Modified lipid compositions and uses thereof

Abstract

Disclosed herein is a modified lipid composition comprising (a) a structural component comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol, and (b) modified with an ionizable lipid. The disclosure also includes a method for producing the modified lipid composition, comprising (a) reconstituting a structural component comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and / or cholesterol in the presence of (b) an ionizable lipid to produce the modified lipid composition, and loading the modified lipid composition with one or more heterologous functional agents.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 441,666, filed January 27, 2023, and U.S. Provisional Patent Application No. 63 / 597,249, filed November 8, 2023, both of which are incorporated by reference herein in their entireties.

[0002] Delivery of heterologous functional agents (such as therapeutic or immunological agents) can be limited by the extent to which the agent can penetrate cellular barriers and thereby effectively act on an organism. Thus, there is a continuing need in the art to develop novel delivery systems that can effectively deliver heterologous functional agents and promote cellular uptake of the agents. Summary of the Invention

[0003] In one aspect, provided herein is a modified lipid composition, the modified lipid composition comprising: (b) ionized lipid, modified by (a) a structural component comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol.

[0004] In some embodiments, one or more lipids listed in (a) increase delivery of the modified lipid composition to the spleen, characterized by an increased spleen-to-liver delivery ratio.

[0005] In some embodiments, the structural component comprises cardiolipin, e.g., cardiolipin derived from bovine heart. In some embodiments, the structural component comprises a sphingolipid, such as a sphingomyelin lipid, e.g., a sphingomyelin lipid derived from animal brain.

[0006] In some embodiments, the one or more lipid-containing structural components are soy-derived lipids.

[0007] Thus, in one aspect, provided herein is a modified lipid composition, the modified lipid composition comprising: (b) ionized lipid, modified by (a) a structural component comprising soy-derived lipids.

[0008] In some embodiments, the soy-derived lipids increase delivery of the modified lipid composition to the spleen and are characterized by an increased spleen-to-liver delivery ratio.

[0009] In some embodiments, the spleen to liver delivery ratio is increased to about 1.0, about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 15.0, about 20.0, or more.

[0010] In some embodiments, increasing the amount of structured lipid components in the modified lipid composition increases the spleen-to-liver delivery ratio.

[0011] In some embodiments, the modified lipid composition may be a complex lipid particle (CLP) comprising natural lipid extracts and derivatives, such as soybean lipid extract or soybean-derived lipids, and ionized lipids.

[0012] In another aspect, provided herein are methods for delivering one or more heterologous functional agents to a cell or a subject, the methods comprising contacting a cell or administering to a subject one or more heterologous functional agents and a modified lipid composition described herein, wherein the heterologous functional agent is encapsulated by, embedded on the surface of, or complexed to the surface of the modified lipid composition.

[0013] In some embodiments, the one or more heterologous functional agents are delivered to spleen cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or bone marrow cells.

[0014] In some embodiments, the modified lipid composition increases transfection of spleen cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or bone marrow cells.

[0015] In some embodiments, lymphoid cells (eg, splenic lymphoid cells) are transfected at a frequency of 1%, 2%, 5%, or more of the parent population.

[0016] In some embodiments, bone marrow cells (eg, splenic bone marrow cells) are transfected at a frequency of 5%, 10%, 15%, or more of the parent population.

[0017] In some embodiments, the hematopoietic stem cells are transfected at a frequency of 5%, 10%, 15%, 20%, 25%, 30%, 35%, or more of the parent population.

[0018] In any of the aspects of the invention, in some embodiments, the soy-derived lipid component comprises soy PC (phosphatidylcholine), soy PE (phosphatidylethanolamine), soy PI (phosphatidylinositol), soy PA (phosphatidic acid), soy lysoPC (LPC), soy lysoPI (LPI), soy PG (phosphatidylglycerol), soy PS (phosphatidylserine), soy lysoPS (LPS), HSPC (hydrogenated soy phosphatidylcholine), or a combination thereof.

[0019] In some embodiments, the structural component is soy PI, soy LPI, soy PG, soy polar, soy LPC, soy PC, soy PE, soy PA, soy PL (phospholipid) mixture, soy PS, soy LPS, HSPC, cardiolipin, or any combination thereof. In some embodiments, the soy-derived lipid is a soy polar lipid composition, a soy PL mixture, or a combination thereof.

[0020] In some embodiments, the soybean polar lipid composition is a polar lipid mixture extracted from soybeans, for example, polar lipids derived by precipitation of soybeans with acetone followed by extraction of the acetone-insoluble material with diethyl ether. In some embodiments, the soybean polar lipid composition comprises a mixture of soybean phospholipids (PL).

[0021] In some embodiments, the soybean polar lipid composition comprises PC, PE, PI, PA, and optionally LPC. In some embodiments, the soybean polar lipid composition comprises: Approximately 40% to 50% PC, Approximately 15% to 30% PE, PI of about 10% to about 25% Approximately 1% to 15% PA, Approximately 0% to approximately 10% LPC, and Contains about 0% to about 15% other lipids. In one embodiment, the soy polar lipid composition comprises PC:PE:PI:PA:LPC:other lipids in a percentage ratio of about 45.7:22.1:18.4:6.9:0:6.9.

[0022] In some embodiments, the soy PL mixture comprises PC, PE, PI, PA, and LPC. In some embodiments, the soy PL mixture comprises: Approximately 30% to 50% PC, Approximately 20% to 40% PE, PI of about 10% to about 25% Approximately 1% to 15% PA, Approximately 1% to approximately 15% LPC, and Contains about 0% to about 10% other lipids. In one embodiment, the soy PL mixture comprises PC:PE:PI:PA:LPC:other lipids in a percentage ratio of about 38:30:18:7:7:0.

[0023] In another aspect, provided herein is a modified lipid composition comprising a plurality of lipid reconstitution structural components, wherein the lipid reconstitution components are produced by a process comprising the steps of: (a) providing a plurality of structural lipids; (b) processing the plurality of lipids to produce a lipid membrane; (c) reconstituting the lipid membrane in an organic solvent selected from the group consisting of acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol, and dimethylformamide:methanol, thereby producing a lipid solution; and (d) processing the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, thereby producing the modified lipid composition.

[0024] In another aspect, provided herein is a method for producing a modified lipid composition. The method comprises reconstituting (a) structural components comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol in the presence of (b) an ionizable lipid to produce a modified lipid composition. The method further comprises loading the modified lipid composition with one or more heterofunctional agents. The ionizable lipid has two or more of the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of which is at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) N:P ratio of at least 10.

[0025] In some alternative embodiments, the ionizable lipid has two or more of the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of which is at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) N:P ratio of at least 3.

[0026] In another aspect, provided herein is a method for delivering a modified lipid composition to a target cell, the method comprising introducing into the target cell a modified lipid composition comprising: (a) a structural component comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramide, glucosylceramide, lactosylceramide, galactosylcholesterol, and glucosylcholesterol; and (b) an ionizable lipid.

[0027] In some embodiments, the structural component comprises isolated natural extracellular vesicles. In some embodiments, the structural component is derived from a natural source. In some embodiments, the natural source is soybeans or animals (e.g., pigs, cows, etc.).

[0028] In some embodiments, the structural component is modified by reconstituting a membrane comprising the structural component in the presence of ionized lipids.

[0029] In some embodiments, the structural component comprises a soy-derived lipid, and the structural lipid is modified by reconstituting a membrane comprising purified soy-derived lipid with an ionized lipid.

[0030] In some embodiments, the ionizable lipid has one or more characteristics selected from the group consisting of: (i) at least two ionizable amines; (ii) at least three lipid tails, each of which is at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) N:P ratio of at least 10.

[0031] In some alternative embodiments, the ionizable lipid has one or more characteristics selected from the group consisting of: (i) at least two ionizable amines; (ii) at least three lipid tails, each of which is at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) N:P ratio of at least 3.

[0032] In some embodiments, the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315. In one embodiment, the ionizable lipid is C12-200.

[0033] In some embodiments, the ionizable lipid is [ka] where R is C to C 14 It is an alkyl group.

[0034] In some embodiments, the ionizable lipid is selected from one of the following groups of compounds: i) Formula [ka] a compound of the formula: A is independently C1 to C 16 branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; B is independently C1 to C 16 branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; each X is independently a biodegradable moiety; and W is [ka] wherein: R5 is OH, SH, or NR 10 R 11 and Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 are taken together to form a heterocycle, R7 and R8 are taken together to form a ring, each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, and Q is O, S, or NR 13 where R 13 are each H or C1-C5 alkyl, ii) Equation [ka] a compound of the formula: [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or [ka] and A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; Each of X and Z is independently absent, —O—, —CO—, —N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, R 7are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, which may be interrupted by heteroatoms or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of l and m is an integer from 1 to 10; t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; iii) Formula [ka] or expression [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, or R 20 and R 30 forms a 3- to 7-membered cyclic ring together with the adjacent N atom, which is R a may be substituted with R ais H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; Each R1 and each R2 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 together form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, n is 0, 1, 2, 3, or 4; Y is O or S; Z is absent, O, S, or N(R 12 ) where R 12 are each independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is present, adjacent R1 and R2 are not OH, NR 10 R 11 , or SH, v is 0, 1, 2, 3, or 4; y is 0, 1, 2, 3, or 4; Each A is independently, C1~C 16 branched or unbranched alkyl, or C2-C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; Each B is independently, C1 to C 16 branched or unbranched alkyl, or C2-C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; and each X is independently a biodegradable moiety; and iv) a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'): [ka] a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing; During the ceremony, E each independently represents -OC(O)-, -C(O)O-, or -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -SS-, or -C(OR 13 )-O-(CH2) r -, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is a branched or unbranched C3 to C 10 is alkyl, r is 1, 2, 3, 4, or 5; R a are each independently C1 to C5 alkyl, C2 to C5 alkenyl, or C2 to C5 alkynyl, u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are each independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, which may be interrupted by heteroatoms or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; and [ka] represents the bond connecting the tail group to the head group, and Lipids have a pKa of about 4 to about 8.

[0035] In all of these aspects of the invention, in some embodiments, the modified lipid composition is a CLP comprising a natural lipid, such as a soy-derived lipid, and an ionizable lipid. Accordingly, all embodiments herein describing features relating to the modified lipid composition apply to CLP formulations.

[0036] In some embodiments, the modified lipid composition may further comprise a sterol. Thus, the reconstitution (or reorganization) of the structural components is carried out in the presence of ionized lipids and a sterol.

[0037] In some embodiments, the modified lipid composition further comprises a polyethylene glycol (PEG) lipid conjugate. Thus, the reconstitution (or reconstitution) of the structural components is carried out in the presence of ionized lipids and PEGylated lipids (or PEG-lipid conjugates).

[0038] In some embodiments, the modified lipid composition further comprises a sterol and a polyethylene glycol (PEG) lipid conjugate. Thus, the reconstitution (or reconstitution) of the structural components is carried out in the presence of ionized lipids, sterols, and / or PEGylated lipids (or PEG-lipid conjugates).

[0039] In some embodiments, the sterol is cholesterol or sitosterol.

[0040] In some embodiments, the PEG-lipid conjugate is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some embodiments, the PEG-lipid conjugate is PEG-DMG or PEG-PE. In some embodiments, the PEG-PEG lipid conjugate is PEG2000-DMG or PEG2000-PE.

[0041] In some embodiments, the modified lipid composition comprises: about 20 mol % to about 50 mol % of ionized lipids; about 10 mol % to about 75 mol % of the structural component; about 0 mol % to about 45 mol % sterols, and About 0.5 mol % to about 3 mol % polyethylene glycol (PEG) lipid conjugate.

[0042] In some embodiments, the modified lipid composition comprises: about 20 mol % to about 50 mol % of ionized lipids; about 20 mol % to about 60 mol % of structural components (e.g., structural lipids); about 7 mol % to about 45 mol % sterol, and About 0.5 mol % to about 3 mol % polyethylene glycol (PEG) lipid conjugate.

[0043] In some embodiments, the modified lipid composition comprises: about 30 mol % to about 40 mol % of ionized lipids; about 20 mol % to about 50 mol % of a structural component (e.g., a structural lipid); about 12 mol % to about 43 mol % sterols, and About 1.5 mol % to about 3 mol % polyethylene glycol (PEG) lipid conjugate.

[0044] In some embodiments, the modified lipid composition comprises: approximately 35 mole percent ionized lipids; about 50 mole % structural components (e.g., structural lipids); about 12.5 mole % sterols, and Approximately 2.5 mol% polyethylene glycol (PEG) lipid conjugate.

[0045] In one embodiment, the modified lipid composition comprises ionizable lipid:structured lipid:sterol:PEG lipid in a molar ratio of about 35:50:12.5:2.5.

[0046] In one embodiment, the modified lipid composition comprises ionizable lipid:structured lipid:sterol:PEG lipid in a molar ratio of about 35:20:42.5:2.5.

[0047] In one embodiment, the modified lipid composition comprises a molar ratio of about 22.5:75:0:2.5 ionizable lipid:structural lipid:sterol:PEG lipid.

[0048] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 35:30:32.5:2.5.

[0049] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 35:16:46.5:2.5.

[0050] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 35:25:37.5:2.5.

[0051] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 35:40:22.5:2.5.

[0052] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 45:10:43.5:1.5.

[0053] In one embodiment, the modified lipid composition comprises ionizable lipid:structured lipid:sterol:PEG lipid in a molar ratio of about 50:20:28.5:1.5.

[0054] In one embodiment, the modified lipid composition comprises an ionizable lipid:structured lipid:sterol:PEG lipid molar ratio of about 50:10:38.5:1.5.

[0055] In some embodiments, the structured lipid comprises a structured lipid 1 and a structured lipid 2, wherein the structured lipid 1 and the structured lipid 2 are different structured lipids.

[0056] In one embodiment, the modified lipid composition comprises an ionizable lipid:(structural lipid 1+structural lipid 2):sterol:PEG lipid molar ratio of about 35:(3+13):46.5:2.5.

[0057] In one embodiment, the modified lipid composition comprises an ionizable lipid:(structural lipid 1+structural lipid 2):sterol:PEG lipid in a molar ratio of about 35:(15+5):42.5:2.5.

[0058] In one embodiment, the modified lipid composition comprises an ionizable lipid:(structural lipid 1+structural lipid 2):sterol:PEG lipid molar ratio of about 35:(45+5):12.5:2.5.

[0059] In some embodiments, the modified lipid composition comprises: a lipid selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramide, glucosylceramide, lactosylceramide, galactosylcholesterol, and glucosylcholesterol; C12-200, Cholesterol, and DMPE-PEG2k.

[0060] In one embodiment, the modified lipid composition comprises: a polar lipid selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, glucosylceramide, lactosylceramide, galactosylcholesterol, and glucosylcholesterol; C12-200, Cholesterol, and DMPE-PEG2k. The modified lipid composition comprises C12-200: structural lipid: cholesterol: DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5 or about 35:20:42.5:2.5.

[0061] In some embodiments, the modified lipid composition may comprise ionized lipids:structured lipids:sterol:PEG lipids in a molar ratio of 22.5:75:0:2.5. In some embodiments, the modified lipid composition may comprise ionized lipids:structured lipids:sterol:PEG lipids in a molar ratio of 35:50:12.5:2.5. In some embodiments, the modified lipid composition may comprise ionized lipids:structured lipids:sterol:PEG lipids in a molar ratio of 35:30:32.5:2.5. In some embodiments, the modified lipid composition may comprise ionized lipids:structured lipids:sterol:PEG lipids in a molar ratio of 35:20:42.5:2.5. In some embodiments, the modified lipid composition may comprise ionized lipids:structured lipids:sterol:PEG lipids in a molar ratio of 35:16:46.5:2.5. In some embodiments, the modified lipid composition may comprise ionizable lipid:(structural lipid 1+structural lipid 2):sterol:PEG lipid in a molar ratio of 35:(3+13):46.5:2.5 (structural lipid 1 may be a structured lipid described herein, structural lipid 2 may be a structured lipid described herein that is different from structural lipid 1, or structural lipid 2 may be any lipid suitable for preparing lipid nanoparticle compositions).

[0062] In some embodiments, the modified lipid composition is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions. In one embodiment, the modified lipid composition is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

[0063] In some embodiments, the modified lipid composition is a lipid nanoparticle.

[0064] In some embodiments, particles of the modified lipid composition have a size of less than about 200 nm. In one embodiment, particles of the modified lipid composition have a size of less than about 150 nm. In one embodiment, particles of the modified lipid composition have a size of less than about 100 nm. In one embodiment, particles of the modified lipid composition have a size of about 55 nm to about 95 nm. In one embodiment, particles of the modified lipid composition have a size of about 85 nm to about 95 nm. In one embodiment, particles of the modified lipid composition have a size of about 85 nm to about 90 nm.

[0065] In some embodiments, the particles of the modified lipid composition have an average polydispersity index in the range of about 0.1 to about 0.5. In some embodiments, the particles of the modified lipid composition have an average PDI in the range of about 0.1 to about 0.4. In some embodiments, the particles of the modified lipid composition have an average PDI in the range of about 0.2 to about 0.3.

[0066] In some embodiments, the modified lipid composition comprises one or more heterofunctional agents. In some embodiments, the heterofunctional agents are encapsulated by the modified lipid composition. In some embodiments, the heterofunctional agents are embedded on the surface of the modified lipid composition. In some embodiments, the heterofunctional agents are complexed to the surface of the modified lipid composition.

[0067] In some embodiments, the heterologous functional agent is a polynucleotide. In some embodiments, the polynucleotide is selected from mRNA, circular RNA (circRNA), siRNA or siRNA precursor, microRNA (miRNA) or miRNA precursor, plasmid, Dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), piwi-interacting RNA (piRNA), ribozyme, deoxyribozyme (DNAzyme), aptamer, guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is circRNA. In some embodiments, the mRNA is derived from a DNA molecule or an RNA molecule (e.g., a self-replicating RNA molecule). In some embodiments, the polynucleotide is an siRNA or a precursor thereof. In some embodiments, the polynucleotide is a plasmid.

[0068] In some embodiments, the efficiency of encapsulation of polynucleotides by the modified lipid composition is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%. In one embodiment, the efficiency of encapsulation of polynucleotides by the modified lipid composition is at least about 90%.

[0069] In some embodiments, the modified lipid composition has a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 50:1 to about 10:1. In some embodiments, the modified lipid composition has a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 44:1 to about 24:1. In some embodiments, the modified lipid composition has a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 40:1 to about 28:1. In some embodiments, the modified lipid composition has a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 38:1 to about 30:1. In some embodiments, the modified lipid composition has a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 37:1 to about 33:1.

[0070] In some embodiments, the modified lipid composition is formulated for delivery to an animal or human. In some embodiments, the modified lipid composition is formulated for delivery to a plant.

[0071] In some embodiments, the modified lipid composition is made by a method comprising lipid extrusion, hi some embodiments, the modified lipid composition is produced by a method comprising processing a solution comprising lipids from the modified lipid composition in a microfluidic device comprising an aqueous phase, thereby producing the modified lipid composition.

[0072] In some embodiments, the heterologous functional agent is formulated into the modified lipid composition via the aqueous phase. In some embodiments, the aqueous phase and the lipid solution (organic phase) are mixed in a volume ratio of 3:1.

[0073] In some embodiments, the aqueous phase comprises a polynucleotide.

[0074] In some embodiments, the modified lipid composition, e.g., the aqueous phase, further comprises a HEPES or TRIS buffer. The HEPES or TRIS buffer may have a pH of about 7.0 to about 8.5. The HEPES or TRIS buffer may have a concentration of about 7 mg / mL to about 15 mg / mL. The aqueous phase may further comprise about 2.0 mg / mL to about 4.0 mg / mL of NaCl.

[0075] In some embodiments, the modified lipid composition, e.g., the aqueous phase, comprises water, PBS, or a citrate buffer. In one embodiment, the aqueous phase comprises a citrate buffer having a pH of about 3.2.

[0076] In some embodiments, the modified lipid composition further comprises one or more cryoprotectants. The one or more cryoprotectants can be sucrose, glycerol, or a combination thereof. In some embodiments, the modified lipid composition comprises sucrose, e.g., at a concentration of about 70 mg / mL to about 110 mg / mL. In some embodiments, the modified lipid composition comprises glycerol, e.g., at a concentration of about 50 mg / mL to about 70 mg / mL. In one embodiment, the modified lipid composition comprises a combination of sucrose (e.g., at a concentration of about 70 mg / mL to about 110 mg / mL) and glycerol (e.g., at a concentration of about 50 mg / mL to about 70 mg / mL).

[0077] In some embodiments, the modified lipid composition is a lyophilized or freeze-dried composition. The lyophilized or freeze-dried modified lipid composition may include one or more lyoprotectants. The freeze-dried modified lipid composition may include a poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the freeze-dried modified lipid composition includes a poloxamer (about 0.01 to about 1.0 wt.% poloxamer). In one embodiment, the poloxamer is poloxamer 188.

[0078] In some embodiments, the modified lipid composition is a lyophilized composition. In some embodiments, the lyophilized modified lipid composition comprises about 0.01 to about 1.0 w / w% heterologous functional agent (e.g., polynucleotide). In some embodiments, the lyophilized modified lipid composition comprises about 1.0 to about 5.0 w / w% lipid. In some embodiments, the lyophilized modified lipid composition comprises about 0.5 to about 2.5 w / w% TRIS buffer. In some embodiments, the lyophilized modified lipid composition comprises about 0.75 to about 2.75 w / w% NaCl. In some embodiments, the lyophilized modified lipid composition comprises about 85 to about 95 w / w% sugar, e.g., sucrose. In some embodiments, the lyophilized modified lipid composition comprises about 0.01 to about 1.0 w / w% poloxamer (e.g., about 0.01 to about 1.0 w / w% poloxamer), e.g., poloxamer 188. In some embodiments, the lyophilized modified lipid composition comprises about 1.0 to about 5.0 w / w% potassium sorbate.

[0079] definition As used herein, the terms "effective amount," "effective concentration," or "concentration that is effective for" refer to an amount of a modified lipid composition or nucleic acid composition sufficient to produce a described result or to reach a target level (e.g., a predetermined level or threshold level) in or for a target organism.

[0080] As used herein, the term "therapeutic agent" refers to an agent that can act on an animal, e.g., a mammal (e.g., a human), an animal pathogen, or a pathogen vector, such as an antifungal agent, an antibacterial agent, a virucide, an antiviral agent, an insecticide, a nematicide, an antiparasitic agent, or an insect repellent.

[0081] As used herein, the term "heterologous" refers to an agent that is exogenous to the organism to which the modified lipid composition is delivered.

[0082] As used herein, the term "functional agent" refers to an agent (e.g., an agricultural agent (e.g., insecticide, fertilizer, herbicide, plant modifier) ​​or therapeutic agent (e.g., an antifungal, antibacterial, virucide, antiviral, insecticide, nematicide, antiparasitic, or insect repellent)) that is associated with or can be associated with a modified lipid composition (e.g., loaded into or onto (e.g., encapsulated by, embedded in, or complexed with) the modified lipid composition) using in vivo or in vitro methods and can produce an enumerated result (e.g., increasing or decreasing the fitness of a plant, plant pest, plant symbiont, animal (e.g., human) pathogen, or animal pathogen vector). In some embodiments, the heterologous functional agent is a polynucleotide.

[0083] As defined herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to RNA or DNA that is linear, branched, or circular, single- or double-stranded, or a hybrid thereof, regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked within a nucleic acid by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs having other types of linkages or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others). Nucleic acids can be single-stranded or double-stranded, or can contain portions of both single-stranded and double-stranded sequences. Nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases, including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0084] As used herein, the terms "linear polyribonucleotide," "linear polyribonucleotide molecule," "linear RNA," and "linRNA" are used interchangeably and refer to a polyribonucleotide molecule having a 5' end and a 3' end. One or both of the 5' and 3' ends may be free or may be attached to another moiety. Linear RNA includes RNA that has not been circularized (e.g., previously circularized) and can be used as starting material for circularization, for example, via splint ligation or chemical, enzymatic, ribozyme, or splicing catalyst circularization methods.

[0085] As used herein, the terms "cyclic polyribonucleotide," "circular polynucleotide molecule," "circular RNA," and "circRNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure with no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide molecule that forms a circular or endless structure via covalent (e.g., covalently closed) or non-covalent bonds. A cyclic polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

[0086] As used herein, the terms "peptide," "protein," or "polypeptide" encompass any chain of natural or unnatural amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more than 1000 amino acids), post-translational modification (e.g., glycosylation or phosphorylation), or the presence of one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide, and include, for example, naturally occurring proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics. A polypeptide can be, for example, at least 0.1 kD, at least 1 kD, at least 5 kD, at least 10 kD, at least 15 kD, at least 20 kD, at least 30 kD, at least 40 kD, at least 50 kD, or greater in size. A polypeptide can be a full-length protein. Alternatively, a polypeptide can comprise one or more domains of a protein.

[0087] As used herein, the term "animal" refers to humans and non-human animals (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, chickens, and non-human primates).

[0088] As used herein, the term "infection" refers to the presence or colonization of a pathogen within an animal (e.g., in one or more parts of the animal), or on an animal (e.g., on one or more parts of the animal), or in the habitat surrounding the animal, particularly when the infection reduces the animal's health, for example, by causing disease, disease symptoms, or an immune (e.g., inflammatory) response.

[0089] As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes disease or disease symptoms in an animal, for example, by (i) directly infecting the animal, (ii) producing an agent that causes the disease or disease symptoms in the animal (e.g., bacteria that produce pathogenic toxins), and / or (iii) eliciting an immune (e.g., inflammatory) response in the animal (e.g., biting insects, such as bedbugs). As used herein, pathogens include, but are not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids, and viruses, or any combination thereof, each of which can cause disease or symptoms in humans, either alone or in concert with another pathogen.

[0090] As used herein, the term "antibody" encompasses natural or partially or fully synthetically produced immunoglobulins, as well as fragments thereof, capable of specifically binding to an antigen. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or partially or fully synthetically produced. "Antibody" further includes polypeptides comprising framework regions from immunoglobulin genes or fragments thereof that specifically bind and recognize an antigen. The use of the term "antibody" is meant to further include whole antibodies; polyclonal, monoclonal, and recombinant antibodies, including fragments thereof; as well as single-chain antibodies (nanobodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, and primate-human monoclonal antibodies; anti-idiotypic antibodies; antibody fragments such as scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments; diabodies; and antibody-related polypeptides. "Antibody" further includes bispecific and multispecific antibodies.

[0091] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, as described in Altschul et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available from the National Biotechnology Center.

[0092] As used herein, the term "unmodified structural component" refers to a composition comprising a structural component (e.g., isolated natural extracellular vesicles, other naturally occurring lipids, or synthetic structural lipids) that lacks a heterologous cell uptake agent that can increase cellular uptake of the structural component (e.g., animal cell uptake, plant cell uptake, native cell uptake, or fungal cell uptake).

