples of steroid alcohols may include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. An ester of a steroid alcohol, or sterol, refers to an ester of a carboxylic acid with the hydroxyl group of the steroid alcohol. Suitable carboxylic acids include, in addition to the carboxyl moiety, a saturated or unsaturated, straight-chain or branched alkyl group. In some embodiments, the alkyl group is C1-C6. 20 It may be an alkyl group. In other embodiments, the carboxylic acid may be a fatty acid.

[0097] As used herein, the terms "prevent," "preventing," or "delay progression" (and grammatical variations thereof) with respect to a disease or disorder relate to prophylactic treatment of the disease, for example, in an individual suspected of having or at risk of developing the disease. Prevention can include, but is not limited to, preventing or delaying the onset or progression of the disease and / or maintaining at least one symptom of the disease at a desired or subpathological level. The term "prevent" does not require 100% elimination of the likelihood or probability of an event occurring. Rather, it means that the likelihood of an event occurring is reduced in the presence of a composition or method described herein.

[0098] The terms "treat" or "treatment" or "therapy" herein refer to the administration or consumption of a composition disclosed herein with the intent to cure, heal, alleviate, relieve, alter, correct, improve, enhance or affect the symptoms of a disorder, condition, or to prevent or delay the onset of symptoms, complications, or otherwise stop or inhibit further development of a disorder in a statistically significant manner.

[0099] As used herein, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of the pathological process under consideration. The specific therapeutically effective amount can be readily determined by a medical practitioner of ordinary skill in the art and may vary depending on factors such as the type and stage of the pathological process under consideration, the patient's medical history and age, and the administration of other therapeutic agents.

[0100] As used herein, "target cell" or "target cell" refers to a cell of interest. The cell may be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, e.g., a mammal, e.g., a human, and e.g., a human patient. In some embodiments, the target cell is a cell isolated from an individual.

[0101] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0103] The lists of sources, ingredients and components as set forth herein below are recited as such, as are combinations and mixtures thereof, which are also contemplated and are within the scope of the present specification.

[0104] It should be understood that every highest numerical limit given throughout this specification includes every lower numerical limit, as if such lower numerical limit were expressly written herein. Every lowest numerical limit given throughout this specification will include every upper numerical limit, as if such upper numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical range were expressly written herein.

[0105] All lists of items, such as, for example, lists of ingredients, are intended to be and should be construed as Markush groups. Accordingly, all lists can be read and construed as a list of items "and combinations and mixtures thereof."

[0106] Trade names for ingredients, including various raw materials, utilized in this disclosure may be referenced herein. The inventors do not intend to be limited herein by materials under any particular trade name. Materials equivalent to those referenced by trade name (e.g., those obtained from different sources under different names or reference numbers) may be substituted and utilized in the descriptions herein.

[0107] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0108] lipid compounds The lipid compounds disclosed herein are ionizable cationic lipid compounds.

[0109] The lipid compounds disclosed herein are amine-containing lipid compounds, and therefore, for example, ionizable. Because such compounds can be easily protonated, their pKa changes depending on the pH value. For example, the compounds disclosed herein have a pKa of less than 7, for example, in the range of 4.5 to 6.7.

[0110] The lipid compounds disclosed herein may have asymmetric centers, chiral axes, and chiral planes (as described in EL Eliel and SH Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and may exist as racemates, racemic mixtures, and individual diastereomers, and all possible isomers, including optical isomers, and mixtures thereof, are included in this disclosure. Furthermore, the cationic lipids disclosed herein may exist as tautomers, and even if only one tautomeric structure is shown, both tautomeric forms are intended to be encompassed within the scope of this disclosure.

[0111] Pharmaceutically acceptable salts of the compounds disclosed herein generally have one or several physiologically acceptable counterions. Possible counterions may include, for example, halides, phosphates, trifluoroacetates, sulfites, nitrates, gluconates, glucuronates, galacturonate radicals, alkylsulfonates, alkylcarboxylates, propionatesulfonates, and methanesulfonates.

[0112] The compounds disclosed herein and their pharmaceutically acceptable salts may also exist as various solvates, including water, methanol, ethanol, dimethylformamide, ethyl acetate, and the like. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in, or adventitious to, the solvent of crystallization, preparation or crystallization. Such solvates are within the scope of the present disclosure.

[0113] For example, the lipid compounds disclosed herein have the formula (I): A-(CH2) n -CX-BZ-R1(I) (In the formula, - R1, C 10 ~C 57 , or C 10 ~C 55 is a lipophilic or hydrophobic tail group of - Z is a spacer arm having 2 to 24, such as 2 to 18, such as 4 to 12 carbon atoms in a branched or unbranched, linear, saturated or unsaturated hydrocarbon chain, interrupted by one or several atoms of oxygen and / or a moiety selected from among -SS-;-(OC)-;-(CO)-O-;-O-(OC)-;-S-;-NH-, -NH-(OC)-;-(OC)-NH- and -NH-(CO)-O-, such as -(CO)-O-;-O-(OC)- and -NH-(CO)-O-, and optionally having an oxygen atom or a moiety selected from among -NH-(OC)-*-O-(OC)--*;-(CO)-O-*; and -(OC)-, the chain being linked at its end to the hydrophobic tail group, * indicating a single bond linking the moiety to the hydrophobic tail group, B represents an oxygen atom or an -NH- group; X is an oxygen atom or a sulfur atom, - n is 0, 1, 2, 3, 4, 5 or 6; - A is - the group R2R3N-, in which R2 and R3 independently of one another represent a linear or branched (C1-C6) alkyl group; the NR2R3-Alk-Y- group, in which Y is an oxygen atom or a nitrogen atom, Alk is a C2-C6 alkylene moiety, and R2 and R3, independently of one another, are a linear or branched (C1-C6) alkyl group; - a 4-8 membered saturated heterocyclic radical containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms, which 4-8 membered saturated heterocyclic radical is linked to the rest of the molecule by a carbon or nitrogen atom and is optionally substituted by 1 to 4 substituents selected, independently of one another, from linear or branched (C1-C6) alkyl groups; represents a group selected from the group consisting of: or one of its pharmaceutically acceptable salts; with the compound being in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0114] According to one embodiment, the compounds disclosed herein are in cationic form.

[0115] According to one embodiment, B is an oxygen atom.

[0116] According to one embodiment, B is a —NH— group.

[0117] According to one embodiment, X is an oxygen atom.

[0118] According to one embodiment, n is 0, 1, 2, 3 or 4.

[0119] In some embodiments, R is C 10 ~C 55 is a lipophilic or hydrophobic tail group.

[0120] According to one particular embodiment, R1 is an optionally substituted, branched or unbranched, linear, saturated or unsaturated, C 10 ~C 57 , or C 10 ~C 55 hydrocarbon radicals, e.g., C 10 ~C 55and a hydrocarbon backbone optionally interrupted by one or several atoms of oxygen or nitrogen and / or one or several moieties -(O-C)-;-(CO)-O-;-O-(O-C)-, and one nitrogen atom, if present in the backbone, can be linked directly or indirectly to the Z radical.

[0121] For example, the hydrophobic or lipophilic tails R1 can each independently be an optionally substituted C8-C 24 , e.g. C 10 ~C 20 , alkyl chains, optionally substituted, variably saturated or unsaturated C to C 24 , e.g. C 10 ~C 20 , alkenyl chains and optionally substituted saturated, variably saturated or unsaturated C-C 24 , e.g. C 10 ~C 20 and the alkyl, alkenyl or acyl chains may be interrupted by one or several oxygen or nitrogen atoms and / or one or several moieties such as -(CO)-O-; -O-(OC)-, preferably by at least one moiety such as -(CO)-O-; -O-(OC)-.

[0122] Each hydrocarbon chain may be substituted with at least one group selected from -OH and -CO2H.

[0123] According to one specific embodiment, the hydrophobic or lipophilic tail is selected from the group consisting of:

[0124] [Table 17]

[0125] [Table 18]

[0126] [Table 19]

[0127] [Table 20]

[0128] [Table 21]

[0129] [Table 22]

[0130] [Table 23]

[0131] [Table 24]

[0132] In one embodiment, the hydrophobic or lipophilic tail of a compound according to the present disclosure does not contain a nitrogen atom, and in this particular embodiment, the hydrophobic or lipophilic tail is specifically selected from among R1a, b, c, d, h, i, j, k, s, cc, dd, ff, gg, and hh.

[0133] In another embodiment, the hydrophobic or lipophilic tail of a compound according to the present disclosure comprises one nitrogen atom, which is directly or indirectly linked to Z. In this particular embodiment, the hydrophobic or lipophilic tail is selected from among Rle, f, g, l, m, n, o, p, q, r, t, u, v, w, x, y, z, aa, bb, ee, ii, and jj, among others.

[0134] In another embodiment, the hydrophobic or lipophilic tail of a compound according to the present disclosure comprises at least three or more hydrocarbon chains, such as hydrophobic or lipophilic tails R1b, e, f, g, h, i, j, k, l, m, n, o, p, qr, s, t, u, v, w, x, y, z, aa, bb, cc, dd, ee, ff, gg, hh, ii, and jj. Each hydrocarbon chain may be substituted C8-C 24 , e.g. C 10 ~C 20 , alkyl chains and substituted variably saturated or unsaturated C8-C 24 , e.g. C 10 ~C 20 , alkenyl chains, where the alkyl or alkenyl chain is optionally and preferably interrupted by one or several moieties such as -(C=O)-, -O-(C=O)-** or -(C=O)-O-**.

[0135] In certain embodiments, the hydrophobic or lipophilic tail of the compounds according to the present disclosure is a tail selected from R1b, R1f, R1h, R1i, R1j, R1bb, R1hh, R1ii, in particular R1.

[0136] According to another embodiment, the cationic and / or ionizable lipid compounds disclosed herein have a Z radical of the formula: -((CH2)2-O) m -(CH2) r (T) q -*(Z) where: - * denotes a single bond connecting the radical to the hydrophobic tail group; m is an integer from 1 to 12, for example from 2 to 4, for example 4; - r is 0 or an integer from 1 to 4, - q is zero or one, - T is selected from the group consisting of -(OC)-; -(CO)-O-**; -O-(OC)-**; and -NH-(CO)-O-**, where ** represents a single bond connecting the group to the hydrophobic tail group.

[0137] In certain embodiments, q and r are different from 0, for example, r is equal to 1 or 2. For example, Z is Za, Zb, Zc, or Zd: [ka] [ka]

[0138] In another particular embodiment, q is equal to 1 and r is equal to 0. For example, Z is Ze or Zf: [ka]

[0139] In another particular embodiment, q is equal to 0 and r is different from 0, for example r is equal to 1 or 2. For example, Z is Zg or Zh: [ka]

[0140] In another specific embodiment, r and q are equal to 0 and Z is —((CH 2 ) 2 —O) m -, and m is as defined herein above.

[0141] In another specific embodiment, Z is: [ka]

[0142] In another specific embodiment, Z is: [ka]

[0143] In certain embodiments, Z is selected from among Za, b, c, d, f, g, h, I, j, and k.

[0144] As regards the radical A, it in certain embodiments represents an R2R3N- group, where R2 and R3 independently of each other represent a linear or branched (C1-C6) alkyl group, for example, A is -N(CH3)2, -N(CH2-CH2-CH3).

[0145] In certain embodiments, compounds of Formula (I) contain one R1-Z moiety selected from among the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0146] According to one embodiment, the radical A is an R2R3N- group, in which R2 and R3 independently of each other represent a linear or branched (C1-C6) alkyl group, for example dimethylamino or dipropylamino.

[0147] According to another embodiment, the radical A represents a group NR2R3-Alk-Y-, where Y is an oxygen atom or a nitrogen atom, Alk is a C2-C6 alkylene, and R2 and R3, independently of each other, represent a linear or branched (C1-C6) alkyl group, for example A is Aa or Ab. [ka]

[0148] According to a particular embodiment, the radical A is selected from the group consisting of -N(CH3)2, -N(CH2CH2-CH3)2, -O-(CH2)2N(CH3)2 and -N-(CH2)2N(CH3)2.

[0149] According to another embodiment, the radical A represents a 4-8 membered saturated heterocyclic radical containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms, which 4-8 membered saturated heterocyclic radical is linked to the rest of the molecule by a carbon or nitrogen atom and is optionally substituted by 1 to 4 substituents, independently of one another, selected from linear or branched (C1-C6) alkyl groups. For example, A is selected from the group consisting of piperidinyl radicals, piperazinyl radicals, and pyrrolidinyl radicals. For example, A is selected from the group consisting of 3-piperidinyl radicals, 4-piperidinyl radicals, 3-piperidinyl radicals substituted with one or two methyl groups, 4-piperidinyl radicals substituted with one or two methyl groups, 1-piperazinyl radicals, 1-piperazinyl radicals substituted with methyl groups, 3-pyrrolidinyl radicals, and 3-pyrrolidinyl radicals substituted with one or two methyl groups.

[0150] According to this particular embodiment, A is, for example, selected from among piperidinyl, 1-methylpiperidinyl, 1,3-dimethylpiperidinyl, pyrrolidinyl and 1,3-dimethylpyrrolidinyl.

[0151] According to one embodiment, the compounds of formula (I) have an apparent pKa of less than 7 or in the range of 4.5-7.

[0152] Another object of the present disclosure is to provide a compound of formula (II) [ka] wherein - Z, n and R1 are as defined herein above, R4 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; R12 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; - p is equal to 0 or 1, for example, p is equal to 0, - R5, R6, R7, R8 and R9 are, independently of one another, a moiety selected from among -CH2-; -CHR12- and -NH-, and one of R5, R6, R7, R8 and R9 involved in the bond to the rest of the molecule, a moiety selected from among -CH-; -CR12- and -N-, with the proviso that only one of R5, R6, R7, R8 and R9 is -NH- or -N-; one compound of formula (II), in which B represents an oxygen atom or an —NH— group, for example an oxygen atom, or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0153] Another object of the present disclosure is to provide a compound of formula (IIa) [ka] wherein R1 is as defined above, - n is 0, 1, 2, 3, 4, 5 or 6, for example 0 to 4, such as 0, 1 or 2; - r is 0, 1, 2, 3 or 4, for example 0, 1 or 2; R4 to R9, R12 and p are as defined above, for example p is equal to 0, one compound of formula (IIa), wherein m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0154] Another object of the present disclosure is to provide a compound of formula (III) [ka] wherein R1 is as defined above, - n is 0, 1, 2, 3, 4, 5 or 6, for example 0 to 4, such as 0, 1 or 2; m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4; - r is 0, 1, 2, 3 or 4, for example 0, 1 or 2; one compound of formula (III), in which R4 to R8, R12 and p are as defined above, for example p is equal to 0, or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0155] Another object of the present disclosure is to provide a compound of formula (IV) [ka] wherein R1 is as defined above, Q is a moiety selected from the group consisting of -O(CO)-*; -(C=O)-O*; -O(C=O)O*; -N(CO)O-* and -O(CO)N-*, * being the moiety (CH2CHO) m indicates the connection to - n is 0, 1, 2, 3, 4, 5 or 6, for example 1 to 5, for example 2, 3 or 4; - r is 0, 1, 2, 3 or 4, for example 0, 1 or 2; R10 and R11 each independently represent a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group or a propyl group; one compound of formula (IV), wherein m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0156] In some embodiments, Q can be a moiety selected from the group consisting of -(C=O)-O*; -O(C=O)O*; -N(C=O)O-* and -O(C=O)N-*, where * is the moiety (CH2CHO) m Indicates a connection to

[0157] Another object of the present disclosure is to provide a compound of formula (V) [ka] wherein one compound of formula (V), wherein R1, R10, R11, n, m and r are as defined herein above, or one of its pharmaceutically acceptable salts; relates to compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0158] According to one embodiment, the compound of formula (II) is selected from the group consisting of the following compounds VI to XLVII in Table 1. In the following formula, the secondary amino moiety may be represented interchangeably as -NH- or -N-.

[0159] [Table 25]

[0160] [Table 26]

[0161] [Table 27]

[0162] [Table 28]

[0163] [Table 29]

[0164] [Table 30]

[0165] [Table 31]

[0166] [Table 32]

[0167] [Table 33]

[0168] or one of its pharmaceutically acceptable salts; a compound in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0169] According to one specific embodiment, the compound of formula (I) is selected from among the compounds listed in Table 2.

[0170] [Table 34]

[0171] [Table 35]

[0172] [Table 36]

[0173] [Table 37]

[0174] or one of its pharmaceutically acceptable salts; a compound in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0175] Compounds according to the present disclosure can be readily prepared from commercially available or described in the literature starting materials using methods and procedures known to those skilled in the art.

[0176] Compositions, lipid nanoparticles, and manufacturing processes The composition or lipid nanoparticle (LNP) can include a lipid component comprising at least one lipid compound disclosed herein.

[0177] The lipid component may further comprise at least one lipid selected from neutral lipids, structured lipids, and optionally PEG-lipids.

[0178] The composition or LNP can include at least one biologically active agent, which can be a prophylactic, therapeutic, immunomodulatory, or diagnostic agent.

[0179] Ionizable cationic lipids The lipid component of the composition or LNP comprises at least one lipid compound described herein, such lipid compound being an ionizable cationic lipid.

[0180] The composition or LNP may comprise a molar amount of a lipid compound disclosed herein, expressed as a percentage of the total molar amount of lipid components, of about 20% to about 60%, or about 25% to about 60%, or about 30% to about 55%, or about 40% to about 55%, or about 40% to about 50%.

[0181] The composition or LNP may comprise a lipid compound disclosed herein in a molar amount of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the total molar amount of the lipid component.

[0182] The lipid compounds disclosed herein may be present in an amount of about 50% by weight based on the total weight of the lipid component of the composition or LNP.

[0183] In one embodiment, a suitable lipid compound may be a lipid compound of formula (I):

[0184] In one embodiment, a suitable lipid compound may be a lipid compound of formula (II):

[0185] In one embodiment, a suitable lipid compound may be a lipid compound of formula (IIa):

[0186] In one embodiment, a suitable lipid compound may be a lipid compound of formula (III):

[0187] In one embodiment, a suitable lipid compound may be a lipid compound of formula (IV):

[0188] In one embodiment, a suitable lipid compound may be a lipid compound of formula (V):

[0189] In one embodiment, suitable lipid compounds may be those shown in Table 1.

[0190] In one embodiment, suitable lipid compounds may be those shown in Table 2.

[0191] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0192] In some embodiments, the lipid compound can be of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0193] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), (XI), or (XII).

[0194] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), or (XII).

[0195] In some embodiments, the lipid compound can be of formula (VII), (XII), (XIV), (XV), (XVI), (XIX), (XX), or (XXI).

[0196] In some embodiments, the lipid compound can be of formula (VII), (XII), (XIV), (XVI), (XIX), or (XXI).

[0197] In some embodiments, the lipid compound can be of formula (VI), (VII), or (XII).

[0198] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0199] In some embodiments, the lipid compound can be of formula (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0200] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), or (XXXVIIII).

[0201] In some embodiments, the lipid compound can be of formula (XVI), (XVIII), (XXXIII), (XXXIV), or (XXXVIII).

[0202] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), (XXXIV), or (XXXVIII).

[0203] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), or (XXXVIII).

[0204] neutral lipid The lipid component of the composition or LNP may include a neutral lipid. The presence of a neutral lipid may improve the structural stability of the lipid nanoparticle. The neutral lipid may be appropriately selected in consideration of the delivery efficiency of a biologically active agent such as a nucleic acid.

[0205] Neutral lipids differ from the lipid compounds disclosed herein in that they are either not ionizable or are zwitterionic compounds that are neutral at a selected pH.

[0206] The neutral lipid may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and ceramide.

[0207] Phosphatidylcholine and phosphatidylethanolamine are zwitterionic lipids. Sphingomyelin and ceramide are not ionizable lipids.

[0208] Examples of phosphatidylcholines include DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and mixtures thereof.

[0209] Examples of phosphatidylethanolamines include DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-s / i-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-SM-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, or 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and mixtures thereof.

[0210] The neutral lipid may be selected from the group consisting of phosphatidylcholines such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines such as DOPE, DPPE, DMPE, DSPE, DLPE, DEPE; sphingomyelin; ceramide; and mixtures thereof.

[0211] In one embodiment, the neutral lipid may be DEPE, DSPC, DOPC, or DOPE, or a mixture thereof, for example, DEPE, DSPC, or DOPE, or a mixture thereof.

[0212] In one embodiment, the neutral lipid can be DSPC, DOPC, or DOPE, or a mixture thereof, for example, DSPC or DOPE, or a mixture thereof.

[0213] In one embodiment, the neutral lipid can be DSPC.

[0214] The composition or LNP may comprise at least one neutral lipid in a molar amount ranging from about 0% to about 50%, about 5% to about 45%, about 8% to about 40%, or about 10% to about 30%, relative to the total molar amount of the lipid components of the composition or LNP.

[0215] The composition or LNP may comprise at least one neutral lipid in a molar amount of about 0%, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50% relative to the total molar amount of the lipid components of the composition or LNP.

[0216] The neutral lipid may be present in the composition or LNP at a molar ratio of lipid compound:neutral lipid that may range from about 70:1 to about 1:2, about 30:1 to about 1:1, about 15:1 to about 2:1, about 10:1 to about 4:1, or about 5:1.

[0217] structured lipids The lipid component of the composition or LNP may include a structured lipid. The presence of a structured lipid, such as a sterol or an ester of a sterol, may improve the structural stability of the lipid nanoparticle.

[0218] LNPs as disclosed herein can include at least one steroid alcohol (ie, sterol) or ester thereof.

[0219] The structured lipid may be selected from the group consisting of sterols or esters thereof, tomatine, alpha-tocopherol, corticosteroids, and mixtures thereof.

[0220] Sterols include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3β-ol), lathosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), and erythrosterol (erythrosterol). ol), campestanol (5a-campestan-3b-ol), fecosterol, brassicasterol, tomatidine, ursolic acid, 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol); BHEM-cholesterol (2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]henanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan-1-aminium bromide; and mixtures thereof.

[0221] Esters of sterols refer to esters of a carboxylic acid with a hydroxyl group of a steroid alcohol. Suitable carboxylic acids contain, in addition to the carboxyl moiety, a saturated or unsaturated, straight or branched alkyl group. In some embodiments, the alkyl group is C1-C 20 Saturated or unsaturated, linear or branched alkyl groups, e.g., C2-C 18 , for example C4~C 16 , for example C8~C 12The alkyl group may be saturated or unsaturated, straight-chain or branched. In other embodiments, the carboxylic acid may be a fatty acid. For example, the fatty acid may be caprylic acid, caproic acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.

[0222] The ester of sterol may be selected from the group consisting of cholesteryl margallate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate; and mixtures thereof.

[0223] In one embodiment, the ester of a sterol may be a cholesteryl ester.

[0224] Sterols or their esters include cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3β-ol), lathosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirosterol), erythrosterol ( ...

[0039] The hydroxybenzoate may be selected from the group consisting of 22,23-dihydrostigmasterol, sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), fecosterol, brassicasterol, tomatidine, ursolic acid, 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and mixtures thereof.

[0225] Alternatively, the sterol may be a cholesterol derivative, such as oxidized cholesterol.

[0226] The oxidized cholesterol suitable for the present disclosure can be 25-hydroxycholesterol, 27-hydroxycholesterol, 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-keto-cholesterol, 7β,25-hydroxycholesterol, 7β-hydroxycholesterol, and combinations thereof. For example, the oxidized cholesterol can be 25-hydroxycholesterol and 20α-hydroxycholesterol, such as 20α-hydroxycholesterol, and mixtures thereof.

[0227] In one embodiment, the sterol, or ester thereof, can be cholesterol, a cholesteryl ester, or a cholesterol derivative, such as oxidized cholesterol.

[0228] In one embodiment, the sterol may be cholesterol or a cholesteryl ester.

[0229] In one embodiment, the sterol can be cholesterol.

[0230] In one embodiment, the sterol can be sitosterol.

[0231] The corticosteroid may be selected from prednisolone, dexamethasone, prednisone, hydrocortisone, or mixtures thereof.

[0232] The structured lipid may be present in the lipid component of the composition or LNP in a molar amount ranging from about 20% to about 55%, or from about 20% to about 50%, or from about 25% to about 45%, % w / w of the total molar amount of the lipid component.

[0233] The structured lipid may be present in the lipid component of the composition or LNP in a molar amount of about 20%, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, about 50, about 52, about 54, or about 55% w / w of the total molar amount of the lipid component.

[0234] In one embodiment, the structured lipid may be present in an amount of about 28.5%, or about 38.5%, or about 40.9%, or about 42.7% by % w / w, relative to the total molar amount of lipid components of the LNP or composition.

[0235] The structured lipid may be present in the lipid component in an amount of 38.5% (% w / w) based on the total molar amount of the lipid component.

