A modified lipid composition and uses thereof

Abstract

Disclosed herein are modified lipid compositions comprising (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol; and modified by (b) an ionizable lipid. The disclosure also includes a method for making a modified lipid composition, comprising reconstructing (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, and / or glucosyl cholesterol in the presence of (b) an ionizable lipid, to produce the modified lipid composition, and loading into the modified lipid composition with one or more heterologous functional agents.

Description

A MODIFIED LIPID COMPOSITION AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 63 / 441 ,666, filed January 27, 2023; and U.S. Provisional Application No. 63 / 597,249, filed November 8, 2023, both of which are herein incorporated by reference in their entirety.BACKGROUND

[0002] The delivery of a heterologous functional agent (such as a therapeutic agent or immunologic agent) can be limited by the degree to which the agent can penetrate cell barriers and thereby effectively act on an organism. Therefore, there is a continuing need in the art for developing novel delivery systems that can effectively deliver the heterologous functional agent and promote cellular uptake of the agent.SUMMARY OF THE INVENTION

[0003] In one aspect, provided herein is a modified lipid composition, comprising:(a) a structural component comprising one or more lipids selected from the group consisting of a soy-derived lipid, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, and glucosyl cholesterol; modified by(b) an ionizable lipid.

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

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

[0006] In some embodiments, the structural component comprising one or more lipids is soy derived lipid.

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

[0008] In some embodiments, the soy-derived lipid increases the delivery of the modified lipid composition to spleen, characiterized 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 greater.

[0010] In some embodiments, an increase in the amount of the structural lipid component 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 soy lipid extracts or soy-derived lipids, and an ionizable lipid.

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

[0013] In some embodiments, the one or more heterologous functional agents is delivered to splenic cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or myeloid cells.

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

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

[0016] In some embodiments, the myeloid cells (e.g., the splenic myeloid 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 this invention, in some embodiments, the soy-derived lipid component comprises soy PC (phosphatidylcholine), soy PE (phosphatidylethanolamine), soy PI (phosphatidylinositol), soy PA (phosphatidic acid), soy lyso PC (LPC), soy lyso PI (LPI), soy PG (phosphatidylglycerol), soy PS (phosphatidylserine), soy lyso PS (LPS), HSPC (hydrogenated soybean 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, a 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, soy PL mixture, or a combination thereof.

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

[0021] In some embodiments, the soy polar lipid composition comprise PC, PE, PI, PA, and optionally LPC. In some embodiments, the soy polar lipid composition comprise about 40% to about 50% of PC, about 15% to about 30% of PE, about 10% to about 25% of PI, about 1% to about 15% of PA, about 0% to about 10% of LPC, and about 0% to about 15% of other lipids.In one embodiment, the soy polar lipid composition comprises PC:PE:PI:PA:LPC:other lipids at 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 comprisesabout 30% to about 50% of PC, about 20% to about 40% of PE, about 10% to about 25% of PI, about 1 % to about 15% of PA, about 1 % to about 15% of LPC, and about 0% to about 10% of other lipids.In one embodiment, the soy PL mixture comprises PC:PE:PI:PA:LPC:other lipids at a percentage ratio of about 38:30:18:7:7:0.

[0023] In another aspect, provided herein is a a modified lipid composition comprising a plurality of lipid reconstructed structural components, wherein the lipid reconstructed 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 film; (c) reconstituting the lipid film in an organic solvent selected from the group consisting of acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-buthanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofurammethanol, dimethyl sulfoxide:methanol, and dimethylformamide:methanol, thereby producing a lipid solution; and (d) processing the lipid solution of step (c) in a microfluidics device comprising an aqueous phase, thereby producing the modified lipid composition.

[0024] In another aspect, provided herein is a method for making a modified lipid composition. The method comprises reconstructing (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol in the presence of (b) an ionizable lipid, to produce the modified lipid composition. The method further comprises loading into the modified lipid composition with one or more heterologous functional agents. The ionizable lipid has two or more of the characteristics listed below:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails, wherein each of the lipid tails 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) an N:P ratio of at least 10.

[0025] In some alternative embodiments, the ionizable lipid has two or more of the characteristics listed below:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails, wherein each of the lipid tails 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) an N:P ratio of at least 3.

[0026] In another aspect, provided herein is a method for delivering a modified lipid composition to atarget cell, the method comprising introducing a modified lipid composition that comprises (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol; and (b) an ionizable lipid to the target cell.