[0093] As used herein, the term "modified" or "modification" with respect to a structural component refers to a modified lipid composition (e.g., one or more exogenous lipids, such as ionized lipids, sterols, and / or PEGylated lipids) comprising a structural component and one or more heterologous agents that can increase cellular uptake (e.g., animal cell uptake, plant cell uptake, natural cell uptake, or fungal cell uptake) of the modified lipid composition, or portions or components thereof, compared to the unmodified structural component, enable or increase delivery of a heterologous functional agent (e.g., agricultural or therapeutic agent) to a cell by the modified lipid composition, and / or enable or increase loading (e.g., loading efficiency or loading capacity) of a heterologous functional agent (e.g., agricultural or therapeutic agent). The structural component may be modified in vitro or in vivo.

[0094] As used herein, the term "cellular uptake" refers to the uptake of a modified lipid composition, or a portion or component thereof (e.g., a polynucleotide carried by the modified lipid composition), by a cell, such as an animal cell, a plant cell, a natural cell, or a fungal cell. For example, uptake can involve the transfer of a portion of the modified lipid composition, or a component thereof, from the extracellular environment to or across the cell membrane, cell wall, extracellular matrix, or into the intracellular environment of the cell. Cellular uptake of a modified lipid composition can occur via active or passive cellular mechanisms. Cellular uptake includes situations in which the entire modified lipid composition is taken up by the cell, for example, by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of a target cell after endocytosis and endosomal escape. In some embodiments, modified lipid compositions comprising ionized lipids (e.g., modified lipid compositions comprising ionized lipids and sterols and / or PEGylated lipids) exhibit increased rates of endosomal escape compared to unmodified structural components (e.g., structural lipids). Cellular uptake also includes situations in which the modified lipid composition fuses with the membrane of a target cell. In some embodiments, the one or more polynucleotides are exposed to the cytoplasm of the target cell after membrane fusion, and in some embodiments, the modified lipid composition has an increased rate of fusion with the membrane of the target cell (e.g., is more fusogenic) compared to a structural component that is not modified by an ionized lipid.

[0095] As used herein, the term "cell membrane permeabilizing agent" refers to an agent that alters the characteristics (e.g., permeability) of the cell wall, extracellular matrix, or cell membrane (e.g., of an animal cell, plant cell, bacterial cell, or fungal cell) in a manner that promotes increased cellular uptake compared to cells not contacted with the agent.

[0096] Described herein are "modified lipid compositions" containing lipid-reconstituted structural components. The structural components are derived from lipid structures (e.g., lipid bilayers, monolayers, multilayers, e.g., vesicular lipid structures) derived from natural sources (e.g., enriched, isolated, or purified from natural sources), and the lipid structures are disrupted (e.g., disrupted by lipid extraction) and reorganized or reconstituted in a liquid phase (e.g., a cargo-containing liquid phase) using standard methods, e.g., by methods including lipid film hydration and / or solvent injection, as described herein, to produce lipid-reconstituted structural components. If desired, the method may further include, for example, sonication, freeze / thaw treatment, and / or lipid extrusion to reduce the size of the reconstituted modified lipid composition. Alternatively, the modified lipid composition may be produced using a microfluidic device (e.g., the NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems)). Alternatively, the structural components are not derived from natural sources but include synthetically obtained structured lipids.

[0097] As used herein, the term "native extracellular vesicles," "native EVs," or "EVs" refers to enclosed lipid bilayer structures that occur naturally in plants. Optionally, natural EVs contain one or more natural EV markers. As used herein, the term "native EV marker" refers to components that are naturally associated with the plant, such as natural proteins, natural nucleic acids, natural small molecules, natural lipids, or combinations thereof.

[0098] As used herein, the term "complex lipid particle" refers to a lipid particle characterized by its complexity, containing a wide variety of lipids, including structured lipids extracted from one or more natural sources and, optionally, at least one exogenous ionized lipid. Complex lipid particles can contain 10% to 99% structured lipids derived from lipid structures from one or more natural sources, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 99% lipid derived from lipid structures from one or more natural sources. In some examples, complex lipid particles incorporating natural lipid extracts may also be referred to as natural messenger packs (NMPs). For example, complex lipid particles incorporating plant lipid extracts may also be referred to as plant messenger packs (PMPs). In some instances, complex lipid particles incorporating a natural lipid extract and at least one exogenous ionizable lipid may also be referred to as lipid-reconstituted natural messenger packs (LNMPs). For example, complex lipid particles incorporating a plant lipid extract and at least one exogenous ionizable lipid may also be referred to as lipid-reconstituted plant messenger packs (LPMPs).

[0099] In some embodiments, the composite lipid particles incorporate soybean lipid extracts or soybean-derived lipids.

[0100] Composite lipid particles can contain 3 to 1,000 lipids extracted from one or more natural sources. Composite lipid particles can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different classes or subclasses of natural lipids from natural sources. Composite lipid particles can contain all or a fraction of the lipid species present in the lipid structures from natural sources, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially 100% of the lipid species present in the lipid structures from natural sources. Composite lipid particles can contain all or a fraction of the lipid species present in the lipid structures from a particular natural source. For example, the composite lipid particles may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially 100% of the lipid species present in the lipid structure derived from natural sources.

[0101] In some embodiments, the composite lipid particles contain three or more lipids extracted or derived from soybeans.

[0102] Complex lipid particles may contain reduced or minimized endogenous protein material relative to one or more natural sources, for example, 0 w / w%, less than 1 w / w%, less than 5 w / w%, less than 10 w / w%, less than 15 w / w%, less than 20 w / w%, less than 30 w / w%, less than 40 w / w%, or less than 50 w / w% of endogenous protein material relative to one or more natural sources. In some examples, the lipid bilayer of a complex lipid particle is protein-free.

[0103] The complex lipid particles may also contain synthetic structural lipids, such as neutral lipids, as structural lipid components. The structural lipid components of the complex lipid particles may contain 10% to 99% structural lipids derived from synthetic lipid structures (as opposed to lipids extracted from natural sources), such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 99% lipids derived from synthetic lipid structures.

[0104] The complex lipid particle can further comprise at least two exogenous lipids.The complex lipid particle can comprise at least 1 wt% of exogenous lipids, at least 2 wt% of exogenous lipids, at least 5 wt% of exogenous lipids, at least 10 wt% of exogenous lipids, at least 15 wt% of exogenous lipids, at least 20 wt% of exogenous lipids, at least 25 wt% of exogenous lipids, at least 30 wt% of exogenous lipids, at least 40 wt% of exogenous lipids, at least 50 wt% of exogenous lipids, at least 60 wt% of exogenous lipids, at least 70 wt% of exogenous lipids, at least 80 wt% of exogenous lipids, or at least 90 wt% of exogenous lipids.Exemplary exogenous lipids include sterol and PEG-lipid complexes.The complex lipid particle can be used to encapsulate one or more exogenous nucleic acids or polynucleotides encoding one or more peptides, polypeptides, or proteins, allowing the delivery of exogenous nucleic acids or polynucleotides to target cells or tissues.

[0105] As used herein, the term "exogenous lipid" refers to a lipid that is exogenous to a natural source, i.e., the lipid is derived from a source other than the natural source from which the lipid was extracted (e.g., a lipid added to a complex lipid particle formulation using the methods described herein). The term "exogenous lipid" does not exclude naturally occurring lipids (such as plant-derived sterols). That is, an exogenous lipid may be a naturally occurring lipid (such as a plant-derived sterol that is exogenous to the plant source from which the lipid was extracted, e.g., the exogenous lipid may be a plant-derived sterol that is added to a complex lipid particle formulation). As another example, an exogenous lipid may be a naturally occurring lipid that is exogenous to the particular natural source from which the lipid was extracted (e.g., a bacterial-derived lipid that is exogenous to the plant source from which the lipid was extracted, or vice versa). An exogenous lipid may be a cell membrane permeabilizing agent, which may increase the delivery of one or more polynucleotides to cells by a complex lipid formulation and / or increase the loading (e.g., loading efficiency or loading capacity) of the polynucleotide. In some embodiments, exogenous lipid can be stabilizing lipid.In some embodiments, exogenous lipid can be structured lipid (for example, synthetic structured lipid).Exemplary exogenous lipid includes ionized lipid, synthetic structured lipid, sterol, and PEGylated lipid.

[0106] As used herein, the term "cationic lipid" refers to a positively charged amphipathic molecule (e.g., a lipid or lipidoid) that contains a cationic group (e.g., a cationic head group).

[0107] As used herein, the term "ionizable lipid" refers to an amphipathic molecule (e.g., a lipid or lipidoid, e.g., a synthetic lipid or lipidoid) that contains a group (e.g., a head group) that can be ionized, e.g., dissociated to generate species having one or more charges, under given conditions (e.g., pH).

[0108] Surprisingly, it has been found that ionizable lipids comprising alkyl chains with multiple unsaturated sites, for example, at least two or three unsaturated sites, are particularly useful for forming lipid particles with increased membrane fluidity.Many ionizable lipids suitable for use herein and related analogs are described in U.S. Patent Application Publication Nos. 20060083780 and 20060240554, U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992, and PCT Application Publication No. WO 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0109] In some embodiments, ionizable lipids can be ionized, so they can dissociate depending on pH and exist in a positively charged form.The ionization of ionizable lipids affects the surface charge of lipid-modified substances containing ionizable lipids under different pH conditions.The surface charge of lipid-modified substances can subsequently affect their plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as their ability to form endosomolytic non-bilayer structures, which can affect the intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8: 1188-1196 (2001)).

[0110] In some embodiments, the ionizable lipid is a lipid that is generally neutral at, for example, physiological pH (e.g., pH about 7), but can carry a net charge at acidic or basic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7, but can carry a net charge at acidic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7, but can carry a net charge at basic pH.

[0111] In some embodiments, ionizable lipids do not include cationic or anionic lipids, which generally carry a net charge at physiological pH (eg, a pH of about 7).

[0112] As used herein, the term "lipid" refers to a molecule that has one or more characteristics of a lipid.

[0113] As used herein, the term "stable structured lipid formulation" or "stable CLP formulation" refers to a formulation that can be maintained for a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days), optionally within a defined temperature range (e.g., at a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, 23°C, ... or at least 20°C (e.g., at least 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C). at least 5% (e.g., at least 5%, 10%, 20%, 23°C), at least 4°C (e.g., at least 5°C, 10°C, or 15°C), at least -20°C (e.g., at least -20°C, -15°C, -10°C, -5°C, or 0°C), or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C)), compared to the number of particles in the structured lipid formulation or CLP formulation (e.g., at the time of manufacture or formulation). 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or optionally within a defined temperature range (e.g., at least 24°C (e.g., 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23°C), at least 4°C (e.g., at least 5°C, 10°C, or 15°C), at least -20°C (e.g., at least -20°C, -15°C, at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity (e.g., cell wall penetrating activity and / or activity of RNA formed within the modified lipid composition) of the modified lipid composition or CLP formulation (e.g., at the time of manufacture or formulation) at a temperature below -10°C, -5°C, or 0°C, or at -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C)), compared to the initial activity of the modified lipid composition or CLP formulation (e.g., at the time of manufacture or formulation),or 100%).

[0114] Alternatively, the expression may refer to a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23°C), at least 4°C (e.g., at least 5°C, 10°C, or 15°C), at least -20°C, over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days). "CLP formulation" refers to a CLP formulation or modified lipid composition that retains at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of its activity at or below -20°C (e.g., at least -20°C, -15°C, -10°C, -5°C, or 0°C), or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C) compared to the initial activity of the CLP formulation or modified lipid composition (e.g., at the time of manufacture or formulation).

[0115] Alternatively, the expression may refer to a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23°C), at least 4°C (e.g., at least 5°C, 10°C, or 15°C), at least -20°C (e.g., at least " refers to a CLP formulation or modified lipid composition that retains its particle size, i.e., does not increase or does not increase by more than 5% (e.g., not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 2-fold, 2.5-fold, or 3-fold) compared to the original particle size of the CLP or modified lipid composition (e.g., at the time of manufacture or formulation) at temperatures below -20°C, -15°C, -10°C, -5°C, or 0°C), or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C).

[0116] In some embodiments, the stable CLP or modified lipid composition continues to encapsulate or remain associated with an exogenous peptide, polypeptide, or protein loaded into the CLP or modified lipid composition, e.g., continues to encapsulate or remains associated with an exogenous peptide, polypeptide, or protein for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, at least 90 days, or more than 90 days.

[0117] As used herein, the term "treating" refers to administering a pharmaceutical composition to an animal for prophylactic and / or therapeutic purposes. "Preventing an infection" refers to the prophylactic treatment of an animal that does not yet have a disease or condition, but is susceptible to or at risk for a particular disease or condition. "Treating an infection" refers to administering treatment to an animal already suffering from a disease to improve or stabilize the animal's condition.

[0118] As used herein, the term "treating an infection" refers to administering a treatment to an individual (e.g., an animal) already suffering from a disease to improve or stabilize the individual's condition. This may involve reducing pathogen colonization in, on, or around the animal by one or more pathogens relative to the starting amount (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) and / or enabling a benefit to the individual (e.g., reducing colonization by an amount sufficient to resolve symptoms). In such instances, a treated infection may manifest as a reduction in symptoms (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some examples, the treated infection is effective to increase the survival chance of an individual (e.g., increase the chance of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or increase the overall survival of a population (e.g., increase the chance of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, the compositions and methods can be effective to "substantially eliminate" the infection, which refers to a reduction in infection sufficient to provide a sustained resolution of symptoms in an animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0119] As used herein, the term "preventing infection" means preventing increased colonization in, on, or around an animal by one or more pathogens (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% compared to an untreated animal) in an amount sufficient to maintain the initial pathogen population (e.g., about the amount found in a healthy individual), prevent the onset of infection, and / or prevent symptoms or conditions associated with infection. For example, an individual (e.g., an animal, e.g., a human) may receive prophylactic treatment to prevent fungal infections while preparing for an invasive medical procedure (e.g., preparing for surgery to receive a transplant, stem cell therapy, graft, prosthetic organ, undergoing prolonged or frequent intravenous catheterization, or receiving treatment in an intensive care unit), in an immunocompromised individual (e.g., an individual with cancer, HIV / AIDS, or taking immunosuppressants), or in an individual receiving long-term antibiotic therapy.

[0120] As used herein, the term "formulated for delivery to an animal" refers to a modified lipid composition comprising a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable" carrier or excipient is, for example, a carrier or excipient that is suitable for administration to an animal (e.g., a human) without undue adverse side effects to the animal (e.g., a human). [Brief explanation of the drawings]

[0121] [Figure 1A] Figure 1A shows the whole-body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of lipid compositions using the structural lipids DOPE, DOPC, DSPC, LPC, brain sphingomyelin (SM), ceramide, glucosylceramide, lactosylceramide, lactosylPE, MGDG, DGDG, galactosylcholesterol, glucosylcholesterol, triglyceride (TG), or cardiolipin (CA) at a lipid molar ratio of 16, 30, 50, or 75% (10 μg / 100 μL; 1:1 FLuc:hEPO mRNA) in mice. N=2 / group. [Figure 1B] FIG. 1B shows the spleen to liver ratio 4 hours after intravenous administration of the same composition (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0122] [Figure 2A] Figure 2A shows the whole body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of lipid compositions using the structural lipids DOPE, DOPS, soy PS, brain PS, LPS, DOPG, soy PG, DOPA, 14:0 PA, soy PA, egg PA, and LPA at 3, 16, 30, or 50% lipid molar ratios (10 μg / 100 μL, 1:1 FLuc:hEPO mRNA) in mice. N=2 / group. [Figure 2B] FIG. 2B shows the spleen to liver ratio 4 hours after intravenous administration of the same composition (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0123] [Figure 3A] Figure 3A shows the whole body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of exemplary modified lipid compositions using the structural lipids soy polar, soy PL blend, soy PC, soy LPC, soy PE, soy PS, soy PG, soy PA, and soy PI at 16, 30, or 50% lipid molar ratios (10 μg / 100 μL, 1:1 FLuc:hEPO mRNA) in mice compared to that of the same molar ratio DOPE LNP composition carrying the same cargo. N=2 / group. [Figure 3B] FIG. 3B shows the spleen to liver ratio 4 hours after intravenous administration of the same composition (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0124] [Figure 4]Figure 4A shows the whole-body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of exemplary modified lipid compositions using the ionized lipid 2213 and the structural lipids galactosylcholesterol, brain sphingomyelin (SM), ceramide, cardiac cardiolipin (CA), soy polar, or soy LPC at a 16 or 50% lipid molar ratio (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA) in mice, compared to that of an LNP composition carrying the same cargo as a control. N=2 / group. Figure 4B shows the spleen-to-liver ratio 4 hours after intravenous administration of the same compositions (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0125] [Figure 5] Figure 5A shows the whole-body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of an exemplary modified lipid composition using the ionized lipid 2231 and structural lipids glucosylcholesterol, brain SM, heart CA, soy PC, soy LPC, soy PS, soy polar, and soy PL mixture at a 16% lipid molar ratio (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA) in mice, compared to that of an LNP composition carrying the same cargo as a control. N=2 / group. Figure 5B shows the spleen-to-liver ratio 4 hours after intravenous administration of the same composition (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0126] [Figure 6] Figure 6A shows the whole-body radiance (mean radiance p / s / cm2 / sr) 4-6 hours after intravenous administration of an exemplary modified lipid composition using ionized lipid 2252 and structural lipids brain SM, CA, soy PC, soy LPC, soy PS, soy polar, and soy PL mixtures at 16% or 50% lipid molar ratios (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA) in mice, compared to that of an LNP composition carrying the same cargo as a control. N=2 / group. Figure 6B shows the spleen-to-liver ratio 4 hours after intravenous administration of the same composition (10 ug / 100 uL, 1:1 FLuc:hEPO mRNA).

[0127] [Figure 7A] Figure 7A shows lymphocyte transfection in Ai9 mice in spleens harvested three days after intravenous administration of modified lipid compositions using ionized lipid 2213 and structural lipids brain SM (16% lipid molar ratio), soy LPC (16% lipid molar ratio), and soy polar (50% lipid molar ratio) (0.375 mg / kg; 4:1 CRE:FLuc mRNA), with PBS and 2213 LNP (0.375 mg / kg; 4:1 CRE:FLuc mRNA) used as controls. N=3 / group. [Figure 7B] Figure 7B shows bone marrow cell transfection in Ai9 mice in spleens harvested three days after intravenous administration of modified lipid compositions using ionized lipid 2213 and structural lipids brain SM (16% lipid molar ratio), soy LPC (16% lipid molar ratio), and soy polar (50% lipid molar ratio) (0.375 mg / kg; 4:1 CRE:FLuc mRNA), with PBS and 2213 LNP (0.375 mg / kg; 4:1 CRE:FLuc mRNA) used as controls. N=3 / group.

[0128] [Figure 8A] FIG. 8A shows the lipid molar ratios of the modified lipid composition derived from soybeans. [Figure 8B] Figure 8B shows the levels of antibodies (IgG) specific to the receptor binding domain (RBD) of SARS-CoV-2 in the blood of mice 28 days after a single intramuscular dose of the soy-derived modified lipid composition from Figure 8A (10 μg S mRNA). N=3 / group. Control was PBS.

[0129] [Figure 9A] Figure 9A shows lymphocyte transfection of tdTomato mice in spleens harvested three days after intravenous administration of a modified lipid composition (Table 10, 1.25 mg / kg CRE mRNA), with untreated mice used as controls. N=4 / experimental group.

[0130] [Figure 9B] Figure 9B shows bone marrow cell transfection of tdTomato mice in spleens harvested three days after intravenous administration of a modified lipid composition (Table 10, 1.25 mg / kg CRE mRNA), with untreated mice used as controls. N=4 / experimental group.

[0131] [Figure 10] Figure 10 shows hematopoietic stem cell (HSC) transfection of tdTomato mice in bone marrow collected three days after intravenous administration of modified lipid compositions (Groups 6-8, 16-18, 1.25 mg / kg CRE mRNA in Table 10), with untreated mice used as controls. N=3-4 / experimental group.

[0132] [Figure 11A] Figure 11A shows the levels of S-specific IgG in the blood of hamsters after a single intramuscular dose of Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 1 ug) 21 days after administration. N=14 / group. Controls were monovalent vaccine (1 ug, n=14) and pooled naive hamsters. [Figure 11B] Figure 11B shows the levels of RBD-specific IgG in the blood of hamsters after a single intramuscular dose of Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 1 ug) 21 days after administration. N=14 / group. Controls were monovalent vaccine (1 ug, n=14) and pooled naive hamsters.

[0133] [Figure 12A] Figure 12A shows the amount of S-specific IgG in the blood of hamsters after a single intramuscular dose of Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 1 ug) 21 days after administration. N=14 / group. Controls were monovalent vaccine (1 ug, n=14) and pooled naive hamsters. [Figure 12B] Figure 12B shows the amount of RBD-specific IgG in the blood of hamsters after a single intramuscular dose of Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 1 ug) 21 days after administration. N=14 / group. Controls were monovalent vaccine (1 ug, n=14) and pooled naive hamsters.

[0134] [Figure 13] Figure 13 shows the percent inhibition in hamsters after a single intramuscular dose of Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 / SARS-CoV-2 (soy, containing S mRNA, 1 ug) 21 days after administration. N=14 / group. The control was the monovalent vaccine (1 ug, n=14).

[0135] [Figure 14A] Figure 14A shows the absolute number of germinal center B cells in pooled lymph nodes 7 or 10 days after a single intramuscular administration of various modified lipid composition formulations (S mRNA 10 μg / 40 μL, see formulations in Table 12). N=6 / group. Controls were LNP formulations (see Table 12) or untreated mice. [Figure 14B] Figure 14B shows the absolute number of follicular helper T cells in pooled lymph nodes 7 or 10 days after a single intramuscular administration of various modified lipid composition formulations (S mRNA 10 μg / 40 μL, see formulations in Table 12). N=6 / group. Controls were LNP formulations (see Table 12) or untreated mice. [Figure 14C] Figure 14C shows the frequency of germinal center B cells among B cells in pooled lymph nodes 7 or 10 days after a single intramuscular administration of various modified lipid composition formulations (S mRNA 10 μg / 40 μL, see formulations in Table 12). N=6 / group. Controls were LNP formulations (see Table 12) or untreated mice. [Figure 14D]Figure 14D shows the frequency of follicular helper T cells among CD4 T cells in pooled lymph nodes 7 or 10 days after a single intramuscular administration of various modified lipid composition formulations (S mRNA 10 μg / 40 μL, see formulations in Table 12). N=6 / group. Controls were LNP formulations (see Table 12) or untreated mice. [Figure 14E] Figure 14E shows the absolute number of class-switched B cells in pooled lymph nodes 7 or 10 days after a single intramuscular administration of various modified lipid composition formulations (S mRNA 10 μg / 40 μL, see formulations in Table 12). N=6 / group. Controls were LNP formulations (see Table 12) or untreated mice.

[0136] [Figure 15A] Figure 15A shows the whole body radiance (mean radiance p / s / cm2 / sr) 4 hours after intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1:1 mRNA FLuc:hEPO administered at 10 μg / 40 μL in mice. N=3 / group. Positive controls used were LNP1 and LNP2 (n=3 / group), and negative controls were mice administered PBS (n=2). [Figure 15B] Figures 15B and 15C show liver (Figure 15B) and spleen (Figure 15C) radiance (mean radiance p / s / cm2 / sr) in mice 4 hours after intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1:1 mRNA FLuc:hEPO administered at 10 μg / 40 μL. N=3 / group. Positive controls used were LNP1 and LNP2 (n=3 / group), and negative controls were mice administered PBS (n=2). [Figure 15C]Figures 15B and 15C show liver (Figure 15B) and spleen (Figure 15C) radiance (mean radiance p / s / cm2 / sr) in mice 4 hours after intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1:1 mRNA FLuc:hEPO administered at 10 μg / 40 μL. N=3 / group. Positive controls used were LNP1 and LNP2 (n=3 / group), and negative controls were mice administered PBS (n=2). [Figure 15D] Figure 15D shows the right and left lymph node radiance (mean radiance p / s / cm2 / sr) 4 hours after intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1:1 mRNA FLuc:hEPO in mice administered at 10 μg / 40 μL. N=3 / group. Positive controls used were LNP1 and LNP2 (n=3 / group), and negative controls were mice administered PBS (n=2). DETAILED DESCRIPTION OF THE INVENTION

[0137] Featured herein are modified lipid compositions comprising (a) a structural component comprising one or more lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipid ceramide, glucosylceramide, lactosylceramide, galactosylcholesterol, and glucosylcholesterol, and (b) an ionized lipid capable of increasing cellular uptake of the structural component. In some embodiments, the modified lipid composition may be a complex lipid particle (CLP) comprising natural lipid extracts and derivatives, such as soybean lipid extracts or soybean-derived lipids, and the ionized lipid.

[0138] These modified lipid compositions may be formulated with one or more heterologous functional agents, such as polynucleotides, and used as delivery vehicles for these heterologous functional agents (eg, polynucleotides).

[0139] composite lipid particles The complex lipid particles (CLPs) described herein comprise a variety of lipids, including structured lipids extracted from one or more natural sources (e.g., plants or bacteria). In some embodiments, the complex lipid particles are natural messenger packs (NMPs) incorporating natural lipid extracts. In some embodiments, the complex lipid particles are lipid-reconstituted natural messenger packs (LNMPs) incorporating natural lipid extracts and at least one exogenous ionizable lipid.

[0140] In some embodiments, the composite lipid particles contain three or more lipids extracted or derived from soybeans.

[0141] The complex lipid particles may also include at least an exogenous ionizable lipid, the ionizable lipid having two or more of the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of which is at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) N:P ratio of at least 3.

[0142] The composite lipid particles may contain from 10% to 99% w / w of structured lipids derived from lipid structures from one or more natural sources, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 99% w / w of lipids derived from lipid structures from one or more natural sources.

[0143] In some embodiments, the composite lipid particles comprise about 10-95 w / w% natural (e.g., plant, bacterial) lipids, for example, about 25-95 w / w%, about 30-95 w / w%, about 35-95 w / w%, about 40-95 w / w%, about 45-95 w / w%, about 50-95 w / w%, about 55-95 w / w%, about 60-95 w / w%, about 65-95 w / w%, about 70-95 w / w%, about 75-95 w / w%, about 80-95 w / w%, or about 85-95 w / w% natural lipids, based on the total lipid content in the composite lipid formulation.