[0236] In one embodiment, the structural lipid can be cholesterol.

[0237] The structured lipid may be present in the lipid component of the composition or LNP in a molar ratio of lipid compound:structured lipid ranging from about 4:1 to about 1:2, such as from about 3.5:1 to about 1:1.8, such as from about 2:1 to about 1:1.5, such as from about 1.5:1 to about 1:1.2, e.g., from about 1.3:1 to about 1:1.3.

[0238] PEG-lipid The lipid component of the composition or LNP can include a PEG-lipid (or a PEGylated lipid).

[0239] Possible PEG-modified lipids include C6-C w These include, but are not limited to, polyethylene glycol chains of 5 kDa or less in length covalently attached to lipids having alkyl chain lengths of 10 kDa or less. The addition of PEG-modified lipids to LNP compositions can prevent complex aggregation and also provide a means for increasing circulation lifetime and delivery of the composition or lipid nanoparticles to target cells.

[0240] Suitable PEG-lipids include, for example, PEGylated diacylglycerols (PEG-DAG), such as λ-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); PEGylated phosphatidylethanolamine (PEG-PE); PEG succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-λ-O-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG). PEGylated ceramide (PEG-cer); DSPC-PEG; DSPE-PEG; PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxyl)propyl)carbamate; 2,3-di(tetradecanoxyl)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and mixtures thereof.

[0241] In one embodiment, a suitable PEG-lipid may be selected from the group consisting of PEG-DAG; PEG-DMG; PEG-PE; PEG-S-DAG; PEG-S-DMG; DSPC-PEG; DSPE-PEG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and mixtures thereof.

[0242] For example, the PEG-lipid can be PEG-DMG, PEG-PE, DSPC-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), and mixtures thereof.

[0243] In one embodiment, the PEG-lipid can be a PEG-PE, such as PEG-2000-PE.

[0244] In one embodiment, the PEG-lipid can be PEG-DMG, such as DMG-PEG-2000.

[0245] In one embodiment, the PEG-lipid can be a DSPC-PEG, such as DSPC-PEG-2000.

[0246] In one embodiment, the PEG-lipid can be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0247] The PEG-lipid may be present in the lipid component of the composition or LNP in a molar amount ranging from about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, or about 1% to about 3.5%, relative to the total molar amount of the lipid component.

[0248] The PEG-lipid may be present in the lipid component of the composition or LNP in a molar amount of about 1%, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15% relative to the total molar amount of the lipid component.

[0249] In one embodiment, the PEG-lipid can be MDG-PEG, such as DMG-PEG-2000, present in an amount of, for example, about 1.5% relative to the total molar amount of the lipid component.

[0250] In one embodiment, the PEG-lipid can be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), present in an amount of about 1.6% or about 1.7%, for example, w / w% based on the total weight of the lipid component of the LNP.

[0251] The PEG-lipid and lipid compounds disclosed herein may be present in the lipid component of a composition or LNP in a molar ratio of lipid compound to PEG-lipid of about 70:1 to about 4:1, about 40:1 to about 10:1, about 35:1 to about 15:1, or about 33:1, or about 14:1.

[0252] LNP formulation In one embodiment, a composition or LNP may comprise a lipid component comprising at least about 30% to about 70% molar amount of a lipid compound disclosed herein, about 0% to about 50% molar amount of a neutral lipid, about 20% to about 50% molar amount of a structured lipid, and optionally about 1% to about 15% molar amount of a PEG-lipid, relative to the total molar amount of the lipid component.

[0253] In one embodiment, a composition or LNP may comprise a lipid component comprising at least about 30% to about 70% molar amount of a lipid compound disclosed herein, about 0% to about 50% molar amount of a neutral lipid, about 20% to about 50% molar amount of a structured lipid, and about 1% to about 15% molar amount of a PEG-lipid, relative to the total molar amount of the lipid component.

[0254] In one embodiment, the composition or LNP may comprise a lipid component comprising a lipid compound disclosed herein, a neutral lipid, a structured lipid, and a PEG-lipid, in molar amounts, relative to the total molar amount of the lipid component, of about 35% to about 55% of the lipid compound disclosed herein, about 5% to about 35% of the neutral lipid, about 25% to about 45% of the structured lipid, and about 1.0% to about 2.5% of the PEG-lipid.

[0255] In one embodiment, a composition or LNP may comprise a lipid component comprising a lipid compound disclosed herein, a neutral lipid, a structured lipid, and a PEG-lipid, in molar amounts, relative to the total molar amount of the lipid component, of about 40% to about 50% lipid compound, about 9% to about 30% neutral lipid, about 28% to about 45% structured lipid, and about 1.5% to about 2.5% PEG-lipid.

[0256] In one embodiment, the composition or LNP may comprise lipid components comprising a lipid compound disclosed herein, a neutral lipid, a structured lipid, and a PEG-lipid in a molar ratio of about 35 / 16 / 46.5 / 1.5, about 50 / 10 / 38.5 / 1.5, about 57.2 / 7.1 / 34.3 / 1.4, about 40 / 15 / 40 / 5, about 50 / 10 / 35 / 4.5 / 0.5, about 50 / 10 / 35 / 5, about 40 / 10 / 40 / 10, about 35 / 15 / 40 / 10, or about 52 / 13 / 30 / 5.

[0257] In one embodiment, the molar ratio of lipid compounds disclosed herein, neutral lipids, structured lipids, and PEG-lipids can be about 35 / 16 / 46.5 / 1.5 or about 50 / 10 / 38.5 / 1.5.

[0258] In one embodiment, the molar ratio of lipid compounds disclosed herein, neutral lipids, structured lipids, and PEG-lipids can be about 50 / 10 / 38.5 / 1.5.

[0259] In one embodiment, a composition or LNP may comprise a lipid component comprising a lipid compound disclosed herein, a neutral lipid, a structured lipid, and a PEG-lipid in molar amounts of about 50% lipid compound, about 10% neutral lipid, about 38.5% structured lipid, and about 1.5% PEG-lipid, based on the total weight of the lipid component of the LNP.

[0260] In one embodiment, the lipid compound can be a lipid compound of formula (I).

[0261] In one embodiment, a suitable lipid compound may be a lipid compound of formula (II):

[0262] In one embodiment, a suitable lipid compound may be a lipid compound of formula (IIa):

[0263] In one embodiment, a suitable lipid compound may be a lipid compound of formula (III):

[0264] In one embodiment, a suitable lipid compound may be a lipid compound of formula (IV):

[0265] In one embodiment, a suitable lipid compound may be a lipid compound of formula (V):

[0266] In one embodiment, suitable lipid compounds may be those shown in Table 1.

[0267] In one embodiment, suitable lipid compounds may be those shown in Table 2.

[0268] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0269] In some embodiments, the lipid compound can be of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0270] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), (XI), or (XII).

[0271] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), or (XII).

[0272] In some embodiments, the lipid compound can be of formula (VII), (XII), (XIV), (XV), (XVI), (XIX), (XX), or (XXI).

[0273] In some embodiments, the lipid compound can be of formula (VII), (XII), (XIV), (XVI), (XIX), or (XXI).

[0274] In some embodiments, the lipid compound can be of formula (VI), (VII), or (XII).

[0275] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0276] In some embodiments, the lipid compound can be of formula (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), or (XXXVIIII).

[0277] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), or (XXXVIIII).

[0278] In some embodiments, the lipid compound can be of formula (XVI), (XVIII), (XXXIII), (XXXIV), or (XXXVIII).

[0279] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), (XXXIV), or (XXXVIII).

[0280] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), or (XXXVIII).

[0281] In one embodiment, the neutral lipid can be DEPE.

[0282] In one embodiment, the neutral lipid can be DOPE.

[0283] In one embodiment, the neutral lipid can be DSPC.

[0284] In one embodiment, the structural lipid can be cholesterol.

[0285] In one embodiment, the PEG-lipid can be PEG-PE (PEG-2000-PE) or PEG-DMG (PEG-2000-DMG).

[0286] In one embodiment, the PEG-lipid can be PEG-DMG (PEG-2000-DMG).

[0287] In one embodiment, the lipid compound can be of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0288] In one embodiment, the lipid compound can be of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0289] In one embodiment, the lipid compound can be of formula (VI), (VII), (VIII), (XI), or (XII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0290] In some embodiments, the lipid compound can be of formula (VI), (VII), (VIII), or (XII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0291] In one embodiment, the lipid compound can be of formula (VII), (XII), (XIV), (XV), (XVI), (XIX), (XX) or (XXI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0292] In some embodiments, the lipid compound can be of formula (VII), (XII), (XIV), (XVI), (XIX), or (XXI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0293] In one embodiment, the lipid compound can be of formula (VI), (VII), or (XII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0294] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0295] In some embodiments, the lipid compound can be of formula (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0296] In some embodiments, the lipid compound can be of formula (XIV), (XVI), (XVIII), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), or (XXXVIIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0297] In some embodiments, the lipid compound can be of formula (XVI), (XVIII), (XXXIII), (XXXIV), or (XXXVIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0298] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), (XXXIV), or (XXXVIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0299] In some embodiments, the lipid compound can be of formula (XVIII), (XXXIII), or (XXXVIII).

[0300] In one embodiment, the lipid compound can be of formula (VI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0301] In one embodiment, the lipid compound can be of formula (VII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0302] In one embodiment, the lipid compound can be of formula (VIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0303] In one embodiment, the lipid compound can be of formula (XII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0304] In one embodiment, the lipid compound can be of formula (XI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0305] In one embodiment, the lipid compound can be of formula (XIV), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0306] In one embodiment, the lipid compound can be of formula (XV), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0307] In one embodiment, the lipid compound can be of formula (XVI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0308] In one embodiment, the lipid compound can be of formula (XIX), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0309] In one embodiment, the lipid compound can be of formula (XX), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0310] In one embodiment, the lipid compound can be of formula (XXI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0311] In one embodiment, the lipid compound can be of formula (XXVII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0312] In one embodiment, the lipid compound can be of formula (XVII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0313] In one embodiment, the lipid compound can be of formula (XVIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0314] In one embodiment, the lipid compound can be of formula (XXX), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0315] In one embodiment, the lipid compound can be of formula (XXXI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0316] In one embodiment, the lipid compound can be of formula (XXXII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0317] In one embodiment, the lipid compound can be of formula (XXXIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0318] In one embodiment, the lipid compound can be of formula (XXXIV), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0319] In one embodiment, the lipid compound can be of formula (XXXV), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0320] In one embodiment, the lipid compound can be of formula (XXXVI), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0321] In one embodiment, the lipid compound can be of formula (XXXVII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0322] In one embodiment, the lipid compound can be of formula (XXXVIII), the neutral lipid can be DSPC or DOPE, the structural lipid can be cholesterol or sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000) or PEG-PE (PE-PEG2000).

[0323] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% of a lipid compound of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0324] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% of a lipid compound of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0325] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (VI), (VII), (VIII), (XI) or (XII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0326] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (VI), (VII), (VIII), or (XII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0327] In one embodiment, the lipid component of the composition or LNP may comprise, in % of the total molar amount of the lipid component, 50% lipid compound of formula (VII), (XII), (XIV), (XV), (XVI), (XIX), (XX) or (XXI), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0328] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (VII), (XII), (XIV), (XVI), (XIX) or (XXI), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0329] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (VI), (VII), or (XII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0330] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% of a lipid compound of formula (XIV), (XVI), (XVIII), (XIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0331] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0332] In one embodiment, the lipid component of the composition or LNP may comprise, in % of the total molar amount of the lipid component, 50% lipid compound of formula (XIV), (XVI), (XVIII), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI) or (XXXVIIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0333] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (XVI), (XVIII), (XXXIII), (XXXIV) or (XXXVIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0334] In one embodiment, the lipid component of the composition or LNP may comprise, in % relative to the total molar amount of the lipid component, 50% lipid compound of formula (XVIII), (XXXIII), (XXXIV) or (XXXVIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0335] In one embodiment, the lipid component of the composition or LNP may comprise, in percentages based on the total molar amount of the lipid component, 50% lipid compound of formula (XVIII), (XXXIII), or (XXXVIII), 10% DSPC or DOPE, 38.5% cholesterol or sitosterol, and 1.5% PEG-DMG (PEG-2000-DMG) or PEG-PE (PE-PEG2000).

[0336] The neutral lipid can be DSPC.

[0337] The structural lipid can be cholesterol.

[0338] The PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0339] In one embodiment, the lipid compound can be of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0340] In one embodiment, the lipid compound can be of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0341] In one embodiment, the lipid compound can be (VI), (VII), (VIII), (XI) or (XII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0342] In one embodiment, the lipid compound can be (VI), (VII), (VIII), or (XII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0343] In one embodiment, the lipid compound can be of formula (VII), (XII), (XIV), (XV), (XVI), (XIX), (XX) or (XXI), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0344] In one embodiment, the lipid compound can be of formula (VII), (XII), (XIV), (XVI), (XIX), or (XXI), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0345] In one embodiment, the lipid compound can be (VI), (VII), or (XII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0346] In one embodiment, the lipid compound can be (XIV), (XVI), (XVIII), (XIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0347] In one embodiment, the lipid compound can be (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0348] In one embodiment, the lipid compound can be (XIV), (XVI), (XVIII), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), or (XXXVIIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0349] In one embodiment, the lipid compound can be (XVI), (XVIII), (XXXIII), (XXXIV) or (XXXVIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0350] In one embodiment, the lipid compound can be (XVIII), (XXXIII), (XXXIV) or (XXXVIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0351] In one embodiment, the lipid compound can be (XVIII), (XXXIII), or (XXXVIII), the neutral lipid can be DSPC, the structural lipid can be sitosterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0352] Manufacturing method of LNPs Methods for producing LNPs are known in the art.

[0353] In one embodiment, the LNPs containing a biologically active agent, e.g., a nucleic acid, comprise at least: i) solubilizing the lipid components of the LNP in a water-miscible organic solvent; ii) mixing the organic solvent obtained in step a) with an aqueous solvent containing a biologically active agent; iii) obtaining LNPs in an aqueous solvent; It can be obtained by a method comprising:

[0354] In one embodiment, a method for producing LNPs includes at least: i) solubilizing at least the lipid compounds disclosed herein, neutral lipids, structured lipids, and optionally PEG-lipids in a water-miscible organic solvent; ii) mixing the organic solvent obtained in step i) with an aqueous solvent containing a biologically active agent; iii) obtaining lipid nanoparticles containing a biologically active agent in an aqueous solvent; may include:

[0355] The biologically active agent can be a nucleic acid.

[0356] Useful water-miscible organic solvents can be any water-miscible organic solvent that can solubilize the lipid compounds disclosed herein and any other added lipids. Examples of suitable organic solvents can include ethanol or methanol, 1-propanol, isopropanol, t-butanol, THF, DMSO, acetone, acetonitrile, diglyme, DMF, 1,4-dioxane, ethylene glycol, glycerin, hexamethylphosphoramide, hexamethylphosphorous triamide. In one embodiment, the organic solvent can be ethanol and isopropanol.

[0357] The lipid compounds disclosed herein may be present in an amount sufficient to structure the lipid nanoparticles and encapsulate any payload to be encapsulated. The amount of lipid compound used may be determined by those skilled in the art according to any known technique and will be adapted according to the nature and amount of the payload and the nature and amount of other lipids likely to be present.

[0358] The lipid compounds, neutral lipids, structured lipids, and optionally PEGylated lipids disclosed herein may each be present in an organic solvent in molar amounts of about 30% to about 70% of the lipid compounds, about 0% to about 50% of the neutral lipids, 20% to about 50% of the structured lipids, and about 1% to about 15% of the PEG-lipids, relative to the total amount of lipid components.

[0359] Aqueous solvents useful in step ii) include aqueous buffers.

[0360] Examples of suitable aqueous buffers may include acidic buffers such as citrate buffer, sodium acetate buffer, succinate buffer, borate buffer or phosphate buffer, etc. For example, the aqueous buffer solvent may be a citrate buffer or an acetate buffer.

[0361] The pH of the aqueous solvent may range from about 3.5 to about 7.0, for example, from about 4.0 to about 6.5, for example, from about 4.5 to about 6.0, for example, about 5.5. In one embodiment, the pH may be about 4.0.

[0362] In step ii), the organic solvent and aqueous solvent may be mixed in a ratio of organic solvent:aqueous solvent ranging from about 1:1 to about 1:6. In one embodiment, this ratio may range from about 1:2 to about 1:4, such as a ratio of about 1:3.

[0363] According to one embodiment, the organic solvent and aqueous solvent may be mixed in step ii) at a flow rate ranging from about 0.01 ml / min to about 12 ml / min. In some embodiments, the flow rate may range from about 0.02 ml / min to about 10 ml / min, from about 0.5 ml / min to about 8 ml / min, from about 1 ml / min to about 6 ml / min, or may be about 4 ml / min.

[0364] The mixing step can be carried out by any method known in the art. For example, both solvents can be mixed in a T-junction or Y-connector. Alternatively, mixing can be carried out by laminar flow mixing in a microfluidic micromixer as described by Belliveau et al. (Mol Ther Nucleic Acids. 2012;1(8):e37).

[0365] As indicated, the aqueous solvent in step ii) comprises a nucleic acid as the biologically active agent. Suitable nucleic acids may be, for example, as detailed below.

[0366] The method may further include, if necessary, raising the pH from acidic to neutral.

[0367] In a further embodiment, the method may comprise step iv) of increasing the pH of the aqueous solvent containing the LNPs obtained in step iii) to a pH in the range of about 5.5 to about 7.5, for example about 6.0 to about 7.5.

[0368] The step of increasing the pH can be carried out by any method known in the art, for example, the pH change can be carried out by a dialysis or diafiltration step.

[0369] Additionally, the method for preparing LNPs may include any further steps suitable for harvesting, purifying, concentrating, and / or sterilizing the lipid nanoparticles for further formulation as a pharmaceutical composition, e.g., as an immunogenic composition.

[0370] Purification can be carried out by dialysis or diafiltration. The dialysis or diafiltration step may be carried out against an aqueous medium having a pH in the range of about 5.5 to about 7.5, for example, about 6.0 to about 7.0, for example, about 6.5 to about 7.0, for example, about 6.5.

[0371] Furthermore, if necessary, the osmolality can be adjusted to reach a final osmolality close to 290 mOsmol / kg for infusing an isotonic solution into the body.

[0372] Lipid nanoparticles can be prepared using a lipid compound of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), a neutral lipid of DSPC or DOPE, a structured lipid of cholesterol, and a PEG-lipid of PEG-PE (PEG2000-PE) or DMG-PEG (DMG-PEG2000).

[0373] Lipid nanoparticles can be prepared using a lipid compound of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), a neutral lipid of DSPC or DOPE, a structured lipid of cholesterol, and a PEG-lipid of PEG-PE (PEG2000-PE) or DMG-PEG (DMG-PEG2000).

[0374] Lipid nanoparticles can be prepared using a lipid compound of formula (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), a neutral lipid of DSPC, a structural lipid of cholesterol, and a PEG-lipid of DMG-PEG (DMG-PEG2000).

[0375] Lipid nanoparticles can be prepared using a lipid compound of formula (VI), (VII), (XII), (XIV), (XVI), (XVIII), (XIX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIII), a neutral lipid of DSPC, a structural lipid of cholesterol, and a PEG-lipid of DMG-PEG (DMG-PEG2000).

[0376] Lipid nanoparticles (LNPs) Lipid nanoparticles (LNPs) can be characterized by several parameters well known in the art, such as mean diameter size, mode diameter size, polydispersity index (PI), which reflects the homogeneity of the size distribution of the LNPs, pKa, and / or zeta potential, which reflects the spherical surface charge of the LNPs.

[0377] LNPs can be used to encapsulate at least one biologically active agent, and the encapsulation rate and total content of such agents can also be used as parameters to characterize the LNPs.

[0378] The modal diameter size, mean diameter size, and PI can be measured using a Malvern Nanoparticles Tracking Analysis (NTA) NS300 equipped with a 96-well plate autosampler or dynamic light scattering (DLS). The pKa can be measured using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The zeta potential can be measured using electrophoretic mobility or dynamic electrophoretic mobility measurements, for example, on a Nicomp 380 ZLS system or a Malvern nanoZS.

[0379] The "mean diameter size" of an LNP can be measured by nanoparticle tracking analysis (NTA) and represents the average diameter of all particles analyzed in a sample. The "modal diameter size" represents the size of the most frequent particle population in a sample. In other words, it is the size of the most frequent particle. With respect to the size distribution profile of a sample, the modal diameter size represents the highest point of the peak seen in the distribution.

[0380] NTA utilizes the properties of both Brownian motion and light scattering to obtain the particle size distribution of a sample in liquid suspension. A laser beam is passed through a sample chamber and the particles in suspension in a beam path that scatters light so that the particles can be viewed through a magnifying microscope equipped with a camera. Particle motion is recorded frame by frame. The center of each observed particle is identified and tracked to obtain the average distance traveled in the x and y planes. This value helps determine the particle diffusion coefficient (Dt), from which, knowing the sample temperature T and solvent viscosity η, the spherical equivalent hydrodynamic diameter d of the particle can be calculated using the Stokes-Einstein equation:

number

[0381] LNPs can have a diameter that makes them suitable for systemic administration, e.g., parenteral administration, or for intramuscular, intradermal, or subcutaneous administration. Typically, lipid nanoparticles have an average diameter size of less than 600 nanometers (nm), e.g., less than 400 nm.

[0382] In one embodiment, the LNPs have a mean diameter size of less than 200 nm. Such a size is advantageously compatible with sterile filtration and is most suitable for transport through lymphatic vessels after intramuscular or subcutaneous administration. This size is also suitable for intravenous administration, as injection of larger particles can induce capillary thrombosis.

[0383] In some embodiments, the LNPs may have an average diameter size ranging from about 20 nm to about 300 nm, e.g., about 25 nm to about 250 nm, e.g., about 30 nm to about 200 nm, about 40 nm to about 180 nm, about 60 nm to about 170 nm, about 70 nm to about 160 nm, and about 80 nm to about 150 nm. In one embodiment, the LNPs may have an average diameter size ranging from about 85 nm to about 140 nm, as measured by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). In the liquid composition (step a) of the methods disclosed herein), the LNPs may have a mode diameter size of about 70 nm to about 250 nm, or about 80 nm to about 200 nm, or about 85 nm to about 140 nm, or about 90 nm to about 120 nm, as measured by DLS and NTA.

[0384] The NTA technique requires the sample to be liquid, therefore, for measurement of the mean diameter size of LNPs after the freezing or lyophilization process, the resulting frozen or lyophilized LNPs are thawed or resuspended in a solution such as an aqueous buffer or water for injection (WFI).

[0385] In some embodiments, the lipid nanoparticles can have a Z-average diameter size ranging from about 20 nm to about 300 nm, e.g., from about 20 nm to about 250 nm, e.g., from about 30 nm to about 200 nm, from about 40 nm to about 180 nm, from about 60 nm to about 170 nm, from about 80 nm to about 160 nm, from about 90 nm to about 150 nm, or from about 90 nm to about 130 nm. In one embodiment, the nanoparticles can have a diameter ranging from about 90 nm to about 150 nm.

[0386] The "Z-average size" of lipid nanoparticles can be determined by dynamic light scattering (DLS). The Z-average size or Z-mean used in dynamic light scattering is a parameter also known as the cumulant mean. It is the primary and most stable parameter generated by this technique. Z-average is defined as the "harmonic intensity average particle size." Z-average size can be measured with a Zetasizer Nano ZS light scattering instrument (Malvern Instruments). For accurate particle sizing with the Nano ZS, the viscosity of the buffer solution and the refractive index of the material needed to be provided to the instrument software (PBS: v = 1.02 cP, RI = 1.45).

[0387] Because slight variations in size may occur during the manufacturing process, variations of up to 20-30% from the stated measurements are acceptable and considered within the stated size. Alternatively, size can be determined by a filtration screening assay. For example, a particle preparation is below a specified size if at least 90%, e.g., at least 95%, e.g., at least 97%, of the particles pass through a "screen-type" filter of the specified size.

[0388] "Polydispersity index" (PI) is a measure of the uniform or non-uniform size distribution of individual lipid nanoparticles in a lipid nanoparticle mixture and indicates the breadth of particle distribution in the mixture. PI can be determined, for example, as described herein.

[0389] In one embodiment, the polydispersity index of the nanoparticles described herein, as measured by dynamic light scattering, is 0.5 or less, such as 0.4 or less, such as 0.3 or less, or even such as 0.2 or less. PI may range from about 0.05 to about 0.2, or from about 0.09 to about 0.17.

[0390] In one embodiment, the lipid nanoparticles are colloidally stable in the sense that no, or substantially no, aggregation, precipitation, or increase in size and polydispersity index as measured by dynamic light scattering can be observed over a given period of time, e.g., over at least 2 hours, over several months, e.g., at least 1, 2, 3, 4, 5, 6, or 12 months.