[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 soybean or animal (e.g. porcine, bovine, etc).

[0028] In some embodiments, the structural component is modified by reconstructing a film comprising the structural component in the presence of the ionizable lipid.

[0029] In some embodiments, the structural component comprises soy-derived lipids, and the structural lipid is modified by reconstructing a film comprising the purified soy-derived lipids with the ionizable lipid.

[0030] In some embodiments, the ionizable lipid has one or more characteristics selected from the group consisting of:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails, wherein each of the lipid tails 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) an N:P ratio of at least 10.

[0031] In some alternative embodiments, the ionizable lipid has one or more of the characteristics selected from the group consisting of:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails, wherein each of the lipid tails 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) an 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, LP01 , 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, wherein R is a C8-C14 alkyl group.

[0034] In some embodiments, the ionizable lipid may be selected from one of the following groups of compounds: i) a compound of formulapharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: each A is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each B is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each X is independently a biodegradable moiety; and,Rs is OH, SH, or NR10R11; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR10R11, wherein each R10 and Rn is independently H, C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring; R7 and Rs 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 NR12, wherein R12 is H, C1-C7 branched orunbranched alkyl, or C2-C7 branched or unbranched alkenyl, andQ is O, S, or NR13, wherein each R13 is H, or C1-C5 alkyl; ii) a compound of formulapharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:a cyclic or heterocyclic moiety;Y is alkyl, hydroxy, hydroxyalkyl orA is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -S-S-, or a bivalent heterocycle; each of X and Z is independently absent, -O-, -CO-, -N(R7)-, -O-alkylene-;-alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(0)-, -C(0)N(R7)-, or -S-; each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; each of R30, R40, R50, Reo, R70, Rso, R90, R100, R110, and R120 is independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of I and m is an integer from 1 to 10; t1 is an integer from 0 to 10; andW is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocylyl or heteroaryl; iii) a compound of formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein:R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, or R20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra;Rais H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, orR1 and R2 are taken together to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R10 and Rn are taken together to form a heterocyclic ring; n is 0, 1 , 2, 3 or 4;Y is O or S;Z is absent, O, S, or N(Ri2>, wherein each R12 is independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH; v is 0, 1 , 2, 3, or 4; y is 0, 1 , 2, 3, or 4; each A is each independently C1-C16 branched or unbranched alkyl, or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; each B is each independently C1-C16 branched or unbranched alkyl, or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with 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 (Tl)pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:E is each independently -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -C(O-RI3)-O-, -C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, -S-S-, or -C(O-Ri3)-O-(CH2)r-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl;R13 is branched or unbranched C3-C10 alkyl; r is 1 , 2, 3, 4, or 5;Rais each independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; u1 and u2 are each independently 0, 1 , 2, 3, 4, 5, 6, or 7;R‘ is each independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; and*> represents the bond connecting the tail group to the head group; and wherein the lipid has a pKa from about 4 to about 8.

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

[0036] In some embodiments, the modified lipid composition further comprises a sterol. The reconstruction (or reconstitution) of the structural component is therefore carried out in the presence of an ionizable lipid and a sterol.

[0037] In some embodiments, the modified lipid composition further comprises a polyethylene glycol (PEG)-lipid conjugate. The reconstruction (or reconstitution) of the structural component is therefore carried out in the presence of an ionizable lipid and a PEGylated lipid (or a PEG-lipid conjugate).

[0038] In some embodiments, the modified lipid composition further comprises a sterol and / or a polyethylene glycol (PEG)-lipid conjugate. The reconstruction (or reconstitution) of the structural component is therefore carried out in the presence of an ionizable lipid, a sterol, and / or a PEGylated lipid (or a PEG-lipid conjugate).

[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 the ionizable lipid, about 10 mol% to about 75 mol% of the structural component, about 0 mol% to about 45 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

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

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

[0044] In some embodiments, the modified lipid composition comprises: about 35 mol% of the ionizable lipid, about 50 mol% of the structural component (e.g., the structural lipids), about 12.5 mol% of the sterol, andabout 2.5 mol% the polyethylene glycol (PEG)-lipid conjugate.

[0045] In one embodiment, the modified lipid composition comprises ionizable lipid:structural li pids:stero I : PEG-li pid at a molar ratio of about 35:50:12.5:2.5.

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

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

[0048] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol:PEG-lipid at a molar ratio of about 35:30:32.5:2.5.

[0049] In one embodiment, the modified lipid composition comprises ionizable lipid:structural li pids:stero I : PEG-li pid at a molar ratio of about 35:16:46.5:2.5.