[0144] The composite lipid particles may contain 3 to 1000 lipids extracted from one or more natural (e.g., plant, bacterial) sources. In some embodiments, the natural source is a plant, plant extract, or plant fragment or part. In some embodiments, the natural source is a bacterium, bacterial fragment, or bacterial part. In some embodiments, the natural source is lemon. In some embodiments, the natural source is soybean. In other embodiments, the natural source is E. coli.

[0145] In some embodiments, the composite lipid particles contain at least 10 naturally occurring lipids belonging to one or more of the species selected from the group consisting of fatty acyl (FA), fatty acyl complex, phospholipid, glycerolipid, glycolipid, glycerophospholipid, sphingolipid, wax, and sterol. For example, composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 naturally occurring lipids belonging to one or more species selected from the group consisting of fatty acyl (FA), fatty acyl complex, phospholipid, glycerolipid, glycolipid, glycerophospholipid, sphingolipid, wax, and sterol. In some embodiments, composite lipid particles contain lipids of at least two or at least three of these different species.

[0146] In some embodiments, composite lipid particles contain at least 10 naturally occurring lipids belonging to one or more species selected from the group consisting of glycerolipids, sphingolipids, and sterols. For example, composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 naturally occurring lipids belonging to one or more species selected from the group consisting of glycerolipids, sphingolipids, and sterols. In some embodiments, composite lipid particles contain at least two or at least three lipids from these different species.

[0147] In some embodiments, the composite lipid particles may contain one or more glycerolipids (GL) or glycerophospholipids (GP), which may also include glycolipids.

[0148] In some embodiments, complex lipid particles may contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids, triacylglycerols (TG), and sulfolipids (SL). In some embodiments, CLPs contain one or more glycerophospholipids (GP) selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI). In some embodiments, complex lipid particles contain one or more sphingolipids (SP) selected from the group consisting of sulfolipids (SL), glycosyl inositol phosphorylceramide (GIPC), glucosylceramide (GCer), ceramide (Cer), and free long-chain bases (LCB). In some embodiments, the complex lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol, β-sitosterol, Δ5-avenasterol, brassicasterol, avenasterol, 4-desmethylsterols, 4α-monomethylsterols, Δ5-sterol, Δ7-sterol, α-spinasterol, Δ5,Δ7-sterol, phytostanol, and sitosterol.

[0149] CLPs are fatty acids, fatty esters, fatty aldehydes, fatty amides, acyclic oxylipins, cyclic oxylipins, glycerolipids, monoadylglycerol, diadylglycerol, triadylglycerol, estolides, glycosylmonoacylglycerol, sulfoquinovosylmonoacylglycerol, monogalactosylmonoacylglycerol, digalactosylmonoacylglycerol, sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, glycosyldiacylglycerol, and glycerophospholipids (glyceropp). The composite lipid particles may contain one or more naturally occurring lipids belonging to one or more classes or subclasses selected from the group consisting of: phospholipids, phospholipids, lysophospholipids, phosphatidylinositol phosphates, n-modified phospholipids, oxygenated / oxidized phospholipids, shingolipids, sphingoid bases, ceramides, phosphocereamides, glycophingolipids, sterols, cholesterol, cholesteryl esters, steryl esters, bile acids, steryl glycosides, and acyl steryl glycosides. The composite lipid particles may contain one or more naturally occurring lipids belonging to one or more classes or subclasses selected from the group consisting of the classes or subclasses listed above. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 naturally occurring lipids belonging to one or more of the classes or subclasses selected from the group consisting of the classes or subclasses listed above.

[0150] In some embodiments, the CLP is selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsterylglycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acid, fatty acid ester of hydroxyl fatty acid, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol. The lipid composition of the present invention contains one or more naturally occurring lipids belonging to one or more of the subclasses selected from the group consisting of phosphoinositides, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, steryl esters, stigmasterol, sulfatides, sulfonolipids, sphingomyelin, sulfoquinovosyl diacylglycerosyl, sterols, and triacylglycerols. In some embodiments, the composite lipid particles contain at least 10 naturally occurring (e.g., plant, bacterial) lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 naturally occurring lipids belonging to one or more of the subclasses selected from the group consisting of the subclasses listed above.

[0151] The complex lipid particles may contain 10 or more naturally occurring lipids belonging to one or more subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemibismonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholines, lysophosphatidylethanolamines, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylethanol, phosphatidylglycerols, phosphatidylinositols, sulfoquinovosyl diacylglycerosyl, and sterols. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 400, at least 500, at least 600, at least 700, or at least 800 naturally occurring lipids belonging to one or more of the subclasses selected from the group consisting of the subclasses listed above.

[0152] Composite lipid particles may contain naturally occurring lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different species or subclasses of lipids from natural sources. In some embodiments, composite lipid particles contain at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different species or subclasses of lipids from a single natural source (e.g., exclusively from plant sources or exclusively from bacterial sources). In some embodiments, CLPs may contain naturally occurring lipids from only one species or only one subclass of lipids from natural sources.

[0153] The identity (and species and subclasses) and quantity of lipids extracted from natural sources can be analyzed in lipidomic analyses by dissolving the lipid extract or complex lipid particles in a compatible solvent and analyzing them by mass spectrometry (e.g., MS / MS). Other known methods, such as charged aerosol detection (CAD) (e.g., HPLC-CAD, normal-phase high-performance liquid chromatography (NP-HPLC-CAD), or reverse-phase high-performance liquid chromatography (RP-HPLC-CAD)), can also be used.

[0154] The composite lipid particles may contain all or a fraction of the lipid species present in the lipid structure from a particular natural source, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially 100% of the lipid species present in the lipid structure from a particular natural source.

[0155] Complex lipid particles may contain reduced or minimized proteinaceous materials endogenous to one or more natural sources. For example, complex lipid particles may contain less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, less than 30 wt.%, less than 25 wt.%, less than 20 wt.%, less than 15 wt.%, less than 10 wt.%, less than 9 wt.%, less than 8 wt.%, less than 7 wt.%, less than 6 wt.%, less than 5 wt.%, less than 4 wt.%, less than 3 wt.%, less than 2 wt.%, less than 1 wt.%, less than 0.5 wt.%, less than 0.1 wt.%, or may be essentially free of proteinaceous materials endogenous to one or more natural sources. In some examples, the lipid bilayer of complex lipid particles does not contain protein. To calculate the w / w % of the remaining protein material that is endogenous to one or more natural sources, divide the protein concentration by the concentration of the natural lipid extract and then multiply by 100. Alternatively, w / w % is calculated as the percentage of the mass of the total protein that is endogenous to one or more natural sources, based on the mass of the total lipid extract.

[0156] Complex lipid particles can comprise reduced or minimized residual dsDNA material that is endogenous to one or more natural sources.For example, complex lipid particles can contain less than 15 w / w%, less than 10 w / w%, less than 5 w / w%, less than 1 w / w%, less than 0.5 w / w%, less than 0.1 w / w%, less than 0.05 w / w%, less than 0.01 w / w%, less than 0.005 w / w%, less than 0.001 w / w%, or can be essentially free of residual dsDNA material that is endogenous to one or more natural sources.In some examples, the lipid bilayer of complex lipid particles does not contain residual dsDNA.To calculate the w / w% residual dsDNA material that is endogenous to one or more natural sources, divide the adjusted total dsDNA concentration by the concentration of natural lipid extract, and then multiply by 100. Alternatively, w / w% is calculated as the percentage of the mass of total remaining dsDNA that is endogenous to one or more natural sources, based on the mass of the total lipid extract.

[0157] In some embodiments, composite lipid particles further incorporate synthetic structured lipids, such as neutral lipids. In some embodiments, the structured lipid component of a composite lipid particle can comprise 10% to 99% w / w of structured lipids derived from synthetic lipid structures, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 99% w / w of lipids derived from synthetic lipid structures.

[0158] In addition to the exogenous ionized lipid, the composite lipid particles may further comprise at least two other exogenous lipids. The composite lipid particles may comprise at least 1 wt.%, at least 2 wt.%, at least 5 wt.%, at least 10 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, or about 95 wt.% of the exogenous lipid. Exemplary exogenous lipids include ionized lipids, synthetic structured lipids, sterols, and PEG-lipid conjugates. The composite lipid particles may further comprise at least two exogenous lipids. In some embodiments, the composite lipid particles contain ionized lipids, sterols, and PEG-lipid conjugates. Additional exogenous lipids suitable for inclusion in composite lipid particles are described below.

[0159] In some embodiments, the CLP comprises a naturally occurring lipid comprising a fatty acid-derived tail, wherein the fatty acid-derived tail of the naturally occurring lipid is Approximately 5-20% fatty acids 16:0 Approximately 0-10% fatty acids 18:1 (C9) Approximately 0-10% fatty acids 18:1 (C7) Approximately 5-30% fatty acids 18:2 Approximately 2-20% fatty acids 18:3.

[0160] In some embodiments, the CLP comprises a phosphatidylcholine (PC) lipid comprising a fatty acid-derived tail, wherein the fatty acid-derived tail of the PC lipid is Approximately 10-20% fatty acids 16:0 Approximately 2-5% fatty acids 18:0 Approximately 7-15% fatty acids 18:1 Approximately 50-75% fatty acids 18:2 Approximately 2-10% fatty acids 18:3.

[0161] In some embodiments, the CLP comprises a phosphatidylethanolamine (PE) lipid comprising a fatty acid-derived tail, wherein the fatty acid-derived tail of the PE lipid comprises: Approximately 0.25-5% fatty acids 14:0 Approximately 25-45% fatty acids 16:0 Approximately 5-15% fatty acids 16:1 Approximately 10-25% fatty acids 17:0 Approximately 25-45% fatty acids 18:1 Approximately 2-7% fatty acid 19:0.

[0162] In some embodiments, the CLP contains natural lipids belonging to the subclasses of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, and is selected from the group consisting of: Approximately 50-75 wt / wt% phosphatidylethanolamine (PE) Approximately 15-30 wt / wt% phosphatidylglycerol (PG) Contains approximately 5-15 wt / wt% cardiolipin (CL).

[0163] In some embodiments, the CLP is Approximately 10-50 wt / wt% phosphatidylcholine (PC) Approximately 5-50 wt / wt% phosphatidylethanolamine (PE) Approximately 0-15 wt / wt% triacylglycerol (TG) Approximately 5-35 wt / wt% hexosylceramide (HexCer) Approximately 0-5 wt / wt% phosphatidylglycerol (PG) Approximately 0-7 wt / wt% phosphatidylserine (PS) Approximately 0-10 wt / wt% phosphatidylinositol (PI) It contains natural lipids, including approximately 0-5 wt / wt% cardiolipin (CL).

[0164] In some embodiments, the composite lipid particles contain less than 12 wt% chloroplasts endogenous to one or more natural sources, hi some embodiments, the composite lipid particles contain less than 20 wt%, less than 15 wt%, less than 10 wt%, less than 5 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.1 wt% chloroplasts endogenous to one or more natural sources.

[0165] In some embodiments, the composite lipid particles contain less than 5% w / w of exogenous antioxidants.

[0166] In some embodiments, the CLP contains natural lipids, including about 0-20 wt / wt% cardiolipin (CL).

[0167] structural components The structural component comprises one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol. The structural component may also comprise one or more of those structural lipids listed in Tables 1-7 and 9-13.

[0168] In some embodiments, the structural component is derived from a natural source. In some embodiments, the natural source may be a plant or an animal. In some embodiments, the animal is a pig or a cow. In some embodiments, the plant is a soybean. In other embodiments, the structural component is synthetically derived.

[0169] The structural component has a lipid (e.g., lipid bilayer, unilamellar, or multilamellar) structure comprising a native extracellular vesicle (EV), or a fragment, portion, or extract thereof (e.g., a lipid extract).

[0170] In some embodiments, the structural component comprises isolated natural extracellular vesicles. Natural EVs refer to enclosed lipid bilayer structures that naturally occur in a source. Natural EVs are derived from natural sources (e.g., plants, bacteria, animals, etc.) that contain structural lipids and may also contain natural proteins and / or natural nucleic acids and / or carbohydrate moieties contained in nanoparticles. Natural EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different lipid species. In one embodiment, the natural source is soybean, pig brain, or beef heart.

[0171] The structural component may comprise a natural EV, or a fragment, portion, or extract thereof. In some embodiments, the plant EV has a diameter of about 5 to 1000 nm. For example, the structural component may have a diameter of about 5 to 50 nm, about 50 to 100 nm, about 100 to 150 nm, about 150 to 200 nm, about 200 to 250 nm, about 250 to 300 nm, about 300 to 350 nm, about 350 to 400 nm, about 400 to 450 nm, about 450 to 500 nm, about 500 to 550 nm, about 550 to 600 nm, about 600 to 650 nm, about 650 to 700 nm, or about 650 to 700 nm. The present invention may include naturally occurring EVs, or fragments, portions, or extracts thereof, having an average diameter of about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1250 nm, about 1250-1500 nm, about 1500-1750 nm, or about 1750-2000 nm. In some examples, the structural component may comprise a native EV, or a fragment, portion, or extract thereof, having an average diameter of about 5-950 nm, about 5-900 nm, about 5-850 nm, about 5-800 nm, about 5-750 nm, about 5-700 nm, about 5-650 nm, about 5-600 nm, about 5-550 nm, about 5-500 nm, about 5-450 nm, about 5-400 nm, about 5-350 nm, about 5-300 nm, about 5-250 nm, about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm, or 5-25 nm. In particular examples, the structural component may comprise a native EV, or a fragment, portion, or extract thereof, having an average diameter of about 50-200 nm, about 50-300 nm, about 200-500 nm, or about 30-150 nm. Various methods standard in the art (e.g., dynamic light scattering) can be used to measure the particle size of native EVs, or fragments, portions, or extracts thereof.

[0172] In some instances, the structural component is at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 x 10 4 nm 2 , at least 1 x 10 5 nm2 , at least 1 x 10 6 nm 2 , or at least 2 × 10 6 nm 2 In some examples, the structural component may comprise a native EV or a fragment, portion, or extract thereof having an average surface area of ​​77 nm 2 ~3.2×10 6 nm 2 (e.g., 77-100 nm 2 , 100~1000nm 2 , 1000 to 1 × 10 4 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~3.2×10 6 nm 2 The present invention may comprise a naturally occurring EV, or a fragment, portion, or extract thereof, having an average surface area of ​​1000 nm to 1000 nm.

[0173] In some instances, the structural component is at least 65 nm 3 (e.g., at least 65 nm 3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 x 10 4 nm 3 , at least 1 x 10 5 nm 3 , at least 1 x 10 6 nm 3 , at least 1 x 10 7 nm 3 , at least 1 x 10 8 nm 3 , at least 2 × 10 8 nm 3 , at least 3 × 10 8 nm 3 , at least 4 × 10 8 nm 3 , or at least 5 × 10 8 nm 3In some examples, the structural component may comprise a native EV, or a fragment, portion, or extract thereof, having an average volume of 65 nm 3 ~5.3×10 8 nm 3 (e.g., 65 to 100 nm 3 , 100~1000nm 3 , 1000 to 1 × 10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~5.3×10 8 nm 3 The present invention may comprise a native EV or a fragment, portion, or extract thereof, having an average volume of 1000 μg / mL or 1000 μg / mL.

[0174] In some examples, the structural component may comprise an entire native EV. Alternatively, the structural component may comprise a fragment, portion, or extract of the complete surface area of ​​a native EV vesicle (e.g., a fragment, portion, or extract comprising less than 100% (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of the complete surface area of ​​the vesicle). The fragment, portion, or extract may be of any shape, such as a circumferential fragment, a spherical fragment (e.g., a hemisphere), a curved fragment, a straight fragment, or a flat fragment. When the fragment is a spherical fragment of a vesicle, the spherical fragment may represent a fragment resulting from the division of the spherical vesicle along a pair of parallel lines or a fragment resulting from the division of the spherical vesicle along a pair of non-parallel lines. Thus, the structural component may contain multiple intact native EVs, multiple fragments, portions, or extracts of native EVs, or a mixture of intact EVs and EV fragments. Those skilled in the art will appreciate that the ratio of intact EVs to fragmented native EVs will depend on the particular isolation method used. For example, grinding or blending native or fragmented EVs can produce structural components containing a higher percentage of fragments, portions, or extracts of native EVs than non-destructive extraction methods such as vacuum infiltration.

[0175] In cases where the structural component comprises a fragment, portion, or extract of a native EV, the EV fragment, portion, or extract has an average surface area that is less than the average surface area of ​​an intact vesicle, e.g., 77 nm 2 , 100 nm 2 , 1000nm 2 , 1×10 4 nm 2 , 1×10 5 nm 2 , 1×10 6 nm 2 , or 3.2 × 10 6 nm 2 In some instances, the structural component may have an average volume less than the average volume of a complete vesicle (e.g., 65 nm 3 , 100 nm 3 , 1000nm 3 , 1×10 4 nm 3, 1×10 5 nm 3 , 1×10 6 nm 3 , 1×10 7 nm 3 , 1×10 8 nm 3 , or 5.3 × 10 8 nm 3 The structural component may comprise native EVs or fragments, portions, or extracts thereof, having an average volume of less than 1000 uL. The structural component may comprise native EV fragments and / or extracted lipids, or a mixture thereof.

[0176] In some embodiments, the structural component is not derived from a natural source, hi some embodiments, the structural component is synthetically derived.

[0177] In some embodiments, the structural component may comprise at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% structural lipids.

[0178] Manufacturing of structural components The structural components may be naturally-occurring lipids produced from native EVs, or fragments, portions, or extracts thereof (e.g., lipid extracts). An exemplary method for producing structural components (including naturally-occurring lipids) includes (a) providing an initial sample from a natural source and (b) isolating a crude native fraction from the initial sample, wherein the crude native fraction has a reduced level of at least one contaminant or undesirable component from the source relative to the level in the initial sample. The method may further include an additional step including (c) purifying the crude native fraction, thereby producing a pure structural component having a reduced level of at least one contaminant or undesirable component from the natural source relative to the level in the unpurified EV fraction.

[0179] In some examples, structural components (including naturally occurring lipids) can be isolated from natural sources by a process comprising the following steps: (a) providing an initial sample from the natural source; (b) isolating a crude natural fraction from the initial sample, wherein the crude natural fraction has a reduced level of at least one contaminant or undesirable component from the natural source compared to the level in the initial sample (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 810%, 820%, 830%, 840%, 85 0%, 90%, 95%, 96%, 98%, 99%, or 100% reduced level), and (c) purifying the crude native fraction, thereby producing pure structural components having a reduced level of at least one contaminant or undesired component from the natural source compared to the level in the crude EV fraction (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced level).

[0180] Structural components (containing naturally occurring lipids) may be produced by whole cell extraction. For example, cells may be first disrupted, and then intracellular and cell membrane / cell wall-associated lipids and extracellular hydrocarbons may be separated from the cell mass, such as by using centrifugation. Intracellular lipids produced in natural sources (e.g., bacteria, plants, animals, etc.) are extracted in some embodiments after lysing the source cells. Additional methods for extracting structural lipids (containing naturally occurring lipids) may be found in U.S. Patent No. 8,592,188, which is incorporated herein by reference in its entirety. Structural components may also be produced by synthetic means.

[0181] Structural components (containing naturally occurring lipids) can be produced from natural sources by various methods. EVs can be separated from the source by either disruptive (e.g., crushing or blending the natural source) or non-destructive (e.g., washing or vacuum infiltration of the natural source) methods. For example, the source can be vacuum infiltrated, crushed, blended, or a combination thereof to isolate EVs from the source. For example, the isolating step can involve vacuum infiltrating the source (e.g., using a vesicle isolation buffer). Alternatively, the isolating step can involve crushing or blending the source to release the EVs.

[0182] Upon isolation of native EVs, structural components can be separated or collected into a crude native fraction (e.g., an apoplastic fraction). For example, the separating step can involve using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the structural components into a crude native fraction, separating the native-containing fraction from larger contaminants, including native tissue debris or cells. Thus, the crude native fraction will have a reduced number of large contaminants compared to the initial sample from the source. Depending on the method used, the crude native fraction can further contain reduced levels of native organelles compared to the initial sample from the source.

[0183] In some examples, the isolation step may involve centrifugation (eg, differential centrifugation or ultracentrifugation) and / or filtration.

[0184] The crude native fraction can be further purified by additional purification methods. For example, the crude native fraction can be purified by ultracentrifugation, for example, using a density gradient (iodixanol or sucrose), and / or other approaches to remove aggregated components (for example, precipitation or size exclusion chromatography). The resulting pure structural components can have reduced levels of contaminants or undesirable components from the natural source (for example, protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof) compared to one or more fractions produced during a previous separation step, or compared to a pre-established threshold level, for example, a commercially published specification. For example, a pure structural component may contain reduced levels (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) of contaminants or other undesirable components, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) of contaminants or other undesirable components compared to the levels in the initial sample, or be substantially free of contaminants or other undesirable components (e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof).

[0185] For example, protein aggregates can be removed from the structural components. For example, the structural components can be taken through a range of pH (e.g., measured using a pH probe) to precipitate protein aggregates in solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9, or pH 11, for example, by adding sodium hydroxide or hydrochloric acid. Once the solution reaches the specified pH, it can be filtered to remove the particles. Alternatively, the structural components can be coagulated using the addition of a charged polymer, such as Polymin-P or Praestol 2640. Briefly, Polymin-P or Praestol 2640 is added to the solution and mixed with an impeller. The solution can then be filtered to remove the particles. Alternatively, the aggregates can be dissolved by increasing the salt concentration. For example, NaCl can be added to the structural components, for example, to 1 mol / L. The solution can then be filtered to isolate the structural components. Alternatively, the aggregates can be dissolved by increasing the temperature. For example, the structural components can be heated with mixing until the solution reaches a uniform temperature of 50°C, for example, for 5 minutes. The structural component mixture can then be filtered. Alternatively, soluble contaminants from the structural components can be separated using a size-exclusion chromatography column according to standard procedures; structural lipids elute in the first fraction, while proteins and ribonucleoproteins, as well as some lipoproteins, elute later. The efficiency of protein aggregate removal can be determined by measuring and comparing the protein concentrations before and after protein aggregate removal via BCA / Bradford protein quantification.

[0186] Alternatively, structured lipids (eg, soy-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, glucosylcholesterol) can be obtained from commercial sources.

[0187] Any of the production methods described herein can be supplemented with any quantitative or qualitative method known in the art to characterize or identify structural components (e.g., structural lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol) at any step of the production process. For example, structural components (e.g., structural lipids selected from the group consisting of soybean-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol) can be characterized by a variety of analytical methods known in the art that allow visualization, quantification, or qualitative characterization (e.g., composition identification), such as various analytical methods for estimating yield, concentration, purity, composition, or size, for example, microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopy (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis).

[0188] During the manufacturing process, the structural components (e.g., structural lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, glucosylcholesterol) can optionally be prepared to provide an increased concentration of the structural components (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) compared to the levels in the control or initial sample. The structural component may comprise from about 0.1% to about 100% of the modified lipid composition, e.g., from about 0.01% to about 100%, from about 1% to about 99.9%, from about 0.1% to about 10%, from about 1% to about 25%, from about 10% to about 50%, from about 50% to about 99%, or from about 75% to about 100%.

[0189] Modified Lipid Composition - Lipid Modification of Structural Components The modified lipid composition comprises (a) a structural component, and (b) an ionizable lipid. The ionizable lipid and / or other exogenous lipids are used to modify the structural component.

[0190] Modification refers to modifying structural components containing lipid structures (e.g., lipid bilayers, monolayers, multilayers, e.g., vesicular lipid structures), which are derived from a natural source (e.g., enriched, isolated, or purified from a natural source), and the lipid structures are disrupted (e.g., disrupted by lipid extraction) and reorganized or reconstituted in a liquid phase (e.g., a liquid phase containing cargo) using standard methods, e.g., reconstituted by methods including lipid film hydration and / or solvent injection, as described herein, to produce a modified lipid composition. In some embodiments, the structural components are modified by reconstituting a membrane containing the structural components in the presence of ionized lipids.

[0191] In some embodiments, the structural component comprises a soy-derived lipid, and the structural lipid is modified by reconstituting a membrane comprising the purified soy-derived lipid of the structural component with an ionized lipid.

[0192] Alternatively, the modified lipid composition can be made using a microfluidic device, such as the NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems).

[0193] In some embodiments, the modified lipid composition is produced by a process comprising the following steps: (a) providing a structural component (e.g., a structural component purified as described above); (b) processing the structural component to produce a lipid membrane; (c) reconstituting the lipid membrane with an organic solvent or combination of solvents, thereby producing a lipid solution; and (d) processing the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, in the presence of ionized lipids, thereby producing the modified lipid composition.

[0194] In some examples, processing structural components to produce lipid membranes includes extracting lipids using the Briigh-Dyer method (Bligh and Dyer, J Biolchem ​​Physiol, 37: 911-917, 1959), which is incorporated herein by reference in its entirety. The extracted lipids can be provided as a stock solution, for example, a solution in chloroform:methanol. Creating lipid membranes can include, for example, evaporating the solvent with a stream of inert gas (e.g., nitrogen).

[0195] The method may further include, if desired, sonication, freeze / thaw treatment, and / or lipid extrusion, for example, to reduce the size of the reconstituted modified lipid composition.

[0196] structural lipids In some embodiments, the structural component comprises structured lipids derived from natural sources. The modified lipid composition may comprise 10% to 100% lipids derived from lipid structures from natural sources, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lipids derived from lipid structures from natural sources. The modified lipid composition may comprise all or a fraction of the lipid species present in the lipid structures from the modified lipid composition, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the lipid species present in the lipid structures from natural sources. The modified lipid composition may not contain any protein species present in the lipid structure from natural source, or may contain a fraction thereof, or all of them, for example, may contain 0%, less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, or 100% of the protein species present in the lipid structure from natural source.In some examples, the lipid bilayer of the modified lipid composition does not contain protein.In some examples, the lipid structure of the modified lipid composition contains a reduced amount of protein compared to the lipid structure from natural source.

[0197] In some embodiments, the structured lipids of the modified lipid composition are selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipids, ceramides, glucosylceramides, lactosylceramides, galactosylcholesterol, and glucosylcholesterol. In some embodiments, the structured lipids of the modified lipid composition comprise one or more of the structured lipids listed in Tables 1-7 and 9-13.

[0198] In some embodiments, the structured lipids of the modified lipid composition are extracted from soybeans.

[0199] exogenous lipids The structural components may be modified to contain a heterologous agent (e.g., a cell membrane permeabilizing agent) that can increase cellular uptake (e.g., animal cell uptake (e.g., mammalian cell uptake, e.g., human cell uptake), plant cell uptake, natural cell uptake, or fungal cell uptake) compared to the unmodified structural component. For example, the modified structural component may include (e.g., loaded, encapsulated, or complexed with) or be formulated with (e.g., suspended or resuspended in a solution containing) a cell membrane permeabilizing agent such as an ionizable lipid. Each of the modified structural components may comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0200] The modified lipid composition can include one or more exogenous lipids, e.g., lipids exogenous to the lipid source (e.g., derived from a source other than the source from which the structural components are produced). The total lipid of the modified lipid composition can include 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipid. In some embodiments, exogenous lipid (e.g., ionizable lipid) is added in an amount of 25% or 40% (w / w) of the total lipid in the preparation. In some embodiments, the exogenous lipid is added to the preparation before step (b), e.g., mixed with the structural lipid before step (b).