[0391] The lipid nanoparticles disclosed herein have a pKa in the range of 4.5 to 6.7.

[0392] This pKa can be determined using preformed LNPs composed of the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and cationic lipid / DOPE / cholesterol / PEG-lipid (35:16:35:2.5 mol%) in PBS at a concentration of approximately 6 mM total lipid. Briefly, TNS is prepared as a 100 μM stock solution in distilled water. LNPs are diluted to 100 μM total lipid in 90 μL of buffer solution (in triplicate) containing 10 mM HEPES, 10 mM 4-morpholineethanesulfonic acid, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.71 to 11.5. Ten microliters of stock TNS is added to the LNP solution and mixed thoroughly in a black 96-well plate. Fluorescence intensity is monitored on a TecanPro200 plate reader using excitation and emission wavelengths of 321 nm and 445 nm. The resulting fluorescence values ​​are used to generate a sigmoidal plot of fluorescence versus buffer pH. The logarithm of the inflection point of this curve is the apparent pKa of the LNP formulation. Such methods are described in detail, for example, in Semple, SC et al., "Rational design of cationic lipids for siRNA delivery." Nat. Biotechnol. 28, 172-176 (2010).

[0393] LNPs may contain or encapsulate at least one nucleic acid as a biologically active agent. In such cases, LNPs may have an overall surface charge, which is the sum of the negative and positive charges on the particle's surface and is represented by the zeta potential. The zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particles. The zeta potential is widely used to quantify the magnitude of the charge in the double layer.

[0394] Zeta potential can be calculated using theoretical models or determined experimentally using electrophoretic mobility or dynamic electrophoretic mobility measurements. Electrophoresis can also be used to estimate the zeta potential of microparticles. In practice, the zeta potential of a dispersion can be measured by applying an electric field across the dispersion. Particles in a dispersion with a zeta potential migrate toward an electrode of opposite charge at a velocity proportional to the magnitude of the zeta potential. This velocity can be measured using laser Doppler anemometry techniques. The frequency or phase shift of an incident laser beam caused by these moving particles is measured as particle mobility, which can be converted to zeta potential by inputting the dispersant viscosity and dielectric constant and applying Smoluchowski theory. Electrophoretic velocity is proportional to electrophoretic mobility, a measurable parameter. Several theories relate electrophoretic mobility to zeta potential.

[0395] Suitable systems, such as the Nicomp 380 ZLS system or the Malvern nanoZS, can be used to determine zeta potential. Such systems typically measure the electrophoretic mobility and stability of charged particles in liquid suspension. These values ​​are predictors of the repulsive forces exerted by particles in suspension, which directly relate to the stability of colloidal systems.

[0396] At neutral pH, the zeta potential of the lipid nanoparticles disclosed herein is near neutral.

[0397] In one advantage, having a near-zero zeta potential facilitates particle movement within the body, reduces opsonization, and enhances access to target tissues.

[0398] In one embodiment, at a pH of 6.0 to 7.5, the zeta potential of the lipid nanoparticles can be about −3 mV to about +3 mV, for example, about −1 mV to about +1 mV, for example, about −0.5 mV to about +0.5 mV.

[0399] The lipid nanoparticles described herein can be prepared by adjusting the charge ratio of the ionizable lipid compounds disclosed herein (cationic charge from the quaternary ammonium: N at the terminal group of Formula (I)) from positive to negative, adding the anionic charge from the phosphate: P to a nucleic acid, and then mixing the nucleic acid with the lipid compounds. The charges of the ionizable lipid compounds and nucleic acid are determined at a selected pH, such as the pH of the formulation process, between about 3.0 and about 4.5.

[0400] The + / - (N / P) charge ratio of the lipid compounds disclosed herein to nucleic acids can be calculated by the following formula: (+ / - charge ratio) = [(amount of cationic lipid (mol)) * (total number of positive charges in the cationic lipid)]: [(amount of nucleic acid (mol)) * (total number of negative charges in the nucleic acid)].

[0401] The amounts of nucleic acid and lipid compound can be easily determined by those skilled in the art, taking into consideration the loading amounts when nanoparticles are prepared.

[0402] In one embodiment, the calculated charge ratio of positive to negative charges may range from about 1:1 to about 14:1, such as from about 2:1 to about 12:1, such as from about 4:1 to about 10:1, such as from about 6:1 to about 8:1, e.g., about 6:1.

[0403] In one embodiment, the lipid nanoparticles encapsulating nucleic acids may have a Z-average size of about 110-130 nm, a PI of about 0.15 to about 0.95, and a calculated charge ratio N / P of about 6:1.

[0404] biologically active agents The biologically active agent can be a prophylactic, therapeutic, or diagnostic agent.

[0405] In some embodiments, the biologically active agent is a nucleic acid.

[0406] In some embodiments, the therapeutic agent is a nucleic acid.

[0407] The nucleic acid may be a therapeutic agent or may encode a therapeutic agent. In some embodiments, the nucleic acid may be an mRNA that encodes a therapeutic agent.

[0408] A "therapeutic agent" is intended to refer to an active ingredient proposed to prevent or reduce the risk of occurrence of a medical condition or symptoms of a medical condition, or to cure or reduce the intensity of a medical condition, or to cure or reduce at least one symptom of a medical condition, in an individual to whom it is administered. "Individual" is intended to refer to humans and animals.

[0409] Therapeutic agents can be peptides, proteins, or nucleic acids. In some embodiments, the therapeutic agent can be a nucleic acid. The nucleic acid can encode a variety of therapeutic peptides or proteins.

[0410] The therapeutic agent can be a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen.

[0411] In one embodiment, the therapeutic agent can be a genome-editing polypeptide. In some embodiments, the genome-editing polypeptide is a CRISPR protein, such as CRISPR / Cas9, a restriction enzyme, a meganuclease, a transcription activator-like effector protein (TALE, such as TALE nuclease, TALEN), or a zinc finger protein (ZF, such as ZF nuclease, ZFN). See, e.g., WO 2020 / 139783.

[0412] The therapeutic agent may be a cytokine or chemokine suitable for stimulating or inhibiting an immune response, stimulating or preventing cell proliferation, or reducing inflammation. Examples of suitable cytokines or chemokines include insulin, insulin-like growth factor, human growth hormone (hGH), tissue plasminogen activator (tPA), interleukins (ILs), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, Examples of cytokines that may be involved include, but are not limited to, cytokines such as IL-33, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega, or IFN tau, tumor necrosis factors (TNF), such as TNF alpha, TNF beta, and TNF gamma, TNF-related apoptosis-inducing ligand (TRAIL), lymphotoxin-β (LT-β), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF). Production of erythropoietin or any other hormone growth factor is also included. Cytokines or chemokines can be encoded by nucleic acids.

[0413] In some embodiments, the therapeutic agent can be an antibody. As used herein, the term "antibody" refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides, or an antigen-binding fragment thereof. An antibody can be a monoclonal antibody (e.g., a full-length monoclonal antibody) exhibiting a single binding specificity and affinity for a particular epitope. The antigen-binding fragment can be a single-chain antibody, a single-chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab' fragment, or an F(ab')2 fragment. An antibody can recognize a tumor antigen or an infectious disease antigen, e.g., an antigen expressed by a tumor cell, against which a protective or therapeutic immune response is desired. Examples of antibodies include, for example, adalimumab, infliximab, rituximab, ipilimumab, tocilizumab, canakinumab, itolizumab, or tralokinumab. An antibody can be encoded by a nucleic acid.

[0414] In some embodiments, the therapeutic peptide or protein may be an enzyme with a desired use for regulating metabolism or growth in a subject. In some embodiments, the enzyme may be administered to replace an absent or dysfunctional endogenous enzyme. In some embodiments, the enzyme may be used to treat metabolic storage diseases. Metabolic storage diseases result from the systemic accumulation of metabolic products due to the absence or dysfunction of endogenous enzymes. Such metabolic products include lipids, glycoproteins, and mucopolycephaly. Examples of enzyme replacement therapy include lysosomal diseases such as Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, and glycogen storage disease type II. The therapeutic peptide or protein may be encoded by a nucleic acid.

[0415] Structural proteins can be, for example, collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin. Structural proteins can be encoded by nucleic acids.

[0416] The blood protein can be, for example, thrombin, serum albumin, factor VII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF), or an anticoagulant, etc. The blood protein can be encoded by a nucleic acid.

[0417] The hormone can be, for example, insulin, thyroid hormone, gonadotropin, trophic hormone, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc. The hormone can be encoded by a nucleic acid.

[0418] Transcription factors (TFs) form a complex system that recognizes specific DNA sequences, controls chromatin and transcription, and directs genome expression. Several families of transcription factors exist, and members of each family may share structural characteristics. Examples of transcription factors may include helix-turn-helix (e.g., Octo-1), helix-loop-helix (e.g., E2A), zinc finger (e.g., glucocorticoid receptor, GATA proteins), basic protein-leucine zipper [cyclic AMP response element binding factor (CREB), activator protein-1 (AP-1)], or β-sheet motifs [e.g., nuclear factor-κB (NF-κB)]. Transcription factors may be encoded by nucleic acids.

[0419] The therapeutic agent may be an antigen suitable for triggering an immune response in the treatment of cancer or in the treatment of an infectious disease (e.g., viral, bacterial, fungal, protozoan, or parasitic infection). The antigen may be encoded by a nucleic acid.

[0420] According to some embodiments, a composition containing an LNP as disclosed herein that comprises an antigen can therefore be an immunogenic composition or a vaccine composition.

[0421] Antigen-containing compositions can vary in their valency. Valency refers to the number of antigenic components in a composition. Immunogenic or vaccine compositions can be monovalent or multivalent, i.e., bivalent, trivalent, or higher. Multivalent compositions can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic components can be present on a single polynucleotide or on separate polynucleotides.

[0422] The compositions as disclosed herein can be used to prevent, treat, or cure infections resulting from contact with infectious agents, such as bacteria, viruses, fungi, protozoa, parasites, etc.

[0423] The compositions as disclosed herein can be used to protect against, treat, or cure cancer diseases.

[0424] The nucleic acid can encode at least one antigen.

[0425] According to some embodiments, the nucleic acid may encode at least one antigen selected from the group consisting of a bacterial antigen, a viral antigen, and a tumor antigen.

[0426] bacterial antigen The bacteria can be gram-positive or gram-negative. Bacterial antigens include Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, and Clostridium botulinum. botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, coagulase-negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, E. coli 0157:H7, Enterobacter sp.), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Proteus mirabilis mirabilis), Proteus sps.), Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0427] Viral antigens Viral antigens include adenovirus; herpes simplex type 1; herpes simplex type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus; hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinitis virus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue fever virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza virus, type A or It may be derived from Type B; Guanarito virus; Junin virus; Lassa fever virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus (RSV); human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; coltivirus; hantavirus, Middle East respiratory coronavirus; SARS-CoV-2 virus; chikungunya virus; Zika virus; parainfluenza virus; human enterovirus; hantavirus; Japanese encephalitis virus; swine vesicular exanthema virus; Eastern equine encephalitis virus; or Banna virus.

[0428] In one embodiment, the antigen is from an influenza A or influenza B virus strain, or a combination thereof. The influenza A or influenza B strain may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0429] The nucleic acid can encode a hemagglutinin protein or a fragment thereof. The hemagglutinin protein can be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. The hemagglutinin protein may or may not include a head domain (HA1). Alternatively, the hemagglutinin protein may or may not include a cytoplasmic domain.

[0430] In embodiments, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein may include a portion of the transmembrane domain.

[0431] In some embodiments, the virus may be selected from the group consisting of H1N1, H3N2, H7N9, H5N1, and H10N8 viruses or type B virus strains.

[0432] In another embodiment, the antigen can be from a respiratory syncytial virus (RSV). Suitable RSV antigens can be from RSV A and / or RSV B strains. The RSV antigen can be, for example, the fusion glycoprotein F protein, or the attachment protein G protein.

[0433] In another embodiment, the antigen can be from a coronavirus, such as SARS-Cov-1 virus, SARS-Cov-2 virus, or MERS-Cov virus. In some embodiments, the antigen can be a SARS-Cov2 antigen, such as the spike protein from SARS-Cov2.

[0434] tumor antigens The antigen may be a tumor antigen, i.e., a component of a cancer cell, such as a protein or peptide expressed in a cancer cell. The term "tumor antigen" refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or at a specific developmental stage, and that is expressed or abnormally expressed in at least one tumor or cancer tissue. Tumor antigens include, for example, differentiation antigens, such as cell type-specific differentiation antigens, i.e., proteins that are specifically expressed under normal conditions in a certain cell type at a certain differentiation stage, and germ line-specific antigens. For example, a tumor antigen is presented by the cancer cells in which it is expressed.

[0435] For example, tumor antigens can include carcinoembryonic antigen, 1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, cc2-H-ferroprotein, and gamma-fetoprotein.

[0436] Other examples of tumor antigens that may be useful in the present disclosure include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / m, CEA, cell membrane surface proteins of the claudin family such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gapl OO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, M UC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVrVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE and WT, for example WT-1.

[0437] Adjuvants The composition or LNP containing the nucleic acid encoding the antigen may further comprise or be co-administered with an adjuvant or immunopotentiator.

[0438] Adjuvants may include, but are not limited to, natural or synthetic adjuvants. They may be organic or inorganic.

[0439] Adjuvants may be selected from classes (1) mineral salts, such as aluminum hydroxide and aluminum phosphate or calcium phosphate gels; (2) emulsions: oil emulsions and surfactant-based formulations, such as microfluidized detergent-stabilized oil-in-water emulsions, purified saponin, oil-in-water emulsions, stabilized water-in-oil emulsions; (3) particulate adjuvants, such as virosomes (unilamellar liposomal vehicles incorporating influenza hemagglutinin), structured complexes of saponin and lipids, polylactide-co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert vehicles such as gold particles; (7) microbial-derived adjuvants; (8) tensile-active compounds; (9) carbohydrates; or combinations thereof.

[0440] The selection of an appropriate adjuvant and an appropriate amount of adjuvant will be apparent to one skilled in the art.

[0441] Specific adjuvants include, but are not limited to, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Alhydrogel, ISCOM™, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit, liposomes, saponin vaccines, DDA adjuvant, squalene-based adjuvant, Etx B subunit adjuvant, IL-12 vaccine adjuvant, LTK63 vaccine mutant adjuvant, TiterMax gold adjuvant, Ribi vaccine adjuvant, Montanide ISA 720 adjuvant, Corynebacterium-derb / ed P40 vaccine adjuvant, MPL™ adjuvant, AS04, AS02, AS01, lipopolysaccharide vaccine adjuvant, muramyl dipeptide adjuvant, CRL1005, killed Corynebacterium parvum vaccine adjuvant, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposomal vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer™, algae glucan, Bay R1005, Theramide®, stearyl tyrosine, Specol, Algammulin, Avridine®, calcium phosphate gel, CTA1-DD gene fusion protein, DOC / Alum complex, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, recombinant hlFN-γ / interferon-g, interleukin-iβ, interleukin-2, interleukin-7, scrabo peptide, Rehydragel LV, Rehydragel HPA, loxoribine, MF59, MTP-PE liposome, murametide, murapalmitin, D-murapalmitin, NAGO, nonionic surfactant vehicle, PMMA, PAA, protein cochleates, QS-21, SPT (AntigenFormulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, Escherichia coli (E. coli) heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, calcium phosphate vaccine adjuvant, Montanide incomplete sepsis adjuvant, imiquimod, resiquimod, AF03, Flagellin, poly(LC), ISCOMATRIX®, Abisco-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, immunoliposomes containing antibodies against costimulatory molecules, SAF-1, Sendai-containing lipid matrices, threonyl muramyl dipeptide (TMDP), Ty Examples of vaccine adjuvants include Particles vaccine adjuvant, bupivacaine vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolide)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, El 12K, a non-toxic variant of cholera toxin mCT-El 12K, and / or Matrix-S.

[0442] Protein expression Compositions or LNPs containing or encapsulating nucleic acids encoding proteins can be used to treat individuals with protein deficiencies. Thus, the compositions or LNPs can be used in methods for treating individuals with protein deficiencies, including administering compositions or LNPs containing at least one nucleic acid, such as mRNA, encoding a functional protein corresponding to the protein the individual is deficient in. In embodiments, the functional protein is produced after expression of the nucleic acid by target cells.

[0443] The present disclosure also relates to methods for intracellular delivery of nucleic acids that can correct an existing genetic defect and / or provide a beneficial function to at least one target cell, wherein after successful delivery to the target tissue and cell, the composition and nucleic acid that transfects the target cell and the nucleic acid (e.g., mRNA) can be translated into a gene product of interest (e.g., a functional protein or enzyme) or otherwise regulate or modulate the presence or expression of the gene product of interest.

[0444] The compositions and methods provided herein are useful for the management and treatment of numerous diseases, e.g., diseases resulting from protein and / or enzyme deficiencies. Individuals suffering from such diseases may have an underlying genetic defect that results in reduced expression of the protein or enzyme, including, for example, no protein synthesis, reduced protein synthesis, or synthesis of a protein that lacks or has reduced biological activity.

[0445] Alternatively, the nucleic acid may encode a full-length antibody or a smaller antibody (e.g., both heavy and light chains) to confer immunity to a subject. In alternative embodiments, the compositions of the present disclosure encode antibodies that can be used to transiently or chronically produce a functional response in a subject. For example, the mRNA nucleic acids of the present disclosure may encode functional monoclonal or polyclonal antibodies that, upon translation (and, optionally, systemic excretion from target cells), may be useful for targeting and / or inactivating a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the mRNA nucleic acids of the present disclosure may encode functional anti-nephritic factor antibodies useful, for example, for treating membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively, may encode anti-vascular endothelial growth factor (VEGF) antibodies useful for treating VEGF-mediated diseases, such as cancer.

[0446] Alternatively, the composition or LNP can comprise or encapsulate a nucleic acid that encodes or expresses an RNAi that can reduce or prevent expression of a protein to treat an individual suffering from overexpression of the protein.

[0447] nucleic acid Nucleic acids suitable for the present disclosure can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules.

[0448] Nucleic acids can be single-stranded or double-stranded molecules, and can be covalently closed to form linear or circular chains. Nucleic acids can be double-stranded RNA (dsRNA); single-stranded RNA (ssRNA); double-stranded DNA (dsDNA); single-stranded DNA (ssDNA); and combinations thereof.

[0449] Nucleic acid-containing compositions or LNPs can be used for the introduction of nucleic acids into cells, i.e., for transfection of cells, e.g., for recombinant protein expression, for gene replacement, to suppress or increase expression of host proteins.

[0450] The nucleic acid may be of eukaryotic or prokaryotic origin, such as human, animal, plant, bacterial, yeast or viral origin. It may be obtained by any technique known to those skilled in the art, such as by screening libraries, by chemical synthesis or alternatively by mixed methods involving chemical or enzymatic modification of sequences obtained by screening libraries. It may be chemically modified.

[0451] The nucleic acid may be contained in a vector. Vectors are known to those skilled in the art and may include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vivo expression system. Cloning vectors are generally used to manipulate and amplify certain desired DNA fragments and may lack functional sequences necessary for expression of the desired DNA fragment.

[0452] The nucleic acid can be messenger RNA (mRNA); microRNA (miRNA); short (or small) interfering RNA (siRNA); small hairpin RNA (shRNA); long non-coding RNA (lncRNA); asymmetric interfering RNA (aiRNA); self-amplifying RNA (saRNA); small nuclear RNA (snRNA); small nucleolar RNA (snoRNA); guide RNA (gRNA); antisense oligonucleotide (ASO); plasmid DNA (pDNA); closed loop DNA (ceDNA), and combinations thereof.

[0453] In some embodiments, the nucleic acid may be RNA.

[0454] In some embodiments, the nucleic acid can be messenger RNA (mRNA); microRNA (miRNA); short (or small) interfering RNA (siRNA); small hairpin RNA (shRNA); long non-coding RNA (lncRNA); asymmetric interfering RNA (aiRNA); self-amplifying RNA (saRNA); guide RNA (gRNA); and combinations thereof.

[0455] In some embodiments, the LNP may contain as nucleic acids an mRNA encoding a CRISPR protein, such as CRISPR / Cas9, and a guide RNA (gRNA). The gRNA may be provided as an rRNA:tracrRNA duplex or as a single guide RNA (sgRNA). In some embodiments, the CRISPR protein may be provided directly as a polypeptide and not as an mRNA encoding the CRISPR protein.

[0456] In some embodiments, the RNA can be messenger RNA (mRNA).

[0457] In some embodiments, the nucleic acid may encode a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen, as described herein.

[0458] messenger RNA (mRNA) mRNA is typically considered a type of RNA that conveys information from DNA to ribosomes. The existence of mRNA is typically very brief and involves processing and translation, followed by degradation. Typically, in eukaryotes, mRNA processing involves the addition of a "cap" onto the N-terminal (5') end and a "tail" onto the C-terminal (3') end.

[0459] A typical cap is a 7-methylguanosine cap, which is a guanosine attached to the first transcribed nucleotide by a 5'-5'-triphosphate bond. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, generating a 5'5'5 triphosphate bond; then, the 7-nitrogen of the guanine is methylated by a methyltransferase.

[0460] A tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of an mRNA molecule. The presence of this "tail" helps protect the mRNA from exonuclease degradation. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0461] In some embodiments, an mRNA comprises a 5' and / or 3' untranslated region (UTR). In some embodiments, an mRNA disclosed herein comprises a 5' UTR that includes one or more elements that affect mRNA stability or translation. In some embodiments, the 5' UTR can be approximately 50-500 nucleotides in length. In some embodiments, an mRNA disclosed herein comprises a 3' UTR that includes one or more polyadenylation signals, binding sites for proteins that affect the stability of the mRNA's location within the cell, or one or more binding sites for miRNAs. In some embodiments, the 3' UTR can be 50-500 nucleotides in length or more. In some embodiments, an mRNA disclosed herein comprises a 5' or 3' UTR that is derived from a gene different from that encoded by the mRNA transcript. In some embodiments, an mRNA disclosed herein comprises a chimeric 5' or 3' UTR.

[0462] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). In vitro transcription methods are known in the art. See, for example, Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14, the contents of which are incorporated by reference. Briefly, IVT is typically performed on a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application. The presence of these reagents is undesirable in the final mRNA product and is considered an impurity or contaminant, which must be purified to provide clean and homogeneous mRNA suitable for therapeutic use. In some embodiments, mRNA provided from an in vitro transcription reaction may be desired, although other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0463] The mRNAs disclosed herein may be modified or unmodified. In some embodiments, the mRNAs disclosed herein contain one or more modifications that typically enhance RNA stability. Exemplary modifications include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNAs may contain purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2 ... Thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxymethylaminomethyl)-2-thio-uracil,

[0033] The nucleotides can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-psodouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.In some embodiments, the disclosed mRNAs comprise at least one chemical modification, including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified nucleotide comprises N1-methylpseudouridine. The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the contents of which are incorporated by reference.

[0464] The term "RNA" refers to a molecule comprising, and for example consisting entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. It includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, or recombinantly produced RNA.

[0465] For clarity, mRNA encompasses any coding RNA molecule that can be translated into a protein by a eukaryotic host. A coding RNA molecule generally refers to an RNA molecule that includes a sequence that encodes a protein of interest and that can be translated by a eukaryotic host, the sequence starting with an initiation codon (ATG) and ending with, for example, a stop codon (i.e., TAA, TAG, TGA).

[0466] The RNA can be naturally occurring RNA or modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of at least one nucleotide. Such alterations can include the addition of non-nucleotide material to the end or within the RNA, for example, to at least one nucleotide of the RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0467] mRNA can be produced by in vitro transcription using a DNA template. Alternatively, RNA can be obtained by chemical synthesis. Such methods are known to those skilled in the art. For example, there are various in vitro transcription kits available commercially.

[0468] RNA can be synthesized in vitro in a cell-free system using an appropriate cell extract and an appropriate DNA template. For example, a cloning vector is applied to generate the transcription product. The promoter for controlling transcription can be any promoter for any RNA polymerase. Some examples of RNA polymerases are T7, T3, and SP6 RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, such as cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA. For example, a cloning vector is generally used to produce a transcription product, which is a designated transcription vector.

[0469] The RNA can encode a protein or peptide. That is, when present in the appropriate environment, e.g., within a cell, such as an antigen-presenting cell, e.g., a dendritic cell, the RNA can be expressed to produce the protein or peptide it encodes. The stability and translation efficiency of the RNA can be modified as needed.

[0470] In some embodiments, the mRNA may encode a genome editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen, such as those described herein.

[0471] In some embodiments, the mRNA may encode an antigen.