[0050] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol:PEG-lipid at a molar ratio of about 35:25:37.5:2.5.

[0051] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol:PEG-lipid at a molar ratio of about 35:40:22.5:2.5.

[0052] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol: PEG-lipid at a molar ratio of about 45:10:43.5:1 .5.

[0053] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol:PEG-lipid at a molar ratio of about 50:20:28.5:1 .5.

[0054] In one embodiment, the modified lipid composition comprises ionizable lipid:structural lipids:sterol: PEG-lipid at a molar ratio of about 50:10:38.5:1 .5.

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

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

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

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

[0059] In some embodiments, the modified lipid composition comprises: lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, and glucosyl cholesterol,C12-200, cholesterol, andDMPE-PEG2k.

[0060] In one embodiment, the modified lipid composition comprises: polar lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, and glucosyl cholesterol,C12-200, cholesterol, andDMPE-PEG2k. The modified lipid composition may comprise C12-200: structural lipids:cholesterol: DMPE-PEG2k at 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 ionizable lipidstructural lipid:sterol:PEG-lipid at a molar ratio of 22.5:75:0:2.5. In some embodiments, the modified lipid composition may comprise ionizable lipid structural li pid sterol: PEG-lipid at a molar ratio of 35:50:12.5:2.5. In some embodiments, the modified lipid composition may comprise ionizable lipidstructural lipid:sterol:PEG-lipid at a molar ratio of 35:30:32.5:2.5. In some embodiments, the modified lipid composition may comprise an ionizable lipid structural lipid :sterol:PEG-lipid at a molar ratio of 35:20:42.5:2.5. In some embodiments, the modified lipid composition may comprise ionizable lipidstructural lipid:sterol:PEG-lipid at 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 at a molar ratio of 35:(3+13):46.5:2.5 (structural lipid 1 may be the structural lipid described herein; structural lipid 2 may be the structural lipid described herein that is different than structural lipid 1 , or structural lipid 2 may be any lipid suitable for preparation of a lipid nanoparticle composition).

[0062] In some embodiments, the modified lipid composition is a lipophilic moiety selected from the group consisting of a lipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion. In one embodiment, the modified lipid composition is a liposome selected from the group consisting of a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide-containing nanoliposome, and a multivesicular liposome.

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

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

[0065] In some embodiments, the average polydispersity index (PDI) of the particles of the modified lipid composition ranges from about 0.1 to about 0.5. In some embodiments, the average PDI of the particles of the modified lipid composition ranges from about 0.1 to about 0.4. In some embodiments, the average PDI of the particles of the modified lipid composition ranges from about 0.2 to about 0.3.

[0066] In some embodiments, the modified lipid composition comprises one or more heterologous functional agents. In some embodiments, the heterologous functional agent is encapsulated by the modified lipid composition. In some embodiments, the heterologous functional agent is embedded on the surface of the modified lipid composition. In some embodiments, the heterologous functional agent is conjugated 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 chosen from an mRNA, a circular RNA (circRNA), an siRNA orsiRNA precursor, a microRNA (miRNA) or miRNA precursor, a plasmid, a Dicer substrate small interfering RNA (dsiRNA), a short hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a peptide nucleic acid (PNA), a morpholino, a locked nucleic acid (LNA), a piwi-interacting RNA (piRNA), a ribozyme, a deoxyribozyme (DNAzyme), an aptamer, a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is a 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 encapsulation efficiency of the polynucleotide by the modified lipid composition is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more than 99%. In one embodiment, the encapsulation efficiency of the polynucleotide by the modified lipid composition is at least about 90%.

[0069] In some embodiments, the modified lipid composition has a total lipid:heterologous functional 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:heterologous functional 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:heterologous functional 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:heterologous functional 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:heterologous functional 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 a human. In some embodiments, the modified lipid composition is formulated for delivery to a plant.

[0071] In some embodiments, the modified lipid composition is produced by a method comprising lipid extrusion. In some embodiments, the modified lipid composition is produced by a method comprising processing a solution comprising the lipids from the modified lipid composition in a microfluidics 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 an aqueous phase. In some embodiments, the aqueous phase and the lipid solution (organic phase) are mixed at a 3:1 volumetric ratio.

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

[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 can be at 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 NaCI.