[0201] Exemplary exogenous lipids include ionizable lipids.

[0202] Exogenous lipids can also include cationic lipids.

[0203] In some examples, the exogenous lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), DLin-MC3-DMA (MC3), dioleoyl-3-trimethylammonium propane (DODAP), DC-cholesterol, DOTAP, ethyl PC, GL67, DLin-KC2-DMA (KC2), MD1 (cKK-E12), OF2, EPC, ZA3-E The ionizable or cationic lipid may be selected from p10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), cationic sulfonamide amino lipids, amphipathic zwitterionic amino lipids, DODAC, DOBAQ, YSK05, DOBAT, DOBAQ, DOPAT, DOMPAQ, DOAAQ, DMAP-BLP, DLinDMA, DODMA, DOTMA, DSDMA, DOSPA, DODAC, DOBAQ, DMRIE, DOTAP cholesterol, GL67A, and 98N12-5, and combinations thereof.

[0204] In some embodiments, the exogenous lipid may be an ionizable lipid or cationic lipid selected from C12-200, MC3, DODAP, DC-cholesterol, DOTAP, ethyl PC, GL67, KC2, MD1, OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), cationic sulfonamide amino lipid, and amphipathic zwitterionic amino lipid, and combinations thereof. In some embodiments, the ionizable lipid is selected from C12-200, MC3, DODAP, and DC-cholesterol, or combinations thereof. In some examples, the ionizable lipid is an ionizable lipid. In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200) or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA (MC3). In some examples, the exogenous lipid is a cationic lipid. In some embodiments, the cationic lipid is DC-cholesterol or dioleoyl-3-trimethylammonium propane (DOTAP).

[0205] In some examples, the modified lipid composition comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionized lipids.

[0206] In some examples, the modified lipid composition comprises at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% ionized lipid, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionized lipid, e.g., about 30%-75% ionized lipid (e.g., about 30%-75% ionized lipid). In some embodiments, the modified lipid composition comprises 25% C12-200 by mole. In some embodiments, the modified lipid composition comprises 35% C12-200 by mole. In some embodiments, the modified lipid composition comprises 50% C12-200 by mole. In some embodiments, the modified lipid composition comprises 40% MC3 by mole. In some embodiments, the modified lipid composition comprises 50% C12-200 by mole. In some embodiments, the modified lipid composition comprises 20% or 40% DC-cholesterol by mole. In some embodiments, the modified lipid composition comprises 25% or 40% DOTAP by mole.

[0207] The agent may increase the uptake of the modified lipid composition as a whole, or may increase the uptake of a portion or component of the modified lipid composition (e.g., a heterofunctional agent) carried by the modified lipid composition. The degree of increased cellular uptake may vary depending on the source from which the composition is delivered, the modified lipid composition, and other modifications made to the modified lipid composition. For example, the modified lipid composition may have at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increased cellular uptake (e.g., animal cell uptake, plant cell uptake, natural cell uptake, or fungal cell uptake) compared to the unmodified structural component. In some examples, the increased cellular uptake is at least 2-fold, 4-fold, 5-fold, 10-fold, 100-fold, or 1000-fold increased cellular uptake compared to the unmodified structural component.

[0208] In some embodiments, modified lipid compositions modified with ionizable lipids encapsulate negatively charged polynucleotides more efficiently than modified lipid compositions not modified with ionizable lipids. In some aspects, modified lipid compositions modified with ionizable lipids alter biodistribution compared to modified lipid compositions not modified with ionizable lipids. In some aspects, modified lipid compositions modified with ionizable lipids alter (e.g., increase) fusion with the endosomal membrane of target cells compared to modified lipid compositions not modified with ionizable lipids.

[0209] Ionized lipids In some embodiments, the ionizable lipid has at least one (e.g., one, two, three, four, or all five) of the following listed characteristics: (i) at least two ionizable amines (e.g., at least two, at least three, at least four, at least five, at least six, or more than six ionizable amines, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ionizable amines); (ii) at least three lipid tails (e.g., at least three, at least four, at least five, at least six, or more than six lipid tails, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 lipid tails), each of which is independently at least 6 carbon atoms in length (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or more than 18 carbon atoms in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 carbon atoms in length); (iii) an acid dissociation constant (pKa) of about 4.5 to about 7.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 (e.g., a pKa of about 6.5 to about 7.5 (e.g., a pKa of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5)); (iv) ionizable amines and heteroorganic groups, and (v) an N:P (amine of ionizable lipid:phosphate of nucleic acid, e.g., mRNA) ratio of at least 10;

[0210] In some embodiments, the modified lipid composition has an N / P ratio of about 12 to about 17, e.g., an N / P ratio of about 15±1, or an N / P ratio of about 15±0.5. In some embodiments, the N / P ratio is about 15. Alternatively, the ionizable lipid is characterized by an N / P ratio of about 3 to about 10, e.g., an N / P ratio of about 6±1, or an N / P ratio of about 6±0.5. In some embodiments, the N / P ratio is about 6.

[0211] In some embodiments, the ionizable lipid is not selected from 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), and 98N12-5.

[0212] In some embodiments, the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0213] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group. In some embodiments, the heteroorganic group is hydroxyl. In some embodiments, the heteroorganic group comprises a hydrogen bond donor. In some embodiments, the heteroorganic group comprises a hydrogen bond acceptor. In some embodiments, the heteroorganic group is -OH, -SH, -(CO)H, -COH, -NH, -CONH, which may be substituted C1-C6 alkoxy or fluorine.

[0214] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

[0215] The ionizable lipid may comprise one of the compounds in groups i) through iv), as discussed below.

[0216] Ionized lipid compounds i) In some embodiments, the ionizable lipid has the following formula I: [ka] a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, A is independently C1 to C 16 branched or unbranched alkyl, or C1-C 16branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; B is independently C1 to C 16 branched or unbranched alkyl, or C1-C 16 branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; each X is independently a biodegradable moiety; and W is [ka] wherein: R5 is OH, SH, or NR 10 R 11 and Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, and Q is O, S, or NR 13 where R 13are H and C1-C5 alkyl, respectively.

[0217] In some embodiments, B is C 20 It is alkyl.

[0218] In some embodiments, W in formula (I) is alternatively: [ka] wherein V is branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 heteroalkylene, which may be substituted with one or more OH, SH, and / or halogen groups; Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, each v is independently 0, 1, 2, 3, 4, or 5; R 17 is OH, SH, or N(CH3)2, and Each u is independently 1, 2, 3, 4, or 5.

[0219] In some embodiments, W in formula (I) is alternatively: [ka] wherein V is C2~C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene, Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; and Each u is independently 1, 2, 3, 4, or 5.

[0220] In some embodiments, W in formula (I) is alternatively: [ka] wherein R 14 is a heterocycle, Each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0221] In some embodiments, W in formula (I) is alternatively: [ka] wherein Z is O, S, -C((CH2) v N(R 15 )2)-, or N(R 15 ) where R 15 is H, C1-C4 branched or unbranched alkyl, and v is 0, 1, 2, 3, 4, or 5; R 10 are each independently H or C1-C3 alkyl, Each u is independently 0, 1, 2, 3, 4, or 5.

[0222] In some embodiments, W in formula (I) is alternatively: [ka] wherein each Y is a divalent heterocycle; Q is O, S, or NH; Each u is independently 1, 2, 3, 4, or 5.

[0223] In some embodiments, W in formula (I) is alternatively: [ka] wherein R 14 is a heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , or C(S)NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, which may be substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle, R 16 is H, ═O, ═S, or CN, Each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0224] In some embodiments, W in formula (I) is alternatively: [ka] wherein T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle which contains one or more -(CH) v OH, -(CH2) v SH, and / or -(CH2) v - optionally substituted with a halogen group, Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, R 17 is OH, SH, or N(CH3)2, Each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0225] In some embodiments, W in formula (I) is alternatively: [ka] wherein T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle; and Each u is independently 1, 2, 3, 4, or 5.

[0226] In some embodiments, when Z is present, adjacent R and R are OH, NR 10 R 11 , or SH.

[0227] In some embodiments, the heterocycle is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic or heteroaromatic.

[0228] In some embodiments, the ionizable lipid has formula (IX): [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 independently represent H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, together with the carbon atoms to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, Each R3 and each R4 independently represent H, C2 to C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; V is branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 heteroalkylene, which may be substituted with one or more OH, SH, and / or halogen groups; Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 wherein each v is independently 0, 1, 2, 3, 4, or 5;17 is OH, SH, or N(CH3)2, and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0229] In some embodiments, V is a branched or unbranched C2-C3 alkylene. In some embodiments, V is a C2-C3 alkylene substituted with OH. In some embodiments, V is a branched or unbranched C2-C3 alkenylene. In some embodiments, each R6 is independently H or methyl.

[0230] In some embodiments, the ionizable lipid has the formula: [ka] and the definitions of the variables are the same as those in formula (X).

[0231] In some embodiments, the present disclosure provides an ionizable lipid of formula (XI): [ka] With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 independently represent H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, together with the carbon atoms to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle, Each R3 and each R4 independently represent H, C2 to C 14 Branched or unbranched alkyl (e.g., C3-C 10branched or unbranched alkyl), or C3-C 10 provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each s is independently 1, 2, 3, 4, or 5; T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle which contains one or more -(CH) v OH, -(CH2) v SH, -(CH2) v - optionally substituted with a halogen group, Each R7 and each R8 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 17 is OH, SH, or N(CH3)2, Each v is independently 0, 1, 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0232] In some embodiments, T is -(CH) v a divalent heterocycle optionally substituted with OH (eg, a divalent piperazine or a divalent dioxopiperazine), where v is independently 0, 1, or 2;

[0233] In some embodiments, in each of the above formulas, X is selected from the group consisting of -OC(O)-, -C(O)O-, -SS-, -N(R 18 )C(O)-, -C(O)N(R 18 )-, -C(OR 13 )-O-, -C(O)O(CH2) a -, -OC(O)(CH2) a -, -C(O)N(R 18 )(CH2) a -, -N(R 18 )C(O)(CH2)a -, -C(OR 13 )-O-(CH2) a -, wherein R 18 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 are each independently, C3 to C 10 and each a is independently 0 to 16. In one embodiment, each X is independently -OCO-, -COO-, -NHCO-, or -CONH-. In one embodiment, at least one X is -SS-.

[0234] Further embodiments of ionizable lipids of formula (I), group i) of ionizable lipid compounds, can be found in PCT Application No. PCT / US22 / 50725, filed November 22, 2022, the contents of which are incorporated herein by reference in their entirety. In particular, all of the ionizable lipids of formulas (I)-(XII) of PCT Application No. PCT / US22 / 50725 are suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

[0235] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table I below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 [Table 1-47] [Table 1-48] [Table 1-49]

[0236] Ionized lipid compounds ii) In some embodiments, the ionizable lipid has the following formula II: [ka] represented by a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or [ka] and A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; Each of X and Z is independently absent, —O—, —CO—, —N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 ,,R 110 , and R 120 are each independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, which may optionally be interrupted by heteroatoms or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of l and m is an integer from 1 to 10; t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl.

[0237] In some embodiments, Y is hydroxy, or [ka] is.

[0238] In some embodiments, [ka] is selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane. [ka] teeth, [ka] is selected from the group consisting of:

[0239] In some embodiments, the ionizable lipid has the formula [ka] All variables in this formula are defined and exemplified as described in the above embodiments.

[0240] In some embodiments, the ionizable lipid has the formula [ka] is expressed as During the ceremony, A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -SS-, or a divalent heterocycle; R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; t1 is an integer from 0 to 10, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; each M is independently a biodegradable moiety; m1 each independently represents an integer of 3 to 6, Each l1 is independently an integer of 4 to 8, m2 and l2 each independently represent an integer of 0 to 3, R 80 and R 90 are each independently an unsubstituted C5-C8 alkyl, or R 80 is H or unsubstituted C1-C4 alkyl, and R 90 is unsubstituted C5-C 11 is alkyl, and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl, or R 110 is H or unsubstituted C1-C4 alkyl, and R 120 is unsubstituted C5-C 11 All other variables in these formulas are defined and exemplified as described in the embodiments above. In some embodiments, in these formulas, R 80 is H or unsubstituted C1-C2 alkyl, and R 90 is unsubstituted C6-C 10 alkyl, and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl. In some embodiments, R 80 , R 90 , R 110 , and R 120 are each independently unsubstituted C5 to C8 alkyl.

[0241] In some embodiments, in the above formula, A is absent, —O—, —N(R 7 )-, N(R 7 )C(O)-, [ka] -OC(O)-, or -C(O)O-, wherein R 6are independently H, alkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-, and R 7 is H or C1-C3 alkyl.

[0242] In some embodiments, in the above formula, t1 is 0, 1, 2, 3, or 4, and t is 0, 1, or 2.

[0243] In some embodiments, in the above formula, W is hydroxyl, hydroxyalkyl, or one of the following: [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 )- and R 6 are each independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; R 8 are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R 8 may form a ring together with the nitrogen atom, q is independently 0, 1, 2, 3, 4, or 5; and Each p is independently 0, 1, 2, 3, 4 or 5.

[0244] In some embodiments, in the above formula: X is absent, -O-, or -C(O)-; Z is -O-, -C(O)O-, or -OC(O)-; M is -OC(O)- or -C(O)O-; Y or [ka] OH, [ka] and R c are each independently H or C1-C3 alkyl, each t1 is independently 1, 2, 3, or 4; R 30 , R 40 , R 50 , and R 60 are each H or C1-C4 branched or unbranched alkyl, R 70 is H and R 80 and R 90 each independently represents H or C1 to C 12 is a branched or unbranched alkyl of R 100 is H and R 110 and R 120 each independently represents H or C1 to C 12 is a branched or unbranched alkyl group, provided that R 80 and R 90 At least one of them is not H and is not R 110 and R 120 At least one of them is not H, l is 3 to 7, and m is 1 to 5.

[0245] In some embodiments, in the above formula, Y or [ka] OH, [ka] is.

[0246] Further embodiments of ionizable lipids of formula (II) in group ii) of ionizable lipid compounds can be found in PCT Application No. PCT / US23 / 16300, filed March 24, 2022, the contents of which are incorporated herein by reference in their entirety. In particular, the ionizable lipids of formulas (I), (IA-1), (IA-2), (IIA)-(IIC), (IIA-1), (IIIA)-(IIIIE), (IIIC-1), (IVA-1)-(IVA-3), (IVC-1)-(IVC-2), (VA-1)-(VA-9), (VC-1)-(VC-6) of PCT Application No. PCT / US23 / 16300 are all suitable for use as ionizable lipids in the present disclosure and are incorporated herein by reference in their entirety.

[0247] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table II below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 [Table 2-27] [Table 2-28] [Table 2-29] [Table 2-30]

[0248] Ionized lipid compounds iii) In some embodiments, the ionizable lipid has the formula [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl; R 20 and R 30 together with the adjacent N atom to form R a forming a 3- to 7-membered cyclic ring which may be substituted with R a is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; Each R1 and each R2 independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 together form a cyclic ring; R 10 and R 11 are each independently a C1-C3 branched or unbranched alkyl or a C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, n is 0, 1, 2, 3, or 4; Y is O or S; Z is absent, O, S, or N(R 12 )(R 12 ) where R 12 are each independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is present, adjacent R1 and R2 are not OH, NR 10 R 11 , or SH, Each A is independently, C1~C 16 branched or unbranched alkyl, or C2-C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; Each B is independently, C1 to C 16 branched or unbranched alkyl, or C2-C 16 branched or unbranched alkenyl, which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; Each X is independently a biodegradable moiety.

[0249] In some embodiments, R 20 and R 30are each independently H or C1-C3 branched or unbranched alkyl. In some embodiments, R 20 and R 30 together with the adjacent N atom to form R a In some embodiments, R a is H, C1-C3 branched or unbranched alkyl, or OH. In one embodiment, R a is H or OH.

[0250] In some embodiments, Z is absent, S, O, or NH. In some embodiments, n is 0, 1, or 2.

[0251] In some embodiments, the ionizable lipid has formula (V): [ka] and pharmaceutically acceptable salts thereof, and any of the stereoisomers described above, wherein: R1 is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 and R2 is H, OH, halogen, SH, or NR 10 R 11 or R1 and R2 together form a cyclic ring; R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 together form a heterocycle, Q is OH or -(OCH2CH2) u NR 20 R 30 and R 20 and R 30are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl; R 20 and R 30 together with the adjacent N atom to form R a forming a 3- to 7-membered cyclic ring which may be substituted with R a is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; u is 0, 1, 2, 3, 4, 5, 6, 7, or 8, v is 0, 1, 2, 3, or 4; y is 0, 1, 2, 3, or 4; A is independently C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; Each B is independently, C1 to C 16 Branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; and Each X is independently a biodegradable moiety.

[0252] In some embodiments, the present disclosure relates to an ionizable lipid of one of the following formulas: [ka] wherein u is 0, 1, 2, 3, 4, 5, 6, 7, or 8, v is 0, 1, 2, 3, or 4, and y is 0, 1, 2, 3, or 4. The other variables are defined as in formulas III) and V) above.

[0253] In some embodiments, in the above formula, X is —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) s -, -OC(O)(CH2) s -, -C(O)N(R 7 )(CH2) s -, -N(R 7 )C(O)(CH2) s -, -C(OR 13 )-O-(CH2) s - and R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 are each independently, C3 to C 10 and each s is independently 0 to 16. In some embodiments, X is -OC(O)-, -C(O)O-, -C(O)O(CH2) s - or -OC(O)(CH2) s In some embodiments, s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0254] Further embodiments of ionizable lipids of formula (III) or (V), in group iii) of ionizable lipid compounds, can be found in PCT Application No. PCT / US22 / 50111, filed November 16, 2022, the contents of which are incorporated herein by reference in their entirety. In particular, the ionizable lipids of formulas (IO)-(VIIO) and formulas (I)-(VIID) of PCT Application No. PCT / US22 / 50111 are all suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

[0255] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table III below. [Table 3-1] Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28 Table 3-29 Table 3-30 Table 3-31 Table 3-32

[0256] Ionized lipid compounds iv) In some embodiments, the ionizable lipid is a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'): [ka] a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, Each E is independently a biodegradable group; R a are each independently C1 to C5 alkyl, C2 to C5 alkenyl, or C2 to C5 alkynyl, u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t are each independently H, C1 to C 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, which may be interrupted by heteroatoms or substituted by OH, SH or halogen, or is cycloalkyl or substituted cycloalkyl; [ka] represents the bond connecting the tail group to the head group, and Lipids have a pKa of about 4 to about 8.

[0257] In some embodiments, each E is independently —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(OR 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -SS-, or -C(OR13 )-O-(CH2) r - and R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is branched or unbranched C3~C 10 alkyl, and r is 1, 2, 3, 4, or 5. In some embodiments, each E is independently —OC(O)—, —C(O)O—, —N(R 7 )C(O)-, or -C(O)N(R 7 )-, wherein R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0258] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII): [ka] wherein u3 and u4 are each independently 0, 1, 2, 3, or 4. The definitions of the other variables in (TII) are the same as those defined above in (TI).

[0259] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII): [ka] (for example, [ka] where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TIII) are the same as those defined above in (TI).

[0260] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV): [ka] wherein u3 and u4 are each independently 0, 1, 2, 3, or 4. The definitions of the other variables in (TIV) are the same as those defined above in (TI).

[0261] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TV): [ka] (for example, [ka] where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; R 7 are each independently H or methyl, and R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TV) are the same as those defined above in (TI).

[0262] In some embodiments, the lipid has the formula (TII'): [ka] and at least one tail group, (TII') (e.g., [ka] where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TII') are the same as defined above in (TI').

[0263] In some embodiments, the lipid has the formula (TII'): [ka] and at least one tail group, (TII') (e.g., [ka] where u3 is 0, 1, 2, 3, 4, 5, 6, or 7; R 7 are each independently H or methyl, and R b is independently at each occurrence H or C1-C4 alkyl. The definitions of the other variables in (TIII') are the same as those defined above in (TI').

[0264] In some embodiments, the lipid comprises at least one tail group of formula (TII), (TIII), (TIV), (TV), (TII'), and (TIII'), wherein: R 7 are each independently H or methyl; R b is independently at each occurrence H or C1-C4 alkyl; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and Lipids have a pKa of about 4 to about 8.

[0265] In some embodiments, the lipid comprises two (tw), three, four, or more tail groups having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'), where each tail group can be the same or different.

[0266] In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI′), (TII′), and / or (TIII′), R aare each independently a C1-C5 branched or unbranched alkyl, a C2-C5 branched or unbranched alkenyl, or a C2-C5 branched or unbranched alkynyl. a are each independently a C1-C3 branched or unbranched alkyl. In one embodiment, R a are each methyl.

[0267] In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u1 is 3, 4, or 5. In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u2 is 0, 1, 2, or 3. In some embodiments, in any of the above formulas (T), (TI), (TII), and (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u3 and u4 are each independently 1 to 7, e.g., u3 and u4 are each independently 1, 2, 3, or 4.

[0268] In some embodiments, the lipid comprises at least one tail of formula (TIII), where R a are methyl, and R b is independently at each occurrence H, ethyl, or butyl, u1 is 3-5, u2 is 0-3, and u3 is 1-7 (e.g., 1-4). In some embodiments, the lipid comprises at least one, two tails of formula (TIII), where the two tails of formula (TIII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIII), where each tail may be the same or different. In some embodiments, the lipid has four tails of formula (TIII), where each tail may be the same or different. In some embodiments, in each tail of formula (TIII), R aare each methyl, u1 is 3, u2 is 2, and u3 is 4.

[0269] In some embodiments, the lipid comprises at least one tail of formula (TII), where R a are each methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4. In some embodiments, the lipid has at least two tails of formula (TII), and the two tails of formula (TII) are the same. In some embodiments, the lipid has at least two tails of formula (TII), and the two tails of formula (TII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TII), and each tail may be the same or different. In some embodiments, the lipid has four tails of formula (TII), and each tail may be the same or different.

[0270] In some embodiments, the lipid comprises at least one tail of formula (TIV), where R a are each methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4. In some embodiments, the lipid comprises at least two tails of formula (TIV), each of which may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), each of which may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), each of which may be the same or different.

[0271] In some embodiments, the lipid comprises at least two tails of formula (TV), each of which may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TV), each of which may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TV), each of which may be the same or different.

[0272] In some embodiments, the lipid has at least two tails of formula (TII'), each of which may be the same or different. In some embodiments, the lipid has at least three tails of formula (TII'), each of which may be the same or different. In some embodiments, the lipid has at least four tails of formula (TII'), each of which may be the same or different.

[0273] In some embodiments, the lipid has at least two tails of formula (TIII'), each of which may be the same or different. In some embodiments, the lipid has at least three tails of formula (TIII'), each of which may be the same or different. In some embodiments, the lipid has at least four tails of formula (TIII'), each of which may be the same or different. In some embodiments, the lipid has at least one tail of formula (TII) and / or at least one tail of formula (TIII), and the lipid further comprises at least one tail that does not have formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail that does not contain a gem-di functional group attached to the same carbon adjacent to E (e.g., -C(O)O-).

[0274] In some embodiments, the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), i.e., the lipid further comprises at least one tail that does not contain a gem-di functional group attached to the same carbon adjacent to E.

[0275] In some embodiments, the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), i.e., the lipid further comprises at least one tail that does not contain a gem-di functional group attached to the same carbon adjacent to E.

[0276] In some embodiments, the lipid further comprises at least one tail of the following formula (TNG-I): [ka] During the ceremony, Each E is independently a biodegradable group as described herein (e.g., —OC(O)—, —C(O)O—, N(R 7 )C(O)-, -SS-, or -C(O)N(R 7 )-) and u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0277] In some embodiments, at least one tail of formula (TNG-I) is [ka] wherein: u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and R b is independently at each occurrence H or C1-C4 alkyl.

[0278] The E, R groups described above for tail groups containing gem-di functional groups attached to the same carbon adjacent to E, having the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), or (TIII') b , Rt All of the above embodiments regarding the definitions of u1, u2, u3 and u4 are also applicable to tail groups having formula (TNG-I), (TNG-II), or (TNG-III) that do not contain a gem-di functional group (gem-di) attached to the same carbon adjacent to E.

[0279] In some embodiments, the lipid further comprises at least two tails that do not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). In some embodiments, the lipid comprises two tail groups of formula (TNG-II) or (TNG-III), each tail group may be the same or different.

[0280] In some embodiments, the lipid further comprises at least three tails that do not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'(TII'), and / or (TIII'). In some embodiments, the lipid comprises three tail groups of formula (TNG-II) or (TNG-III), where each tail group may be the same or different:

[0281] In some embodiments, the head group of the lipid has the structure of formula (HA-I): [ka] During the ceremony, R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, which may be interrupted by one or more heteroatoms or substituted with OH, SH, halogen, or cycloalkyl groups; or R 20 and R 30together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which may be optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; Each of R1 and R2 independently represents H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 together form a cyclic ring; R 10 and R 11 each independently is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, n is 0, 1, 2, 3, or 4; Z is absent or is O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is present, the adjacent R1 and R2 are OH, NR 10 R 11 , SH should not be.

[0282] In some embodiments, R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which may be substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups.

[0283] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-I): [ka] During the ceremony, R1 and R2 each independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11 or R1 and R2 together form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7, or 8; n is 0, 1, 2, 3, or 4; Z is absent, O, S, or NR 12 where R 12 is H or C1-C7 branched or unbranched alkyl, provided that when Z is present, adjacent R1 and R2 are OH, NR 10 R 11 , or SH, [ka] represents the bond connecting the head group to the tail group.

[0284] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-III): [ka] wherein Z is absent, O, S, or NR 12 and R 12 is H or C1-C7 branched or unbranched alkyl. The definitions of the other variables in (HA-III) are the same as those defined above in (HA-IA).

[0285] In some embodiments, the head group is [ka] wherein: Rc is H or alkyl optionally substituted with OH, and m1 is 1, 2, or 3.

[0286] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-V): [ka] During the ceremony, R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R2 is OH, halogen, SH, or NR 10 R 11 or R1 and R2 together can form a cyclic ring; R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 can be taken together to form a heterocycle, R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or R 20 and R 30 can be taken together to form a cyclic ring, and Each of v and y is independently 1, 2, 3, or 4.