[0472] The RNA molecules can be of varying lengths. Thus, they can be short RNA molecules, for example, shorter than about 100 nucleotides, or long RNA molecules, for example, longer than about 100 nucleotides, or even longer than about 300 nucleotides.

[0473] The mRNA may be at least 30 nucleotides in length.

[0474] The mRNA may comprise a 5' cap structure, a 5'-UTR sequence, an ORF sequence encoding a protein or peptide, a 3'-UTR sequence, and a poly(A) tail.

[0475] Typically, the mRNA may comprise or consist of the following general formula: [5' cap]w-[5' UTR]x-[gene of interest]-[3' UTR]y-[poly A]z wherein [5' cap] contains a methylguanine nucleotide attached to the mRNA by a 5'-5' linkage; In the formula, [5'UTR] and [3'UTR] are untranslated regions (UTRs), wherein [5'UTR] contains a Kozak sequence, where [gene of interest] is any gene encoding a protein of interest, where [polyA] is the poly(A) tail; where w, x, y, and z are the same or different and equal to 0 or 1.

[0476] Kozak sequence refers to a sequence that occurs in eukaryotic mRNA and is generally a consensus sequence and plays a major role in the initiation of the translation process. Kozak sequences and Kozak consensus sequences are well known in the art.

[0477] The 3'UTR does not express any protein. The purpose of the 3'UTR is to increase the stability of the mRNA. In one embodiment, the α-globin UTR is selected because it is known not to be unstable.

[0478] The sequence corresponding to the gene of interest may be codon optimized for satisfactory protein production in the host under consideration.

[0479] Poly(A) tails are composed of multiple adenosine monophosphates, as is well known in the art. Poly(A) tails are generally produced during a process called polyadenylation, a post-translational modification that commonly occurs during the production of mature messenger RNA. Such poly(A) tails contribute to the stability and half-life of mRNA and can be of variable length. For example, a poly(A) tail can be 10 A or more nucleotides, it can contain 20 A or more nucleotides, it can contain 100 A or more nucleotides, for example, about 120 A nucleotides.

[0480] RNA molecules are (i) capped and unmodified RNA molecules; (ii) capped modified RNA molecules; (iii) uncapped, unmodified RNA molecules; (iv) Uncapped modified RNA molecule may include:

[0481] Capped and uncapped RNA molecules A "capped RNA molecule" refers to an RNA molecule having at its 5' end a guanosine or modified guanosine, such as 7-methylguanosine (m7G), that is linked to a 5'-5' triphosphate linkage or analog. This definition corresponds to the most widely accepted definition of a 5' cap.

[0482] "Cap analog" includes a cap that is biologically equivalent to 7-methylguanosine (m7G) linked to a 5'-5' triphosphate linkage and therefore can also be substituted for without impairing protein expression of the corresponding messenger RNA in a eukaryotic host.

[0483] Examples of caps include m7GpppN, m7GpppG, m7GppspG, m7GppspspG, m7GppspspG, m7Gppppm7G, m27',3'-OGpppG, m27',2'-OGpppG, m27',2'-OGppspsG, or m27',2'-OGpppspsG.

[0484] Examples of cap analogs can be glyceryl, an inverted deoxynucleotide abasic residue (moiety), a 4',5' methylene nucleotide, a 1-(beta-D-erythrofuranosyl) nucleotide, a 4'-thionucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a threo-pentofuranosul nucleotide, an acyclic 3',4'-seconucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5 dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted nucleotide abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted nucleotide abasic moiety, 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3' phosphorothioate, a phosphorodithioate, or a bridged or non-bridged methylphosphonate moiety.

[0485] Other examples of cap analogs include anti-reversal cap analog (ARCA), N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0486] Of note, among cap analogs, some are favorable for protein expression, whereas others may interfere with protein expression. Such differences are understood by those skilled in the art.

[0487] Providing a 5'-cap or 5'-cap analog to RNA can be achieved by in vitro transcription of a DNA template in the presence of a 5'-cap or 5'-cap analog, where the 5'-cap can be incorporated into the resulting RNA strand by co-transcription, or the RNA can be produced, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme, such as cowpox virus capping enzyme.

[0488] An "uncapped RNA molecule" refers to any RNA molecule that does not fall within the definition of a "capped RNA molecule."

[0489] Thus, according to a general embodiment, "uncapped mRNA" may refer to an mRNA whose 5' end is not linked to 7-methylguanosine via a 5'-5' triphosphate linkage, or an analog as previously defined.

[0490] Uncapped RNA molecules, such as messenger RNA, can be uncapped RNA molecules with a (5')ρρρ(5'), (5')ρρ(5'), (5')ρ(5'), or even a (5')OH tip. Such RNA molecules can be abbreviated as 5'ρρRNA; 5'ρρRNA; 5'ρRNA; 5'OHRNA, respectively.

[0491] Without limitation, the first base of the uncapped RNA molecule can be either adenosine, guanosine, cytosine, or uridine.

[0492] The RNA may be free of uncapped 5'-triphosphates, and removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0493] Modified and unmodified RNA molecules The RNA may include further modifications, such as an extension or truncation of the naturally occurring poly(A) tail, or alteration of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR not related to the coding region of the RNA, e.g., replacement of an existing 3'-UTR with at least one, e.g., two copies, of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0494] A "modified RNA molecule" refers to an RNA molecule that contains at least one modified nucleotide, nucleoside sugar, or base, such as a modified purine or modified pyrimidine. The modified nucleoside or base can be any nucleoside or base that is not A, U, C, or G (adenosine, uridine, cytidine, or guanosine, respectively, with respect to the nucleoside; and adenine, uracil, cytosine, or guanine when referring to only the sugar moiety).

[0495] "Unmodified RNA molecule" refers to any RNA molecule that does not meet the definition of a modified RNA molecule.

[0496] The terms "modified and unmodified" are considered differently from the terms "capped and uncapped" as the latter relates specifically to bases at the 5'-end of the RNA.

[0497] The presence of modified nucleotides can increase the stability and / or reduce the cytotoxicity of nucleic acids. The term RNA stability is related to the half-life of RNA, which is the period required to eliminate half of the activity, amount, or number of molecules. The half-life of RNA can indicate its stability. The half-life of RNA can affect the duration of RNA expression. RNA with a long half-life can be expected to be expressed for an extended period of time.

[0498] Non-limiting examples of modified nucleotides, nucleosides, and bases are disclosed in WO 2015 / 024667A1. Modified RNAs may contain modified nucleotides, nucleosides, or bases, including backbone modifications, sugar modifications, or base modifications. Modified bases and / or modified RNA molecules are known in the art and are taught, for example, in Warren et al. ("Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA"; Cell Stem Cell; 2010), the contents of which are incorporated by reference.

[0499] Sugar modifications include chemical modifications of the sugar of a nucleotide. Sugar modifications can consist in the substitution or modification of the 2' hydroxy (OH) group, which can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0500] Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CHCHO)CHCHOR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0501] The "deoxy" modification includes hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar via a linker, where the linker includes at least one of the atoms C, N, and O.

[0502] The sugar group can also contain at least one carbon that has the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified RNAs can include, for example, nucleotides that contain arabinose as the sugar.

[0503] Backbone modifications include modifications in which the backbone phosphate of a nucleotide is chemically modified. The backbone phosphate group can be modified by replacing at least one of the oxygen atoms with a different substituent. Furthermore, modified nucleosides and nucleotides can include complete replacement of unmodified phosphates with modified phosphates as described herein.

[0504] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.Phosphorodithioate has both non-bonding oxygens replaced by sulfur.Phosphate linker can also be modified by replacing the bonded oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene-phosphonate).

[0505] Base modifications include chemical modifications of the base portion of a nucleotide. In this regard, nucleotide analogs or modifications can be selected from nucleotide analogs suitable for transcription and / or translation of RNA molecules in eukaryotic cells, for example. Modified nucleosides and nucleotides can be modified at the nucleobase portion. For example, nucleosides and nucleotides can be chemically modified at the major groove surface. Major groove chemical modifications can include amino groups, thiol groups, alkyl groups, or halo groups. Modified bases can be modified purine bases or pyrimidine bases. Examples of modified purine bases include modified adenosines and / or guanosines, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthosine, and 7-methylguanosine. Modified pyrimidine bases include modified cytidines and / or uridines, such as 5,6-dihydrouracil; pseudouridine; 5-methylcytidine; 5-hydroxymethylcytidine; dihydrouridine, and 5-methylcytidine.

[0506] For example, nucleotide analogs / modifications include the following base modifications: 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate. 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine- 5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxy Uridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole- It may be selected from riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, and xanthosine-5'-triphosphate.

[0507] Modified nucleosides include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, and l-taurinomethyl-4-thio-uridine. , 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine / 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0508] Modified nucleosides and nucleotides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, and 5-methyl-cytidine. pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0509] Modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0510] Modified nucleosides can include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0511] RNA with an unmasked poly-A sequence can be translated more efficiently than RNA with a masked poly-A sequence. An "unmasked poly-A sequence" means that the poly-A sequence at the 3' end of the RNA molecule ends with an A in the poly-A sequence and is not followed by any nucleotides downstream other than the A located at the 3' end of the poly-A sequence. Furthermore, a long poly-A sequence of about 120 base pairs provides optimal transcript stability and RNA translation efficiency.

[0512] Therefore, to enhance RNA stability and / or expression, the poly-A sequence may be modified, for example, to have a length of 10 to 500, for example 30 to 300, for example 65 to 200, for example 100 to 150 adenosine residues. The poly-A sequence may have a length of approximately 120 adenosine residues. To further enhance RNA stability and / or expression, the poly-A sequence may be unmasked.

[0513] The incorporation of a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can result in improved translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated regions can be autologous or heterologous to the RNA into which they are introduced. The 3'-untranslated region can be derived from the human β-globin gene.

[0514] The combination of the above modifications, i.e., incorporation of a poly-A sequence, unmasking of a poly-A sequence and incorporation of at least one 3'-untranslated region, may have a synergistic effect on improving RNA stability and translation efficiency.

[0515] RNA expression can also be increased by modifying the sequence encoding the peptide or protein, for example by increasing the GC content to increase mRNA stability and / or by codon optimization to increase translation in the cell.

[0516] Pharmaceutical compositions and uses thereof The compositions or LNPs disclosed herein can be used in pharmaceutical compositions, which can include the compositions or LNPs disclosed herein and at least one pharmaceutically acceptable excipient.

[0517] The composition or LNP can include a biologically active agent disclosed herein.

[0518] The biologically active agent can be a nucleic acid as disclosed herein.

[0519] Also disclosed are immunogenic compositions comprising at least one composition comprising a nucleic acid encoding at least one antigen or LNP.

[0520] In some embodiments, compositions or LNPs disclosed herein containing at least one biologically active agent, such as a nucleic acid, may be for use as a pharmaceutical.

[0521] In some embodiments, the compositions or LNPs disclosed herein containing at least one biologically active agent, e.g., a nucleic acid, may be for use in methods for preventing and / or treating a disease selected from the group consisting of an infectious disease, an allergy, an autoimmune disease, a blood disorder, a metabolic disease, a neurological disease, and a cancer disease.

[0522] Also disclosed are methods of making a medicament or pharmaceutical composition comprising at least the step of mixing a composition or LNP disclosed herein comprising at least one biologically active agent, e.g., a nucleic acid, with at least one pharmaceutically acceptable excipient.

[0523] The method of producing a medicament or pharmaceutical composition can further comprise preparing a composition of LNPs as described above.

[0524] The method may further comprise suspending or diluting the composition or LNP in a pharmaceutically acceptable solvent.

[0525] A "pharmaceutically acceptable solvent" can be any solvent suitable for resuspending or dissolving lyophilized LNPs and that is pharmaceutically acceptable for enteral or parenteral administration to an individual in need thereof. The pharmaceutically acceptable solvent can be water for injection or a buffer such as saline, citrate buffer, histidine buffer, or phosphate buffer.

[0526] The pharmaceutical or immunogenic composition may be sterile.

[0527] General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are found, for example, in Remington's The Science and Practice of Pharmacy, 21 St Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Any pharmaceutically acceptable excipient may be used in the pharmaceutical composition, except insofar as the excipient may be incompatible with one or more components of the LNP.

[0528] The compositions disclosed herein may be formulated into solid, semi-solid, or liquid form preparations such as powders, solutions, suspensions, or injectables.

[0529] Exemplary pharmaceutically acceptable excipients that may be used may be selected from diluents such as water for injection or saline, such as amino acid buffer (histidine, arginine, glycine, proline, glycylglycine), saline buffer (inorganic salts NaCl, calcium chloride), phosphate buffer, acetate buffer, citrate buffer, succinate buffer, etc.; sugars or polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol, etc.; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, etc., and combinations thereof. In many cases, it will be desirable to include an isotonic agent in the composition, such as a sugar, a polyhydric alcohol, or sodium chloride. The formulation may also contain other additives, such as antioxidants such as tryptamine and stabilizers such as Tween 20 or 80, other solvents such as monohydric alcohols, such as ethanol or isopropanol, and polyhydric alcohols, such as glycols, and edible oils, such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like, oily esters, such as ethyl oleate, isopropyl myristate, and the like; binders, adjuvants, solvents, etc. They may contain solubilizers, thickeners, stabilizers, disintegrants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, colorants, flavorings, preservatives, antioxidants, processing agents, drug delivery modifiers, and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, or polyethylene glycol. Pharmaceutically acceptable excipients may also include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.

[0530] Appropriate concentrations and dosages can be readily determined by one skilled in the art.

[0531] Administration of the pharmaceutical and immunogenic compositions disclosed herein can be via any of the accepted modes of administration for compositions to provide similar utilities.

[0532] Typical routes of administration of such pharmaceutical and immunogenic compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques.

[0533] Pharmaceutical and immunogenic compositions may be administered by any suitable route, depending on parameters known in the art, such as the form of the composition (solid or liquid), the individual being treated, and the nature of the therapeutic agent contained in the LNP.

[0534] For example, the pharmaceutical or immunogenic composition can be administered systemically, orally, sublingually, intranasally, intradermally, or subcutaneously.

[0535] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art.

[0536] In some embodiments, the pharmaceutical or immunogenic composition may be suitable for subcutaneous administration.

[0537] In some embodiments, the pharmaceutical or immunogenic composition may be suitable for intramuscular administration.

[0538] Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0539] The composition may contain at least one inert diluent or carrier.

[0540] In one embodiment, the composition may be in the form of a liquid, such as a solution, emulsion, or suspension. The liquid may be for delivery by injection. A composition intended to be administered by injection may contain at least one of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent. The liquid composition disclosed herein may contain at least one of the following: water for injection, saline, such as saline, Ringer's solution, isotonic sodium chloride, a sterile diluent such as fixed oil, synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as a solvent or suspension medium; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and a tonicity adjuster such as sodium chloride or dextrose; or an agent for acting as a cryoprotectant, such as sucrose or trehalose.

[0541] The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are, for example, sterile.

[0542] The pharmaceutical compositions and immunogenic compositions disclosed herein can be prepared by methods well known in the pharmaceutical field. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining the lipid nanoparticles disclosed herein with sterile, distilled water or other carriers to form a solution. A surfactant may be added to promote the formation of a homogeneous solution or suspension.

[0543] The compositions disclosed herein are administered in therapeutically effective amounts, which vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated.

[0544] The compositions disclosed herein can also be administered simultaneously with, before, or after the administration of at least one other therapeutic agent.Such combination therapy includes the administration of a single pharmaceutical dosage formulation of the compositions disclosed herein and at least one additional active agent, as well as the administration of the compositions disclosed herein and each active agent in their own separate pharmaceutical dosage formulations.When separate dosage formulations are used, the compositions disclosed herein and at least one additional active agent can be administered essentially simultaneously, i.e., simultaneously, or separately, staggered, i.e., sequentially.Combination therapy is understood to include all of these regimens.

[0545] The pharmaceutical or immunogenic composition may be administered via a drug combination device such as a multi-chamber syringe, in which at least one chamber contains the pharmaceutical composition in solid form and at least one chamber contains a pharmaceutically acceptable solvent for suspending or dissolving the composition.

[0546] In some embodiments, the LNPs disclosed herein may comprise at least one nucleic acid encoding an antigen from an influenza A virus and / or an influenza B virus. Such LNPs may be for use in preventing or treating influenza A and / or influenza B virus infection. Such LNPs may be for use as immunogenic compositions against influenza A virus and / or influenza B virus.

[0547] In some embodiments, the present disclosure relates to LNPs comprising lipid components including cationic ionizable lipids, neutral lipids, structured lipids, and optionally PEG-lipids, and comprising at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus. Such LNPs may be for use in preventing or treating influenza A and / or influenza B virus infection. Such LNPs may be for use as immunogenic compositions against influenza A virus and / or influenza B virus.

[0548] In some embodiments, the LNPs disclosed herein may comprise at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus. Such LNPs may be for use in preventing or treating respiratory syncytial A virus and / or respiratory syncytial B virus infection. Such LNPs may be for use as immunogenic compositions against respiratory syncytial A virus and / or respiratory syncytial B virus.

[0549] In some embodiments, the present disclosure relates to LNPs comprising lipid components including cationic ionizable lipids, neutral lipids, structured lipids, and optionally PEG-lipids, and comprising at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus. Such LNPs may be for use in preventing or treating respiratory syncytial A virus and / or respiratory syncytial B virus infection. Such LNPs may be for use as immunogenic compositions against respiratory syncytial A virus and / or respiratory syncytial B virus.

[0550] In some embodiments, the LNPs disclosed herein can comprise at least one nucleic acid encoding a SARS-Cov2 antigen. Such LNPs can be for use in preventing or treating SARS-Cov-2 infection. Such LNPs can be for use as immunogenic compositions against SARS-Cov-2.

[0551] In some embodiments, the present disclosure relates to LNPs comprising lipid components including cationic ionizable lipids, neutral lipids, structured lipids, and optionally PEG-lipids, and comprising at least one nucleic acid encoding a SARS-Cov2 antigen. Such LNPs may be for use in preventing or treating SARS-Cov-2 infection. Such LNPs may be for use as immunogenic compositions against SARS-Cov-2.

[0552] In some embodiments, the compositions or LNPs disclosed herein that comprise at least one nucleic acid can be used in the manufacture of a medicament.

[0553] Some embodiments relate to the use of a composition or LNP disclosed herein comprising at least one biologically active agent in the manufacture of a pharmaceutical composition.

[0554] Treatment method In some embodiments, the present disclosure also relates to methods of preventing and / or treating disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition or LNP disclosed herein comprising at least one biologically active agent. For example, the compositions or LNPs disclosed herein may be for use in therapeutic methods for preventing and / or treating infectious diseases, allergies, autoimmune diseases, blood disorders, metabolic diseases, neurological diseases, and tumor or cancer diseases.

[0555] In some embodiments, the hematological, metabolic, or neurological disorder can be a rare disease. A rare disease is a disease that affects a small percentage of the population, for example, with an incidence ranging from about 1 in 1,000 to about 1 in 200,000.

[0556] For example, a disease of concern according to the present disclosure may be an infectious disease such as a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. A disease of concern according to the present disclosure may also be a cancer or tumor disease.

[0557] Viral infections include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie disease, infectious mononucleosis, Burkitt's lymphoma, acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis, keratoconjunctivitis in adults), latent HSV-1 infection (e.g., herpes labialis and herpes simplex), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, inclusion body disease, Kaposi's sarcoma, multicentric kyphosis, and cytoplasmic leukemia. The lesion may be Castleman's disease, primary effusion lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, hyperplastic epithelial lesions (e.g., common flat plantar and anogenital warts, laryngeal papillomas, epidermodysplasia verruciformis), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchiolitis, the common cold, polio, rabies, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, rubella, congenital rubella, chickenpox, COVID-19, respiratory syncytial virus (RSV) infection, and shingles.

[0558] In one embodiment, the disease is influenza, respiratory syncytial virus (RSV) infection, or COVID-19, e.g., influenza.

[0559] Bacterial infections include abscesses, actinomycosis, acute prostatitis, aerobic bacteria, annual ryegrass toxicity, anthrax, bacterial aeruginosa, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterial-associated skin conditions, bartonellosis, BCG-oma, botulism, botulism, Brazilian suppurative fever, Brody's abscess, brucellosis, Buruli ulcer, campylobacteriosis, dental caries, carrier disease, feline pruritus, cellulitis, chlamydial infection, cholera, chronic bacterial prostatitis, chronic recurrent multifocal osteomyelitis, clostridial necrosis, complex periodontal endodontic lesions, contact bovine pleuropneumonia, diphtheria Leprosy, diphtheria gastritis, ehrlichiosis, erythema, erysipelas, epiglottitis (piglottitis), erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, foot rot (infectious foot dermatitis), Galle's sclerosing osteomyelitis, gonorrhea, granuloma inguinale, human granulocytic aplasia, human monocytic ehrlichiosis, 100-day cough, impetigo, late congenital syphilitic ophthalmopathy, Legionnaires' disease, Lemire's syndrome, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, meningitis, meningococcal disease, meningococcal septicemia, methicillin-resistant Staphylococcus aureus (MRS) A) Infections, Mycobacterium avium (MAI), Mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, noma (testicular cancer or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, overwhelming post-splenectomy infection (OPSI), ovine brucellosis, pasteurellosis, periorbital cellulitis, whooping cough, plague, pneumococcal pneumonia, Pott's disease, proctitis, pseudomonal infection, psittacosis, suppurative disease, pyomyositis, Q fever, relapsing fever (typhus), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsiosis, salmonellosis, scarlet fever, The illness may be septicemia, Serratia infection, Shigella disease, Southern tick-associated erythema, Staphylococcal scalded skin syndrome, Streptococcal pharyngitis, swimming pool granulomatosis, porcine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhoid fever, typhus fever, urogenital tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Friderichsen syndrome, pseudotuberculosis (Yersinia) disease, and yersiniosis.

[0560] The parasitic infection may be amebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (kala-azar), balantidiosis, toxoplasmosis, malaria, acanthamoeba keratitis, and babesiosis.

[0561] Fungal infections can include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetoma, paracoccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are prone to diseases caused by fungal genera, such as Aspergillus, Candida, Cryptococcus, Histoplasma, and Pneumocystis. Other fungi attack the eyes, nails, hair, and especially the skin, so-called dermatophytes and keratotic fungi, and can cause a variety of symptoms, among which ringworm, such as athlete's foot, is common. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxa can induce allergic reactions in some people.

[0562] Cancer or tumor diseases include, for example, melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, blastoma, renal cancer, gastrointestinal tumors, glioma, prostate tumor, bladder cancer, rectal tumor, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, breast cancer (= breast cancer), uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), liver cancer, various virus-induced tumors, for example papillomavirus-induced carcinoma (e.g. cancer of the cervix = cervical carcinoma), adenocarcinoma, herpesvirus-induced tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphoma, acoustic neuroma, lung cancer (= lung cancer = cancer of the bronchus), small cell lung cancer, leukemia ... Alveolar lung cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumor, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schöneberger's disease, pituitary tumor, mycosis fungoides, carcinoid, schwannoma, acanthoma, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, corpus cancer, bone cancer, non-Hodgkin's lymphoma, urethral cancer , CUP syndrome, head and neck tumors, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, cancer of the esophagus (= esophageal cancer), wart lesions, tumors of the small intestine, craniopharyngioma, ovarian cancer, reproductive organ tumors, cancer of the ovaries (= ovarian cancer), cancer of the pancreas (= pancreatic cancer), endometrial cancer, liver metastasis, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumor, prostate cancer (= prostate tumor).

[0563] Diseases for which the present disclosure may be useful as a therapeutic intervention include SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders, including cystinuria; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Mersbacher disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS). FMR1-related disorders, including fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid-lipofuscinosis-related disorders, including juvenile neuronal ceroid-lipofuscinosis (JNCL), juvenile Batten disease, Santabu-Orichalcia disease, Jansky-Bielschowski disease, and PTT-1 and TPP1 deficiency; childhood ataxia with central nervous system hypomyelination / white matter loss, associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5; and CACNA1A- and CACNB4-associated episodic ataxia type 2. MECP2-related disorders, including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A- and SCN1B-related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy, dysarthria and ophthalmoplegia, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson's disease.

[0564] In one embodiment, the nucleic acids, e.g., mRNA, of the present disclosure can encode a functional protein or enzyme. For example, a composition of the present disclosure can include mRNA encoding erythropoietin (EPO), alpha-1-antitrypsin, carboxypeptidase N, alpha-galactosidase (GLA), ornithine carbamoyltransferase (OTC), or human growth hormone (hGH).

[0565] In other embodiments, the present disclosure relates to a method of transfecting at least one isolated target cell with a nucleic acid, comprising contacting the at least one target cell with an effective amount of at least one nucleic acid polynucleotide and (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one composition described herein containing a nucleic acid, or (iii) at least one lipid nanoparticle containing a nucleic acid described herein, to transfect the at least one target cell with the nucleic acid.