[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 may 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 freeze-dried or lyophilized composition. The freeze-dried or lyophilized modified lipid composition may comprise one or more lyoprotectants. The lyophilized modified lipid composition may comprise a poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized modified lipid composition comprises a poloxamer (e.g., about 0.01 to about 1.0 % w / w of a 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 of the heterologous functional agents (e.g., polynucleotides). In some embodiments, the lyophilized modified lipid composition comprises about 1 .0 to about 5.0 % w / w lipids. In some embodiments, the lyophilized modified lipid composition comprises about 0.5 to about 2.5 % w / w of TRIS buffer. In some embodiments, the lyophilized modified lipid composition comprises about 0.75 to about 2.75 % w / w of NaCI. In some embodiments, the lyophilized modified lipid composition comprises about 85 to about 95 % w / w of a sugar, e.g., sucrose. In some embodiments, the lyophilized modified lipid composition comprises about 0.01 to about 1.0 % w / w of a poloxamer (e.g., about 0.01 to about 1.0 % w / w of a poloxamer) such as poloxamer 188. In some embodiments, the lyophilized modified lipid composition comprises about 1 .0 to about 5.0 % w / w of potassium sorbate.Definitions

[0079] As used herein, the term “effective amount,” “effective concentration,” or “concentration effective to” refers to an amount of a modified lipid composition, or nucleic acid composition, sufficient to effect the recited result or to reach a target level (e.g., a predetermined or threshold level) in or on 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 virucidal agent, an anti-viral agent, an insecticidal agent, a nematicidal agent, an antiparasitic agent, or an insect repellent.

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

[0082] As used herein, the term “functional agent” refers to an agent (e.g., an agricultural agent (e.g., pesticidal agent, fertilizing agent, herbicidal agent, plant-modifying agent) or a therapeutic agent (e.g., an antifungal agent, an antibacterial agent, a virucidal agent, an anti-viral agent, an insecticidal agent,a nematicidal agent, an antiparasitic agent, or an insect repellent)) that is or can be associated with the modified lipid composition (e.g., loaded into or onto the modified lipid composition (e.g., encapsulated by, embedded in, or conjugated to the modified lipid composition)) using in vivo or in vitro methods and is capable of effecting the recited 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 term “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 in a nucleic acid by phosphodiester bonds, although the term “nucleic acid” also encompasses nucleic acid analogs having other types of linkages or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others). The nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequence. A nucleic acid can contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-canonical bases (including, e.g., 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 mean polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety. Linear RNA includes RNA that has not undergone circularization (e.g., is pre-circularized) and can be used as a starting material for circularization through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing-catalyzed circularization methods.

[0085] As used herein, the terms “circular polyribonucleotide,” “circular polynucleotide molecule,” “circular RNA,” and “circRNA” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e. , no free 3’ and / or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent (e.g., covalently closed) or non- covalent bonds. The circular polyribonucleotide may be, e.g., a covalently closed polyribonucleotide.

[0086] As used herein, the term “peptide,” “protein,” or “polypeptide” encompasses any chain of naturally or non-naturally occurring 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), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, e.g., one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics. The polypeptide may be, e.g., at least 0.1 , at least 1 , at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, atleast 50, or more than 50 kD in size. The polypeptide may be a full-length protein. Alternatively, the polypeptide may 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 in an animal (e.g., in one or more parts of the animal), on an animal (e.g., on one or more parts of the animal), or in the habitat surrounding an animal, particularly where the infection decreases the fitness of the animal, e.g., by causing a 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 an invertebrate, which causes disease or disease symptoms in an animal by, e.g., (i) directly infecting the animal, (ii) producing agents that causes disease or disease symptoms in an animal (e.g., bacteria that produce pathogenic toxins and the like), and / or (iii) by eliciting an immune (e.g., inflammatory response) in animals (e.g., biting insects, e.g., bedbugs). As used herein, pathogens include, but are not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease or symptoms in humans.

[0090] As used herein, the term “antibody” encompasses an immunoglobulin, whether natural or partly or wholly synthetically produced, and fragments thereof, capable of specifically binding to an antigen. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partly or wholly synthetically produced. “Antibody” further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. Use of the term “antibody” is meant to include whole antibodies; polyclonal, monoclonal and recombinant antibodies; fragments thereof; and further includes single-chain antibodies (nanobodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; anti-idiotype antibodies; antibody fragments, such as, e.g., 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 antibodies and multispecific antibodies.