[0287] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-VI): [ka] The definitions of all variables in (HA-VI) are the same as those defined above in (HA-V).

[0288] In some embodiments, in either of the above formulas of (HA-V) or (HA-VI), R 20 and R 30 are each independently C1-C3 alkyl. In one embodiment, R 20 and R 30 are each independently methyl.

[0289] In some embodiments, the head group of the ionizable lipid has the structure of formula (HB-I): [ka] In the formula, W is [ka] and During the ceremony, R5 is OH, SH, (CH2) s OH or NR 10 R 11 and R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; R7 and R8 each independently represent H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 where R 10 and R 11 are each independently H or C1-C3 alkyl, or R 10 and R 11 are taken together to form a heterocycle, or R7 and R8 are taken together to form a ring; R 20 are each independently H or C1-C3 branched or unbranched alkyl, R 14 is a heterocycle, NR 10 R 11, C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, which may be substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle, R 16 is H, ═O, ═S, or CN, each of s, u, and t is independently 1, 2, 3, 4, or 5; each v is independently 0, 1, 2, 3, 4, or 5; each Y is a divalent heterocycle; Each Z is independently absent, O, S, or NR 12 and R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, Q is O, S, CH2, or NR 13 and R 13 are each H or C1-C5 alkyl, V is a branched or unbranched C2 to C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene or C2-C 10 heteroalkylene, which may be substituted with one or more OH, SH, and / or halogen groups; and T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocycle.

[0290] In some embodiments, in Formula (HB-I), W is [ka] wherein: R6, R7, and R8 are each independently H or methyl; and u and t are each independently 1, 2, or 3.

[0291] In some embodiments, in Formula (HB-I), W is [ka] wherein: R 16 is H or =O, R 14 is a nitrogen-containing 5- or 6-membered heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 and R 10 and R 11 are each independently H or C1-C3 alkyl, and u and v are each independently 1, 2, or 3;

[0292] In some embodiments, in Formula (HB-I), W is [ka] wherein: Each R6 is independently H or methyl; Each u is independently 1, 2, or 3; and V is C2-C6 alkylene or C2-C6 alkenylene.

[0293] In some embodiments, in Formula (HB-I), W is [ka] wherein: Each R6 is independently H or methyl; Each R7 is independently H; Each R8 is methyl; Each u is independently 1, 2, or 3; and V is C2-C6 alkylene or C2-C6 alkenylene.

[0294] In some embodiments, in Formula (HB-I), W is [ka] wherein: Each u is independently 1, 2, or 3; and T is a divalent nitrogen-containing 5- or 6-membered heterocycle.

[0295] In some embodiments, in Formula (HB-I), W is [ka] and During the ceremony, each u is independently 1, 2, or 3; Q is O, Z is independently NR 12 and R 12 is H or C1-C3 alkyl.

[0296] In some embodiments, the head group is [ka] wherein each of u and t is independently 1 or 2.

[0297] In some embodiments, the head group of the ionizable lipid has the structure of formula (HC-I): [ka] During the ceremony, [ka] is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, [ka] and A is absent or -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, or -SS-; Each of X and Z is independently absent, —O—, —C(O)—, —N(R 7 )-, -O-alkylene, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-, R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl.

[0298] In some embodiments, the head group has the formula [ka] It has the following structure.

[0299] In some embodiments, in the above formula: A is absent or -O-, -N(R 7 )-, -OC(O)-, or -C(O)O-; X is absent, -O-, or -C(O)-, and Z is —O—, —C(O)O—, or —OC(O)—.

[0300] In some embodiments, the head group has the formula [ka] wherein t1 is 0, 1, 2, or 3.

[0301] In some embodiments, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, or any one of the following moieties: [ka] During the ceremony, Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, or -(CH 2) qC(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )-, or -N(R 7 ) and R 6 are each independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene eN(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N + (R 7 ) 3-alkylene-Q-; R 8are each independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, or thiolalkyl, heterocyclyl, heteroaryl, or two R 8 may be taken together with the nitrogen atom to form a ring which may be substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl; q is 0, 1, 2, 3, 4, or 5; p is 0, 1, 2, 3, 4, or 5.

[0302] In some embodiments, W is one of the following: OH, [ka]

[0303] In some embodiments, the ionizable lipid is [ka] and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: Each R1 is independently H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R1 and R2 can be joined together to form a cyclic ring, and R 10 and R 11 are each independently H or C1-C3 alkyl, and R 10 and R 11 can be taken together to form a heterocycle, R2 is independently H, C1-C3 alkyl, OH, halogen, SH, or NR 10 R 11 and R1 and R2 can be joined together to form a cyclic ring, and R 10 and R 11 are each independently H or C1-C3 alkyl, and R 10 and R 11can be taken together to form a heterocycle, m is 1, 2, 3, 4, 5, 6, 7 or 8; n is 0, 1, 2, 3, or 4; each r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; R3 is independently H or C3 to C 10 is alkyl, R4 is independently H or C3 to C 10 alkyl, with the proviso that at least one of R3 and R4 is not H; Z is absent, O, S, or NR 12 where R 12 is a C1-C7 alkyl, X', [ka] However, at least one X in the formula is [ka] and X' is a biodegradable moiety.

[0304] In some embodiments, each X is [ka] In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(OR 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s -, or -C(OR 13 )-O-(CH2) s -, wherein R 7 is H or C1-C3 alkyl, and R 13 is C3~C 10 It is alkyl.

[0305] In some embodiments, at least one X in the formula is [ka] where R 7 is H or methyl. In one embodiment, each X is [ka] In one embodiment, each X is [ka] where R 7 is H or methyl.

[0306] In some embodiments, m=3. In some embodiments, n=0 or 1. In some embodiments, R R1 and R2 are each H. In some embodiments, Z is absent.

[0307] In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z is NH. In some embodiments, r is 3. In some embodiments, r is 4.

[0308] Further embodiments of the above ionizable lipids comprising at least one head group (e.g., a head group of formula (HA-I), (HA-III), (HA-V), (HA-VI), (HB-I), and (HC-I)) and at least one tail group of formula (TI) or (T1') (e.g., a tail group of formula (TII), (TIII), TIV, TV, TII', or TIII') in group iv) of ionizable lipid compounds can be found in PCT Application PCT / US23 / 31669, filed August 31, 2023, the contents of which are incorporated herein by reference in their entirety. Additionally, the ionizable lipids of formulae (LA-I) through (LA-VII), (LB-I)-(LB-VII), (LC-IA)-(LC-IC), (LC-IIA)-(LC-IIC), and (LC-IIIA) through (LC-IIIE) of PCT Application No. PCT / US23 / 31669, filed August 31, 2023, are all suitable for use as ionizable lipids in the present disclosure and are incorporated herein by reference in their entireties.

[0309] Certain exemplary ionizable lipid compounds disclosed herein are listed in Table IV below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 Table 4-25 Table 4-26 Table 4-27 Table 4-28 Table 4-29 Table 4-30 Table 4-31 Table 4-32 Table 4-33 Table 4-34 Table 4-35 Table 4-36 Table 4-37 Table 4-38 Table 4-39 Table 4-40 Table 4-41 Table 4-42 Table 4-43 Table 4-44 Table 4-45 Table 4-46 Table 4-47 Table 4-48 Table 4-49 Table 4-50 Table 4-51 Table 4-52 Table 4-53 Table 4-54 Table 4-55 Table 4-56 Table 4-57 Table 4-58 Table 4-59 Table 4-60 Table 4-61 Table 4-62 Table 4-63 Table 4-64 Table 4-65 Table 4-66 Table 4-67 Table 4-68 Table 4-69 Table 4-70 Table 4-71 Table 4-72 Table 4-73 Table 4-74 Table 4-75 Table 4-76 Table 4-77 Table 4-78 Table 4-79 Table 4-80 Table 4-81 Table 4-82 Table 4-83 Table 4-84 Table 4-85 Table 4-86 Table 4-87 Table 4-88 Table 4-89 Table 4-90 Table 4-91 Table 4-92 Table 4-93 Table 4-94 Table 4-95 Table 4-96 Table 4-97 Table 4-98 Table 4-99 Table 4-100 Table 4-101 Table 4-102 Table 4-103 Table 4-104 Table 4-105 Table 4-106 Table 4-107

[0310] Other ionizable lipids The modified lipid composition can use more than one ionizable lipid in the ionizable lipid component, i.e., one or more ionizable lipids from the compounds of formulae in groups i) to iv) can be used alone or in combination with different ionizable lipids from the compounds of formulae in groups i) to iv).

[0311] In some embodiments, the ionizable lipid does not include 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), and 98N12-5.

[0312] In some embodiments, the modified lipid composition comprises an ionizable lipid selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0313] In some embodiments, the modified lipid composition comprises an ionizable lipid represented by Formula III: [ka] In the formula, R is C8 to C 14 It is an alkyl group.

[0314] In some embodiments, the lipid membrane of the modified lipid composition comprises at least 35% lipids of Formula I, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or greater than 90% lipids of Formula I, e.g., 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% lipids of Formula I.

[0315] In some embodiments, the lipid membrane of the modified lipid composition comprises at least 35% lipids of Formula II, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or greater than 90% lipids of Formula II, e.g., 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% lipids of Formula II.

[0316] In some embodiments, the lipid membrane of the modified lipid composition comprises at least 35% lipids of Formula III, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or greater than 90% lipids of Formula III, e.g., 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% lipids of Formula III.

[0317] In some examples, the modified lipid composition comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionized lipids.

[0318] In some examples, the modified lipid composition comprises, by mole, at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% ionized lipid, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionized lipid, e.g., about 25%-75% ionized lipid (e.g., about 25%-75% ionized lipid).

[0319] The ionizable lipids described herein can comprise an amine core described herein substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) lipid tails. In some embodiments, the ionizable lipids described herein comprise at least three lipid tails. The lipid tails can be C8 to C9. 18 Hydrocarbons (e.g., C6-C 18 -Alkyl or C6-C 18 -alkanoyl). The amine core can be substituted at the nitrogen atom with one or more lipid tails (e.g., one hydrogen atom attached to the nitrogen atom can be replaced with a lipid tail).

[0320] In some embodiments, the amine core is [ka] It has the following structure.

[0321] In some embodiments, the amine core is [ka] It has the following structure.

[0322] In some embodiments, the amine core is [ka] It has the following structure.

[0323] In some embodiments, the amine core is [ka] It has the following structure.

[0324] In some embodiments, the amine core is [ka] It has the following structure.

[0325] In some embodiments, the amine core is [ka] It has the following structure.

[0326] In some embodiments, the amine core is [ka] It has the following structure.

[0327] In some embodiments, the amine core is [ka] It has the following structure.

[0328] The modified lipid composition may further comprise a cationic lipid.

[0329] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2016 / 118725, which is incorporated herein by reference in its entirety.

[0330] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0331] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2016 / 118724, which is incorporated herein by reference in its entirety.

[0332] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0333] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0334] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publications WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated by reference in its entirety.

[0335] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R L Each example is independently an optionally substituted C6-C40 alkenyl.

[0336] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0337] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0338] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0339] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0340] Other lipids suitable for use in the modified lipid compositions and methods of making and using same include those described in International Patent Publication WO 2015 / 184256, which is incorporated herein by reference in its entirety. In some embodiments, the modified lipid compositions and methods of making and using same comprise lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and R Aare each independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen; and each R B is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen.

[0341] In certain embodiments, the modified lipid compositions and methods of making and using the same comprise "Target 23," a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0342] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference in its entirety.

[0343] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0344] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0345] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0346] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference in its entirety.

[0347] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are each independently H or a C1-C6 aliphatic group, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or arylene, and L 1 are each independently an ester, thioester, disulfide, or anhydride group; L 2 are each independently a C2 to C10 aliphatic group, and X 1 are each independently H or OH, and R 3 are each independently a C6 to C20 aliphatic.

[0348] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0349] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0350] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0351] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference in their entirety.

[0352] In certain embodiments, the lipids of the modified lipid compositions and methods of making and using same are lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof.

[0353] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2015 / 199952, which is incorporated herein by reference in its entirety.

[0354] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0355] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0356] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0357] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0358] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0359] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0360] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0361] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0362] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0363] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0364] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0365] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0366] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0367] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2017 / 004143, which is incorporated herein by reference in its entirety.

[0368] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0369] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0370] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0371] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0372] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0373] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0374] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0375] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0376] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0377] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0378] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0379] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0380] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0381] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0382] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0383] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0384] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0385] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2017 / 075531, which is incorporated herein by reference in its entirety.

[0386] In some embodiments, modified lipid compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, and L 1 or L 2The other of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond, and G 1 and G 2 are each independently unsubstituted C1 to C 12 Alkylene or C1-C 12 alkenylene, G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a is H or C1~C 12 alkyl, and R 1 and R 2 are each independently, C6 to C 24 Alkyl or C6-C 24 alkenyl, and R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 is C1~C 12 alkyl, and R 5 is H or C1-C6 alkyl, and x is 0, 1, or 2.

[0387] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference in its entirety. In some embodiments, the modified lipid compositions and methods of making and using them include lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof.

[0388] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0389] In some embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0390] Other lipids suitable for use in the modified lipid compositions and methods of making and using them include those described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference in its entirety.

[0391] In some embodiments, the lipid of the modified lipid composition and methods of making and using same is a compound of one of the following formulas: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) nand n is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), and heterocycle;

[0392] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0393] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0394] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0395] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0396] Other lipids suitable for use in the modified lipid compositions and methods of making and using same include those described in International Patent Applications WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference in its entirety. In certain embodiments, the modified lipid compositions and methods of making and using same comprise lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof.

[0397] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0398] In certain embodiments, the modified lipid compositions and methods of making and using same comprise a lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0399] In certain embodiments, the modified lipid compositions and methods of making and using the same comprise a lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0400] In some embodiments, the modified lipid compositions described herein may comprise, be prepared as described, comprise, or consist of an ionizable lipid described in WO2016118724, WO2016118725, WO2016187531, WO2017176974, WO2018078053, WO2019027999, WO2019036030, WO2019089828, WO2019099501, WO2020072605, WO2020081938, WO2020118041, WO2020146805, or WO2020219876, each of which is incorporated herein by reference in its entirety.

[0401] Other lipids and other drugs The exogenous lipid may be a cell membrane permeabilizing agent, and may be capable of increasing the delivery of polypeptide to cells by the modified lipid composition, and / or may be capable of increasing the loading (e.g., loading efficiency or loading capacity) of polypeptide. Further exemplary exogenous lipids include sterols and PEGylated lipids.

[0402] The modified lipid composition can include other components (e.g., lipids, e.g., sterols, e.g., cholesterol, or small molecules) to further alter the functional and structural characteristics of the modified lipid composition. For example, the modified lipid composition can further include a stabilizing molecule that increases the stability of the modified lipid composition (e.g., stable at room temperature for at least one day and / or at least one week at 4°C).

[0403] In some embodiments, the modified lipid composition further comprises a sterol, such as sitosterol, sitostanol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or plant-isolated cholesterol), stigmasterol, campesterol, fucosterol, or an analog of any sterol (e.g., a glycoside, ester, or peptide). In some examples, the exogenous sterol is added to the preparation prior to step (b), e.g., mixed with the structured lipid prior to step (b). The exogenous sterol can be added in an amount of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to more than 90% (w / w) of the total lipid and sterol in the preparation.

[0404] In some embodiments, the sterol is cholesterol or sitosterol. In some examples, the modified lipid composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% sterol (e.g., cholesterol or sitosterol), for example, a molar ratio of sterol of 1% to 10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60%. In some embodiments, the modified lipid composition comprises about 35% to 50% sterol (e.g., cholesterol or sitosterol), for example, a molar ratio of sterol of about 36%, 38.5%, 42.5%, or 46.5%. In some embodiments, the modified lipid composition comprises a molar ratio of sterol of about 20% to 40%.

[0405] In some embodiments, modified lipid compositions modified with sterols have altered stability (e.g., increased stability) compared to modified lipid compositions that are not modified with sterols, hi some aspects, modified lipid compositions modified with sterols fuse with target cell membranes more quickly compared to modified lipid compositions that are not modified with sterols.

[0406] In some examples, the modified lipid composition comprises an exogenous lipid and an exogenous sterol.

[0407] In some embodiments, the modified lipid composition further comprises a PEGylated lipid. The length of the polyethylene glycol (PEG) can vary from 1 kDa to 10 kDa, and in some aspects, PEGs having a length of 2 kDa are used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some examples, the modified lipid composition comprises at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 20%, 30%, 40%, 50%, or greater than 50% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2.5%, 2.5%-3.5%, 3.5%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, or 30%-50% PEGylated lipid, all by mole percent. In some embodiments, the modified lipid composition comprises about 0.1% to 10% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., about 1% to 3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid, all by mole %.

[0408] In some embodiments, modified lipid compositions modified with PEGylated lipids have altered stability (e.g., increased stability) compared to modified lipid compositions not modified with PEGylated lipids. In some embodiments, modified lipid compositions modified with PEGylated lipids have altered particle size compared to modified lipid compositions not modified with PEGylated lipids. In some embodiments, modified lipid compositions modified with PEGylated lipids are less likely to be phagocytosed than modified lipid compositions not modified with PEGylated lipids. The addition of PEGylated lipids may also affect stability in the gastrointestinal tract and enhance particle movement through mucus. PEG can be used as a method of attaching targeting moieties.

[0409] In some embodiments, the modified lipid composition may include an ionizable lipid (e.g., C12-200 or MC3), and one or both of a sterol (e.g., cholesterol or sitosterol) and a PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k). In embodiments, the modified lipid composition comprises about 5% to 50% naturally derived lipid (e.g., about 10% to 20% naturally derived lipid, e.g., about 10%, 12.5%, 16%, or 20% naturally derived lipid), about 30% to 75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 35% to 50% sterol (e.g., about 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1% to 10% PEGylated lipid (e.g., about 1% to 3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid), all in molar percentages.

[0410] In some embodiments, the modified lipid composition comprises about 5% to 60% naturally derived lipids (e.g., about 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60% naturally derived lipids, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60% naturally derived lipids), about 25% to 75% ionized lipids (e.g., about 35% or about 50% ionized lipids), and about 10% to 125% ionic lipids (e.g., about 10% to 125% ionic lipids). PEGylated lipid), about 10% to 50% sterol (e.g., about 10%, 12.5%, 14%, 16%, 18%, 20%, 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1% to 10% PEGylated lipid (e.g., about 0.5% to 5% PEGylated lipid, e.g., about 1% to 3% PEGylated lipid, or about 1.5% or about 2.5% PEGylated lipid), all in molar ratios.

[0411] In some embodiments, the ionizable lipids, structured lipids, sterols, and PEGylated lipids comprise about 25%-75%, about 20%-60%, about 10%-45%, and about 0.5%-5%, respectively, of the lipids in the modified lipid composition.

[0412] In some embodiments, the ionizable lipids, structured lipids, sterols, and PEGylated lipids comprise about 30%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the modified lipid composition.

[0413] In some embodiments, the ionizable lipids, structured lipids, sterols, and PEGylated lipids comprise about 35%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the modified lipid composition.

[0414] In some embodiments, the ionizable lipid, structured lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:50:12.5:2.5.

[0415] In some embodiments, the ionizable lipid, structured lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:50:11.5:3.5.

[0416] In some embodiments, the ionizable lipid, structured lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:20:42.5:2.5.

[0417] In some embodiments, the modified lipid composition may comprise a molar ratio of ionizable lipid:structured lipid:sterol:PEG lipid of 22.5:75:0:2.5.

[0418] In some embodiments, the modified lipid composition may comprise a molar ratio of ionizable lipid:structured lipid:sterol:PEG lipid of 35:30:32.5:2.5.

[0419] In some embodiments, the modified lipid composition may comprise a molar ratio of ionizable lipid:structured lipid:sterol:PEG lipid of 35:16:46.5:2.5.

[0420] In some embodiments, the modified lipid composition may comprise ionizable lipid:(structural lipid 1 + structural lipid 2):sterol:PEG lipid in a molar ratio of 35:(3 + 13):46.5:2.5 (structural lipid 1 may be a structured lipid described herein, structural lipid 2 may be a structured lipid described herein that is different from structural lipid 1, or structural lipid 2 may be any lipid suitable for preparing lipid nanoparticle compositions).

[0421] In some embodiments, modified lipid compositions modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids encapsulate negatively charged cargo (e.g., nucleic acids) more efficiently than modified lipid compositions not modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids. The modified lipid compositions may have an encapsulation efficiency of cargo (e.g., heterologous functional agents such as nucleic acids, e.g., RNA or DNA) of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%, e.g., 5%-30%, 30%-50%, 50%-70%, 70%-80%, 80%-90%, 90%-95%, or 95%-100%.

[0422] Cellular uptake of modified lipid compositions can be measured by various methods known in the art. For example, the modified lipid composition or a component thereof can be labeled with a marker (e.g., a fluorescent marker) that can be detected in isolated cells to confirm uptake.

[0423] In some embodiments, the modified lipid compositions provided herein comprise two or more different structural components, e.g., structural components derived from two or more different natural sources. In some embodiments, the modified lipid compositions provided herein comprise two or more different types of modifications, e.g., different types and / or ratios of ionizable lipids, sterols, and / or PEGylated lipids.

[0424] In some examples, the organic solvent that dissolves lipid film is dimethylformamide:methanol (DMF:MeOH).Alternatively, the organic solvent or solvent combination can be, for example, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol, or dimethylformamide:methanol.

[0425] The aqueous phase can be any suitable solution, such as a citrate buffer (e.g., a citrate buffer having a pH of about 3.2), water, or phosphate buffered saline (PBS). The aqueous phase can further include a heterologous functional agent (e.g., an agricultural or therapeutic agent) or a small molecule.

[0426] The lipid solution and aqueous phase can be mixed in the microfluidic device in any suitable ratio. In some examples, the aqueous phase and lipid solution are mixed in a 3:1 volume ratio.

[0427] The modified lipid composition can optionally contain an additional agent, such as a cell membrane permeabilizing agent, a therapeutic agent, a polynucleotide, a polypeptide, or a small molecule. The modified lipid composition can carry or bind the additional agent in various ways to enable delivery of the heterologous functional agent to target cells, for example, by encapsulating the heterologous functional agent, incorporating the heterologous functional agent into the lipid bilayer structure, or associating the heterologous functional agent with the surface of the lipid bilayer structure (e.g., by complexation). The heterologous functional agent can be incorporated into the modified lipid composition either in vivo or in vitro (e.g., in tissue culture, cell culture, or synthetically incorporated).

[0428] Zeta potential A modified lipid composition comprising an ionizable lipid (e.g., C12-200 or MC3) and optionally a cationic lipid (e.g., DC-cholesterol or DOTAP) may have a zeta potential of, for example, greater than -30 mV in the absence of cargo, greater than -20 mV, greater than -5 mV, greater than 0 mV, or about 30 mV in the absence of cargo. In some examples, the modified lipid composition has a negative zeta potential, for example, a zeta potential of less than 0 mV, less than -10 mV, less than -20 mV, less than -30 mV, less than -40 mV, or less than -50 mV in the absence of cargo. In some examples, the modified lipid composition has a positive zeta potential, for example, a zeta potential of greater than 0 mV, greater than 10 mV, greater than 20 mV, greater than 30 mV, greater than 40 mV, or greater than 50 mV in the absence of cargo. In some examples, the modified lipid composition has a zeta potential of about 0.

[0429] The zeta potential of modified lipid composition can be measured by any method known in the art.Zeta potential is generally measured indirectly, for example, by using theoretical models to calculate the data obtained by methods and techniques known in the art, such as electrophoretic mobility or dynamic electrophoretic mobility.Electrophoretic mobility is typically measured by microelectrophoresis, electrophoretic light scattering, or TRPS (tunable resistive pulse sensing).Electrophoretic light scattering is based on dynamic light scattering.Typically, zeta potential can be accessed by photon correlation spectroscopy or dynamic light scattering (DLS), also known as quasi-elastic light scattering.

[0430] Natural EV markers The structural components (e.g., structural lipids) in the modified lipid compositions and methods for making and using them can have various markers that identify the structural components that are produced. In some embodiments, the structural components are natural. As used herein, the term "native EV marker" refers to a component that is naturally associated with a natural source, such as a natural protein, a natural nucleic acid, a natural small molecule, a natural lipid, or a combination thereof, and is incorporated into or attached to the surface of natural EVs.

[0431] Drug loading The modified lipid composition may include a heterologous functional agent, such as those described herein, e.g., a cell membrane permeabilizing agent and / or a heterologous agricultural agent (e.g., an insecticide, a fertilizer, a herbicide, a plant modifying agent), a heterologous therapeutic agent (e.g., an antifungal agent, an antibacterial agent, a virucide, an antiviral agent, an insecticide, a nematicide, an antiparasitic agent, or an insect repellent)).

[0432] The modified lipid compositions can carry or bind such agents by a variety of means to enable delivery of the agent to a target organism (e.g., a target animal, plant, bacterium, or fungus), for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure, or binding (e.g., by complexation) the agent to the surface of the lipid bilayer structure of the modified lipid composition. In some examples, heterologous functional agents (e.g., cell membrane permeabilizing agents) are included in formulations made with the modified lipid composition formulations, as described herein.

[0433] Heterologous functional agents can be incorporated into or loaded onto the modified lipid composition by any method known in the art that allows for direct or indirect binding between the modified lipid composition and the agent. The agent can be incorporated into the modified lipid composition by in vivo or in vitro (e.g., tissue culture or cell culture) or by both in vivo and in vitro methods.

[0434] In some examples, the modified lipid composition is loaded in vitro. Heterologous functional agents can be loaded into or onto the modified lipid composition (e.g., encapsulated by the modified lipid composition) using, but not limited to, physical, chemical, and / or biological methods (e.g., in tissue culture or cell culture). For example, agents can be introduced into the modified lipid composition by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction, or extrusion. In some examples, agents are incorporated into the modified lipid composition using a microfluidic device, e.g., a method in which lipids are provided in an organic phase and an agent is provided in an aqueous phase, and the organic and aqueous phases are combined in the microfluidic device to create a modified lipid composition containing a heterologous functional agent. The loaded modified lipid composition can be evaluated to confirm the presence or level of the loaded agent using various methods, such as HPLC (e.g., to evaluate small molecules), immunoblotting (e.g., to evaluate proteins), and / or quantitative PCR (e.g., to evaluate nucleotides). However, it should be understood by those skilled in the art that loading of the modified lipid composition with heterologous functional agents of interest is not limited to the methods described above.