[0566] Target cells include, but are not limited to, lymph node, liver cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal, astrocytes, motor neurons, cells of the dorsal root ganglion and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen presenting cells such as dendritic cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0567] In one embodiment, the targeted cells may be spleen, liver, lung, heart, and kidney cells, hi another embodiment, the targeted cells may be spleen cells and kidney cells, for example, spleen cells.

[0568] In some embodiments, lipid nanoparticles or compositions disclosed herein that allow for avoidance of hepatic clearance may be of particular interest.

[0569] For example, after transfection of at least one target cell with a nucleic acid encapsulated in a lipid nanoparticle, the production of a polypeptide or protein encoded by such nucleic acid can be stimulated, for example, enhancing the ability of such target cells to express the nucleic acid and, for example, produce a polypeptide or protein of interest. For example, transfection of a target cell with a composition encapsulating mRNA enhances (i.e., increases) the production of a protein or enzyme encoded by such mRNA.

[0570] In other embodiments, the present disclosure relates to a method of producing a polypeptide in at least one target cell, comprising contacting at least one target cell with an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one composition described herein containing a nucleic acid, or (iii) at least one lipid nanoparticle containing a nucleic acid described herein, such that the at least one target cell is transfected with a nucleic acid that operably encodes the polypeptide.

[0571] The present disclosure should be understood to encompass all variations, combinations, and permutations in which at least one limitation, element, clause, descriptive term, etc. from at least one of the enumerated claims is introduced into another claim dependent on the same base claim (or any other related claim, etc.), unless otherwise specified or unless a contradiction or inconsistency would be apparent to one skilled in the art. When elements are presented as a list, e.g., a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed, and that any element can be removed from the group. In general, when the present disclosure, or aspects of the present disclosure, are said to include certain elements, features, etc., it should be understood that they also encompass embodiments consisting of, or consisting essentially of, such elements, features, etc. For the sake of brevity, those embodiments have not in every instance been specifically set forth herein. It should also be understood that any embodiment or aspect of the present disclosure can be specifically excluded from the scope of the claims, regardless of whether the specific exclusion is recited in the specification. Publications and other reference materials mentioned herein to describe the background of this disclosure and to provide additional details regarding its practice are hereby incorporated by reference.

[0572] The following examples are offered for purposes of illustration and not limitation. [Example]

[0573] Materials and Methods Nuclear magnetic resonance spectroscopy (H,C NMR) - H and C NMR spectra were recorded at room temperature on the following spectrometer: Brucker Advance 400 (NMR H: 400 MHz and NMR C: 75 MHz).

[0574] The recorded shifts are reported in parts per million (δ) and were calibrated using residual undeuterated 3: H 7.26 ppm; C 77.16 ppm, MeOH H 3.31 ppm; C 49.0 ppm. Data are expressed as chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), coupling constant (J (Hz)), integral, and assignment.

[0575] NMR spectra were obtained using the commercially available software NMRnotebook.

[0576] - High-resolution mass spectra (HRMS) were obtained using an Agilent Q-TOF (time of flight) 6520, and low-resolution mass spectra (LCMS) were obtained using an Agilent MSD 1200SL (ESI / APCI) and an Agilent HPLC 1200 SL.

[0577] Example 1: Synthesis of 2-[2-[2-[2-[2,3-bis(8-nonoxy-8-oxo-octoxy)propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound VI) [ka] Compound VI is prepared according to the synthetic schematic presented in FIG.

[0578] Synthesis of intermediate LE-1-IJ0858-1 [ka] Under nitrogen, 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and triethylamine (35.6 g, 352 mmol) in dry dichloromethane (500 mL) were cooled to −5° C. Methanesulfonyl chloride (30.2 g, 264 mmol) in dry DCM (20 mL) was added dropwise to this solution at 0° C. The mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. Triethylamine hydrochloride was filtered off, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (69.1 g, 175 mmol, quantitative) as a pale yellow oil, which was used without further purification. 1H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.56(s,2H),4.39-4.33(m,2H),3.78-3.73(m,2H),3.69-3.60(m,12H),3.06(s,3H).

[0579] Synthesis of intermediate LE-1-IJ0858-2 [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.4 g, 175 mmol) in THF (500 mL) was added NaH (14 g, 351 mmol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (69.1 g, 175 mmol) was added under nitrogen, and the reaction was heated at 80° C. for 24 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 70% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),4.28(t,J=5.9Hz,1H),4.05(dd,J=8.3,6.4Hz,1H),3.72(dd,J=8.3,6.4Hz,1 H),3.70-3.61(m,16H),3.57(dd,J=10.0,5.8Hz,1H),3.49(dd,J=10.0,5.5Hz,1H),1.42(s,3H),1.35(s,3H).

[0580] Synthesis of intermediate LE-1-IJ0858-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 123 mmol) in AcOH (200 mL) and HO (200 mL) was stirred at room temperature for 18 h. TLC (EA / PE1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxy)ethoxy]ethoxy]ethyl methanesulfonate (49 g, 123 mmol, quantitative) was obtained as a pale yellow oil and used without further purification. 1 H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),3.88-3.81(m,1H),3.70-3.51(m,21H).

[0581] Synthesis of intermediate LE-1-IJ0858-4 [ka] To a solution of 3-[2-[2-[2-(2-benzyloxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen, NaH (9.64 g, 241 mmol) was added, and the mixture was heated at 80° C. for 15 minutes. The reaction was then cooled to room temperature, and 9-bromon-1-ene (31.9 g, 151 mmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 minutes and then at 80° C. for 18 hours. TLC (EA / PE=1 / 1, Rf: 0.5) showed the formation of a new spot. The reaction was quenched with water (50 mL) and then partitioned between ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20%–50% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol, 24.2% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),5.89-5.72(m,2H),5.04-4.89(m,4H),4.57(s,2H),3.71-3.60(m,1 7H),3.59-3.38(m,9H),2.03(q,J=6.7Hz,4H),1.60-1.49(m,4H),1.43-1.23(m,16H).

[0582] Synthesis of intermediate LE-1-IJ0858-5 [ka] To a solution of 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol) in MeCN (80 mL), CCl (80 mL), and water (80 mL) was added NaIO (24.9 g, 116 mmol) and RuCl (656 mg, 2.91 mmol). The reaction mixture was stirred at room temperature for 24 h. LCMS indicated the title compound was the major product along with a partial monoaldehyde product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aqueous HCl (400 mL). The organic layer was washed with NaSO solution, then dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 12.4 mmol) as a yellow oil, which was used without further purification.

[0583] 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-oxooctoxy)propoxy]octanoic acid (10 g, 8 mmol) was dissolved in t-BuOH:NaH2PO4.2H2O (3.73 g, 24 mmol), 2-methyl-2-butene (40 mL), and H2O (3:1, 160 mL) containing sodium chlorite (2.71 mg, 24 mmol). The reaction was stirred at room temperature for 2 hours, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 3.22 mmol, quantitative) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),3.71-3.61(m,17H),3.59-3.37(m,9H),2.33(t,J=7.3Hz,4H),1.69-1.51(m,8H),1.39 -1.28(m,14H).

[0584] Synthesis of intermediate LE-1-IJ0858-6 [ka] To a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 14.8 mmol) and 1-nonanol (5.12 g, 35.5 mmol) in dry dichloromethane (200 mL) under nitrogen, N,N-diisopropylethylamine (11.5 g, 88.7 mmol), 4-dimethylaminopyridine (DMAP) (0.722 g, 5.91 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (7.37 g, 38.4 mmol) were added. The mixture was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20% to 55% ethyl acetate in petroleum ether to give nonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (5 g, 35.9%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),4.05(t,J=6.8Hz,4H),3.70-3.61(m,16H),3.59-3.39(m, 9H),2.28(t,J=7.5Hz,4H),1.67-1.50(m,12H),1.37-1.21(m,36H),0.88(t,J=6.8Hz,6H).

[0585] Synthesis of intermediate LE-1-IJ0858-7 [ka] To a solution of nonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (5 g, 5.31 mmol) in ethyl acetate (100 mL) was added Pd / C (1.13 g, 20% w / w). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (ethyl acetate / petroleum ether 1 / 1) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give nonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (4.22 g, 4.98 mmol, 93.8%) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.05(t,J=6.8Hz,4H),3.74-3.38(m,27H),2.28(t,J=7.5Hz,4H),1.68-1.50(m,12H),1.39-1.21(m,37H),0.88(t,J=6.8Hz,6H).

[0586] Synthesis of Compound VI To a solution of 1-methylpiperidine-4-carboxylic acid (0.152 g, 1.06 mol) and nonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (0.6 g, 0.708 mol) in dichloromethane (15 ml), N,N-diisopropylethylamine (0.11 g, 0.85 mmol) and DMAP (8.65 mg, 0.07 mol) were added in portions, followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.163 g, 0.85 mmol). The reaction was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated in vacuo, and the residue was purified by silica gel chromatography (0–15% methanol in dichloromethane) to give 2-[2-[2-[2-[2,3-bis(8-nonoxy-8-oxo-octoxy)propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (0.281 g, 0.29 mmol, 41% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.27-4.20(m,2H),4.05(t,J=6.8Hz,4H),3.72-3.61(m,14H),3.59-3.39(m,9H),2.84(d,J=11.5Hz,2H),2.38-2.23(m, 8H),2.06(s,1H),1.99-1.87(m,3H),1.86-1.77(m,2H),1.67-1.52(m,12H),1.37-1.23(m,36H),0.88(t,J=6.8Hz,6H). MS(ESI)m / z=930.8(M+H)+

[0587] Example 2: Synthesis of 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound VII) [ka] Compound VII is prepared according to the synthetic schematic presented in FIG.

[0588] Synthesis of intermediate EXP-21-IJ0476-6 [ka] To a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (1.26 g, 1.96 mmol) and heptadecan-8-ol (1.51 g, 5.88 mmol) in dry dichloromethane (20 mL) was added DIPEA (1.52 g, 11.8 mmol), DMAP (0.096 g, 0.784 mmol), and EDCI (0.977 g, 5.10 mmol) in portions under an ice bath. The mixture was stirred at room temperature for 18 hours. The reaction was quenched with NaHCO3 (30 mL) and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 5% (2%) CHOH in DCM to give 1-heptyldecyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.600 g, 0.482 mmol, 24.6% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 8.08-8.04(m,1H),7.56(t,J=7.4Hz,1H),7.48-7.43(m,1H),7.36-7.26(m,5H),4.90-4.82(m,2H),4.57(s,2H),4.50-4.47(m, 1H),3.86-3.82(m,1H),3.73-3.38(m,24H),2.30-2.24(m,4H),1.62-1.46(m,16H),1.35-1.22(m,60H),0.88(t,J=6.9Hz,12H).

[0589] Synthesis of intermediate EXP-21-IJ0476-7 [ka] A solution of 1-heptyldecyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.600 g, 0.536 mmol) in EtOAc (10 mL) was purged with N for 10 minutes. Subsequently, Pd / C (180 mg) was added, and the reaction continued to be purged with N. The reaction was then evacuated under vacuum and filled with H three times. The reaction was then stirred overnight at room temperature under an H atmosphere. TLC (4% CHOH in DCM) indicated the reaction was complete. The slurry was filtered through Celite, and the Celite was rinsed several times with EtOAc. The combined organic layers were then concentrated under vacuum to give 1-heptyldecyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.537 g, 0.495 mmol, 92.5% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 4.85(dd,J=12.5,6.2Hz,2H),3.79-3.32(m,25H),2.32-2.23(m,4H),1.64-1.46(m,16H),1.35-1.20(m,60H),0.88(t,J=6.9Hz,12H).

[0590] Synthesis of Compound VII To a solution of 1-heptyldecyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnononoxy)-8-oxo-octoxy]propoxy]octanoate (0.537 g, 0.522 mmol) and 1-methylpiperidine-4-carboxylic acid (0.299 g, 2.09 mmol) in dichloromethane (20 mL) was added DIPEA (0.270 g, 2.09 mmol) and DMAP (0.026 g, 0.209 mmol), followed by EDCI (0.400 g, 2.09 mmol) in portions at 0 °C. The reaction was stirred at room temperature for 32 hours. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated in vacuo, and the residue was purified by silica gel chromatography (0–10% CH3OH in DCM (4%)) to give 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (0.370 g, 0.308 mmol, 59.0% yield) as an orange oil. LCMS.MS(ESI) m / z=1155.9(M+H)+ 1H NMR(500MHz,CDCl3)δ 4.92-4.79(m,2H),4.29-4.19(m,2H),3.72-3.67(m,2H),3.64(d,J=6.4Hz,12H),3.59-3.39(m,9H),2.85(s, 2H),2.39-2.22(m,8H),1.89(d,J=56.9Hz,6H),1.60-1.47(m,14H),1.34-1.21(m,62H),0.91-0.84(m,12H).

[0591] Example 3: Synthesis of 2-[2-[2-[2-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[8-[(Z)-oct-2-enoxy]-8-oxooctoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound VIII) [ka] Compound VIII is prepared according to the synthetic schematic detailed in FIG.

[0592] Synthesis of LE-1-IJ0470-1 [ka] To a mixture of 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (SM1) (50 g, 0.257 mol), N,N-dimethylpyridin-4-amine (1.57 g, 12.9 mmol), and [chloro(diphenyl)methyl]benzene (57.4 g, 0.206 mol) in DCM (400 mL) cooled to 0 °C, N,N-diethylethanamine (52.1 g, 0.515 mol) was added. The reaction mixture was stirred at ambient temperature for 16 h. TLC (EA:PE = 2:1, Rf = 0.5) showed the formation of a new spot. The mixture was poured into water (600 mL) and extracted with DCM (2 × 400 mL). The organic layer was washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by flash column chromatography on silica eluting with 1:1 EA / PE to give 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (45.7 g, 40.7% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 7.46(dt,J=3.4,1.9Hz,6H),7.32-7.26(m,6H),7.24-7.18(m,3H),3.73-3 .63(m,12H),3.61-3.56(m,2H),3.27-3.21(m,2H),2.56(t,J=6.0Hz,1H).

[0593] Synthesis of LE-1-IJ0470-2 [ka] To a mixture of 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (45.7 g, 0.105 mol) and N,N-diethylethanamine (21.2 g, 0.209 mol) in DCM (600 mL) was slowly added methanesulfonyl chloride (14.4 g, 0.126 mol) at 0 °C. The mixture was stirred at room temperature overnight. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was removed to give 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (53.5 g, 99.3%) as a yellow oil. 1H NMR(400MHz,CDCl3)δ 7.46(dt,J=3.4,1.9Hz,6H),7.32-7.26(m,6H),7.25-7.19(m,3H),4.35-4.30(m ,2H),3.75-3.71(m,2H),3.70-3.64(m,10H),3.23(t,J=5.2Hz,2H),2.98(s,3H).

[0594] Synthesis of LE-1-IJ0470-1 [ka] To a solution of trityl chloride (45.5 g, 163 mmol), glycerol (50 g, 543 mmol), and N,N-dimethylpyridin-4-amine (0.663 g, 5.43 mmol) in 500 mL of THF, triethylamine (16.48 g, 163 mmol) was added, and the mixture was stirred at room temperature for 22 h. Then, 300 mL of ethyl acetate and 150 mL of HO were added to the solution. The organic phase was collected, and the aqueous layer was extracted with 2 × 300 mL of ethyl acetate. The combined organic phases were washed with 200 mL of 10% (w / v) NaHCO, followed by 200 mL of brine, and dried over NaSO. The solvent was evaporated, and the residue was purified on a silica gel column (eluted with CHCl / MeOH) to give 3-trityloxypropane-1,2-diol as a white solid (50.4 g, 27.8% yield). 1H NMR(400MHz,CDCl3)δ 7.42(dt,J=3.4,1.9Hz,6H),7.35-7.26(m,7H),7.26-7.21(m,2H),3.91-3.82(m,1H),3.74-3. 56(m,2H),3.25(m,J=15.7,9.6,5.2Hz,2H),2.49(d,J=5.1Hz,1H),1.96(dd,J=7.1,5.3Hz,1H).

[0595] Synthesis of LE-1-IJ0470-3 [ka] To a suspension of NaH (5.98 g, 5.0 eq) in 500 mL of anhydrous N,N-dimethylformamide was added 3-trityloxypropane-1,2-diol (10.0 g, 1.0 eq). The mixture was heated at 80 °C for 15 min and cooled to room temperature. 9-Bromo-1-ene (15.3 g, 2.5 eq) in 50 mL of anhydrous DMF was added dropwise to the mixture, which was then heated at 80 °C for 18 h. After cooling to room temperature, 500 mL of HO was added. The mixture was extracted with ethyl acetate, and the organic layer was washed successively with 500 mL of 5% (w / v) NaHCO and 500 mL of brine and dried over NaSO. The solvent was evaporated under reduced pressure, and the residue was purified on a silica gel column eluted with petroleum ether / ethyl acetate (0% to 20% ethyl acetate in petroleum ether) to give a colorless oil (8.4 g, 48.2% yield). 1H NMR(400MHz,CDCl3)δ 7.59-7.16(m,17H),5.86-5.74(m,3H),4.95(dd,J=24.2,13.6Hz,6H),3.61- 3.35(m,12H),3.24-3.10(m,2H),2.03(d,J=6.4Hz,6H),1.46-1.24(m,27H).

[0596] Synthesis of LE-1-IJ0470-4 [ka] To a solution of ((2,3-bis(non-8-en-1-yloxy)propoxy)methanetriyl)tribenzene (20.0 g, 34.3 mmol) in methanol / THF (400 mL, 1 / 1 v / v) was added 4-methylbenzenesulfonic acid (29.5 g, 172 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 18 h. TLC (4% ethyl acetate in petroleum ether) showed complete disappearance of the starting material. The reaction was quenched by the addition of 50 mL of triethylamine, and the solvent was removed under vacuum. The residue was purified by flash chromatography eluting with 20%–30% ethyl acetate in petroleum ether (21%) to give 2,3-bis(non-8-enoxy)propan-1-ol (8.6 g, 73.6% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.88-5.74(m,2H),5.04-4.90(m,4H),3.77-3.38(m,8H),2.20(s,1H),2 .04(q,J=7.0Hz,4H),1.56(dt,J=13.6,6.8Hz,4H),1.43-1.24(m,17H).

[0597] Synthesis of LE-1-IJ0470-5 [ka] To a mixture of 2,3-bis(non-8-enoxy)propan-1-ol (8.6 g, 25.3 mmol) in 300 mL of dry THF, NaH (60% mineral oil dispersion, 2.02 g, 50.5 mmol) was added and then stirred at 80° C. for 15 minutes. The reaction was cooled to room temperature, and 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (15.6 g, 30.3 mmol) dissolved in 50 mL of dry THF was added to the above reaction. The reaction mixture was stirred at reflux (80° C.) overnight. The reaction mixture was cooled to room temperature, and water (200 mL) was added. EtOAc (400 mL) was added, the mixture was shaken, the layers were separated, and the organic layer was collected. The aqueous layer was extracted with EtOAc (400 mL × 2). The combined organic layers were washed with brine and dried over Na2SO4. The residue was purified by flash column chromatography on silica gel eluting with ethyl acetate (0–15%) in petroleum ether (14%) to give [2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (15.7 g, 81.9% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.49-7.44(m,7H),7.32-7.26(m,7H),7.24-7.19(m,4H),5.88-5.72(m,2H),5.0 2-4.99(m,1H),4.98-4.95(m,1H),4.93(d,J=1.2Hz,1H),4.91(d,J=1.2Hz,1H),3 .70-3.64(m,12H),3.62(d,J=4.2Hz,4H),3.59-3.39(m,9H),3.23(t,J=5.2Hz,2H ),2.03(dt,J=7.8,3.9Hz,4H),1.54(dd,J=13.1,6.5Hz,4H),1.38-1.25(m,16H).

[0598] Synthesis of LE-1-IJ0470-6 [ka] To a solution of [2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (15.7 g, 20.7 mmol) in MeCN (200 mL), CCl (200 mL), and water (200 mL) was added NaIO (35.4 g, 165 mmol) and RuCl (0.933 g, 4.14 mmol). The reaction mixture was stirred at room temperature for 24 hours. The reaction was filtered through Celite, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aqueous HCl (900 mL). The organic layer was washed with NaSO solution (700 mL × 2), then dried over sodium sulfate, filtered, and concentrated to give 8-[2-(7-carboxyheptoxy)-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid (9.4 g, 57.2% yield) as a yellow oil, which was used without further purification. 1H NMR(400MHz,CDCl3)δ 7.48-7.44(m,6H),7.29(dd,J=10.1,4.7Hz,6H),7.24-7.20(m,3H),3.72-3.38(m,24H),3 .23(t,J=5.2Hz,2H),2.35(dt,J=24.3,7.4Hz,4H),1.68-1.49(m,8H),1.38-1.26(m,12H).

[0599] Synthesis of LE-1-IJ0470-7 [ka] 8-[2-(7-carboxyheptoxy)-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy] To a solution of [propoxy]octanoic acid (1.0 g, 1.26 mmol) and (Z)-non-2-en-1-ol (0.43 g, 3.02 mmol) in dry dichloromethane (50 mL), DIPEA (0.98 g, 7.55 mmol), DMAP (0.062 g, 0.503 mmol) were added, followed by the addition of EDCI (2.56 g, 13.4 mmol) in portions under an ice-water bath. The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 20% (15%) ethyl acetate in petroleum ether to give [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (0.39 g, 29.7% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 7.47-7.45(m,6H),7.29(t,J=7.5Hz,6H),7.22(dd,J=8.3,6.2Hz,3H),5.6 4(dd,J=18.4,7.6Hz,2H),5.56-5.47(m,2H),4.61(d,J=6.8Hz,4H),3.69- 3.39(m,23H),3.23(t,J=5.2Hz,2H),2.29(dd,J=10.7,4.4Hz,4H),2.14-2 .03(m,4H),1.61-1.50(m,8H),1.40-1.22(m,28H),0.88(t,J=6.9Hz,6H).

[0600] Synthesis of LE-1-IJ0470-8 [ka] To a solution of [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (0.39 g, 0.374 mmol) in methanol / THF (40 mL, 1 / 1 v / v) was added p-toluenesulfonic acid (0.322 g, 1.87 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 2 hours. TLC (30% ethyl acetate in petroleum ether) showed complete disappearance of the starting material. The reaction was quenched by the addition of 5 mL of triethylamine, and the solvent was removed under vacuum. The residue was purified by flash chromatography eluting with DCM containing 0% to 20% (10%) methanol to give 2,3-bis(non-8-enoxy)propan-1-ol (0.270 g, 90.2% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.70-5.59(m,2H),5.58-5.47(m,2H),4.62(d,J=6.7Hz,4H),3.77-3.37(m,27H),2.70(s,1H),2.37 -2.24(m,4H),2.10(q,J=6.8Hz,4H),1.66-1.50(m,8H),1.41-1.20(m,28H),0.88(t,J=6.9Hz,6H).

[0601] Synthesis of Compound VIII To a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (570 mg, 0.711 mmol) and 1-methylpiperidine-4-carboxylic acid (0.153 g, 1.07 mmol) in dry dichloromethane (15 mL) was added DIPEA (0.110 g, 0.854 mmol), DMAP (0.009 g, 0.071 mmol), and EDCI (0.164 g, 0.854 mmol) under ice bath. The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 20% MeOH in DCM to give 2-[2-[2-[2-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[8-[(Z)-oct-2-enoxy]-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (0.414 g, 63.8% yield) as a colorless oil. 1H NMR (400MHz, CDCl3) δ 5.64(dt,J=10.9,7.4Hz,2H),5.52(dt,J=11.0,6.8Hz,2H),4.62(d,J=6.8Hz, 4H),4.27-4.21(m,2H),3.72-3.61(m,14H),3.58-3.39(m,9H),2.85(d,J=11.2 Hz,2H),2.39-2.26(m,8H),2.10(dd,J=14.0,6.9Hz,5H),1.94(s,2H),1.82(d ,J=10.6Hz,3H),1.66-1.50(m,9H),1.41-1.24(m,30H),0.88(t,J=6.9Hz,6H).

[0602] Example 4 Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[3-(4-methylpiperazin-1-yl)propanoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxooctoxy]propoxy]octanoate (Compound IX) [ka] Compound IX is prepared from compound LE616IJ0470-8 (see Example 3 for the synthesis of LE616IJ0470-8).