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

[0092] As used herein, the term “unmodified structural component” refers to a composition including a structural component (e.g., isolated natural extracellular vesicles, other nature-derived lipids, or synthetic structural lipids) that lack a heterologous cell uptake agent capable of increasing cell uptake (e.g., animal cell uptake, plant cell uptake, natural cell uptake, or fungal cell uptake) of the structural component.

[0093] As used herein, the term “modified” or “modification” to the structural component refers to amodified lipid composition including a structural component and one or more heterologous agents (e.g., one or more exogenous lipids, such as an ionizable lipid, sterol and / or a PEGylated lipid) capable of increasing cell uptake (e.g., animal cell uptake, plant cell uptake, natural cell uptake, or fungal cell uptake) of the modified lipid composition, or a portion or component thereof, relative to an unmodified structural component; capable of enabling or increasing delivery of a heterologous functional agent (e.g., an agricultural or therapeutic agent) by the modified lipid composition to a cell, and / or capable of enabling or increasing loading (e.g., loading efficiency or loading capacity) of a heterologous functional agent (e.g., an agricultural or therapeutic agent). The structural component may be modified in vitro or in vivo.

[0094] As used herein, the term “cell uptake” refers to 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, natural cell, or fungal cell. For example, uptake can involve transfer of the modified lipid composition or a portion of component thereof from the extracellular environment into or across the cell membrane, the cell wall, the extracellular matrix, or into the intracellular environment of the cell). Cell uptake of modified lipid composition may occur via active or passive cellular mechanisms. Cell uptake includes aspects in which the entire modified lipid composition is taken up by a cell, e.g., taken up by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of the target cell following endocytosis and endosomal escape. In some embodiments, a modified lipid composition comprising an ionizable lipid, e.g., a modified lipid composition comprising an ionizable lipid and a sterol and / or a PEGylated lipid) has an increased rate of endosomal escape relative to an unmodified structural component (e.g., structural lipids). Cell uptake also includes aspects in which the modified lipid composition fuses with the membrane of the target cell. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of the target cell following membrane fusion. In some embodiments, a modified lipid composition has an increased rate of fusion with the membrane of the target cell (e.g., is more fusogenic) relative to a structural component that is not modified by an ionizable lipid.

[0095] As used herein, the term “cell-penetrating agent” refers to agents that alter properties (e.g., permeability) of the cell wall, extracellular matrix, or cell membrane of a cell (e.g., an animal cell, a plant cell, a bacterial cell, or a fungal cell) in a manner that promotes increased cell uptake relative to a cell that has not been contacted with the agent.

[0096] A “modified lipid composition” comprising a lipid reconstructed structural component is described herein. The structural component has been derived from a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure), which may be derived from (e.g., enriched, isolated or purified from) a natural source, and the lipid structure is disrupted (e.g., disrupted by lipid extraction) and reassembled or reconstituted in a liquid phase (e.g., a liquid phase containing a cargo) using standard methods, e.g., reconstituted by a method comprising lipid film hydration and / or solvent injection, to produce the lipid reconstructed structural component, as is described herein. The method may, if desired, further comprise sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted modified lipid composition. Alternatively, the modified lipid composition may be produced using a microfluidic device (such as aNanoAssemblr® IGNITE™ microfluidic instrument (Precision NanoSystems)). Alternatively, the structural component comprises structural lipid that is not derived from a natural source, but is synthetically derived.

[0097] As used herein, the term “natural extracellular vesicle”, “natural EV”, or “EV” refers to an enclosed lipid-bilayer structure naturally occurring in a natural. Optionally, the natural EV includes one or more natural EV markers. As used herein, the term “natural EV marker” refers to a component that is naturally associated with a natural, such as a natural protein, a natural nucleic acid, a natural small molecule, a natural lipid, or a combination thereof.

[0098] As used herein, the term “complex lipid particle” refers to a lipid particle that has a complexity characterized by comprising a wide variety of lipids, including structural lipids extracted from one or more natural sources, and optionally at least one exogenous ionizable lipid. The complex lipid particle may comprise between 10% w / w and 99% w / w structural lipids derived from a lipid structure from one or more natural sources, e.g., it may contain at least 10% w / w, at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or about 99% w / w lipids derived from a lipid structure from one or more natural sources. In some instances, a complex lipid particle incorporating natural lipid extracts may also be referred to as a natural messenger pack (NMP). For instance, a complex lipid particle incorporating plant lipid extracts may also be referred to as a plant messenger pack (PMP). In some instances, a complex lipid particle incorporating natural lipid extracts and at least one exogenous ionizable lipid may also be referred to as a lipid reconstructed natural messenger pack (LNMP). For instance, a complex lipid particle incorporating plant lipid extracts and at least one exogenous ionizable lipid may also be referred to as a lipid reconstructed plant messenger pack (LPMP).