[0435] In some instances, heterofunctional agents may be complexed with the modified lipid composition, in which the agent is indirectly or directly connected or bound to the modified lipid composition. For example, one or more agents may be chemically linked to the modified lipid composition, such that the one or more agents are directly bound (e.g., by covalent or ionic bonds) to the lipid bilayer of the modified lipid composition. In some instances, complexation of various agents to the modified lipid composition may be achieved by first mixing one or more agents with a suitable cross-linking agent (e.g., N-ethylcarbodiimide ("EDC"), which is generally used as a carboxyl activating agent for amide bonds with primary amines and also reacts with phosphate groups) in a suitable solvent. After an incubation period sufficient to allow the agent to attach to the cross-linking agent, the cross-linking agent / agent mixture may be combined with the modified lipid composition, and after a further incubation period, the mixture may be subjected to a sucrose gradient (e.g., 8, 30, 45, and 60% sucrose gradient) to separate the free agent and the free modified lipid composition from the agent complexed to the modified lipid composition. As part of combining the mixture with a sucrose gradient and accompanying centrifugation step, the modified lipid composition complexed to the drug is then seen as a band in the sucrose gradient, and the resulting complexed modified lipid composition can be recovered, washed, and dissolved in a solution suitable for use.

[0436] In some instances, the modified lipid composition is stably associated with the heterologous functional agent before and after delivery of the modified lipid composition, hi other instances, the modified lipid composition is associated with the agent such that the agent dissociates from the modified lipid composition after delivery of the modified lipid composition.

[0437] Depending on the particular agent or use, the modified lipid compositions may be loaded with, or formulated with, various concentrations of heterologous functional agents. For example, in some examples, the modified lipid compositions disclosed herein are loaded or formulated to contain about 0.001, 0.01, 0.1, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 95 (or any range between about 0.001 and 95) or more wt% of agent. In some examples, the modified lipid composition is loaded or formulated to contain less than about 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.1, 0.01, 0.001 (or any range between about 95 and 0.001) wt% of the agent. For example, the modified lipid composition may contain about 0.001 to about 0.01 wt%, about 0.01 to about 0.1 wt%, about 0.1 to about 1 wt%, about 1 to about 5 wt%, or about 5 to about 10 wt%, or about 10 to about 20 wt% of the agent. In some examples, the modified lipid composition may be loaded or formulated with more than about 1, 5, 10, 50, 100, 200, or 500, 1,000, 2,000 (or any range between about 1 and 2,000) μg / ml of drug. The modified lipid composition may be loaded or formulated with less than about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range between about 2,000 and 1) μg / ml of drug.

[0438] In some examples, the modified lipid composition is loaded or formulated to contain at least 0.001 wt%, at least 0.01 wt%, at least 0.1 wt%, at least 1.0 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, or at least 95 wt% heterologous functional agent. In some examples, the modified lipid composition may be loaded or formulated with at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 200 μg / ml, at least 500 μg / ml, at least 1,000 μg / ml, or at least 2,000 μg / ml of agent.

[0439] In some instances, the modified lipid composition is formulated with a heterologous functional agent, for example, by suspending the modified lipid composition in a solution containing or consisting of the agent by vigorous mixing. The agent (e.g., a cell membrane permeabilizing agent, such as a nucleic acid, an enzyme, a surfactant, an ionic, fluorescent, or zwitterionic liquid, or an ionizable lipid) may comprise, for example, less than 1% or at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the solution.

[0440] formulation agricultural formulations The modified lipid compositions described herein can be formulated into agricultural compositions.

[0441] To facilitate application, handling, transportation, storage, and effective activity, modified lipid compositions can be formulated with other substances. The modified lipid compositions can be formulated, for example, into baits, concentrated emulsions, powders, emulsifiable concentrates, fumigants, gels, granules, microcapsules, seed treatments, suspension concentrates, suspoemulsions, tablets, water-soluble liquids, water-dispersible granules or dry flowables, wettable powders, and ultra-low-volume sprays. For details on formulation types, see "Catalogue of Pesticide Formulation Types and International Coding System" Technical Monograph no. 2,5th Edition by CropLife International (2002).

[0442] The modified lipid composition can be applied as an aqueous suspension or emulsion prepared from a concentrated formulation of such a drug. Such water-soluble, water-suspendable, or emulsifiable formulations can be either solids, commonly known as wettable powders, or water-dispersible granules; liquids, commonly known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which can be compressed to form water-dispersible granules, contain an intimate mixture of the modified lipid composition, a carrier, and a surfactant. The carrier is typically selected from attapulgite clay, montmorillonite clay, diatomaceous earth, or purified silicates. Effective surfactants, comprising about 0.5% to about 10% of the wettable powder, are found among nonionic surfactants such as sulfonated lignin, condensed naphthalene sulfonates, naphthalene sulfonates, alkylbenzene sulfonates, alkyl sulfates, and ethylene oxide adducts of alkylphenols.

[0443] Emulsifiable concentrates can contain a suitable concentration of the modified lipid composition, such as about 50 to about 500 grams per liter of liquid, dissolved in a carrier, which can be either a water-miscible solvent or a mixture of a water-immiscible organic solvent and an emulsifier. Useful organic solvents include aromatics, particularly xylenes, and petroleum fractions, especially the high-boiling naphthalene and olefinic portions of petroleum, such as heavy aromatic naphtha. Other organic solvents, such as terpene solvents including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol, can also be used. Suitable emulsifiers for emulsifiable concentrates can be selected from conventional anionic and nonionic surfactants.

[0444] The aqueous suspension comprises a suspension of the water-insoluble modified lipid composition dispersed in an aqueous carrier at a concentration ranging from about 5% to about 50% by weight. The suspension is prepared by vigorously mixing the water-insoluble modified lipid composition into a carrier consisting of water and a surfactant. Ingredients such as inorganic salts and synthetic or natural gums may also be added to increase the density and viscosity of the aqueous carrier.

[0445] The modified lipid composition may also be applied as a granular composition, particularly useful for application to soil. Granular compositions typically contain about 0.5% to about 10% by weight of the modified lipid composition dispersed in a carrier comprising clay or a similar material. Such compositions are typically prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier preformed to the appropriate particle size, ranging from about 0.5 to about 3 mm. Such compositions may also be formulated by making a dough or paste of the carrier and compound, grinding, and drying to obtain the desired granular particle size.

[0446] Powders containing the modified lipid composition are prepared by intimately mixing the powdered form of the modified lipid composition with a suitable dusty agricultural carrier, such as kaolin clay, crushed volcanic rock, etc. The powders may suitably contain from about 1% to about 10% of the packet. These may be applied as seed dressings or as foliar applications using a dust blower.

[0447] It is equally practical to apply the formulation in the form of a solution in a suitable organic solvent, usually a petroleum oil such as spray oil, which is widely used in agricultural chemistry.

[0448] The modified lipid composition can also be applied in the form of an aerosol composition.In this composition, the packet is dissolved or dispersed in a carrier that is a pressure-generating propellant mixture.The aerosol composition is packaged in a container, and the mixture is sprayed through an atomizing valve.

[0449] Another embodiment is an oil-in-water emulsion, the emulsion comprising oily globules, each provided with a lamellar liquid crystal coating, dispersed in an aqueous phase, each oily globule comprising at least one agriculturally active compound, individually coated with monolamellar or oligolamellar layers, comprising (1) at least one non-ionic lipophilic surfactant, (2) at least one non-ionic hydrophilic surfactant, and (3) at least one ionic surfactant, the globules having an average particle diameter of less than 800 nanometers. Further information regarding this embodiment is disclosed in U.S. Patent Publication No. 20070027034, published February 1, 2007. For ease of use, this embodiment is referred to as "OIWE."

[0450] In addition, generally, when the molecules disclosed above are used in formulations, such formulations may also contain other components.These components include but are not limited to wetting agents, nucleic acid agents, adhesives, penetrating agents, buffers, sequestering agents, drift reducing agents, compatibility agents, antifoaming agents, detergents and emulsifiers (this is a non-exhaustive and non-mutually exclusive list).Some components will be described below.

[0451] Wetting agents are substances that, when added to a liquid, increase the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface onto which it spreads. Wetting agents serve two main functions in pesticide formulations: increasing the wetting rate of powders in water to make concentrates for soluble liquids or suspension concentrates during processing and manufacturing, and mixing the product with water in the spray tank to reduce the wetting time of wettable powders and improve water penetration into water-dispersible granules. Examples of wetting agents used in wettable powder, suspension concentrate, and water-dispersible granule formulations are sodium lauryl sulfate, sodium dioctyl sulfosuccinate, alkylphenol ethoxylates, and fatty alcohol ethoxylates.

[0452] Dispersants are substances that adsorb onto particle surfaces, helping to preserve particle dispersion and prevent particle reagglomeration. Dispersants are added to pesticide formulations to facilitate dispersion and suspension during manufacturing and ensure particle redispersion in the water in the spray tank. They are widely used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb onto particle surfaces and provide a charge or steric barrier against particle reagglomeration. The most commonly used surfactants are anionic, nonionic, or a mixture of the two types. For wettable powder formulations, the most common dispersant is sodium lignosulfonate. For suspension concentrates, very good adsorption and stabilization are achieved using polyelectrolytes such as sodium naphthalene sulfonate formaldehyde condensates. Tristyrylphenol ethoxylate phosphate esters are also used. Nonionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersants for suspension concentrates. In recent years, new types of ultra-high molecular weight polymeric surfactants have been developed as dispersants. These have a very long hydrophobic "backbone" and numerous ethylene oxide chains that form the "teeth" of a surfactant "comb." These high molecular weight polymers can provide excellent long-term stability to suspension concentrates because the hydrophobic backbone provides many anchoring points on the particle surface. Examples of dispersants used in pesticide formulations include sodium lignosulfonate, sodium naphthalenesulfonate formaldehyde condensate, tristyrylphenol ethoxylate phosphate esters, fatty alcohol ethoxylates, alkyl ethoxylates, EO-PO (ethylene oxide-propylene oxide) block copolymers, and graft copolymers.

[0453] An emulsifier is a substance that stabilizes the suspension of droplets of one liquid phase in another. Without an emulsifier, the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain alkylphenols or aliphatic alcohols with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzenesulfonic acid. A hydrophile-lipophile balance ("HLB") value range of 8 to 18 usually provides a well-stabilized emulsion. Emulsion stability can be improved by adding a small amount of an EO-PO block copolymer surfactant.

[0454] Solubilizers are surfactants that form micelles in water at concentrations above the critical micelle concentration. The micelles can then dissolve or solubilize water-insoluble materials within the hydrophobic compartment of the micelle. The types of surfactants commonly used for solubilization are nonionic, sorbitan monooleate, sorbitan monooleate ethoxylate, and methyl oleate.

[0455] Surfactants may be used alone or with other additives, such as mineral or vegetable oils, as adjuvants in spray tank mixtures to improve the biological performance of modified lipid compositions on targets. The type of surfactant used for bioenhancement generally depends on the nature and mode of action of the modified lipid composition. However, they are often non-ionic, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates.

[0456] Carriers or diluents in agricultural formulations are materials added to modified lipid compositions to obtain the required strength of the product. Carriers are usually materials with high absorption capacity, and diluents are usually materials with low absorption capacity. Carriers and diluents are used in the formulation of powders, wettable powders, granules, and water-dispersible granules.

[0457] Organic solvents are primarily used in the formulation of emulsifiable concentrates, oil-in-water emulsions, suspoemulsions, and ultra-microdosage formulations, and to a lesser extent, granular formulations. Mixtures of solvents may also be used. The first major group of solvents are aliphatic paraffin oils, such as kerosene or refined paraffin. The second major group (and most common) includes aromatic solvents, such as xylene, and the high molecular weight fractions of C9 and C10 aromatic solvents. Chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of the modified lipid composition when the formulation is emulsified in water. Alcohols are sometimes used as cosolvents to increase solvent output. Other solvents include vegetable oils, seed oils, and esters of vegetable and seed oils.

[0458] Thickeners or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheological or flow properties of liquids and prevent separation and settling of dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents generally fall into two categories: water-insoluble particles and water-soluble polymers. Clay and silica can be used to produce suspension concentrate formulations. Examples of these types of materials include, but are not limited to, montmorillonite, bentonite, magnesium aluminum silicate, and attapulgite. Water-soluble polysaccharides have been used as thickening and gelling agents for many years. The most commonly used types of polysaccharides are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum, locust bean gum, carrageenan, alginate, methylcellulose, sodium carboxymethylcellulose (SCMC), and hydroxyethylcellulose (HEC). Other types of anti-settling agents are based on modified starch, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0459] Microorganisms can cause spoilage of formulated products. Therefore, preservatives are used to eliminate or reduce their effects. Examples of such agents include, but are not limited to, propionic acid and its sodium salt, sorbic acid and its sodium or potassium salt, benzoic acid and its sodium salt, p-hydroxybenzoic acid sodium salt, methyl p-hydroxybenzoate, and 1,2-benzisothiazolin-3-one (BIT).

[0460] The presence of surfactants often causes foaming in aqueous formulations during the mixing process during manufacturing and application through spray tanks. To reduce the tendency for foaming, antifoaming agents are often added during the manufacturing stage or before filling into bottles. Generally, there are two types of antifoaming agents: silicone and non-silicone. Silicone antifoaming agents are usually aqueous emulsions of dimethylpolysiloxane, while non-silicone antifoaming agents are water-insoluble oils such as octanol and nonanol, or silica. In both cases, the function of the antifoaming agent is to remove the surfactant from the air-water interface.

[0461] "Green" agents (e.g., adjuvants, surfactants, solvents) can reduce the overall environmental footprint of crop protection formulations. Green agents are biodegradable and generally derived from natural and / or sustainable sources, such as plant and animal sources. Specific examples are vegetable oils, seed oils, and their esters, as well as alkoxylated alkyl polyglucosides.

[0462] In some instances, the modified lipid composition can be freeze-dried or lyophilized. See U.S. Patent No. 4,311,712. The modified lipid composition can be subsequently reconstituted upon contact with water or another liquid. Other components can be added to the freeze-dried or reconstituted modified lipid composition, such as other heterologous functional agents according to the formulations described herein, agriculturally acceptable carriers, or other materials.

[0463] Other optional features of the composition include a carrier or delivery vehicle that protects the modified lipid composition from UV and / or acidic conditions. In some examples, the delivery vehicle contains a pH buffer. In some examples, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, any one of the following pH ranges: about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.

[0464] For more information on agricultural formulations, see "Chemistry and Technology of Agrochemical Formulations" edited by D.A. Knowles, copyright 1998 by Kluwer Academic Publishers. Also see "Insecticides in Agriculture and Environment - Retrospects and Prospects" by A.S. Perry, I. Yamamoto, I. Ishaaya, and R. Perry, copyright 1998 by Springer-Verlag.

[0465] Pharmaceutical preparations The modified lipid composition is formulated into a pharmaceutical composition (i.e., a modified lipid composition composition) for administration to, for example, an animal (e.g., a human). The pharmaceutical composition can be administered to an animal (e.g., a human) together with a pharmaceutically acceptable diluent, carrier, and / or excipient. Depending on the mode of administration and dosage, the pharmaceutical composition of the methods described herein will be formulated into a suitable pharmaceutical composition for easy delivery. A single dose can be in the form of a unit dose, if necessary.

[0466] The modified lipid compositions can be formulated, for example, for oral administration, intravenous administration (e.g., injection or infusion), intramuscular administration, or subcutaneous administration to an animal. For injectable formulations, a variety of effective pharmaceutical carriers are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22 nd ed.,(2012)and ASHP Handbook on Injectable Drugs,18 th ed., (2014).

[0467] Suitable pharmaceutically acceptable carriers and excipients are non-toxic to recipients at the dosage and concentration employed.Acceptable carriers and excipients can include buffers such as phosphate, citrate, HEPES and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran and immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine and lysine, and carbohydrates such as glucose, mannose, sucrose and sorbitol.Modified lipid compositions can be formulated according to conventional pharmaceutical practice.The concentration of compound in the formulation will vary depending on many factors, including the dosage of the active agent (for example, modified lipid composition and nucleic acid) to be administered and the route of administration.

[0468] For oral administration to animals, the modified lipid composition can be prepared in the form of an oral formulation. Formulations for oral use can include tablets, caplets, capsules, syrups, or oral liquid dosage forms containing the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may include, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients may be colorants, flavoring agents, plasticizers, wetting agents, buffering agents, etc. Formulations for oral use may also be provided in unit dosage form as chewable tablets, non-chewable tablets, caplets, capsules (e.g., hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with a water or oil medium). The compositions disclosed herein may also further comprise immediate-release, sustained-release, or delayed-release formulations.

[0469] For parenteral administration to animals, modified lipid compositions can be formulated in the form of liquid solution or suspension and administered by parenteral administration route (for example, subcutaneous, intravenous, or intramuscular). Pharmaceutical compositions can be formulated for injection or infusion. Pharmaceutical compositions for parenteral administration can be formulated using sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, or cell culture medium (for example, Dulbecco's modified Eagle's medium (DMEM), alpha modified Eagle's medium (alpha-MEM), and F-12 medium). Formulation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor & Francis Group, CRC Press (2009).

[0470] xenofunctional drugs The modified lipid composition may further comprise a heterofunctional agent, such as, for example, a heterofunctional agent (e.g., a heterofunctional agricultural agent (e.g., an insecticide, fertilizer, herbicide, plant modifying agent) or a heterofunctional therapeutic agent (e.g., an antifungal agent, an antibacterial agent, an antiviral agent, an insecticide, a nematicide, an antiparasitic agent, or an insect repellent)). For example, the modified lipid composition may encapsulate the heterofunctional agent. Alternatively, the heterofunctional agent may be embedded on or complexed to the surface of the modified lipid composition. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different heterofunctional agents. The heterofunctional agents may be added at any step in the manufacturing process effective to introduce the agent into the modified lipid composition.

[0471] In certain examples, a heterologous functional agent (e.g., a heterologous agricultural agent (e.g., an insecticide, a fertilizer, a herbicide, a plant modifier, a heterologous nucleic acid, a heterologous polypeptide, or a heterologous small molecule) or a heterologous therapeutic agent (e.g., an antifungal agent, an antibacterial agent, a virucide, an antiviral agent, a nematicide, an antiparasitic agent, or an insect repellent)) can be modified. For example, the modification can be a chemical modification, e.g., conjugation with a marker, e.g., a fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation or operative linkage with a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of the agent, e.g., a lipid, a glycan, a polymer (e.g., PEG), or a cationic moiety.

[0472] Examples of heterofunctional agents are outlined below.

[0473] Different agricultural chemicals The modified lipid composition may include a heterologous agricultural agent (e.g., an agent that affects plants or organisms associated with plants) such as, for example, a pesticide, herbicide, fertilizer, or plant modifier, which may be loaded into the modified lipid composition.

[0474] For example, in some instances, the modified lipid composition may contain an insecticide. The insecticide may be an antifungal agent, an antibacterial agent, an insecticide, a molluscicide, a nematicide, a viricide, or a combination thereof. The insecticide may be a chemical agent, such as those known in the art. Alternatively or additionally, the insecticide may be a peptide, a polypeptide, a nucleic acid, a polynucleotide, or a small molecule. The insecticide may be an agent that can reduce the fitness of various plant pests, or may be an agent that targets one or more specific target plant pests (e.g., a specific species or genus of plant pests).

[0475] In some examples, the modified lipid composition may contain one or more heterologous fertilizer agents. Examples of heterologous fertilizer agents include, for example, plant nutrients or plant growth regulators, such as those known in the art. Alternatively, or in addition, the fertilizer agent may be a peptide, polypeptide, nucleic acid, or polynucleotide that can increase the fitness of plant symbionts. The fertilizer agent may be a drug that can increase the fitness of various plants or plant symbionts, or may be a drug that targets one or more specific target plants or plant symbionts (e.g., a specific species or genus of plant or plant symbiont).

[0476] In other instances, the modified lipid composition may include one or more heterologous plant modifying agents. In some instances, the plant modifying agent may include a peptide or a nucleic acid.

[0477] antibacterial agents The modified lipid compositions described herein may further comprise an antimicrobial agent. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antimicrobial agents. For example, the antimicrobial agent can reduce the fitness (e.g., reduce growth or kill) of a natural plant pest (e.g., a natural plant pathogen). The modified lipid composition containing an antibiotic can be contacted with a target pest or its infested plant in an amount and for a time sufficient to (a) achieve a target level of antibiotic concentration (e.g., a predetermined level or threshold level) within or on the target pest, and (b) reduce the fitness of the target pest. The antimicrobial agent may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0478] As used herein, the term "antimicrobial agent" refers to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of bacteria, such as plant pathogenic bacteria, and includes a bactericide (e.g., a disinfectant compound, antiseptic compound, or antibiotic) or a bacteriostatic agent (e.g., a compound or antibiotic). Bactericidal antibiotics kill bacteria, while bacteriostatic antibiotics only slow the growth or reproduction of bacteria.

[0479] Sanitizers may include disinfectants, antiseptics, or antibiotics. The most commonly used disinfectants include active chlorine (i.e., hypochlorites (e.g., sodium hypochlorite), chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide, etc.), active oxygen (peroxides, e.g., peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea perhydrate). perhydrate), iodine (iodopovidone (povidone-iodine, Betadine), Lugol's solution, iodine tincture, iodinated nonionic surfactants), concentrated alcohol (mainly ethanol, 1-propanol also called n-propanol, and 2-propanol called isopropanol, and their mixtures, in addition, 2-phenoxyethanol and 1- and 2-phenoxypropanol are used), phenolic substances (phenol (also called carbolic acid), cresol (combined with liquid potassium soap called lysol), halogenated (chlorinated, brominated) phenols, e.g., hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophen, These include chlorophenols, dibromophenols, and their salts, cationic surfactants, such as some quaternary ammonium cations (e.g., benzalkonium chloride, cetyltrimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, and benzethonium chloride), non-quaternary compounds, such as chlorhexidine, glucoprotamine, and octenidine dihydrochloride, strong oxidizing agents such as ozone and permanganate solutions, and heavy metals and their salts, such as colloidal silver, silver nitrate, mercury chloride, phenylmercuric salts, copper sulfate, copper oxide chloride, copper hydroxide, copper octanoate, copper oxychloride sulfate, copper sulfate, and copper sulfate pentahydrate. Heavy metals and their salts are the most toxic and environmentally harmful disinfectants, and their use is therefore strongly discouraged or canceled, as are appropriately concentrated strong acids (phosphoric acid, nitric acid, sulfuric acid, amidosulfonic acid, and toluenesulfonic acid) and alkalis (sodium, potassium, and calcium hydroxide).

[0480] As disinfectants (i.e., germicides that can be used on the human or animal body, skin, mucous membranes, wounds, etc.), under appropriate conditions (mainly concentration, pH, temperature and toxicity to humans / animals), some of the disinfectants mentioned above can be used. Among these, the most important are appropriately diluted chlorine preparations (i.e., Daquin solution, 0.5% sodium or potassium hypochlorite solution, pH adjusted to 7-8, or 0.5-1% benzenesulfochloramide sodium solution (chloramine B)), some iodine preparations, such as iodopovidone in various galenic preparations (ointments, solutions, wound dressings), and in the past, peroxides such as Lugol's solution, urea perhydrate solution, and pH-buffered 0.1-0.25% peracetic acid solutions; alcohols, with or without disinfectant additives, primarily used for skin disinfection; weak organic acids such as sorbic acid, benzoic acid, lactic acid, and salicylic acid; phenolic compounds such as hexachlorophene, triclosan, and dibromide; and cationically active compounds, such as 0.05-0.5% benzalkonium, 0.5-4% chlorhexidine, and 0.1-2% octenidine solutions.

[0481] The modified lipid composition may include an antibiotic. Any antibiotic known in the art may be used. Antibiotics are generally classified based on their mechanism of action, chemical structure, or spectrum of activity.

[0482] The antibiotics described herein may target any natural function or growth process and may be either bacteriostatic (e.g., slow or prevent natural growth) or bacteriocidal (e.g., kill bacteria). In some examples, the antibiotic is a bactericidal antibiotic. In some examples, bactericidal antibiotics target natural cell walls (e.g., penicillins and cephalosporins), target cell membranes (e.g., polymyxins), or inhibit essential natural enzymes (e.g., rifamycins, lipiarmycins, quinolones, and sulfonamides). In some examples, the bactericidal antibiotic is an aminoglycoside (e.g., kasugamycin). In some examples, the antibiotic is a bacteriostatic antibiotic. In some examples, the bacteriostatic antibiotic targets protein synthesis (e.g., macrolides, lincosamides, and tetracyclines). Additional classes of antibiotics that may be used herein include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin). Examples of antibiotics include rifampicin, ciprofloxacin, doxycycline, ampicillin, and polymyxin B. The antibiotics described herein can have any level of target specificity (e.g., narrow spectrum or broad spectrum). In some examples, the antibiotic is a narrow-spectrum antibiotic, thus targeting a specific type of bacteria, such as gram-negative or gram-positive bacteria. Alternatively, the antibiotic may be a broad-spectrum antibiotic that targets a wide range of bacteria.

[0483] Other non-limiting examples of antibiotics can be found in Table 1 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. One of ordinary skill in the art will understand that the appropriate concentration of each antibiotic in the composition will depend on factors such as antibiotic efficacy, stability, number of distinct antibiotics, formulation, and method of application of the composition.