[0603] To a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (400 mg, 0.500 mmol) and 3-(4-methylpiperazin-1-yl)propanoic acid (0.129 g, 0.750 mmol) in dry dichloromethane (10 mL) was added DIPEA (0.078 g, 0.600 mmol), DMAP (0.006 g, 0.050 mmol), and EDCI (0.115 g, 0.600 mmol) in an ice bath. The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 20% (9%) MeOH in DCM to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[3-(4-methylpiperazin-1-yl)propanoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (0.378 g, 79.2% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 5.69-5.47(m,4H),4.62(d,J=6.8Hz,4H),4.26-4.21(m,2H),3.75-3.60(m,17H),3.60-3.38(m,11H),2.72(t,J=7.4Hz,2H), 2.57-2.49(m,6H),2.33-2.27(m,8H),2.10(q,J=6.9Hz,4H),1.67-1.51(m,10H),1.41-1.09(m,32H),0.88(t,J=6.8Hz,6H).

[0604] Example 5: Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-(1-methyl-4-piperidyl)acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxooctoxy]propoxy]octanoate (Compound X) [ka] Compound X is prepared from the synthetic scheme in FIG.

[0605] To a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (400 mg, 0.500 mmol) and 2-(1-methyl-4-piperidyl)acetic acid (0.118 g, 0.749 mmol) in dry dichloromethane (10 mL) was added DIPEA (0.078 g, 0.600 mmol), DMAP (0.006 g, 0.050 mmol), and EDCI (0.115 g, 0.600 mmol) under ice bath conditions. The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 20% (9%) MeOH in DCM to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[(1-methyl-4-piperidyl)acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (0.271 g, 57.7% yield) as a pale yellow oil. 1H NMR (400MHz, CDCl3) δ 5.64(dt,J=10.9,7.5Hz,2H),5.57-5.47(m,2H),4.62(d,J=6.8Hz,4H),4.26 -4.20(m,2H),3.76-3.60(m,16H),3.60-3.37(m,11H),3.02(s,2H),2.43(s, 3H),2.28(dd,J=24.3,16.8Hz,9H),2.10(dd,J=14.3,7.1Hz,4H),1.80(d,J= 13.8Hz,4H),1.65-1.51(m,11H),1.40-1.20(m,31H),0.88(t,J=6.8Hz,6H).

[0606] Example 6: Synthesis of 2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound XI) [ka] Compound XI is prepared according to the synthetic scheme in FIG.

[0607] Synthesis of intermediates [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (5 g, 8.43 mmol) in DCM (100 mL) at 0 °C was added Dess-Martin periodinane (5.36 g, 12.6 mmol). The reaction was stirred at room temperature for 2 h. TLC indicated that the starting material had been consumed. The mixture was filtered and concentrated. The crude product was diluted with ethyl acetate (50 mL), washed with NaSO / NaHCO (25 mL / 25 mL × 3), brine (50 mL), and dried over NaSO. The organics were concentrated to give 2,3-bis[(Z)-octadec-9-enoxy]propanal (5 g, 8.29 mmol, 98.3% yield) as a colorless oil, which was taken directly into the next step. 1H NMR(500MHz,CDCl3)δ 9.72(d,J=1.1Hz,1H),5.40-5.31(m,3H),3.84-3.54(m,5H),3.49-3.39(m,2H),2.04-1.92(m,7 H),1.63(dd,J=14.2,7.1Hz,2H),1.58-1.53(m,2H),1.34-1.24(m,44H),0.88(t,J=6.9Hz,6H).

[0608] Synthesis of intermediate LE-1-IY5612-2 2,3-Bis[(Z)-octadec-9-enoxy]propanal (5 g, 8.46 mmol) was dissolved in HO (3:1, 60 mL) containing t-BuOH:NaHPO (3.05 g, 25.4 mmol), 2-methyl-2-butene (17.8 mL), and sodium chlorite (2.3 g, 25.4 mmol). The reaction was stirred at room temperature for 1 hour, diluted with ethyl acetate (150 mL), and washed with water (200 mL × 2). The aqueous layer was extracted with ethyl acetate (50 mL). The combined extracts were dried over sodium sulfate and concentrated to give 2,3-bis[(Z)-octadec-9-enoxy]propanoic acid (4.7 g, 7.6 mmol, 89.7% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 5.43-5.31(m,3H),4.04(dd,J=4.9,3.2Hz,1H),3.81-3.41(m,6H),2.04-1 .92(m,6H),1.65-1.55(m,4H),1.34-1.24(m,44H),0.88(t,J=6.9Hz,6H).

[0609] Synthesis of intermediate LE-1-IY5612-1 [ka] 2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (11 g, 17.7 mmol) was added to octan-1-amine (44 ml), and the mixture was stirred at 80 °C for 18 hours. LCMS showed that SM was consumed and the product was formed. The mixture was diluted with ethyl acetate (EA) (500 ml), washed with water (500 ml × 2), brine (500 ml), and dried over Na SO . The organic layer was concentrated and purified by flash chromatography column (10% MeOH in DCM) to give N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (10 g, 16.6 mmol, 93.4% yield) as a yellow oil. 1H NMR(500MHz,CDCl3)δ 7.48-7.43(m,6H),7.29(dd,J=10.3,4.8Hz,6H),7.25-7.20(m,3H),3.75-3.53(m,16H),3.24(t,J=5.2Hz,2H ),2.86(t,J=5.1Hz,2H),2.70-2.63(m,2H),1.62-1.51(m,2H),1.26(d,J=5.0Hz,10H),0.87(t,J=6.9Hz,3H).

[0610] Synthesis of intermediate LE-1-IY5612-3 To a solution of N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (1.38 g, 1.98 mmol) in DCM (20 mL) was added 2,3-bis[(Z)-octadec-9-enoxy]propanoic acid (1 g, 1.65 mmol), 4-dimethylaminopyridine (20 mg), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (940 mg, 2.47 mmol), and TEA (333 mg, 3.29 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was then diluted with DCM (50 mL), washed with water (250 mL × 2), brine (250 mL), and dried over Na2SO4. The organic layer was purified by flash chromatography column (5% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (1.15 g, 0.95 mmol, 57.9% yield) as a yellow oil. 1H NMR(500MHz,CDCl3)δ 7.46(d,J=7.6Hz,6H),7.28(dd,J=12.5,4.6Hz,6H),7.22(t,J=7.2Hz,3H),5.39-5.31(m,3H),4.43-4 .28(m,1H),3.83-3.08(m,28H),1.99(dd,J=15.5,9.2Hz,7H),1.56-1.19(m,60H),0.92-0.84(m,9H).

[0611] Synthesis of intermediate LE-1-IY5612-4 [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (1.15 g, 0.97 mmol) in THF / MeOH (10 ml, 1 / 1) was added toluene-4-sulfonic acid (371 mg, 1.95 mmol). The mixture was stirred for 25 min.o The mixture was stirred at RT for 2 h. The mixture was diluted with ethyl acetate (EA) (50 ml), washed with NaHCO (50 ml), brine (50 ml), and dried over NaSO. The organic layer was concentrated and purified by flash chromatography column (10% MeOH in DCM) to give N-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-propanamide (730 mg, 0.78 mmol, 79.9% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.43-5.31(m,3H),4.45-4.30(m,1H),4.00-3.19(m,28H),2.10-1.90(m,8H),1.67-1.16(m,60H),0.95-0.82(m,9H).

[0612] Synthesis of Compound XI To a solution of 1-methylpiperidine-4-carboxylic acid (111 mg, 0.78 mmol) in DCM (10 ml) was added N-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-propanamide (730 mg, 0.78 mmol), DIEA (251 mg, 1.94 mmol), DMAP (19 mg) and EDC HCl (298 mg, 1.56 mmol). The mixture was stirred for 25 min. o The mixture was stirred at RT for 18 hours at room temperature. The mixture was then diluted with ethyl acetate (50 ml), washed with water (50 ml), brine (50 ml), and dried over NaSO. The organic layer was concentrated, and the residue was purified by flash chromatography column (50% ethyl acetate in PE) to give 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (318 mg, 0.29 mmol, 37.6% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.35(dd,J=12.4,7.2Hz,3H),4.44-4.30(m,1H),4.29-4.19(m,2H),3.74-3.23(m,26H) ,2.83(s,2H),2.30(s,3H),2.11-1.68(m,14H),1.62-1.15(m,60H),0.94-0.82(m,9H).

[0613] Example 7: Synthesis of 2-[2-[2-[2-[2-[2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound XII) [ka] Compound XII is prepared according to the synthetic schematic in FIG.

[0614] Synthesis of intermediate LE-1-IY5614-8 [ka] To a solution of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-hydroxy-propoxy]hexyl 2-hexyldecanoate (1.5 g, 1.95 mmol) in acetonitrile (AcCN, 20 mL) and pH 4 buffer solution (10 mL, AcOH:AcONa:water = 92 mL:33 g:1000 mL), sodium chlorite (0.97 g, 10.7 mmol) and sodium hypochlorite (0.0726 g, 0.975 mmol) were added, followed by TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) (0.152 g, 0.0975 mmol). The reaction turned black and was stirred at 20 °C for 4 hours. LCMS indicated a clean reaction. The reaction was quenched with 20 drops of methanol, poured into water (40 mL), and extracted with ethyl acetate (60 ml × 3). The organic layers were combined, washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 5% to 10% methanol in dichloromethane to give 2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propanoic acid (1.23 g, 80.5% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 4.11-4.02(m,5H),3.82-3.41(m,6H),2.36-2.26(m,2H),1.70-1.52(m,12H),1.47-1.34(m,12H),1.25(s,40H),0.87(dd,J=6.9,6.2Hz,12H).

[0615] Synthesis of intermediate LE-1-IY5614-9 [ka] A mixture of 2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propanoic acid (1.23 g, 1.57 mmol), EDC HCl (0.452 g, 2.36 mmol), and N-hydroxysuccinimide (0.271 g, 2.36 mmol) in DCM (20 mL) was stirred at room temperature for 2 hours. Then, N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (1.12 g, 1.88 mmol) and DIEA (0.61 g, 4.71 mmol) were added. The mixture was stirred at room temperature for 16 hours. The mixture was poured into DCM (200 mL), washed with NaHCO3 solution and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography eluting with 2-5% methanol in dichloromethane (3%) to give 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (1.27 g, 59.6% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.69-6.99(m,15H),4.46-4.27(m,1H),4.06(q,J=6.6Hz,4H),3.89-3.19(m,26H), 2.35-2.26(m,2H),1.75-1.33(m,28H),1.25(s,50H),0.88(dd,J=8.3,4.3Hz,15H).

[0616] Synthesis of intermediate LE-1-IY5614-8-10 [ka] To a solution of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (1.27 g, 0.936 mmol) in methanol / THF (30 mL, 1 / 1 v / v) was added 4-methylbenzenesulfonic acid (0.89 g, 4.68 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 18 hours. TLC (4% ethyl acetate in petroleum ether) showed complete disappearance of the starting material. The reaction was quenched by the addition of 30 mL of triethylamine, and the solvent was removed in vacuo. The residue was purified by flash chromatography eluting with 0% to 5% MeOH (4%) in DCM to give 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl-octylamino]-3-oxo-propoxy]hexyl-hexyldecanoate (0.78 g, 74.8% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.46-4.29(m,1H),4.05(td,J=6.6,2.1Hz,4H),3.78-3.24(m,28H),2.35-2 .25(m,2H),1.69-1.32(m,28H),1.25(s,50H),0.87(dd,J=6.7,5.8Hz,15H).

[0617] Synthesis of Compound XII To a solution of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl-octylamino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (0.4 g, 0.359 mmol) and 1-methylpiperidine-4-carboxylic acid (0.103 g, 0.718 mmol) in dry dichloromethane (10 mL) was added DIPEA (0.056 g, 0.431 mmol), DMAP (0.004 g, 0.036 mmol), followed by the portionwise addition of EDCI (0.08 g, 0.431 mmol) in an ice bath. The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 20% methanol in dichloromethane to give 2-[2-[2-[2-[2-[2-[2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propanoyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (0.278 g, 62.5% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.43-4.30(m,1H),4.27-4.21(m,2H),4.05(td,J=6.6,2.1Hz,4H),3.72-3.29(m,26H),2.84(d,J=11.2Hz,2H),2.38-2.23(m,6H) ),2.05(s,2H),1.95(d,J=15.4Hz,2H),1.86-1.74(m,3H),1.67-1.52(m,14H),1.46-1.33(m,12H),1.25(s,50H),0.87(t,15H).

[0618] Example 8: Synthesis of -[2-[2-[2-[2-[2,3-bis[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound XIII) [ka] Compound XIII is prepared according to the synthetic schematic in FIG.

[0619] Synthesis of LE-1-IY5613 [ka] Trityl chloride (96.6 g, 347 mmol), glycerol (129 g, 1400 mmol), and N,N-dimethylpyridin-4-amine (966 mg, 7.91 mmol) were dissolved in 300 mL of THF. After adding triethylamine (42.5 g, 420 mmol), the mixture was stirred at room temperature for 22 hours. Then, 300 mL of ethyl acetate and 300 mL of HO were added to the solution. The organic phase was collected and extracted with 2 × 300 mL of ethyl acetate. The combined organic phases were washed with 200 mL of 10% (w / v) NaHCO3, then 200 mL of brine, and dried over Na2SO4. The solvent was evaporated, and the residual oil was recrystallized in benzene / hexane. The resulting product was further purified on a silica gel column (elution gradient CH2Cl2 / MeOH, 5% MeOH in DCM) to give 3-trityloxypropane-1,2-diol as a white solid (67.9 g, 203 mmol, 63.7% yield). 1H NMR(400MHz,CDCl3)δ 7.42(d,J=7.8Hz,6H),7.31(t,J=7.5Hz,6H),7.24(dd,J=8.9,5.5Hz,3H),3.85(s,1H),3.62(d t,J=11.3,8.5Hz,2H),3.24(ddd,J=15.6,9.6,5.3Hz,2H),2.53(d,J=3.7Hz,1H),2.02(s,1H).

[0620] Synthesis of IY5613-B-1 [ka] To a suspension of NaH (24.4 g, 5.0 eq) in 500 mL of anhydrous N,N-dimethylformamide was added 3-trityloxypropane-1,2-diol (67.9 g, 1.0 eq). The mixture was heated at 80° C. for 1 h and cooled to room temperature. 9-Bromo-1-ene (104 g, 2.5 eq) in 50 mL of anhydrous DMF was added 0.05 g. o C. to the mixture, which was then heated at 80°C for 18 hours. After cooling to room temperature, 1000 mL of HO was added, and the mixture was washed successively with 500 mL of 5% (w / v) NaHCO. and 500 mL of brine, and dried over Na.sub.2SO.sub.4. The solvent was evaporated under reduced pressure, and the resulting oil was purified on a silica gel column eluted with petroleum ether / ethyl acetate (0% to 20% ethyl acetate in petroleum ether) to give a colorless oil (20 g, 16.6% yield). 1H NMR(500MHz,CDCl3)δ 7.46(dd,J=5.2,3.4Hz,6H),7.31-7.26(m,6H),7.25-7.20(m,3H),5.80(ttd,J=13.1,6.7,2.7Hz,2 H),5.02-4.89(m,4H),3.59-3.35(m,7H),3.21-3.11(m,2H),2.08-1.98(m,4H),1.55-1.24(m,20H).

[0621] Synthesis of IY5613-C [ka] To a solution of [2,3-bis(non-8-enoxy)propoxy-diphenyl-methyl]benzene (27 g, 46.3 mmol) in THF / MeOH (100 ml / 100 ml) was added toluene-4-sulfonic acid (6.53 g, 34.3 mmol). oThe mixture was stirred at RT for 2 h at RT. The mixture was then concentrated, diluted with ethyl acetate (100 ml), washed with aqueous NaHCO (100 × 2 ml), brine (100 ml), and dried over NaSO. The organic layer was concentrated and purified by flash chromatography column (20% EA in PE) to give 2,3-bis(non-8-enoxy)propan-1-ol (12.8 g, 35.7 mmol, 77.1% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.81(ddt,J=16.9,10.2,6.7Hz,2H),5.06-4.88(m,4H),3.76-3.41(m,9H) ,2.04(q,J=6.9Hz,4H),1.56(dt,J=13.6,6.9Hz,4H),1.40-1.27(m,16H).

[0622] Synthesis of IY5613-D [ka] To a solution of 2,3-bis(non-8-enoxy)propan-1-ol (12.8 g, 37.6 mmol) in DCM (200 ml) was added Dess-Martin periodinane (23.9 g, 56.4 mmol) for 0.5 min. o The mixture was then added at 25°C for 5 minutes. o The mixture was stirred at RT for 2 h. The mixture was concentrated, diluted with ethyl acetate (EA) (500 mL), washed with aqueous NaSO / NaHCO (500 mL / 500 mL), brine (500 mL), and dried over NaSO. The organic layer was concentrated to give 2,3-bis(non-8-enoxy)propanal (11.7 g, crude, 90.1% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 9.72(d,J=1.3Hz,1H),5.81(ddt,J=16.9,10.2,6.7Hz,2H),5.05-4.87(m,4H),3.86-3.33(m,7H),2.09-1.97(m,4H),1.61-1.20(m,20H).

[0623] Synthesis of IY5613-E [ka] To a solution of 2,3-bis(non-8-enoxy)propanal (11.7 g, 34.6 mmol) in tert-butyl alcohol / water (180 ml / 60 ml) was added sodium chlorite (9.38 g, 104 mmol), 2-methyl-2-butene (60.6 g, 864 mmol), and sodium dihydrogen phosphate (9.38 g, 104 mmol). The mixture was stirred for 25 o The mixture was stirred at RT for 2 h at RT. The mixture was then diluted with ethyl acetate (EA) (500 mL), washed with water (500 mL × 2), brine (300 mL), and dried over NaSO. The organic layer was concentrated to give 2,3-bis(non-8-enoxy)propanoic acid (9.75 g, 27 mmol, 78% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.81(ddt,J=16.9,10.2,6.7Hz,2H),5.07-4.87(m,4H),4.04(dd,J=5.1,3.3Hz,1H),3.81-3.4 4(m,6H),2.04(dd,J=13.1,6.5Hz,4H),1.66-1.54(m,4H),1.32(ddd,J=12.7,9.1,5.4Hz,16H).

[0624] Synthesis of IY5613-F [ka] To a solution of N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (9.52 g, 16.1 mmol) in DCM (150 ml) was added 2,3-bis(non-8-enoxy)propanoic acid (5 g, 14.1 mmol), [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium;hexafluorophosphate (8.04 g, 21.2 mmol), and N,N-diethylethanamine (2.85 g, 28.2 mmol). The mixture was stirred for 25 minutes. oThe mixture was stirred at RT for 18 h at RT. The mixture was then treated with ethyl acetate (EA) (300 ml), washed with water (300 ml × 2), brine (300 ml), and dried over NaSO. The organic layer was concentrated and purified by flash chromatography column (25% EA in PE) to give 2,3-bis(non-8-enoxy)-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (8.22 g, 8.69 mmol, 61.5% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 7.46(d,J=7.5Hz,6H),7.29(t,J=7.6Hz,6H),7.22(t,J=7.3Hz,3H),5.86-5.74(m,2H),5.03-4.89(m,4H),4.43-4.29( m,1H),3.70-3.22(m,29H),2.03(dd,J=13.5,6.5Hz,4H),1.57-1.51(m,4H),1.40-1.23(m,28H),0.88(q,J=6.9Hz,3H).

[0625] Synthesis of IY5613-G [ka] To a solution of 2,3-bis(octa-7-enoxy)-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (8.22 g, 9.13 mmol) in ACN / CCl4 / HO (80 ml / 80 ml / 80 ml) was added NaIO4 (15.6 g, 73 mmol) and ruthenium(III) chloride hydrate (412 mg, 1.83 mmol). The mixture was stirred for 25 min. oThe mixture was stirred at RT for 18 hours. The mixture was filtered, treated with ethyl acetate (EA) (500 ml), washed with aqueous NaSO (300 ml), brine (300 ml), and dried over NaSO. The organic layer was concentrated and treated with tert-butyl alcohol / water (120 ml / 40 ml). Sodium chlorite (2.48 g, 27.4 mmol), 2-methyl-2-butene (16 g, 228 mmol), and sodium dihydrogen phosphate (3.29 g, 27.4 mmol) were added to the mixture. The mixture was stirred for 25 hours. o The mixture was stirred at RT for 2 h. The mixture was then treated with ethyl acetate (EA) (500 ml), washed with water (500 ml), brine (300 ml), and dried over NaSO. The organics were concentrated and purified by flash chromatography (10% MeOH in DCM) to give 7-[2-(6-carboxyhexoxy)-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]heptanoic acid (5.11 g, 5.19 mmol, 56.8% yield) as a grey oil. 1H NMR(400MHz,CDCl3)δ 7.48(dd,J=15.2,13.8Hz,6H),7.29(dd,J=10.1,4.8Hz,6H),7.22(dd,J=8.3,6.1Hz,3H),4.38(ddd,J=35.0,7.3,4 .4Hz,1H),3.80-3.33(m,26H),3.23(t,J=5.2Hz,2H),2.50-2.24(m,4H),1.56-1.18(m,28H),0.87(q,J=6.8Hz,3H).

[0626] Synthesis of IY5613-7 To a solution of 7-[2-(6-carboxyhexoxy)-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]heptanoic acid (5.1 g, 5.45 mmol) in DCM (80 ml) was added (Z)-non-2-en-1-ol (1.86 mg, 13.1 mmol), EDC HCl (3.13 g, 16.3 mmol), DIEA (2.46 g, 19.1 mmol) and DMAP (333 mg). The mixture was stirred for 25 min. o The mixture was stirred at C for 18 h. The mixture was then concentrated and purified by flash chromatography column (25% EA in PE) to give [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]octanoate (2.27 g, 1.83 mmol, 33.7% yield) as a colorless oil. 1H NMR(500MHz,CDCl3)δ 7.46(d,J=7.4Hz,6H),7.29(t,J=7.5Hz,6H),7.22(t,J=7.3Hz,3H),5.64(dd,J=18.3,7.5Hz,2H),5.55-5.47(m,2H),4.61(d,J=6.9Hz,4H),4.42 -4.28(m,1H),3.69-3.38(m,26H),3.23(t,J=5.2Hz,2H),2.29(t,J=7.5H z,4H),2.09(q,J=7.3Hz,4H),1.52-1.24(m,48H),0.88(t,J=6.8Hz,9H).

[0627] Synthesis of IY5613-8 To a solution of [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]octanoate (2.38 g, 1.96 mmol) in THF / MeOH (15 ml / 15 ml) was added toluene-4-sulfonic acid (560 mg, 2.94 mmol). The mixture was stirred for 25 min. o The mixture was stirred at RT for 2 hours. The mixture was then diluted with ethyl acetate (150 ml), washed with water (150 ml), brine (150 ml), and dried over NaSO. The mixture was concentrated and purified by flash chromatography column (5% MeOH in DCM) to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl-octylamino]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-oxo-propoxy]octanoate (1.51 g, 1.52 mmol, 77.7% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.69-5.46(m,4H),4.62(d,J=6.8Hz,4H),4.43-4.29(m,1H),3.74-3.40(m,28H),2.29(td,J=7 .7,1.5Hz,4H),2.10(dd,J=14.1,7.0Hz,4H),1.61-1.21(m,48H),0.88(td,J=6.7,4.3Hz,9H).

[0628] Synthesis of Compound XIII To a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl-octylamino]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]-3-oxo-propoxy]octanoate (400 mg, 0.41 mmol) in DCM (6 ml) was added 1-methylpiperidine-4-carboxylic acid (88.5 mg, 0.62 mmol), EDCI (158 mg, 0.82 mmol), DIEA (133 mg, 1.03 mmol) and DMAP (10 mg). The mixture was stirred for 25 min. o The mixture was stirred at RT for 18 h. The mixture was then diluted with ethyl acetate (50 mL), washed with water (50 mL), brine (50 mL), and dried over NaSO. The organics were concentrated and purified by flash chromatography column (5% MeOH in DCM) to afford 2-[2-[2-[2-[2-[2-[2,3-bis[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propanoyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (269 mg, 0.24 mmol, 58.3% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 5.64(dd,J=18.3,7.5Hz,2H),5.56-5.47(m,2H),4.62(d,J=6.9Hz,4H),4.41-4.3 1(m,1H),4.26-4.20(m,2H),3.71-3.56(m,20H),3.43(ddd,J=18.7,12.5,7.2Hz, 6H),2.84(s,2H),2.49-2.24(m,9H),2.10(dd,J=14.2,6.8Hz,5H),1.89(d,J=44. 2Hz,4H),1.60(d,J=6.8Hz,8H),1.40-1.19(m,40H),0.88(dt,J=6.9,4.3Hz,9H).

[0629] Example 9: Synthesis of 2-[2-[2-[2-[2,3-bis[6-(2-octyldecanoyloxy)hexoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound XIV) [ka] Compound XIV is prepared according to the synthetic scheme in FIG.