[0099] In some embodiments, the complex lipid particle incorporates soy lipid extracts or soy-derived lipids.

[0100] The complex lipid particle may contain 3-1000 lipids extracted from one or more natural sources. The complex lipid particle may contain natural lipids from at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of lipids from the natural source. The complex lipid particle may comprise all or a fraction of the lipid species present in the lipid structure from the natural source, e.g., it 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 virtually 100% of the lipid species present in the lipid structure from the natural source. The complex lipid particle may comprise all or a fraction of the lipid species present in the lipid structure from a particular natural source. For instance, it 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 virtually 100% of the lipid species present in the lipid structure from a particular natural source.

[0101] In some embodiments, the complex lipid particle contains 3 or more lipids extracted from soy or derived from soy.

[0102] The complex lipid particle may comprise reduced or minimized protein matter endogenous to the one or more natural sources, e.g., it may contain 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, orless than 50% w / w of the protein matter endogenous to the one or more natural sources. In some instances, the lipid bilayer of the complex lipid particle does not contain proteins.

[0103] The complex lipid particle may also include synthetic structural lipids such as neutral lipids as the structural lipid component. The structural lipid component of the complex lipid particle may comprise between 10% w / w and 99% w / w structural lipids derived from a synthetic lipid structure (as opposed to the lipids extracted from a natural source), e.g., it may contain at least 10% w / w, at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or about 99% w / w lipids derived from a synthetic lipid structure.

[0104] The complex lipid particle may further comprise at least two exogenous lipids. The complex lipid particle may include at least 1 % w / w, at least 2% w / w, at least 5% w / w, at least 10% w / w, at least 15% w / w, at least 20% w / w, at least 25% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, or about 90% w / w exogenous lipids. Exemplary exogenous lipids include sterols and PEG-lipid conjugate. The complex lipid particle may be used to encapsulate one or more exogenous nucleic acids or polynucleotides encoding one or more peptides, polypeptides, or proteins, to enable delivery of the exogenous nucleic acids or polynucleotides to a target cell or tissue.

[0105] As used herein, the term “exogenous lipid” refers to a lipid that is exogenous to the natural source, i.e., a lipid originates from a source that is not the natural source from which the lipids are extracted (e.g., a lipid that is added to the complex lipid particle formulation using method described herein). The term “exogenous lipid” does not exclude a natural-derived lipid (such as a plant-derived sterol). That is to say, an exogenous lipid can be a natural-derived lipid (such as a plant-derived sterol that is exogenous to the plant source from which the lipids are extracted, e.g., an exogenous lipid can be a plant derived sterol that is added to the complex lipid particle formulation). As another example, an exogenous lipid can be a natural-derived lipid that is exogenous to the particular natural source from which the lipids are extracted (e.g., a bacteria-derived lipid that is exogenous to the plant source from which the lipids are extracted, or vice versa). An exogenous lipid may be a cellpenetrating agent, may be capable of increasing delivery of one or more polynucleotides by the complex lipid formulation to a cell, and / or may be capable of increasing loading (e.g., loading efficiency or loading capacity) of a polynucleotide. In some embodiments, the exogenous lipid may be a stabilizing lipid. In some embodiments, the exogenous lipid may be a structural lipid (e.g., a synthetic structural lipid). Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and PEGylated lipids.

[0106] As used herein, the term “cationic lipid” refers to an amphiphilic molecule (e.g., a lipid or a lipidoid) that is positively charged, containing a cationic group (e.g., a cationic head group).

[0107] As used herein, the term “ionizable lipid” refers to an amphiphilic molecule (e.g., a lipid or a lipidoid, e.g., a synthetic lipid or lipidoid) containing a group (e.g., a head group) that can be ionized, e.g., dissociated to produce one or more electrically charged species, under a given condition (e.g., pH).

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

[0109] In some embodiments, ionizable lipids are ionizable such that they can dissociate to exist in a positively charged form depending on pH. The ionization of an ionizable lipid affects the surface charge of a lipid modified comprising the ionizable lipid under different pH conditions. The surface charge of the lipid modifiedin turn can influence its plasma protein absorption, blood clearance, and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as its ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) that can influence the intracellular delivery of nucleic acids.