[0484] antifungal agents The modified lipid composition may further comprise an antifungal agent. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different antifungal agents. For example, the antifungal agent can reduce the fitness of a fungal plant pest (e.g., reduce growth or kill). The modified lipid composition comprising the antifungal agent can be contacted with a target fungal pest, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level of antibiotic concentration (e.g., a predetermined level or threshold level) within or on the target fungus, and (b) reduce the fitness of the target fungus. The antifungal agent may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0485] As used herein, the terms "fungi" or "antifungal agent" refer to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of fungi, such as plant pathogenic fungi. Many different types of antifungal agents are manufactured and commercially available. Non-limiting examples of antifungal agents include azoxystrobin, mancozeb, prothioconazole, folpet, tebuconazole, difenoconazole, captan, bupirimate, or fosetyl-Al. Additional exemplary fungicides include, but are not limited to, strobilurins, azoxystrobin, dimoxystrobin, enstrobulin, fluoxastrobin, kresoxim-methyl, metominostrobin, picoxystrobin, pyraclostrobin, trifloxystrobin, orysastrobin, carboxamides, carboxanilides, benalaxyl, benalaxyl-M, benodanil, carboxin, mebenil, mepronil, Fenfuram, fenhexamid, flutolanil, furalaxyl, furcarbanil, furametpyr, metalaxyl, metalaxyl-M (mefenoxam), metofuroxam, metsulfovax, ofrace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbollide, salicillanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamide, bixafen, boscalid, morpholone, dimethicone Fluf, Flumorph, Benzamide, Flumetober, Fluopicolide (Picobenzamide), Zoxamide, Carboxamide, Carpropamid, Diclocymet, Mandipropamide, Silthiofam, Azoles, Triazoles, Bitertanol, Bromuconazole, Cyproconazole, Difenoconazole, Diniconazole, Enilconazole, Epoxiconazole, Fenbuconazole, Flusilazole, Fluquinconazole, Fluto Liafoll, hexaconazole, imibenconazole, ipconazole, methonazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimenol, triadimefon, triticonazole, imidazole, cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, benzimidazole, benomyl, carbendazim,Fuberidazole, thiadiabendazole, ethaboxam, etridiazole, hymexazole, nitrogen-containing heterocyclyl compounds, pyridine, fuazinam, pyrifenox, pyrimidine, bupirimate, cyprodinil, ferimzone, fenarimol, mepanipyrim, nuarimol, pyrimethanil, piperazine, triforine, pyrrole, fludioxonil, fenpiclonil, morpholine, aldimorph, dodemorph, fenpropimorph, tridemorph, dicarboximide, iprodione, procymidone, vinclozolin, acibenzola-S-methyl Anilazine, captan, captafol, dazomet, diclomezine, fenoxanil, folpet, fenpropizine, famoxadone, fenamidone, octhilinone, probenazole, proquinazide, pyroquilon, quinoxyfen, tricyclazole, carbamate, dithiocarbamate, ferbam, mancozeb, maneb, metiram, metam, propineb, thiram, zineb, ziram, dietofencarb, flubenchivabricarb, iprovalicarb, propamocarb, guanidine, dodine, iminoctadine, guazatine, kasugamycin, Polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing heterocyclyl compounds, isoprothiolane, dithianon, organophosphorus compounds, edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, organochlorine compounds, thiophanate-methyl, chlorothalonil, dichlofluanid, tolylfluanid, flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton, s Pyroxamine, cyflufenamid, cymoxanil, metrafenone, N-2-cyanophenyl-3,4-dichloroisothiazole-5-carboxamide (isothianil), N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-4-carboxamide, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]-pyridine, N-(3',4'-dichloro-4-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-e-4-carboxamide,5-chloro-7-(4-methylpiperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]tria-zolo[1,5-a]pyrimidine, 2-butoxy-6-iodo-3-propylchromen-4-one, N,N-dimethyl-3-(3-bromo-6-fluoro-2-methylindole-1-sulfonyl)-[1,2,4]triazole-1-sulfonamide, methyl-(2-chloro-5-[1-(3-methylbenzyloxyimino)-ethyl]benzyl)carbamate bamate, methyl-(2-chloro-5-[1-(6-methylpyridin-2-ylmethoxy-imino)ethyl]benzyl)carbamate, methyl 3-(4-chlorophenyl)-3-(2-isopropoxycarbonylamino-3-methylbutyryl-amino)propionate, 4-fluorophenyl N-(1-(1-(4-cyanophenyl)ethanesulfonyl)but-2-yl)carbamate, N-(2-(4-[3-(4-chlorophenyl)prop-2-yloxy]-3-methoxyphenyl)propionate N-(4'-bromobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide, N-(4'-trifluoromethylbi ... Examples of suitable antifungal agents include benzophenone-4-yl benzoate, N-(4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methyl-thiazole-5-carboxamide ... or methyl 2-(ortho-((2,5-dimethylphenyloxy-methylene)phenyl)-3-methoxyacrylate. One of ordinary skill in the art will understand that the suitable concentration of each antifungal agent in the composition will depend on factors such as the efficacy and stability of the antifungal agents, the number of individual antifungal agents, the formulation, and the method of application of the composition.

[0486] insecticides The modified lipid composition may further comprise an insecticide. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different insecticides. For example, the insecticide can reduce the fitness of an insect plant pest (e.g., reduce growth or kill). The modified lipid composition containing the insecticide can be contacted with a target pest, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level of insecticide concentration (e.g., a predetermined level or threshold level) within or on the target insect, and (b) reduce the fitness of the target insect. The insecticide may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0487] As used herein, the terms "insecticide" or "pesticide" refer to a substance that kills or inhibits the growth, growth, reproduction, or spread of insects, such as agricultural insect pests. Non-limiting examples of insecticides are set forth in Table 2 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. Further non-limiting examples of suitable insecticides include biologicals, hormones, or pheromones, such as azadirachtin, Bacillus species, Beauveria species, Codlemone, Metarhizium species, Paecilomyces species, Thuringiensis, and Verticillium species, as well as active compounds with unknown or unidentified mechanisms of action, such as fumigants (such as aluminum phosphide, methyl bromide, and sulfuryl fluoride) and selective antifeedants (such as cryolite, flonicamid, and pymetrozine). Those skilled in the art will understand that the suitable concentration of each pesticide in the composition will depend on factors such as pesticide efficacy, stability, number of separate pesticides, formulation, and method of application of the composition.

[0488] nematicides The modified lipid composition may further comprise a nematicide. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different nematicides. For example, the nematicide can reduce the fitness of nematode plant pests (e.g., reduce growth or kill them). The modified lipid composition containing the nematicide can be contacted with a target nematode pest, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level of nematicide concentration (e.g., a predetermined level or threshold level) inside or on the target nematode, and (b) reduce the fitness of the target nematode. The nematicide may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0489] As used herein, the terms "nematicide" or "nematicide" refer to a substance that kills or inhibits the growth, growth, reproduction, or spread of nematodes, such as agricultural nematode pests. Non-limiting examples of nematicides are set forth in Table 3 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. One of ordinary skill in the art will understand that the appropriate concentration of each nematicide in the composition will depend on factors such as nematicide efficacy, stability, number of separate nematicides, formulation, and method of application of the composition.

[0490] Molluscicide The modified lipid composition may further comprise a molluscicide. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different molluscicides. For example, the molluscicide can reduce the fitness of a mollusc plant pest (e.g., reduce growth or kill). The modified lipid composition comprising a molluscicide can contact a target mollusc pest, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level of molluscicide concentration (e.g., a predetermined level or threshold level) within or on the target mollusc, and (b) reduce the fitness of the target mollusc. The molluscicide may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0491] As used herein, the term "molluscicide" or "molluscicide" refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of mollusks, such as agricultural mollusc pests. Numerous chemicals can be used as molluscicides, including, for example, metal salts such as iron(III) phosphate, aluminum sulfate, and iron sodium EDTA(III),[3][4] metaldehyde, methiocarb, or acetylcholinesterase inhibitors. Those skilled in the art will understand that the appropriate concentration of each molluscicide in the composition will depend on factors such as the efficacy and stability of the molluscicide, the number of distinct molluscicides, the formulation, and the method of application of the composition.

[0492] Virucidal agents The modified lipid composition may further comprise a virucide. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different virucides. For example, the virucide can reduce (e.g., reduce or eliminate) the fitness of a viral plant pathogen. A modified lipid composition comprising a virucide described herein can be contacted with a target virus, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level of virucide concentration (e.g., a predetermined level or threshold level) and (b) reduce or eliminate the target virus. The virucides described herein may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0493] As used herein, the terms "virucide" or "antiviral agent" refer to a substance that kills or inhibits the growth, proliferation, reproduction, development, or spread of viruses, such as agricultural viral pathogens. Numerous agents can be used as virucidal agents, including chemicals or biological agents (e.g., nucleic acids, e.g., dsRNA). Those skilled in the art will understand that the appropriate concentration of each virucidal agent in the composition will depend on factors such as virucidal efficacy, stability, the number of distinct virucidal agents, the formulation, and the method of application of the composition.

[0494] herbicides The modified lipid composition can further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) herbicides. For example, the herbicides can reduce (e.g., decrease or eliminate) the fitness of the weed. The modified lipid composition comprising the herbicide can be contacted with the target weed in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of herbicide concentration on the plant and (b) reduce the fitness of the weed. The herbicide may be formulated in the modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0495] As used herein, the term "herbicide" refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of weeds. Many chemicals can be used as herbicides, including glufosinate, propaquizafop, metamitron, metazachlor, pendimethalin, flufenacet, diflufenican, clomazone, nicosulfuron, mesotrione, pinoxaden, sulcotrione, prosulfocarb, sulfentrazone, bifenox, quinmerac, triallate, terbuthylazine, atrazine, oxyfluorfen, diuron, trifluralin, or chlorotoluron. Further examples of herbicides include, but are not limited to, benzoic acid herbicides such as dicamba esters, phenoxyalkanoic acid herbicides such as 2,4-D, MCPA, and 2,4-DB esters, aryloxyphenoxypropionic acid herbicides such as clodinafop, cyhalofop, fenoxaprop, fluazifop, haloxifop, and quizalofop esters, pyridine carboxylic acid herbicides such as aminopyralid, picloram, and clopyralid esters, pyrimidine carboxylic acid herbicides such as aminocyclopyrachlor esters, pyridyloxyalkanoic acid herbicides, pyridyloxyalkanoic acid herbicides such as fluoroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides such as bromoxynil and ioxynil esters, esters of arylpyridine carboxylic acids, and the herbicides described in U.S. Pat. No. 7,314,849, U.S. Pat. No. 7,300,907, and U.S. Pat. No. 7,642,220, which is incorporated herein by reference in its entirety.In particular embodiments, the herbicide is 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butyrate, carfentrazone, chloramben, chlorimuron, chlorproham, chlorsulfuron, clethodim, clomazone, clopyralid, chloransulam, cyanazine, cycloate, DCPA, desmedipham, dichlobenil, diclofop, Diclosulam, diethathyl, difenzoquat, diflufenzopyr, dimethenamid-p, diquat, diuron, DSMA, endosole, EPTC, ethalfluralin, ethametsulfolone, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluthiacet, fomsafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, haloxifop, hexazinone, Imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, methazole, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxasulfuron, oxyflufen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, pramine Selected from the group consisting of rhodiamine, prometryn, pronamide, propachlor, propanil, prosulfuron, pyrazone, pyridate, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, and vernolate.Those skilled in the art will understand that the suitable concentration of each herbicide in the composition will depend on factors such as herbicide efficacy, stability, the number of separate herbicides, the formulation, and the method of application of the composition.

[0496] insect repellent The modified lipid composition may further comprise an insect repellent. In some examples, the modified lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different insect repellents. For example, the insect repellent may repel any of the pests (e.g., insects, nematodes, or mollusks), microorganisms (e.g., plant pathogens or endophytes such as bacteria, fungi, or viruses), or weeds described herein. The modified lipid composition comprising the insect repellent may be contacted with a target plant or an infested plant in an amount and for a time sufficient to (a) achieve a target level of insect repellent concentration (e.g., a predetermined level or threshold level) and (b) reduce the level of pests on the plant compared to an untreated plant. The insect repellent may be formulated in the modified lipid composition for any of the methods described herein, and in certain examples, may be associated with the modified lipid composition.

[0497] In some examples, the repellent is an insect repellent. Some examples of well-known insect repellents include benzyl, benzyl benzoate, 2,3,4,5-bis(butyl-2-ene)tetrahydrofurfural (MGK repellent 11), butoxypolypropylene glycol, N-butylacetanilide, normal-butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate (indanone), dibutyl adipate, dibutyl phthalate, di-normal-butyl succinate (Talbatrex); Included are N,N-diethyl-meta-toluamide (DEET), dimethyl carbonate (endo,endo)-dimethylbicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate), dimethyl phthalate, 2-ethyl-2-butyl-1,3-propanediol, 2-ethyl-1,3-hexanediol (Rutger 612), di-n-propyl isocinchomeronate (MGK insect repellent 326), 2-phenylcyclohexanol, p-methane-3,8-diol, and n-propyl N,N-diethyl succinamate. Other insect repellents include citronella oil, dimethyl phthalate, normal-butyl mesityl oxide oxalate, and 2-ethylhexanediol-1,3 (see Kirk-Othmer Encyclopedia of Chemical Technology, 2nd Ed., Vol. 11:724-728, and The Condensed Chemical Dictionary, 8th Ed., p 756).

[0498] The insect repellent may be synthetic or non-synthetic. Examples of synthetic insect repellents include methyl anthranilate and other anthranilic acid-based insect repellents, benzaldehyde, DEET (N,N-diethyl-m-toluamide), dimethyl carbate, dimethyl phthalate, icaridin (i.e., picaridin, Baylepel, and KBR 3023), indalone (e.g., "6-2-2" mixture (60% dimethyl phthalate, 20% indalone, 20% ethyl hexanediol), IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester), metofluthrin, permethrin, SS220, or tricyclodecenyl allyl ether. Examples of natural insect repellents include spider berry (Callicarpa) leaves, birch bark, and Myrica Gale), catnip oil (e.g., nepetalactone), citronella oil, essential oil of lemon eucalyptus (Corymbia citriodora, e.g., p-menthane-3,8-diol (PMD)), neem oil, lemongrass, tea tree oil from the leaves of Melaleuca alternifolia, tobacco, or extracts thereof.

[0499] fertilizer The modified lipid composition may further comprise a heterogeneous fertilizer agent. In some instances, the heterogeneous fertilizer agent is associated with the modified lipid composition. For example, the modified lipid composition may encapsulate the heterogeneous fertilizer agent. Additionally or alternatively, the heterogeneous fertilizer agent may be embedded in or complexed to the surface of the modified lipid composition.

[0500] Examples of heterologous fertilizer agents include plant nutrients or plant growth regulators, such as those known in the art. Alternatively, or in addition, the fertilizer agent may be a peptide, polypeptide, nucleic acid, or polynucleotide that can enhance the fitness of plant symbionts. The fertilizer agent may be an agent that can enhance the fitness of various plants or plant symbionts, or may be an agent that targets one or more specific target plants or plant symbionts (e.g., a specific species or genus of plant or plant symbiont).

[0501] In some examples, the heterologous fertilizer agent may be modified. For example, the modification may be a chemical modification, such as conjugation with a marker, such as a fluorescent or radioactive marker. In other examples, the modification may include conjugation or operative linkage with a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of a drug, such as a lipid, glycan, polymer (e.g., PEG), or cationic moiety.

[0502] In some examples, heterogeneous fertilizers include any material of natural or synthetic origin that is applied to soil or plant tissue to provide one or more plant nutrients essential for plant growth. Plant nutrients can include macronutrients, micronutrients, or combinations thereof. Plant macronutrients include nitrogen, phosphorus, potassium, calcium, magnesium, and / or sulfur. Plant micronutrients include copper, iron, manganese, molybdenum, zinc, boron, silicon, cobalt, and / or vanadium. Examples of plant nutrient fertilizers include urea, ammonium nitrate, ammonium sulfate, non-pressurized nitrogen solution, aqueous ammonia, anhydrous ammonia, ammonium thiosulfate, sulfur-coated urea, urea-formaldehyde, IBDU, polymer-coated urea, calcium nitrate, ureaform, or methylene urea; phosphate fertilizers such as diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, concentrated superphosphate, and triple superphosphate; or potassium fertilizers such as potassium chloride, potassium sulfate, potassium magnesium sulfate, and potassium nitrate. These compositions may exist as free salts or ions within the composition. Fertilizers may be designated by the content of one or more of their components, such as nitrogen, phosphorus, or potassium. The content of these components in a fertilizer may be indicated by an NPK value (where N = nitrogen content in weight percent, P = phosphorus content in weight percent, and K = potassium content in weight percent).

[0503] On the other hand, inorganic fertilizers are made from non-biological materials and include, for example, ammonium nitrate, ammonium sulfate, urea, potassium chloride, potassium, ammonium phosphate, anhydrous ammonia, and other phosphates. Inorganic fertilizers are readily available on the market and contain nutrients in soluble forms that are readily available to plants. Inorganic fertilizers are generally inexpensive and have low unit values ​​for the desired elements. Those skilled in the art will understand that the exact amount of a given element in a fertilizer formulation can be calculated and administered to plants or soil.

[0504] Fertilizers may be further classified as either organic or inorganic. Organic fertilizers include fertilizers with molecular frameworks that have a carbon skeleton, such as compositions derived from biological materials. Organic fertilizers are made from materials derived from living organisms. Animal manure, compost, bone meal, feather meal, and blood meal are common examples of organic fertilizers. However, organic fertilizers are typically not readily available to plants and require soil microorganisms to break down the fertilizer components into simpler structures before they can be used by plants. In addition, organic fertilizers not only induce plant growth responses, as observed with common inorganic fertilizers, but natural organic fertilizers may also stimulate the growth and activity of soil microbial populations. Increasing soil microbial populations (e.g., plant symbionts) can have significant beneficial effects on the physical and chemical properties of the soil and increase resistance to diseases and pests.

[0505] In one aspect, the modified lipid composition comprising a plant nutrient can be contacted with a plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of plant nutrient concentration in or on the plant, and (b) increase the fitness of the plant compared to an untreated plant.

[0506] In another aspect, the modified lipid composition comprising a plant nutrient can be contacted with the plant symbiont in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of plant nutrient concentration within or on the plant symbiont (e.g., a bacterial or fungal endosymbiont), and (b) increase the fitness of the plant symbiont compared to an untreated plant symbiont.

[0507] The heterologous fertilizer agent may include a plant growth regulator. Exemplary plant growth regulators include auxins, cytokines, gibberellins, and abscisic acid. In some examples, the plant growth regulator is abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6-dimethylpurine, ethephon, flumetralin, fluprimizole, fluthiacet, forchlorfunuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmone, thidiazuron, tripentenol, tributyl phosphorothioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl, and uniconazole. Other plant growth regulators that may be incorporated into the seed coating composition are described in U.S. Patent Application Publication No. 2012 / 0108431, which is incorporated by reference in its entirety.

[0508] Plant Modifiers The modified lipid compositions described herein include one or more heterologous plant modifying agents. For example, the modified lipid composition may encapsulate the heterologous plant modifying agent. Additionally or alternatively, the heterologous plant modifying agent may be embedded in or complexed to the surface of the modified lipid composition.

[0509] In some examples, the plant modifier may comprise a peptide or a nucleic acid. The plant modifier may be an agent that enhances the fitness of various plants or may be an agent that targets one or more specific plants (e.g., a specific species or genus of plants). Furthermore, in some examples, the modified lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different plant modifiers.

[0510] Additionally, in some instances, heterologous plant modifiers (e.g., agents comprising nucleic acid molecules or peptides) can be modified. For example, the modification can be a chemical modification, such as conjugation with a marker, e.g., a fluorescent marker or a radioactive marker. In other instances, the modification can include conjugation or operative linkage with a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of the agent, e.g., a lipid, a glycan, a polymer (e.g., PEG), or a cationic moiety.

[0511] Polypeptides The modified lipid composition may comprise a polypeptide. In some instances, the modified lipid composition comprises a polypeptide or a functional fragment or derivative thereof.

[0512] Exemplary polypeptides can include enzymes (e.g., metabolic recombinases, helicases, integrases, ribonucleases, deoxyribonucleases, or ubiquitinating proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene-editing proteins (e.g., CRISPR-Cas systems, TALENs, or zinc fingers), riboproteins, protein aptamers, or chaperones.

[0513] The polypeptide may include a naturally occurring polypeptide or a recombinantly produced variant. In some examples, the polypeptide may be a functional fragment or variant thereof (e.g., an enzymatically active fragment or variant thereof). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide described herein or a naturally occurring polypeptide, e.g., over a specific region or over the entire sequence. In some examples, a polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

[0514] Polypeptides may be formulated in modified lipid compositions. The compositions disclosed herein can contain any number or type (e.g., class) of polypeptides, for example, at least about any one of one polypeptide, two, three, four, five, ten, fifteen, twenty, or more polypeptides. The appropriate concentration of each polypeptide in the composition depends on factors such as the efficacy and stability of the polypeptide, the number of distinct polypeptides in the composition, the formulation, and the method of application of the composition. In some examples, each polypeptide in a liquid composition is about 0.1 ng / mL to about 100 mg / mL. In some examples, each polypeptide in a solid composition is about 0.1 ng / g to about 100 mg / g.

[0515] Methods for producing polypeptides are routine in the art. See generally, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0516] Although methods for producing polypeptides involve expression in plant cells, recombinant proteins can also be produced using insect cells, yeast, bacteria, mammalian cells, or other cells under the control of an appropriate promoter. Mammalian expression vectors can include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as early promoters, enhancer sites, splice sites, and polyadenylation sites of SV40 origin, can be used to provide other genetic elements required for expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with natural, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0517] Various mammalian cell culture systems can be used to express and produce recombinant polypeptide drugs. Examples of mammalian expression systems include CHO cells, COS cells, HeLA, and BHK cell lines. Host cell culture processes for the production of protein therapeutics are described, for example, in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Protein purification is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Protein therapeutic formulation is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0518] In some examples, the modified lipid composition comprises an antibody or its antigen-binding fragment. For example, the agent described herein may be an antibody that blocks or enhances the activity and / or function of a component. An antibody can act as an antagonist or agonist of a polypeptide (e.g., an enzyme or a cellular receptor). The production and use of antibodies against target antigens are known in the art. For example, see Zhiqiang An (Ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1st Edition, Wiley, 2009, and Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 2013, for antibody engineering, the use of degenerate oligonucleotides, methods for producing recombinant antibodies, including 5'-RACE, phage display, and mutagenesis, antibody testing and characterization, antibody pharmacokinetics and pharmacodynamics, antibody purification and storage, and screening and labeling techniques.

[0519] nucleic acid In some examples, the modified lipid composition comprises a nucleic acid (polynucleotide or polyribonucleotide). A large number of nucleic acids are useful in the modified lipid compositions and methods described herein. The modified lipid composition can comprise any number or type (e.g., class) of heterologous nucleic acids (e.g., DNA molecules (e.g., plasmids) or RNA molecules, such as mRNA, circRNA, guide RNA (gRNA), or inhibitory RNA molecules or their precursors (e.g., siRNA, shRNA, or miRNA, or precursors of any of these), or hybrid DNA-RNA molecules), for example, at least about one class or variant of nucleic acid, or 2, 3, 4, 5, 10, 15, 20 or more classes or variants of nucleic acid. The appropriate concentration of each nucleic acid in the composition depends on factors such as the efficacy and stability of the nucleic acid, the number of distinct nucleic acids, formulation, and the method of application of the composition. Examples of nucleic acids useful herein include DNA molecules (e.g., plasmids), mRNA, circRNA, siRNA, Dicer substrate small interfering RNA (dsiRNA), antisense RNA, small interfering RNA (siRNA) or siRNA precursors (e.g., one or more RNA strands that hybridize intermolecularly or intramolecularly to form an at least partially double-stranded RNA having at least about 20 consecutive base pairs), short hairpins (shRNA), microRNA (miRNA) or miRNA precursors, asymmetric interfering RNA (aiRNA), peptide nucleic acids (PNAs), morpholinos, locked nucleic acids (LNAs), piwi-interacting RNA (piRNA), ribozymes, deoxyribozymes (DNAzymes), aptamers (DNA, RNA), circular RNAs (circRNAs), guide RNAs (gRNAs), or DNA molecules encoding any of these RNAs.

[0520] Nucleic acid encoding a peptide In some examples, the modified lipid composition comprises a nucleic acid encoding a polypeptide, the nucleic acid encoding the polypeptide being about 10 to about 50,000 nucleotides (nts), about 25 to about 100 nts, about 50 to about 150 nts, about 100 to about 200 nts, about 150 to about 250 nts, about 200 to about 300 nts, about 250 to about 350 nts, about 300 to about 500 nts, about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 to about 1000 nts, about 1000 to about 2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, about 5 ... The length may be from about 10,000 to about 6,000 nts, from about 6,000 to about 7,000 nts, from about 7,000 to about 8,000 nts, from about 8,000 to about 9,000 nts, from about 9,000 to about 10,000 nts, from about 10,000 to about 15,000 nts, from about 10,000 to about 20,000 nts, from about 10,000 to about 25,000 nts, from about 10,000 to about 30,000 nts, from about 10,000 to about 40,000 nts, from about 10,000 to about 45,000 nts, from about 10,000 to about 50,000 nts, or any range therebetween.

[0521] Modified lipid compositions can also comprise the active variant of the nucleic acid sequence of interest.In some examples, nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence of the nucleic acid of interest, for example, over a specific region or over the entire sequence.In some examples, modified lipid compositions comprise the active polypeptide encoded by nucleic acid variant. In some examples, the active polypeptide encoded by the nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide of interest or a naturally occurring polypeptide sequence, for example, over a specific region or over the entire amino acid sequence.

[0522] A specific method for expressing a nucleic acid encoding a protein can involve expression in cells, including insects, yeast, plants, bacteria, or other cells, under the control of an appropriate promoter. Expression vectors can include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as early promoters, enhancer sites, splice sites, and polyadenylation sites of SV40 origin, can be used to provide other genetic elements required for the expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with natural, fungal, yeast, and mammalian cell hosts are described in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

[0523] Genetic modification using recombinant methods is generally known in the art.The nucleic acid sequence encoding desired gene can be obtained using recombinant methods known in the art, for example, by screening a library from cells that express gene, by extracting gene from a vector that is known to contain gene, or by directly isolating gene from cells and tissues that contain gene, using standard techniques.Alternatively, gene of interest can be produced synthetically instead of being cloned.

[0524] The expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector.The expression vector may be suitable for replication and expression in bacteria.The expression vector may also be suitable for replication and integration in eukaryotes.A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expressing the desired nucleic acid sequence.

[0525] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 base pairs (bp) upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0526] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), the long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.

[0527] Alternatively, the promoter can be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on the expression of the operably linked polynucleotide sequence when such expression is desired, or can turn off the expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0528] The expression vector to be introduced can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells desired to be transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include antibiotic resistance genes, such as neo.

[0529] Reporter genes can be used to identify potentially transformed cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient source, and encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., FEBS Letters 479:79-82, 2000). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the smallest 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0530] In some examples, an organism can be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins can be modified for a specific time, such as during the development or differentiation state of the organism. In one example, the invention includes compositions that alter the expression of one or more proteins, e.g., proteins that affect activity, structure, or function. The expression of one or more proteins can be restricted to a specific location or can be widespread throughout the organism.

[0531] mRNA The modified lipid composition can include an mRNA molecule, for example, an mRNA molecule encoding a polypeptide. The mRNA molecule can be, for example, chemically synthesized and modified. The mRNA molecule can be chemically synthesized or transcribed in vitro. The mRNA molecule can be placed on a plasmid, for example, a viral vector, a natural vector, or a eukaryotic expression vector. In some embodiments, the mRNA molecule can be delivered to cells by transfection, electroporation, or transduction (e.g., adenoviral or lentiviral transduction).

[0532] In some examples, the modified RNA agent described herein has modified nucleoside or nucleotide.Such modifications are known and are described, for example, in International Publication No. 2012 / 019168.Further modifications are described, for example, in International Publication No. 2015 / 038892, International Publication No. 2015 / 038892, International Publication No. 2015 / 089511, International Publication No. 2015 / 196130, International Publication No. 2015 / 196118 and International Publication No. 2015 / 196128A2, which are incorporated herein by reference in their entirety.