[0630] Synthesis of EXP-21-IJ5617 To a solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol (1.6 g, 2.86 mmol) in dry DCM (20 mL) was added 2-octyldecanoic acid (2.44 g, 8.59 mmol), DIPEA (2.22 g, 17.2 mmol), and DMAP (0.14 g, 1.15 mmol) at room temperature. The mixture was then cooled to 0°C, and EDCI (1.43 g, 7.45 mmol) was added portionwise over 15 minutes. The reaction was stirred at room temperature for 17 hours. TLC (5% CHOH in DCM) indicated the reaction was complete. The reaction was poured into water and extracted with DCM. The water was extracted once more with DCM. The combined organic layers were washed with brine, dried over NaSO, and concentrated in vacuo, then purified by column chromatography using CHOH in DCM (0–4%) (3%) to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-octyldecanoyloxy)hexoxy]propoxy]hexyl 2-octyldecanoate (1.65 g, 52.8% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.57(s,2H),4.10-4.01(m,4H),3.71-3.38(m,25H),2.35- 2.26(m,2H),1.67-1.34(m,24H),1.33-1.17(m,50H),0.87(t,J=6.8Hz,12H).

[0631] A solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-octyldecanoyloxy)hexoxy]propoxy]hexyl 2-octyldecanoate (1.6 g, 1.47 mmol) in EtOAc (20 mL) was purged with N for 10 minutes. Subsequently, Pd / C (20% wt / wt, 0.4 g) was added, and the reaction continued to be purged with N. The reaction was then evacuated under vacuum and filled with H three times. The reaction was then stirred under an H atmosphere at room temperature overnight. TLC indicated the reaction was complete. The slurry was filtered through Celite, and the Celite was rinsed several times with EtOAc. The combined organic layers were then concentrated under vacuum to give 6-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-octyldecanoyloxy)hexoxy]propoxy]hexyl 2-octyldecanoate (1.38 g, 94.0% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 4.15-4.02(m,4H),3.75-3.40(m,25H),2.35-2.26(m,2H),1.68-1.53(m,1 2H),1.46-1.34(m,12H),1.27(d,J=16.3Hz,48H),0.88(t,J=6.7Hz,12H).

[0632] Synthesis of compound XIV To a solution of 6-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[6-(2-octyldecanoyloxy)hexoxy]propoxy]hexyl 2-octyldecanoate (1.0 g, 1.00 mmol) in dry DCM (20 mL) was added 1-methylpiperidine-4-carboxylic acid (0.21 g, 1.50 mmol), DIPEA (0.39 g, 3.00 mmol), and DMAP (0.02 g, 0.20 mmol) at room temperature. The mixture was then cooled to 0° C., and EDCI (0.29 g, 1.50 mmol) was added portionwise over 15 minutes. The reaction was stirred at room temperature for 17 hours. TLC indicated the reaction was complete. The reaction was poured into water and extracted with DCM. The water was extracted once more with DCM. The combined organic phase was washed with brine, dried over NaSO, concentrated in vacuo, and then purified by column chromatography using CHOH (0–10%) in DCM (8%) to give 2-[2-[2-[2-[2,3-bis[6-(2-octyldecanoyloxy)hexoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (476 mg, 42.3% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 4.27-4.20(m,2H),4.10-4.02(m,4H),3.71-3.39(m,23H),2.82(d,J=11.6Hz,2H),2.32-2.26(m,5H),2.13(s,2H),2.0 2-1.90(m,3H),1.84-1.73(m,2H),1.68-1.52(m,12H),1.49-1.34(m,12H),1.32-1.21(m,48H),0.88(t,J=6.8Hz,12H).

[0633] Example 10: Synthesis of 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpyrrolidine-3-carboxylate (Compound XV) [ka] Compound XV is prepared according to the synthetic scheme in FIG.

[0634] Synthesis of the following compounds [ka] To a mixture of 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and EtN (35.6 g, 352 mmol) in DCM (400 mL) was added methanesulfonyl chloride (30.2 g, 264 mmol) for 0.5 min. o C. The mixture was stirred overnight at room temperature. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The mixture was shaken, the layers were separated, and the organic layer was collected. The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was then removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 172 mmol, 97.5% yield) as an orange oil. 1 H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.56(s,2H),4.40-4.33(m,2H),3.78-3.72(m,2H),3.69-3.60(m,12H),3.06(s,3H).

[0635] Synthesis of EXP-21-IY5625-2 [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.6 g, 177 mmol) in THF (500 mL) was added NaH (14.1 g, 354 mmol), and the mixture was stirred for 30 min at 80 oThe reaction was heated to 80°C. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 177 mmol) was added under nitrogen, and the reaction was heated at 80°C for 24 hours. TLC showed that the starting material had been consumed. The reaction was quenched with water (300 mL) and extracted with ethyl acetate (600 mL). The aqueous layer was extracted again with ethyl acetate (EA) (600 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 116 mmol, 65.6% yield) as a pale yellow oil. LCMS: Peak at 2.154 m / z MS (ESI) m / z = 421.4 (M+23) + Find 1 H NMR(500MHz,CDCl3)δ 7.36-7.26(m,5H),4.57(s,2H),4.32-4.22(m,1H),4.07-4.01(m,1H),3.75-3.70(m,1H ),3.69-3.60(m,16H),3.59-3.55(m,1H),3.51-3.47(m,1H),1.42(s,3H),1.35(s,3H).

[0636] Synthesis of EXP-21-IY5625-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 122 mmol) in AcOH (200 mL) and HO (200 mL) was stirred at room temperature for 18 h. TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxy)ethoxy]ethoxy]ethyl methanesulfonate (43.7 g, 116 mmol, quantitative) was obtained as a pale yellow oil and used without further purification. 1H NMR(400MHz,CDCl3)δ 7.36-7.23(m,5H),4.56(s,2H),3.89-3.80(m,1H),3.72-3.49(m,21H).

[0637] Synthesis of EXP-21-IY5625-4 [ka] To a solution of 3-[2-[2-[2-(2-benzyloxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen, NaH (9.64 g, 241 mmol) was added, and the mixture was heated at 80° C. for 15 minutes. The reaction was then cooled to room temperature, and 9-bromon-1-ene (31.9 g, 151 mmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 minutes and then at 80° C. for 18 hours. TLC (EA / PE=1 / 1, Rf: 0.5) showed the formation of a new spot. The reaction was quenched with water (50 mL) and then partitioned between ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20%–50% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol, 32.6% yield) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.36-7.27(m,5H),5.86-5.75(m,2H),5.03-4.89(m,4H),4.57(s,2H),3.69-3.61(m ,17H),3.58-3.40(m,9H),2.08-1.99(m,4H),1.61-1.51(m,4H),1.42-1.27(m,16H).

[0638] Synthesis of EXP-21-IY5625-5 [ka] To a solution of 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol) in MeCN (120 mL), CCl (120 mL), and water (120 mL) was added NaIO (35.5 g, 166 mmol) and RuCl (935 mg, 4.15 mmol). The reaction mixture was stirred at room temperature for 18 h. LCMS indicated the title compound was the major product along with a partial monoaldehyde product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aqueous HCl (400 mL). The organic layer was washed with NaSO solution, then dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (13.3 g, 8.28 mmol, 39.9% yield) as a pale green oil, which was used without further purification. 1H NMR(500MHz,CDCl3)δ 9.79-9.72(m,1H),7.36-7.27(m,5H),4.57(s,2H),3.72-3.37(m,26H),2 .45-2.39(m,1H),2.32(t,J=7.3Hz,2H),1.68-1.49(m,8H),1.32(s,12H).

[0639] 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-oxooctoxy)propoxy]octanoic acid (13.3 g, 12.7 mmol, 60% purity) was dissolved in t-BuOH:NaH2PO4.2H2O (7.64 g, 63.7 mmol), 2-methyl-2-butene (60 mL), and H2O (3:1, 100 mL) containing sodium chlorite (7.2 g, 63.7 mmol). The reaction was stirred at room temperature for 2 hours, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined extracts were dried over sodium sulfate. The residue was purified by flash chromatography eluting with 0% to 5% CHOH in DCM to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 9.83 mmol, 77.2% yield) and the partial oxidation product (2.78 g) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.37-7.27(m,5H),4.57(s,2H),3.69-3.61(m,16H),3.58-3.40(m,10H),2.38-2.25(m,4H),1.68-1.48(m,8H),1.34(d,J=14.9Hz,12H).

[0640] Synthesis of EXP-21-IY5625-6 [ka] A mixture of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 10.3 mmol), heptadecan-9-ol (7.96 g, 31 mmol), DMAP (506 mg, 4.14 mmol), EDCI HCl (5.16 g, 26.9 mmol), and DIEA (8.02 g, 62.1 mmol) in dry DCM (100 mL). The mixture was stirred at room temperature for 16 hours. DCM (500 mL) was added to the mixture, which was washed with 1 N HCl, NaCl, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with CHOH in DCM (0–4%) to give 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.05 mmol, 30.5% yield) as a pale yellow oil. 1 H NMR(400MHz,CDCl3)δ 7.36-7.27(m,5H),4.90-4.81(m,2H),4.57(s,2H),3.71-3.61(m,16H),3.59-3.39(m,9H) ,2.27(t,J=7.1Hz,4H),1.62-1.46(m,16H),1.28(d,J=22.7Hz,60H),0.91-0.84(m,12H).

[0641] Synthesis of EXP-21-IY5625-7 [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.22 mmol) in ethyl acetate (50 mL) was added Pd / C (1.1 g, 30% w / w). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (CHOH / DCM (3%)) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.187 g, 3.1 mmol, 96.3% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ 4.91-4.80(m,2H),3.75-3.71(m,2H),3.70-3.39(m,23H),2.27(t,J=7 .5Hz,4H),1.67-1.45(m,16H),1.35-1.20(m,60H),0.92-0.81(m,12H).

[0642] Synthesis of compound XV To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.500 g, 0.486 mmol) and 1-methylpyrrolidine-3-carboxylic acid (0.188 g, 1.46 mmol) in dry dichloromethane (15 mL) was added DIPEA (0.188 g, 1.46 mmol) and DMAP (0.018 g, 0.146 mmol). oTo the reaction mixture was added EDCI (0.279 g, 1.46 mmol). The reaction was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over NaSO. The organic layer was filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (DCM (4%) with 0-5% CHOH) to give 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpyrrolidine-3-carboxylate (0.3149 g, 0.271 mmol, 55.7% yield). LCMS: Peak MS (ESI) m / z = 1141.9 (M+H) + found at 2.54. 1H NMR(400MHz,CDCl3)δ 4.92-4.80(m,2H),4.29-4.22(m,2H),3.75-3.37(m,23H),3.18-3.06(m,1H),2.92(s,1H),2.78-2.51(m,3H),2 .41(s,3H),2.30-2.24(m,4H),2.20-2.09(m,2H),1.64-1.45(m,16H),1.35-1.20(m,60H),0.93-0.83(m,12H).

[0643] Example 11: Synthesis of 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-3-carboxylate (Compound XVI) [ka] Compound XVI is prepared according to the synthetic scheme in FIG.

[0644] Synthesis of EXP-21-IY5625-1 [ka] To a mixture of 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and EtN (35.6 g, 352 mmol) in DCM (400 mL) was added methanesulfonyl chloride (30.2 g, 264 mmol) for 0.5 min. o C. The mixture was stirred overnight at room temperature. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The mixture was shaken, the layers were separated, and the organic layer was collected. The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was then removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 172 mmol, 97.5% yield) as an orange oil. 1 H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.56(s,2H),4.40-4.33(m,2H),3.78-3.72(m,2H),3.69-3.60(m,12H),3.06(s,3H)

[0645] Synthesis of EXP-21-IY5625-2 [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.6 g, 177 mmol) in THF (500 mL) was added NaH (14.1 g, 354 mmol), and the mixture was heated at 80°C for 30 min. o The reaction was then heated to C. The reaction was then cooled to room temperature and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethylmethane was added.

[0646] The sulfonate (64.1 g, 177 mmol) was added under nitrogen, and the reaction was heated at 80 °C for 24 h. TLC indicated that the starting material had been consumed. The reaction was quenched with water (300 mL) and extracted with ethyl acetate (600 mL). The aqueous layer was extracted again with ethyl acetate (EA) (600 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 116 mmol, 65.6% yield) as a pale yellow oil. -LCMS: Peak at 2.154 m / z MS (ESI) m / z = 421.4 (M+23) + Find. 1 H NMR(500MHz,CDCl3)δ 7.36-7.26(m,5H),4.57(s,2H),4.32-4.22(m,1H),4.07-4.01(m,1H),3.75-3.70(m,1H ),3.69-3.60(m,16H),3.59-3.55(m,1H),3.51-3.47(m,1H),1.42(s,3H),1.35(s,3H).

[0647] Synthesis of EXP-21-IY5625-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 122 mmol) in AcOH (200 mL) and HO (200 mL) was stirred at room temperature for 18 h. TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxy)ethoxy]ethoxy]ethyl methanesulfonate (43.7 g, 116 mmol, quantitative) was obtained as a pale yellow oil and used without further purification. 1H NMR(400MHz,CDCl3)δ 7.36-7.23(m,5H),4.56(s,2H),3.89-3.80(m,1H),3.72-3.49(m,21H).

[0648] Synthesis of EXP-21-IY5625-4 [ka] To a solution of 3-[2-[2-[2-(2-benzyloxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen, NaH (9.64 g, 241 mmol) was added, and the mixture was heated at 80° C. for 15 minutes. The reaction was then cooled to room temperature, and 9-bromon-1-ene (31.9 g, 151 mmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 minutes and then at 80° C. for 18 hours. TLC (EA / PE=1 / 1, Rf: 0.5) showed the formation of a new spot. The reaction was quenched with water (50 mL) and then partitioned between ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20%–50% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol, 32.6% yield) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.36-7.27(m,5H),5.86-5.75(m,2H),5.03-4.89(m,4H),4.57(s,2H),3.69-3.61(m ,17H),3.58-3.40(m,9H),2.08-1.99(m,4H),1.61-1.51(m,4H),1.42-1.27(m,16H).

[0649] Synthesis of EXP-21-IY5625-5 [ka] To a solution of 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol) in MeCN (120 mL), CCl (120 mL), and water (120 mL) was added NaIO (35.5 g, 166 mmol) and RuCl (935 mg, 4.15 mmol). The reaction mixture was stirred at room temperature for 18 h. LCMS indicated the title compound was the major product along with a partial monoaldehyde product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aqueous HCl (400 mL). The organic layer was washed with NaSO solution, then dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (13.3 g, 8.28 mmol, 39.9% yield) as a pale green oil, which was used without further purification. 1H NMR(500MHz,CDCl3)δ 9.79-9.72(m,1H),7.36-7.27(m,5H),4.57(s,2H),3.72-3.37(m,26H),2 .45-2.39(m,1H),2.32(t,J=7.3Hz,2H),1.68-1.49(m,8H),1.32(s,12H).

[0650] 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-oxooctoxy)propoxy]octanoic acid (13.3 g, 12.7 mmol, 60% purity) was dissolved in t-BuOH:NaH2PO4.2H2O (7.64 g, 63.7 mmol), 2-methyl-2-butene (60 mL), and H2O (3:1, 100 mL) containing sodium chlorite (7.2 g, 63.7 mmol). The reaction was stirred at room temperature for 2 h, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined extracts were dried over sodium sulfate. The residue was purified by flash chromatography eluting with 0% to 5% methanol in DCM to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 9.83 mmol, 77.2% yield) and the partial oxidation product (2.78 g) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.37-7.27(m,5H),4.57(s,2H),3.69-3.61(m,16H),3.58-3.40(m,10H),2.38-2.25(m,4H),1.68-1.48(m,8H),1.34(d,J=14.9Hz,12H).

[0651] Synthesis of EXP-21-IY5625-6 [ka] A mixture of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 10.3 mmol), heptadecan-9-ol (7.96 g, 31 mmol), DMAP (506 mg, 4.14 mmol), EDCI HCl (5.16 g, 26.9 mmol), and diisopropylethylamine (DIEA) (8.02 g, 62.1 mmol) in dry DCM (100 mL) was stirred at room temperature for 16 hours. DCM (500 mL) was added to the mixture, which was washed with 1N HCl, NaCl, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with CHOH in DCM (0–4%) to give 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.05 mmol, 30.5% yield) as a pale yellow oil. 1 H NMR(400MHz,CDCl3)δ 7.36-7.27(m,5H),4.90-4.81(m,2H),4.57(s,2H),3.71-3.61(m,16H),3.59-3.39(m,9H) ,2.27(t,J=7.1Hz,4H),1.62-1.46(m,16H),1.28(d,J=22.7Hz,60H),0.91-0.84(m,12H).

[0652] Synthesis of EXP-21-IY5625-7 [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.22 mmol) in ethyl acetate (50 mL) was added Pd / C (1.1 g, 30% w / w). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (CHOH / DCM (3%)) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.187 g, 3.1 mmol, 96.3% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ 4.91-4.80(m,2H),3.75-3.71(m,2H),3.70-3.39(m,23H),2.27(t,J=7 .5Hz,4H),1.67-1.45(m,16H),1.35-1.20(m,60H),0.92-0.81(m,12H).

[0653] Synthesis of Compound XVI To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.500 g, 0.486 mmol) and 1-methylpyrrolidine-3-carboxylic acid (0.297 g, 1.46 mmol) in dry dichloromethane (15 mL) was added DIPEA (0.377 g, 2.97 mmol) and DMAP (0.018 g, 0.146 mmol). EDCI (0.279 g, 1.46 mmol) was then added at 0° C. The reaction was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (0–5% CH3OH in DCM (4%)) to give 2-[2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1,4-dimethylpiperidine-4-carboxylate (0.4171 g, 0.350 mmol, 72.0% yield) as a pale yellow oil. LCMS: Peak MS (ESI) m / z = 1169.1 (M+H) at 2.3 minutes + Find. 1 H NMR(400MHz,CDCl3)δ 4.91-4.81(m,2H),4.31-4.25(m,2H),3.72-3.39(m,23H),2.78(s,2H),2.36(s,3H) ),2.32-2.14(m,8H),1.64-1.47(m,16H),1.34-1.20(m,65H),0.92-0.84(m,12H).

[0654] Example 12: Synthesis of 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1,3-dimethylpyrrolidine-3-carboxylate (Compound XVII) [ka] Compound XVII is prepared according to the synthetic scheme in FIG.

[0655] Synthesis of EXP-21-IY5627 [ka] To a mixture of 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and EtN (35.6 g, 352 mmol) in DCM (400 mL) was added methanesulfonyl chloride (30.2 g, 264 mmol) for 0.5 min. o C. The mixture was stirred overnight at room temperature. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The mixture was shaken, the layers were separated, and the organic layer was collected. The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was then removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 172 mmol, 97.5% yield) as an orange oil. 1 H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.56(s,2H),4.40-4.33(m,2H),3.78-3.72(m,2H),3.69-3.60(m,12H),3.06(s,3H).

[0656] Synthesis of EXP-21-IY5625-2 [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.6 g, 177 mmol) in THF (500 mL) was added NaH (14.1 g, 354 mmol), and the mixture was stirred for 30 min at 80 oThe reaction was heated to 80°C. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 177 mmol) was added under nitrogen, and the reaction was heated at 80°C for 24 hours. TLC showed that the starting material had been consumed. The reaction was quenched with water (300 mL) and extracted with ethyl acetate (600 mL). The aqueous layer was extracted again with ethyl acetate (EA) (600 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 116 mmol, 65.6% yield) as a pale yellow oil. LCMS: Peak at 2.154 m / z MS (ESI) m / z = 421.4 (M+23) + Find. 1 H NMR(500MHz,CDCl3)δ 7.36-7.26(m,5H),4.57(s,2H),4.32-4.22(m,1H),4.07-4.01(m,1H),3.75-3.70(m,1H ),3.69-3.60(m,16H),3.59-3.55(m,1H),3.51-3.47(m,1H),1.42(s,3H),1.35(s,3H).

[0657] Synthesis of EXP-21-IY5625-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 122 mmol) in AcOH (200 mL) and HO (200 mL) was stirred at room temperature for 18 h. TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxy)ethoxy]ethoxy]ethyl methanesulfonate (43.7 g, 116 mmol, quantitative) was obtained as a pale yellow oil and used without further purification. 1H NMR(400MHz,CDCl3)δ 7.36-7.23(m,5H),4.56(s,2H),3.89-3.80(m,1H),3.72-3.49(m,21H).

[0658] Synthesis of EXP-21-IY5625-4 [ka] To a solution of 3-[2-[2-[2-(2-benzyloxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen, NaH (9.64 g, 241 mmol) was added, and the mixture was heated at 80° C. for 15 minutes. The reaction was then cooled to room temperature, and 9-bromon-1-ene (31.9 g, 151 mmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 minutes and then at 80° C. for 18 hours. TLC (EA / PE=1 / 1, Rf: 0.5) showed the formation of a new spot. The reaction was quenched with water (50 mL) and then partitioned between ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20%–50% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol, 32.6% yield) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.36-7.27(m,5H),5.86-5.75(m,2H),5.03-4.89(m,4H),4.57(s,2H),3.69-3.61(m ,17H),3.58-3.40(m,9H),2.08-1.99(m,4H),1.61-1.51(m,4H),1.42-1.27(m,16H).

[0659] Synthesis of EXP-21-IY5625-5 [ka] To a solution of 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (13.24 g, 20.7 mmol) in MeCN (120 mL), CCl (120 mL), and water (120 mL) was added NaIO (35.5 g, 166 mmol) and RuCl (935 mg, 4.15 mmol). The reaction mixture was stirred at room temperature for 18 h. LCMS indicated the title compound was the major product along with a partial monoaldehyde product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aqueous HCl (400 mL). The organic layer was washed with NaSO solution, then dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (13.3 g, 8.28 mmol, 39.9% yield) as a pale green oil, which was used without further purification. 1 H NMR(500MHz,CDCl3)δ 9.79-9.72(m,1H),7.36-7.27(m,5H),4.57(s,2H),3.72-3.37(m,26H),2 .45-2.39(m,1H),2.32(t,J=7.3Hz,2H),1.68-1.49(m,8H),1.32(s,12H).

[0660] 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-oxooctoxy)propoxy]octanoic acid (13.3 g, 12.7 mmol, 60% purity) was dissolved in t-BuOH:NaH2PO4.2H2O (7.64 g, 63.7 mmol), 2-methyl-2-butene (60 mL), and H2O (3:1, 100 mL) containing sodium chlorite (7.2 g, 63.7 mmol). The reaction was stirred at room temperature for 2 h, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined extracts were dried over sodium sulfate. The residue was purified by flash chromatography eluting with 0% to 5% CHOH in DCM to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 9.83 mmol, 77.2% yield) and the partial oxidation product (2.78 g) as a pale yellow oil. 1 H NMR(500MHz,CDCl3)δ 7.37-7.27(m,5H),4.57(s,2H),3.69-3.61(m,16H),3.58-3.40(m,10H),2.38-2.25(m,4H),1.68-1.48(m,8H),1.34(d,J=14.9Hz,12H).

[0661] Synthesis of EXP-21-IY5625-6 [ka] A mixture of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (6.65 g, 10.3 mmol), heptadecan-9-ol (7.96 g, 31 mmol), DMAP (506 mg, 4.14 mmol), EDCI HCl (5.16 g, 26.9 mmol), and DIEA (8.02 g, 62.1 mmol) in dry DCM (100 mL). The mixture was stirred at room temperature for 16 hours. DCM (500 mL) was added to the mixture, which was washed with 1 N HCl, NaCl, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with CHOH in DCM (0–4%) to give 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.05 mmol, 30.5% yield) as a pale yellow oil. 1 H NMR(400MHz,CDCl3)δ 7.36-7.27(m,5H),4.90-4.81(m,2H),4.57(s,2H),3.71-3.61(m,16H),3.59-3.39(m,9H) ,2.27(t,J=7.1Hz,4H),1.62-1.46(m,16H),1.28(d,J=22.7Hz,60H),0.91-0.84(m,12H).

[0662] Synthesis of EXP-21-IY5625-7 [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.6 g, 3.22 mmol) in ethyl acetate (50 mL) was added Pd / C (1.1 g, 30% w / w). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (CHOH / DCM (3%)) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.187 g, 3.1 mmol, 96.3% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ 4.91-4.80(m,2H),3.75-3.71(m,2H),3.70-3.39(m,23H),2.27(t,J=7 .5Hz,4H),1.67-1.45(m,16H),1.35-1.20(m,60H),0.92-0.81(m,12H).