[0110] In some embodiments, ionizable lipids are those that are generally neutral, e.g., at physiological pH (e.g., pH about 7), but can carry net charge(s) at an acidic pH or basic pH. In one embodiment, ionizable lipids are those that are generally neutral at pH about 7, but can carry net charge(s) at an acidic pH. In one embodiment, ionizable lipids are those that are generally neutral at pH about 7, but can carry net charge(s) at a basic pH.

[0111] In some embodiments, ionizable lipids do not include those cationic lipids or anionic lipids that generally carry net charge(s) at physiological pH (e.g., pH about 7).

[0112] As used herein, the term “lipidoid” refers to a molecule having one or more characteristics of a lipid.

[0113] As used herein, the term “stable structural lipid formulation” or “stable CLP formulation” refers to a modified lipid composition or a CLP formulation that 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) 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 the initial number of the particles of the modified lipid composition or the CLP (e.g., particles per mL of solution) relative to the number of the particles in the structural lipid formulation or CLP formulation (e.g., at the time of production or formulation) optionally at a defined temperature range (e.g., 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 -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)); or 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 (e.g., cell wall penetrating activity and / or activity of the mRNA formulated within the modified lipid composition) relative to the initial activity of the modified lipid composition or or CLP formulation (e.g., at the time of production or formulation) optionally at a defined temperature range (e.g., 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 -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)).

[0114] Alternatively, the expression refers to a CLP formulation or modified lipid composition that 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) 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 relative to the initial activity of the CLP formulation or modified lipid composition (e.g., at the time of production or formulation) optionally at a defined temperature range (e.g., 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 -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)).

[0115] Alternatively, the expression refers to a CLP formulation or modified lipid composition that 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) retains their particle size, i.e., the particle size does not increase, or has an increase of no more than 5% (e.g., no 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) relative to the initial particle size of the CLPs or modified lipid composition (e.g., at the time of production or formulation) optionally at a defined temperature range (e.g., 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 -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 remains associated with an exogenous peptide, polypeptide, or protein with which the CLP or modified lipid composition has been loaded, 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 90 or more days.

[0117] As used herein, the term “treatment” refers to administering a pharmaceutical composition to an animal for prophylactic and / or therapeutic purposes. To “prevent an infection” refers to prophylactic treatment of an animal that does not yet have a disease or condition, but which is susceptible to, or otherwise at risk of, a particular disease or condition. To “treat 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 “treat an infection” refers to administering treatment to an individual (e.g., an animal) already having a disease to improve or stabilize the individual’s condition. This may involve reducing colonization of a pathogen in, on, or around an animal by one or more pathogens (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to a starting amount and / or allow benefit to the individual (e.g., reducing colonization in an amountsufficient to resolve symptoms). In such instances, a treated infection may manifest as a decrease in symptoms (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some instances, a treated infection is effective to increase the likelihood of survival of an individual (e.g., an increase in likelihood 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., an increase in likelihood of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, the compositions and methods may be effective to “substantially eliminate” an infection, which refers to a decrease in the infection in an amount sufficient to sustainably resolve symptoms (e.g., for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months) in the animal.

[0119] As used herein, the term “prevent an infection” refers to preventing an increase in colonization in, on, or around an animal by one or more pathogens (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to an untreated animal) in an amount sufficient to maintain an initial pathogen population (e.g., approximately the amount found in a healthy individual), prevent the onset of an infection, and / or prevent symptoms or conditions associated with infection. For example, an individual (e.g., an animal, e.g., a human) may receive prophylaxis treatment to prevent a fungal infection while being prepared for an invasive medical procedure (e.g., preparing for surgery, such as receiving a transplant, stem cell therapy, a graft, a prosthesis, receiving long-term or frequent intravenous catheterization, or receiving treatment in an intensive care unit), in immunocompromised individuals (e.g., individuals with cancer, with HIV / AIDS, or taking immunosuppressive agents), or in individuals undergoing long term antibiotic therapy.

[0120] As used herein, the term “formulated for delivery to an animal” refers to a modified lipid composition that includes a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable" carrier or excipient is one that is suitable for administration to an animal (e.g., human), e.g., without undue adverse side effects to the animal (e.g., human).BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1A shows whole body radiance (average radiance p / s / cm2 / sr) 4-6 hours post-dose of intravenous administration of lipid compositions using the structural lipids DOPE, DOPC, DSPC, LPC, Brain Sphingomyelin (SM), Ceramide, Glucosyl Ceramide, Lactosyl Ceramide, Lactosyl PE, MGDG, DGDG, Galactosyl Cholesterol, Glucosyl Cholesterol, triglyceride (TG), or Cardiolipin (CA) at 16, 30, 50, or 75% lipid molar ratio (10ug / 1 OOuL; 1 :1 FLuc:hEPO mRNA) in mice. N=2 / group. Figure 1 B shows the spleen to liver ratio 4 hours post-intravenous dose of the same compositions (10ug / 100uL; 1 :1 FLuc:hEPO mRNA).