[0533] In some instances, the modified RNA encoding the polypeptide of interest has one or more terminal modifications, such as a 5' cap structure and / or a poly-A tail (e.g., 100-200 nucleotides in length). The 5' cap structure may be selected from the group consisting of CapO, CapI, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some instances, the modified RNA also contains a 5' UTR and a 3' UTR that contain at least one Kozak sequence. Such modifications are known and are described, for example, in International Publication Nos. WO 2012 / 135805 and WO 2013 / 052523, which are incorporated herein by reference in their entireties. Additional terminal modifications are described, for example, in WO 2014 / 164253 and WO 2016 / 011306, WO 2012 / 045075, and WO 2014 / 093924, which are incorporated by reference in their entireties. Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNAs) that may contain at least one chemical modification are described in WO 2014 / 028429, which is incorporated by reference in its entirety.

[0534] In some instances, modified mRNAs can be circularized or ligated to generate translationally competent molecules that facilitate interaction between polyA-binding proteins and 5'-end binding proteins. The cyclization or ligation mechanism can occur via at least three different pathways: 1) chemical, 2) enzymatic, and 3) ribozyme-catalyzed. The newly formed 5'-linkage / 3'-linkage can be intramolecular or intermolecular. Such modifications are described, for example, in International Publication No. 2013 / 151736.

[0535] The method of producing and purifying modified RNA is known and disclosed in the art.For example, modified RNA is produced only by in vitro transcription (IVT) enzyme synthesis.The method of producing IVT polynucleotide is known in the art, and is described in International Publication No. WO2013 / 151666, WO2013 / 151668, WO2013 / 151663, WO2013 / 151669, WO2013 / 151670, WO2013 / 151664, WO2013 / 151665, WO2013 / 151671, WO2013 / 151672, WO2013 / 151667 and WO2013 / 151736. Purification methods include purifying RNA transcripts containing polyA tails by contacting the sample with a surface bound to multiple thymidines or derivatives thereof and / or multiple uracils or derivatives thereof (polyT / U) under conditions such that the RNA transcripts bind to the surface, and eluting the purified RNA transcripts from the surface (WO 2014 / 152031), using ion (e.g., anion) exchange chromatography, which allows for the separation of longer RNAs up to 10,000 nucleotides in length, via a scalable method (WO 2014 / 144767), and subjecting the modified mRNA sample to DNase treatment (WO 2014 / 152030).

[0536] Formulations of modified RNA are known and are described, for example, in WO 2013 / 090648. For example, formulations can be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA), microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin sealants, fibrinogen, thrombin, rapidly clearing lipid nanoparticles, and combinations thereof.

[0537] Modified RNAs encoding polypeptides in the areas of human disease, antibodies, viruses, and various in vivo environments are known and are disclosed, for example, in WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, Table 6 of WO 2013 / 151736, Tables 6 and 7 of WO 2013 / 151672, Tables 6, 178, and 179 of WO 2013 / 151671, and Table 6, 185, and 186 of WO 2013 / 151667. Any of the foregoing may be synthesized as an IVT polynucleotide, a chimeric polynucleotide, or a circular polynucleotide, each of which may contain one or more modified nucleotides or terminal modifications.

[0538] cyclic polyribonucleotide The modified lipid composition may include a cyclic polyribonucleotide comprising one or more expression sequences encoding a polypeptide.

[0539] The polyribonucleotide may include an IRES (internal ribosome entry site) operably linked to the expression sequence encoding the polypeptide. The circular polyribonucleotide may include, for example, a splice junction joining a 5' exon fragment and a 3' exon fragment. The circular polyribonucleotide may include any one or more of the elements described herein. In some embodiments, the circular polyribonucleotide includes any feature or any combination of features disclosed in International Publication Nos. 2019 / 118919, WO2020 / 023655, WO2020 / 180751, WO2020 / 180752, WO2020 / 181013, WO2020 / 198403, WO2020 / 257730, WO2020 / 257727, and 2020 / 252436, each of which is incorporated herein by reference in its entirety.

[0540] In some embodiments, the circular polynucleotide further comprises a polyribonucleotide cargo. In some embodiments, the polyribonucleotide cargo comprises an expressed (or coding) sequence, a non-coding sequence, or a combination of expressed (coding) and non-coding sequences. In some embodiments, the polyribonucleotide cargo comprises an expressed (coding) sequence that encodes a polypeptide. In some embodiments, the polyribonucleotide comprises an IRES operably linked to the expressed sequence that encodes the polypeptide. In some embodiments, the IRES is located upstream of the expressed sequence. In some embodiments, the IRES is located downstream of the expressed sequence. In some embodiments, the circular polyribonucleotide further comprises a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The spacer region may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. The spacer region may be, for example, 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides. In some embodiments, the spacer region comprises a polyA sequence. In some embodiments, the spacer region comprises a polyAC sequence. In some embodiments, the spacer region comprises a polyAG sequence. In some embodiments, the spacer region comprises a polyAT sequence. In some embodiments, the spacer region comprises a random sequence. In some embodiments, the first annealing region and the second annealing region are combined, thereby forming a circular polyribonucleotide.

[0541] ...

Claims

1. 1. A modified lipid composition comprising: (b) an ionizable lipid, modified by (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipids, ceramide, glucosylceramide, lactosylceramide, galactosylcholesterol, and glucosylcholesterol; The modified lipid composition, wherein the one or more lipids increase delivery of the modified lipid composition to the spleen, characterized by an increased spleen-to-liver delivery ratio.

2. 2. The modified lipid composition of claim 1, wherein the structural component comprises cardiolipin derived from bovine heart.

3. 2. The modified lipid composition of claim 1, wherein the structural components comprise sphingolipids, the spingolipids being sphingomyelin lipids derived from animal brains.

4. (b) an ionizable lipid, modified by A modified lipid composition comprising: (a) a structural component comprising a soy-derived lipid.

5. 5. The modified lipid composition of claim 1 or 4, wherein the structured lipid is soy PI, soy LPI, soy PG, soy polar, soy LPC, soy PC, soy PE, soy PA, soy PL (phospholipid) mixture, soy PS, soy LPS, HSPC, cardiolipin, or a combination thereof.

6. 10. The modified lipid composition of claim 1 or 4, wherein the soy-derived lipid is a soy polar lipid composition, a soy PL mixture, or a combination thereof.

7. The soybean PL mixture about 30% to about 50% PC, about 20% to about 40% PE; about 10% to about 25% PI; about 1% to about 15% PA; about 1% to about 15% LPC, and 7. The modified lipid composition of claim 5 or 6, comprising about 0% to about 10% of other lipids.

8. 8. The modified lipid composition of claim 7, wherein the soy PL mixture comprises PC:PE:PI:PA:LPC:other lipids in a percentage ratio of approximately 38:30:18:7:7:

0.

9. The soy polar lipid composition comprises: about 40% to about 50% PC, about 15% to about 30% PE; about 10% to about 25% PI; about 1% to about 15% PA; about 0% to about 10% LPC, and 7. The modified lipid composition of claim 5 or 6, comprising from about 0% to about 15% of other lipids.

10. 10. The modified lipid composition of claim 9, wherein the soy polar lipid composition comprises PC:PE:PI:PA:LPC:other lipids in a percentage ratio of approximately 46:22:18:7:0:

7.

11. 5. The modified lipid composition of claim 4, wherein the soy-derived lipid increases delivery of the modified lipid composition to the spleen and is characterized by an increased spleen-to-liver delivery ratio.

12. 12. The modified lipid composition of claim 1 or 11, wherein the spleen-to-liver delivery ratio is 1.0 or greater.

13. 12. The modified lipid composition of claim 1 or 11, wherein increasing the amount of said structured lipid component present in said modified lipid composition increases said spleen to liver delivery ratio.

14. The modified lipid composition of claim 1 , wherein the structural component is modified by reconstituting the structural component with the ionized lipid.

15. 10. The modified lipid composition of claim 1 or 4, wherein the structural component comprises a purified soybean-derived lipid, and the structural component is modified by reconstituting the purified soybean-derived lipid with the ionized lipid.

16. The ionizable lipid may be: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain of at least two atoms; and (v) an N:P ratio of at least 3. The modified lipid composition of claim 1 or 4, having one or more characteristics selected from the group consisting of:

17. 5. The modified lipid composition of claim 1 or 4, wherein the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

18. The ionizable lipid is 【Chemistry 1】 wherein each R is independently selected from the group consisting of: 8 ~C 14 The modified lipid composition of claim 1 or 4, wherein the modified lipid composition is an alkyl group.

19. The ionizable lipid is selected from the group consisting of the following compounds: i) Formula 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: A is independently C 1 ~C 16 branched or unbranched alkyl of C 1 ~C 16 branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; B is independently C 1 ~C 16 branched or unbranched alkyl of C 1 ~C 16 branched or unbranched alkenyl of the formula: which may be substituted with heteroatoms or with OH, SH, or halogen; Each X is independently a biodegradable moiety; and W is 【Transformation 3】 where: R 5 is OH, SH, or NR 10 R 11 and R 6 are each independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 is a branched or unbranched alkenyl or cycloalkyl of the formula Each R 7 and each R 8 are independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl, halogen, OH, SH, or NR 10 R 11 wherein R 10 and R 11 are each independently H, C 1 ~C 3 alkyl or R 10 and R 11 together form a heterocycle, each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 wherein R 12 is H, C 1 ~C 7 branched or unbranched alkyl of C 2 ~C 7 is a branched or unbranched alkenyl of the formula: Q is O, S, or NR 13 wherein R 13 are H or C, respectively. 1 ~C 5 which is alkyl, ii) Formula 【Chemistry 4】 or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: 【Transformation 5】 is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, or 【Transformation 6】 and A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ) -, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S-, or a divalent heterocycle; Each of X and Z is independently absent, —O—, —CO—, —N(R 7 )-, -O-alkylene, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 , and R 120 each independently represents a C which may be interrupted by H, a heteroatom, or substituted by OH, SH, or a halogen. 1 ~C 16 Branched or unbranched alkyl or C 1 ~C 16 is a branched or unbranched alkenyl, or a cycloalkyl or substituted cycloalkyl of the formula: each of l and m is an integer from 1 to 10; t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; iii) Formula 【Transformation 7】 or expression 【Transformation 8】 and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: R 20 and R 30 are each independently H, C 1 ~C 5 branched or unbranched alkyl of C 2 ~C 5 or R 20 and R 30 forms a 3- to 7-membered cyclic ring together with the adjacent N atom, which is represented by R a may be substituted with R a is H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl, halogen, OH, or SH; Each R 1 and each R 2 are independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl of the formula (I), OH, halogen, SH, or NR 10 R 11 or R 1 and R 2 together form a circular ring, R 10 and R 11 are each independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 or R 10 and R 11 together form a heterocycle, n is 0, 1, 2, 3, or 4; Y is O or S; Z is absent, O, S, or N(R 12 ) wherein R 12 are each independently H, C 1 ~C 7 branched or unbranched alkyl of C 2 ~C 7 and wherein when Z is present, the adjacent R 1 and R 2 OH, NR 10 R 11 , or SH, v is 0, 1, 2, 3, or 4; y is 0, 1, 2, 3, or 4; Each A is independently 1 ~C 16 or a branched or unbranched alkylene of C 2 ~C 16 branched or unbranched alkenylene of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; Each B is independently 1 ~C 16 branched or unbranched alkyl of C 2 ~C 16 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH or halogen; and Each X is independently a biodegradable moiety; and iv) a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'); 【Chemistry 9】 a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, Each E is independently —OC(O)—, —C(O)O—, or —N(R 7 )C(O)-, -C(O)N(R 7 ) -, -C(O-R 13 )-O-, -C(O)O(CH 2 ) r -, -C(O)N(R 7 ) (CH 2 ) r -, -S-S-, or -C(O-R 13 )—O—(CH 2 ) r -, wherein R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R 13 is a branched or unbranched C 3 ~C 10 is alkyl, r is 1, 2, 3, 4, or 5; R a are each independently, C 1 ~C 5 Alkyl, C 2 ~C 5 alkenyl, or C 2 ~C 5 is alkynyl, u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t each independently represents C which may be interrupted by H, a heteroatom, or substituted by OH, SH, or a halogen; 1 ~C 16 Branched or unbranched alkyl or C 1 ~C 16 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl; and 【Chemistry 10】 represents the bond connecting the tail group to the head group; and 10. The modified lipid composition of claim 1 or 4, wherein the lipid has a pKa of about 4 to about 8.

20. The ionizable lipid is 【Chemistry 11】 and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: Each R 1 and each R 2 are independently H, C 1 ~C 3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or Each R 1 and each R 2 independently, taken together with the carbon atoms to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C 1 ~C 3 or R 10 and R 11 together form a heterocycle, Each R 3 and each R 4 are independently H, C 2 ~C 14 Branched or unbranched alkyl (e.g., C 3 ~C 10 branched or unbranched alkyl), or C 3 ~C 10 is a branched or unbranched alkenyl of the formula 3 and R 4 At least one of them is not H, Each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; V is a branched or unbranched C 2 ~C 10 Alkylene, C 2 ~C 10 Alkenylene, C 2 ~C 10 Alkynylene, or C 2 ~C 10 heteroalkylene, which may be substituted with one or more OH, SH, and / or halogen groups; R 6 are each independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 is a branched or unbranched alkenyl or cycloalkyl of the formula Each R 7 and each R 8 are independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl, halogen, OH, SH, (CH 2 ) v R 17 , or NR 10 R 11 wherein each v is independently 0, 1, 2, 3, 4, or 5; 17 is OH, SH, or N(CH 3 ) 2 and 20. The modified lipid composition of claim 19, wherein each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. V is a branched or unbranched C 2 ~C 3 alkylene, and R 6 21. The modified lipid composition of claim 20, wherein each is independently H or methyl.

22. The ionizable lipid is 【Chemistry 12】 and pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: Each R 1 and each R 2 are independently H, C 1 ~C 3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or Each R 1 and each R 2 independently, taken together with the carbon atoms to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C 1 ~C 3 or R 10 and R 11 together form a heterocycle, Each R 3 and each R 4 are independently H, C 2 ~C 14 Branched or unbranched alkyl (e.g., C 3 ~C 10 branched or unbranched alkyl), or C 3 ~C 10 is a branched or unbranched alkenyl of the formula 3 and R 4 At least one of them is not H, Each X is independently a biodegradable moiety; each s is independently 1, 2, 3, 4, or 5; T is —NHC(O)O—, —OC(O)NH—, or a divalent heterocycle which contains one or more —(CH 2 ) v OH, -(CH 2 ) v SH, -(CH 2 ) v - optionally substituted with halogen groups, Each R 7 and each R 8 are independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl, halogen, OH, SH, (CH 2 ) v R 17 , or NR 10 R 11 wherein R 17 is OH, SH, or N(CH 3 ) 2 and Each v is independently 0, 1, 2, 3, 4, or 5; and 20. The modified lipid composition of claim 19, wherein each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. 23. The modified lipid composition of claim 22, wherein T is a divalent piperazine or a divalent dioxopiperazine.

24. The modified lipid composition of any one of claims 20 to 23, wherein X is -OCO-, -COO-, -CONH-, or -NHCO-.

25. The ionizable lipid is a compound of group ii) represented by one of the following formulas: 【Chemistry 13】 During the ceremony, A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ) -, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S-, or a divalent heterocycle; R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; t1 is an integer from 0 to 10, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; each M is independently a biodegradable moiety; Each m1 is independently an integer of 3 to 6, Each l1 is independently an integer from 4 to 8, m2 and l2 each independently represent an integer of 0 to 3; R 80 and R 90 are each independently an unsubstituted C 5 ~C 8 alkyl or alkenyl, or R 80 is H or unsubstituted C 1 ~C 4 alkyl or alkenyl, R 90 is unsubstituted C 5 ~C 11 alkyl or alkenyl, and R 110 and R 120 are each independently an unsubstituted C 5 ~C 8 alkyl or alkenyl, or R 110 is H or unsubstituted C 1 ~C 4 alkyl or alkenyl, R 120 is unsubstituted C 5 ~C 11 20. The modified lipid composition of claim 19, wherein the lipid is alkyl or alkenyl.

26. M is —OC(O)— or —C(O)O—; 【Chemistry 14】 Ha, OH, 【Chemistry 15】 and R c are each independently H or C 1 ~C 3 is alkyl, and The modified lipid composition of claim 25, wherein each t1 is independently 1, 2, 3, or 4.

27. The ionizable lipid is a compound of group iii), wherein R 1 and R 2 are each H or R 1 are H and R 2 20. The modified lipid composition of claim 19, wherein one of the variables is OH and X is -OC(O)- or -C(O)O-.

28. The ionizable lipid is a compound of group iii) represented by formula III), wherein: R 20 and R 30 are each independently H or C 1 ~C 3 or R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered cyclic ring, which is represented by R a may be substituted with R a is H or OH, Z is absent, S, O, or NH; and 28. The modified lipid composition of claim 27, wherein n is 0, 1, or 2.

29. 28. The modified lipid composition of claim 27, wherein the ionizable lipid is a compound of group iii) represented by formula V).

30. The ionizable lipid is a compound of group iv), wherein the ionizable lipid comprises at least one head group and at least one tail group; the tail group has the structure of formula (TI) (or TI'), and The head group has a structure of one of the following formulas: i) 【Chemistry 16】 During the ceremony, R 20 and R 30 are each independently H, C 1 ~C 5 or C 2 ~C 5 branched or unbranched alkenyl of the formula: which may be interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl groups; or R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which may be optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; R 1 and R 2 each independently represents H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl of the formula (I), OH, halogen, SH, or NR 10 R 11 or R 1 and R 2 together form a circular ring, R 10 and R 11 each independently represents H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 or R 10 and R 11 together form a heterocycle, n is 0, 1, 2, 3, or 4; and Z is absent, O, S, or NR 12 wherein R 12 is H or C 1 ~C 7 and when Z is present, the adjacent R 1 and R 2 OH, NR 10 R 11 , or SH, ii) 【Chemistry 17】 During the ceremony, R 1 is H, C 1 ~C 3 Alkyl, OH, halogen, SH, or NR 10 R 11 and R 2 is OH, halogen, SH, or NR 10 R 11 or R 1 and R 2 can join together to form a cyclic ring, R 10 and R 11 are each independently H or C 1 ~C 3 alkyl or R 10 and R 11 can be taken together to form a heterocycle, R 20 and R 30 are each independently H, C 1 ~C 5 branched or unbranched alkyl of C 2 ~C 5 or R 20 and R 30 can be taken together to form a cyclic ring, and each of v and y is independently 1, 2, 3, or 4; iii) [Chemistry 18] In the formula, W is 【Chemistry 19】 and During the ceremony, R 5 OH, SH, (CH 2 ) s OH or NR 10 R 11 and R 6 are each independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 is a branched or unbranched alkenyl or cycloalkyl of R 7 and R 8 are each independently H, C 1 ~C 3 branched or unbranched alkyl of C 2 ~C 3 branched or unbranched alkenyl, halogen, (CH 2 ) v OH, (CH 2 ) v SH, (CH 2 ) s N (CH 3 ) 2 , or NR 10 R 11 wherein R 10 and R 11 are each independently H or C 1 ~C 3 alkyl or R 10 and R 11 together form a heterocycle, or R 7 and R 8 come together to form a ring, R 20 are each independently H or C 1 ~C 3 is a branched or unbranched alkyl of the formula R 14 is a heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , N.R. 10 C(O)NR 10 R 11 , or NR 10 C(S)NR 10 R 11 wherein R 10 and R 11 are each independently H, C 1 ~C 3 Alkyl, C 3 ~C 7 Cycloalkyl, C 3 ~C 7 cycloalkenyl, which may be substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle, R 16 is H, ═O, ═S, or CN, each of s, u, and t is independently 1, 2, 3, 4, or 5; Each v is independently 0, 1, 2, 3, 4, or 5; Each Y is a divalent heterocycle; Each Z is independently absent, O, S, or NR 12 and R 12 is H, C 1 ~C 7 or C 2 ~C 7 is a branched or unbranched alkenyl of the formula: Q is O, S, CH 2 , or NR 13 and R 13 are H and C, respectively. 1 ~C 5 is alkyl, V is a branched or unbranched C 2 ~C 10 Alkylene, C 2 ~C 10 Alkenylene, C 2 ~C 10 Alkynylene or C 2 ~C 10 heteroalkylene, which may be substituted with one or more OH, SH, and / or halogen groups; and T is —NHC(O)O—, —OC(O)NH—, or a divalent heterocycle; and iv) 【Chemistry 20】 During the ceremony, 【Chemistry 21】 is a cyclic or heterocyclic moiety, Y is alkyl, hydroxy, hydroxyalkyl, 【Chemistry 22】 and A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 ) -, -N(R 7 )C(O)N(R 7 )-, -S-, or -S-S-; Each of X and Z is independently absent, —O—, —C(O)—, —N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; R 7 are each independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl; t is 0, 1, 2, or 3; t1 is an integer from 0 to 10, and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; and 20. The modified lipid composition of claim 19, wherein the lipid has a pKa of from about 4 to about 8.

31. wherein the ionizable lipid is a compound of group iv) and at least one tail group of the lipid has one of the following formulas: 【Chemistry 23】 During the ceremony, R 7 are each independently H or methyl; R b is independently H or C for each occurrence 1 ~C 4 is alkyl, u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and The head group has the formula: i) 【Chemistry 24】 wherein m is 1, 2, 3, 4, 5, 6, 7, or 8; ii) 【Chemistry 25】 iii) 【Chemistry 26】 and iv) 【Chemistry 27】 31. The modified lipid composition of claim 30, having one of the following structures:

32. At least one tail group has a structure of formula (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'), where u1 is 3 to 5, u2 is 0 to 3, u3 and u4 are each independently 1 to 7, and R a The modified lipid composition of claim 31 , wherein each is independently methyl.

33. 20. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of Table I, Table II, Table III, or Table IV.

34. The ionizable lipid is 【Chemistry 28-1】 【Chemistry 28-2】 【Chemistry 28-3】 34. The modified lipid composition of claim 33, wherein:

35. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition further comprises a sterol and / or a polyethylene glycol (PEG) lipid conjugate.

36. The modified lipid composition of claim 35, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

37. The modified lipid composition of claim 36, wherein the PEG-lipid conjugate is PEG2000-DMG or PEG2000-PE.

38. The modified lipid composition comprises: about 20 mol% to about 50 mol% of said ionized lipid; about 10 mol % to about 75 mol % of said structural component; from about 0 mol % to about 45 mol % of said sterol; and 36. The modified lipid composition of claim 35, comprising about 0.5 mol% to about 3 mol% of said polyethylene glycol (PEG) lipid conjugate.

39. The modified lipid composition comprises: about 20 mol% to about 50 mol% of said ionized lipid; about 20 mol % to about 60 mol % of said structural component; about 7 mol % to about 45 mol % of said sterol, and The modified lipid composition of claim 35, comprising about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG) lipid conjugate.

40. 39. The modified lipid composition of claim 38, wherein the modified lipid composition comprises ionizable lipid:structured lipid:sterol:PEG lipid in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 22.5:75:0:2.5, about 35:30:32.5:2.5, about 35:16:46.5:2.5, about 35:25:37.5:2.5, about 35:40:22.5:2.5, about 45:10:43.5:1.5, about 50:20:28.5:1.5, or about 50:10:38.5:1.

5.

41. 36. The modified lipid composition of claim 35, wherein the structured lipids comprise structured lipid 1 and structured lipid 2, and wherein structured lipid 1 and structured lipid 2 are different structured lipids.

42. 42.5:2.5, or about 35:(45+5):12.5:2.

5.

43. The modified lipid composition of claim 41, wherein the modified lipid composition comprises ionizable lipid:(structural lipid 1 + structural lipid 2):sterol:PEG lipid in a molar ratio of about 35:(3+13):46.5:2.5, about 35:(15+5):42.5:2.5, or about 35:(45+5):12.5:2.

5.

43. 5. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions.

44. 5. The modified lipid composition of claim 1, wherein the modified lipid composition is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

45. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition is a lipid nanoparticle.

46. 46. ​​The modified lipid composition of claim 45, wherein the lipid nanoparticles have a particle size of less than about 200 nm.

47. 46. ​​The modified lipid composition of claim 45, wherein the lipid nanoparticles have a size of less than about 100 nm.

48. 46. ​​The modified lipid composition of claim 45, wherein the lipid nanoparticles have an average polydispersity index (PDI) in the range of about 0.1 to about 0.

4.

49. 46. ​​The modified lipid composition of claim 45, wherein the lipid nanoparticles have an average PDI in the range of about 0.2 to about 0.

3.

50. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition further comprises one or more heterologous functional agents.

51. 51. The modified lipid composition of claim 50, wherein the heterologous functional agent is encapsulated by, embedded on the surface of, or complexed to the surface of the modified lipid composition.

52. 51. The modified lipid composition of claim 50, wherein the heterologous functional agent comprises a polynucleotide.

53. 53. The modified lipid composition of claim 52, wherein the polynucleotide is mRNA or circRNA.

54. 51. The modified lipid composition of claim 50, wherein the modified lipid composition has a total lipid:heterogeneous functional agent weight ratio of about 50:1 to about 10:

1.

55. 55. The modified lipid composition of claim 54, wherein the modified lipid composition has a total lipid:heterologous functional agent weight ratio of about 40:1 to about 28:

1.

56. 55. The modified lipid composition of claim 54, wherein the modified lipid composition has a total lipid:heterologous functional agent weight ratio of about 37:1 to about 33:

1.

57. 1. A method for delivering one or more heterologous functional agents to a cell or a subject, the method comprising:

57. A method comprising contacting the cells with or administering to the subject one or more heterologous functional agents and modified lipid compositions according to any one of claims 1 to 56, wherein the heterologous functional agent is encapsulated by, embedded on the surface of, or complexed to the surface of the modified lipid composition.

58. 58. The method of claim 57, wherein the one or more heterologous functional agents are delivered to spleen cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or bone marrow cells.

59. 58. The method of claim 57, wherein the modified lipid composition increases transfection of the spleen cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or bone marrow cells.

60. 60. The method of claim 59, wherein the splenic lymphocytes are transfected at a frequency of at least 1%, at least 2%, or at least 5% of the parent population.

61. 60. The method of claim 59, wherein the splenic bone marrow cells are transfected at a frequency of at least 5%, at least 10%, or at least 15% of the parent population.

62. 60. The method of claim 59, wherein the hematopoietic stem cells are transfected at a frequency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the parent population.

63. 58. The method of claim 57, wherein the heterologous functional agent comprises a polynucleotide.

64. 64. The method of claim 63, wherein the polynucleotide is mRNA or circRNA.

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