[0663] Synthesis of Compound XVI To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.500 g, 0.486 mmol) and 1,3-dimethylpyrrolidine-3-carboxylic acid (0.209 g, 1.46 mmol) in dry dichloromethane (15 mL) was added DIPEA (0.188 g, 1.46 mmol) and DMAP (0.018 g, 0.146 mmol). oTo the reaction mixture was added EDCI (0.279 g, 1.46 mmol) at 37°C. The reaction was stirred at room temperature for 18 h. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (0-5% CH3OH in DCM (4%)) to give 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1,3-dimethylpyrrolidine-3-carboxylate (0.2761 g, 0.230 mmol, 47.3% yield) as a pale yellow oil. LCMSA; Peak MS (ESI) m / z = 1155.9 (M+H) at 4.60 min + Find. 1 H NMR(500MHz,CDCl3)δ 4.91-4.82(m,2H),4.31-4.22(m,2H),3.70(t,J=4.9Hz,2H),3.64(d,J=6.9Hz,12H),3.60-3.39(m,9H),3.03(d,J=8.2Hz,1H),2.66(t,J=25 .5Hz,2H),2.55-2.41(m,2H),2.38(s,3H),2.30-2.23(m,4H),1.64-1.47(m,17H),1.38(s,3H),1.34-1.21(m,60H),0.88(t,J=6.9Hz,12H).

[0664] Example 13: Synthesis of 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-3-carboxylate (Compound XVIII) [ka] Compound XVIII is prepared from the intermediate 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate according to the synthetic scheme in FIG. 12.

[0665] Synthesis of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (3.6 g, 3.22 mmol) in ethyl acetate (50 mL) was added Pd / C (1.1 g, 30% w / w). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (CHOH / DCM (3%)) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (3.187 g, 3.1 mmol, 96.3% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.91-4.80(m,2H),3.75-3.71(m,2H),3.70-3.39(m,23H),2.27(t,J=7 .5Hz,4H),1.67-1.45(m,16H),1.35-1.20(m,60H),0.92-0.81(m,12H).

[0666] Synthesis of Compound XVIII To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (0.500 g, 0.486 mmol) and 1-methylpiperidine-3-carboxylic acid (0.209 g, 1.46 mmol) in dry dichloromethane (15 mL) was added DIPEA (0.188 g, 1.46 mmol) and DMAP (0.018 g, 0.146 mmol). EDCI (0.279 g, 1.46 mmol) was then added at 0° C. The reaction was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated in vacuo. The residue was purified by silica gel chromatography (0–5% CH3OH in DCM (4%)) to give 2-[2-[2-[2-[2,3-bis[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-3-carboxylate (0.3595 g, 0.299 mmol, 61.5% yield) as a colorless oil. At 3.88 min, a peak MS(ESI) m / z=1155.0 (M+H)+ is found. 1H NMR(500MHz,CDCl3)δ 4.91-4.81(m,2H),4.29-4.19(m,2H),3.74-3.60(m,14H),3.59-3.36(m, 9H),2.96(d,J=9.7Hz,1H),2.68(d,J=36.1Hz,2H),2.34-2.22(m,7H),2. 17(d,J=10.7Hz,1H),2.03-1.92(m,2H),1.77-1.71(m,1H),1.65-1.45(m ,17H),1.42(d,J=6.7Hz,1H),1.34-1.22(m,60H),0.88(t,J=6.9Hz,12H).

[0667] Example 14: Synthesis of 2-[2-[2-[2-[2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (Compound XIX) [ka] Compound XIX is prepared according to the synthetic scheme in FIG.

[0668] Synthesis of IY5616-SM1 [ka] To a solution of 6-bromohexan-1-ol (18 g, 0.10 mol) and 3,4-dihydro-2H-pyran (8.61 g, 0.1 mol) in DCM (500 mL), pyridinium p-toluenesulfonate (PPTS) (2.75 g, 0.01 mol) was added and stirred at room temperature for 3 h. TLC (EA / PE 1 / 9, SM Rf: 0.2; product Rf: 0.7) indicated that all starting material had been consumed. The solvent was concentrated and purified by flash chromatography column (0-10% EA in PE (5%)) to give 2-(6-bromohexoxy)tetrahydropyran (20.3 g, 77.0% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ 4.57(t,J=3.6Hz,1H),3.92-3.82(m,1H),3.79-3.69(m,1H),3.56-3.46(m,1H) ,3.46-3.34(m,3H),1.94-1.77(m,3H),1.75-1.69(m,1H),1.66-1.35(m,10H).

[0669] Synthesis of IY5616-1 [ka] 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (10 g, 35.2 mmol) and triethylamine (7.12 g, 70.3 mmol) in dry dichloromethane (100 mL) at 0 °C. Methanesulfonyl chloride (6.04 g, 52.8 mmol) in dry DCM (10 mL) was added dropwise to this solution at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. Triethylamine hydrochloride was filtered off, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (12.8 g, quantitative) as a pale yellow oil, which was used without further purification. 1H NMR(400MHz,CDCl3)δ 7.42-7.27(m,5H),4.57(s,2H),4.41-4.31(m,2H),3.79-3.72(m,2H),3.70-3.60(m,12H),3.05(d,J=9.8Hz,3H).

[0670] Synthesis of IY5616-2 [ka] Bromomethylbenzene (12.8 g, 35.5 mmol) in THF (100 ml) was dissolved in NaH (4.24 g, 0.106 mol) in THF (50 ml) for 0 o The mixture was added at 0 o The mixture was stirred at RT for 30 min. (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (4.67 g, 35.3 mmol) was added to the mixture. o Stir at 75°C for 1 hour, then o The mixture was heated to 50°C for 18 hours. The mixture was quenched with water and extracted with ethyl acetate (EA) (500 ml). The organics were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography column (5% to 10% (8%) MeOH in DCM) to give 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (10.64 g, 75.6% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.37-7.23(m,5H),4.56(s,2H),4.31-4.21(m,1H),4.08-4.00(m,1H),3.75-3.70(m,1H),3.69 -3.61(m,16H),3.60-3.54(m,1H),3.53-3.46(m,1H),1.45-1.40(m,3H),1.36(d,J=7.7Hz,3H).

[0671] Synthesis of IY5616-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (10.6 g, 26.7 mmol) in AcOH (50 mL) and water (50 mL) was prepared. The mixture was stirred at ambient temperature for 16 h. TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (10.0 g, quantitative) was obtained as a pale yellow oil, which was used without further purification. 1H NMR(400MHz,CDCl3)δ 7.34-7.13(m,5H),4.56(s,2H),3.89-3.81(m,1H),3.68-3.52(m,20H).

[0672] Synthesis of IY5616-4 [ka] To a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (10.0 g, 27.9 mmol) in dry DMF (120 mL) was added NaH (5.58 g, 139 mmol) for 0.5 min. oC several times, and then the mixture was heated to 80°C for 30 minutes. The reaction was then cooled to room temperature, and 2-(6-bromohexoxy)tetrahydropyran (18.5 g, 69.7 mmol) in dry DMF (30 mL) was added under nitrogen, and the reaction was heated at 80°C for 18 hours. TLC showed that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with DCM containing 0–5% (1%) methanol to give 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexoxy)ethoxy]hexoxy]tetrahydropyran (8.0 g, 39.4% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.37-7.24(m,5H),4.57(s,4H),3.90-3.83(m,2H),3.78-3.33(m,31H),1.79-1.67(m,3H),1.62-1.50(m,17H),1.41-1.34(m,8H).

[0673] Synthesis of IY5616-5 [ka] To a solution of 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexoxy)ethoxy]hexoxy]tetrahydropyran (8.0 g, 11.0 mmol) in EtOH (120 mL) was added p-toluenesulfonic acid (2.1 g, 12.1 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 24 h. TLC (4% CH3OH in DCM) showed complete disappearance of the starting material. The reaction was quenched with dilute sodium bicarbonate solution (150 mL), and the solvent was extracted with EA (2 × 100 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 10% CHOH (8%) in DCM to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol (3.23 g, 52.5% yield) as a pale yellow oil. 1H NMR(500MHz,CDCl3)δ 7.40-7.20(m,5H),4.57(s,2H),3.72-3.38(m,29H),1.87(s,2H),1.63-1.47(m,8H),1.42-1.28(m,8H).

[0674] Synthesis of IY5616-6 [ka] To a solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol (1.6 g, 2.86 mmol) in dry DCM (20 mL) was added 2-hexyldecanoic acid (2.2 g, 8.59 mmol), DIPEA (2.22 g, 17.2 mmol), and DMAP (0.14 g, 1.15 mmol) at room temperature. The mixture was then cooled to 0° C., and EDCI (1.43 g, 7.45 mmol) was added portionwise over 15 minutes. The reaction was stirred at room temperature for 17 hours. TLC (5% CH3OH in DCM) indicated the reaction was complete. The reaction was poured into water and extracted with DCM. The water was extracted once more with DCM. The combined organic layers were washed with brine, dried over NaSO, and concentrated in vacuo. The residue was purified by column chromatography using CHOH (0–4%) in DCM (3%) to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-hexyldecanoyloxy)hexoxy]propoxy]hexyl 2-hexyldecanoate (2.9 g, 97.8% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),4.09-4.03(m,4H),3.69-3.40(m,25H),2. 34-2.27(m,2H),1.63-1.34(m,24H),1.25(s,40H),0.87(t,J=6.5Hz,12H).

[0675] Synthesis of IY5616-7 [ka] A solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-hexyldecanoyloxy)hexoxy]propoxy]hexyl 2-hexyldecanoate (2.90 g, 2.80 mmol) in EtOAc (30 mL) was purged with N for 10 minutes, followed by the addition of Pd / C (20% wt / wt, 0.6 g), and the reaction continued to be purged with N. The reaction was then evacuated under vacuum and filled with H three times. The reaction was then stirred overnight at room temperature under an H atmosphere. TLC indicated the reaction was complete. The slurry was filtered through Celite, and the Celite was rinsed several times with EtOAc. The combined organic layers were then concentrated under vacuum to give 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-hexyldecanoate (1.7 g, 64.2% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 7.38-7.27(m,5H),4.57(s,2H),4.09-4.03(m,4H),3.69-3.40(m,25H),2. 34-2.27(m,2H),1.63-1.34(m,24H),1.25(s,40H),0.87(t,J=6.5Hz,12H).

[0676] Synthesis of compound XIX To a solution of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-hexyldecanoate (1.0 g, 1.06 mmol) in dry DCM (20 mL) was added 1-methylpiperidine-4-carboxylic acid (0.23 g, 1.59 mmol), DIPEA (0.41 g, 3.17 mmol), and DMAP (0.03 g, 0.21 mmol) at room temperature. The mixture was then cooled to 0° C., and EDCI (0.30 g, 1.59 mmol) was added portionwise over 15 minutes. The reaction was stirred at room temperature for 17 hours. TLC indicated the reaction was complete. The reaction was poured into water and extracted with DCM. The water was extracted once more with DCM. The combined organic layers were washed with brine, dried over NaSO, and concentrated in vacuo, then purified by column chromatography using CHOH (0–10%) in DCM (8%) to give 2-[2-[2-[2-[2,3-bis[6-(2-hexyldecanoyloxy)hexoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl 1-methylpiperidine-4-carboxylate (614 mg, 54.2% yield) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ 4.28-4.21(m,2H),4.10-4.01(m,4H),3.72-3.61(m,14H),3.60-3.40(m,9H),2.83(d,J=11.5Hz,2H),2.33-2.28(m,5H),2. 05(s,3H),1.96-1.90(m,2H),1.84-1.76(m,2H),1.65-1.54(m,12H),1.47-1.35(m,12H),1.25(s,40H),0.90-0.85(m,12H).

[0677] Example 15: Synthesis of 1-octylnonyl 8-[3-[2-[2-[2-[2-[5-(dimethylamino)pentanoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (Compound XX) [ka] Compound XX is prepared according to the synthetic scheme in FIG.

[0678] Synthesis of EXP-21-IY5625-1 [ka] To a mixture of 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and EtN (35.6 g, 352 mmol) in DCM (400 mL) was added methanesulfonyl chloride (30.2 g, 264 mmol) for 0.5 min. o C. The mixture was stirred overnight at room temperature. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The mixture was shaken, the layers were separated, and the organic layer was collected. The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was then removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 172 mmol, 97.5% yield) as an orange oil. 1 H NMR(400MHz,CDCl3)δ 7.37-7.27(m,5H),4.56(s,2H),4.40-4.33(m,2H),3.78-3.72(m,2H),3.69-3.60(m,12H),3.06(s,3H).

[0679] Synthesis of EXP-21-IY5625-2 [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.6 g, 177 mmol) in THF (500 mL) was added NaH (14.1 g, 354 mmol), and the mixture was stirred for 30 min at 80 oThe reaction was heated to 80°C. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (64.1 g, 177 mmol) was added under nitrogen, and the reaction was heated at 80°C for 24 hours. TLC showed that the starting material had been consumed. The reaction was quenched with water (300 mL) and extracted with ethyl acetate (600 mL). The aqueous layer was extracted again with ethyl acetate (EA) (600 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 116 mmol, 65.6% yield) as a pale yellow oil. LCMS: Peak at 2.154 m / z MS (ESI) m / z = 421.4 (M+23) + Find. 1 H NMR(500MHz,CDCl3)δ 7.36-7.26(m,5H),4.57(s,2H),4.32-4.22(m,1H),4.07-4.01(m,1H),3.75-3.70(m,1H ),3.69-3.60(m,16H),3.59-3.55(m,1H),3.51-3.47(m,1H),1.42(s,3H),1.35(s,3H).

[0680] Synthesis of EXP-21-IY5625-3 [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (48.635 g, 122 mmol) in AcOH (200 mL) and HO (200 mL) was stirred at room temperature for 18 h. TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxy)ethoxy]ethoxy]ethyl methanesulfonate (43.7 g, 116 mmol, quantitative) was obtained as a pale yellow oil and used without further purification. 1 H NMR(400MHz,CDCl3)δ 7.36-7.23(m,5H),4.56(s,2H...

Claims

1. Lipid compounds of formula (I): A-(CH 2 ) n -CX-B-Z-R1(I) wherein - R1 is C 10 ~C 57 R represents an optionally substituted, branched or unbranched, linear, saturated or unsaturated, C 10 ~C 55 with a hydrocarbon skeleton optionally interrupted by one or several atoms of oxygen or nitrogen and / or one or several moieties -(C=O)-, -O-(C=O)- or -(C=O)-O-, and one nitrogen atom, if present in said skeleton, may be linked directly or indirectly to a Z radical, Z is a spacer arm having 2 to 24, for example 2 to 18, for example 4 to 12 carbon atoms in one or more unbranched, linear, saturated or unsaturated hydrocarbon chains, containing one or several atoms of oxygen and / or -S-S-; -(O-C)-; -(C-O)-O-;-O-(OC)-;-S-;-NH-, -NH-(O-C)-; said chain interrupted by a moiety selected from among -(O-C)-NH- and -NH-(C-O)-O-, such as -(C-O)-O-; -O-(O-C)- and -NH-(C-O)-O-, and optionally having an oxygen atom or a moiety selected from among -NH-(O-C)-*-O-(O-C)-*; -(C-O)-O-*; and -(O-C)-, is connected at its end to said hydrophobic tail group, * representing a single bond connecting said moiety to said hydrophobic tail group; B represents an oxygen atom or an —NH— group; X is an oxygen atom or a sulfur atom, n is 0, 1, 2, 3, 4, 5 or 6, - A is, R2 and R3 are independently linear or branched (C 1 ~C 6 ) an R2R3N- group, representing an alkyl group; Y is an oxygen atom or a nitrogen atom, and Alk is C 2 ~C 6 and R2 and R3 are independently linear or branched (C 1 ~C 6 ) an NR2R3-Alk-Y- group, representing an alkyl group; - a 4-8 membered saturated heterocyclic radical containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms, said 4-8 membered saturated heterocyclic radical being separated by a carbon or nitrogen atom; a 4- to 8-membered saturated heterocyclic radical linked to the remainder of the molecule and optionally substituted by 1 to 4 substituents, independently of one another, selected from straight-chain or branched (C1-C6) alkyl groups represents a group selected from the group consisting of A lipid compound of formula (I), or one of its pharmaceutically acceptable salts; a compound in all possible racemic, enantiomeric and diastereomeric isomeric forms.

2. 10. The compound of claim 1 in cationic form.

3. R1 is, Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 represents a group selected from the group consisting of The compound of claim 1.

4. Z represents a radical of the formula: -((CH 2 ) 2 -O) m -(CH 2 ) r (T) q -*(Z) During the ceremony, * denotes a single bond connecting the radical to the hydrophobic tail group; m is an integer from 1 to 12, for example from 2 to 4, for example 4, r is 0 or an integer from 1 to 4, q is zero or one, T is selected from the group consisting of: -(O-C)-; -(C-O)-O-**; -O-(O-C)-**; and -NH-(C-O)-O-**, where ** represents a single bond connecting said group to said hydrophobic tail group; The compound of claim 1.

5. The compound of claim 1 , wherein B is an oxygen atom.

6. The compound of claim 1, wherein B is an -NH- group.

7. The compound of claim 1 , wherein X is an oxygen atom.

8. 2. The compound of claim 1, wherein n is 0, 1, 2, 3, or 4.

9. A is -N(CH 3 ) 2 , -N(CH 2 -CH 2 -CH 3 ) 2 , O-(CH 2 ) 2 N (CH 3 ) 2 , N-(CH 2 ) 2 N (CH 3 ) 2 and NR2R3-Alk-Y- groups, wherein Y is an oxygen atom or a nitrogen atom, Alk is a C2-C6 alkylene moiety, and R2 and R3, independently of each other, represent a linear or branched (C1-C6) alkyl group.

10. A is a 4- to 8-membered saturated heterocyclic ring containing 3 to 7 carbon atoms and 1 or 2 nitrogen atoms.

2. The compound of claim 1, wherein the 4-8 membered saturated heterocyclic radical represents a 4-8 membered saturated heterocyclic radical linked to the remainder of the molecule by a carbon atom or a nitrogen atom and is optionally substituted by 1 to 4 substituents, independently of each other, selected from linear or branched (C1-C6) alkyl groups.

11. 2. The compound of claim 1 having an apparent pKa of less than 7 or in the range of 4.5 to 7.

12. The compound is represented by formula (II) 【Chemistry 1】 wherein Z, n and R1 are as defined in claim 1, R4 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; R12 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; p is equal to 0 or 1, for example p is equal to 0, R5, R6, R7, R8 and R9 are, independently of one another, a moiety selected from among -CH2-; -CHR12- and -NH-, and one of R5, R6, R7, R8 and R9 involved in the bond to the rest of the molecule, a moiety selected from among -CH-; -CR12- and -N-, with the proviso that only one of R5, R6, R7, R8 and R9 is -NH- or -N-, B represents an oxygen atom or an —NH— group, for example an oxygen atom, A compound of formula (II) or one of its pharmaceutically acceptable salts, said compound in all possible racemic, enantiomeric and diastereomeric isomeric forms, The compound of claim 1.

13. The compound is represented by formula (IIa) 【Chemistry 2】 wherein R1 is as defined in claim 1, n is 0, 1, 2, 3, 4, 5 or 6, for example 0 to 4, such as 0, 1 or 2; r is 0, 1, 2, 3 or 4, for example 0, 1 or 2, R4 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; R12 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; p is equal to 0 or 1, for example p is equal to 0, R5, R6, R7, R8 and R9 are, independently of one another, a moiety selected from among -CH2-; -CHR12- and -NH-, and one of R5, R6, R7, R8 and R9 involved in the bond to the rest of the molecule, a moiety selected from among -CH-; -CR12- and -N-, with the proviso that only one of R5, R6, R7, R8 and R9 is -NH- or -N-, m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, A compound of formula (IIa) or one of its pharmaceutically acceptable salts, which is said compound in all possible racemic, enantiomeric and diastereomeric isomeric forms, The compound of claim 1.

14. The compound is of formula (III) 【Transformation 3】 wherein R1 is as defined in claim 1, n is 0, 1, 2, 3, 4, 5 or 6, for example 0 to 4, such as 0, 1 or 2; m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, r is 0, 1, 2, 3 or 4, for example 0, 1 or 2, R4 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; R12 is a (C1-C5) alkyl group, for example a (C1-C4) alkyl group such as a methyl group; p is equal to 0 or 1, for example p is equal to 0, R5, R6, R7 and R8 are, independently of one another, a moiety selected from among -CH2-; -CHR12- and -NH-, and one of R5, R6, R7 and R8 involved in the bond to the rest of the molecule, a moiety selected from among -CH-; -CR12- and -N-, with the proviso that only one of R5, R6, R7 and R8 is -NH- or -N-, A compound of formula (III) or one of its pharmaceutically acceptable salts; said compound in all possible racemic, enantiomeric and diastereomeric isomeric forms, The compound of claim 1.

15. The compound is represented by formula (IV) 【Chemistry 4】 wherein R1 is as defined in claim 1, Q is a moiety selected from the group consisting of --(C=O)--O*; --O(C=O)O*; --N(C=O)O-* and --O(C=O)N-*, * being a moiety (CH 2 CH 2 O) m indicates the connection to - n is 0, 1, 2, 3, 4, 5 or 6, for example 1 to 5, for example 2, 3 or 4, - r is 0, 1, 2, 3 or 4, for example 0, 1 or 2, R10 and R11 are independently selected from the group consisting of (C 1 ~C 5 ) alkyl groups, such as methyl or propyl groups (C 1 ~C 4 ) represents an alkyl group, m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, A compound of formula (IV) or one of its pharmaceutically acceptable salts; said compound in all possible racemic, enantiomeric and diastereomeric isomeric forms, The compound of claim 1.

16. The compound is represented by formula (V) 【Transformation 5】 wherein R1 is as defined in claim 1, - n is 0, 1, 2, 3, 4, 5 or 6, for example 1 to 5, for example 2, 3 or 4, - r is 0, 1, 2, 3 or 4, for example 0, 1 or 2, R10 and R11 are independently selected from the group consisting of (C 1 ~C 5 ) alkyl groups, such as methyl or propyl groups (C 1 ~C 4 ) represents an alkyl group, m is an integer from 1 to 12, for example an integer from 2 to 6, for example 4, A compound of formula (V) or one of its pharmaceutically acceptable salts; said compound in all possible racemic, enantiomeric and diastereomeric isomeric forms, The compound of claim 1.

17. The compound is Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 or one of the pharmaceutically acceptable salts thereof; in all possible racemic, enantiomeric and diastereomeric isomeric forms, in particular the compounds (VI), (VII), (VIII), (XI), (XII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIIIII), The compound of claim 1.

18. A composition or lipid nanoparticle (LNP) comprising a lipid component comprising at least one lipid compound according to any one of claims 1 to 17.

19. 20. The composition or LNP of claim 18, wherein the lipid component further comprises at least one lipid selected from a neutral lipid, a structured lipid, and optionally a PEG-lipid.

20. 20. The composition or LNP of claim 19, wherein the neutral lipid is selected from the group consisting of phosphatidylcholines, such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines, such as DOPE, DPPE, DMPE, DSPE, DLPE; DEPE; DPPS; DOPG; sphingomyelin; and ceramide; and mixtures thereof.

21. 20. The composition or LNP of claim 19, wherein the structured lipid is selected from the group consisting of a sterol or ester thereof, tomatine, alpha-tocopherol, and a corticosteroid, and mixtures thereof.

22. 22. The composition or LNP of claim 21, wherein the sterol or ester thereof is selected from the group consisting of cholesterol and its derivatives, ergosterol, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, diosgenin, sitosterol, sitostanol, campesterol, fecosterol, brassicasterol, tomatidine, ursolic acid, 24-methylenecholesterol, cholesteryl margallate, cholesteryl oleate, and cholesteryl stearate, and mixtures thereof.

23. 20. The composition or LNP of claim 19, wherein the PEG-lipid is selected from the group consisting of PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, DSPC-PEG, DSPE-PEG, PEG-cer, mPEG-N,N-ditetradecylacetamide, PEG-dialkyloxypropylcarbamate, and mixtures thereof.

24. The composition or LNP of claim 19, comprising, relative to the total molar amount of the lipid components, at least about 30% to about 70% of a lipid compound, about 0% to about 50% of a neutral lipid, about 20% to about 50% of a structured lipid, and about 1% to about 15% of a PEG-lipid.

25. 20. The composition or LNP of claim 19, further comprising at least one biologically active agent.

26. 26. The composition or LNP of claim 25, wherein the biologically active agent is a nucleic acid.

27. 27. The composition or LNP of claim 26, wherein the nucleic acid encodes at least one antigen.

28. 27. A pharmaceutical composition comprising at least one composition or LNP of claim 26 and a pharmaceutically acceptable excipient.

29. 29. An immunogenic composition comprising at least one composition or LNP of claim 28.

30. 27. The composition or LNP of claim 26 for use as a pharmaceutical.

31. 27. The composition or LNP of claim 26 for use in a method for preventing and / or treating a disease selected from the group consisting of an infectious disease, an allergy, an autoimmune disease, a blood disorder, a metabolic disease, a neurological disease, and a cancer disease.

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