[0122] Figure 2A shows whole body radiance (average radiance p / s / cm2 / sr) 4-6 hours post-dose of 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 ratio (10ug / 100uL; 1 :1 FLuc:hEPO mRNA) in mice. N=2 / group. Figure 2B shows the spleen to liver ratio 4 hours post-intravenous dose of the same compositions (10ug / 100uL; 1 :1 FLuc:hEPO mRNA).

[0123] Figure 3A shows whole body radiance (average radiance p / s / cm2 / sr) 4-6 hours post-dose ofintravenous administration of exemplified modified lipid compositions using the structural lipids Soy Polar, Soy PL mix, Soy PC, Soy LPC, Soy PE, Soy PS, Soy PG, Soy PA, and Soy PI at 16, 30, or 50% lipid molar ratio (10ug / 100uL; 1 :1 FLuc:hEPO mRNA) in mice, as compared to those of the DOPE LNP compositions at the same molar ratios carrying the same cargo. N=2 / group. Figure 3B shows the spleen to liver ratio 4 hours post-intravenous dose of the same compositions (10ug / 100uL; 1 :1 FLuc:hEPO mRNA).

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

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

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

[0127] Figure 7A shows lymphoid cell transfection in Ai9 mice in spleens collected three days post- intravenous dose of modified lipid compositions using ionizable lipid 2213 and the 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) as controls. N=3 / group. Figure 7B shows myeloid cell transfection in Ai9 mice in spleens collected three days post-intravenous dose of modified lipid compositions using ionizable lipid 2213 and the 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) as controls. N=3 / group.

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

[0129] Figure 9A shows lymphoid cell transfection in tdTomato mice in spleens collected three dayspost-intravenous dose of modified lipid compositions (Table 10, 1.25 mg / kg CRE mRNA ), with naive mice as controls. N=4 / experimental group.

[0130] Figure 9B shows myeloid cell transfection in tdTomato mice in spleens collected three days post-intravenous dose of modified lipid compositions (Table 10, 1.25 mg / kg CRE mRNA ), with naive mice as controls. N=4 / experimental group.

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

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

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

[0134] Figure 13 shows the percentage of inhibition in hamsters after one intramuscular dose of Formulation 19 I SARS-CoV-2 (soy, containing S mRNA, 10 ug) or Formulation 19 I SARS-CoV-2 (soy, containing S mRNA, 1 ug) on Day 21 post-dose. N=14 / group. Controls were the monovalent vaccine (1 ug, n=14).

[0135] Figure 14A shows the absolute number of germinal center B cells in pooled lymph nodes seven or ten days after one intramuscular dose of various modified lipid composition formulations (S mRNA 10 pg / 40 pL, see the formulations in Table 12). N=6 / group. Controls were the LNP formulation (see Table 12) or naive mice. Figure 14B shows the absolute number of T follicular helper cells in pooled lymph nodes seven or ten days after one intramuscular dose of various modified lipid composition formulations (S mRNA 10 pg / 40 pL, see the formulations in Table 12). N=6 / group. Controls were the LNP formulation (see Table 12) or naive mice. Figure 14C shows the frequency of germinal center B cells among B cells in pooled lymph nodes seven or ten days after one intramuscular dose of various modified lipid composition formulations (S mRNA 10 pg / 40 pL, see the formulations in Table 12). N=6 / group. Controls were the LNP formulation (see Table 12) or naive mice. Figure 14D shows the frequency of T follicular helper cells among CD4 T cells in pooled lymph nodes seven or ten days after one intramuscular dose of various modified lipid compositionformulations (S mRNA 10 pg / 40 pL, see the formulations in Table 12). N=6 / group. Controls were the LNP formulation (see Table 12) or naive mice. Figure 14E shows the absolute number of class switched B cells in pooled lymph nodes seven or ten days after one intramuscular dose of various modified lipid composition formulations (S mRNA 10 pg / 40 pL, see the formulations in Table 12). N=6 / group. Controls were the LNP formulation (see Table 12) or naive mice.

[0136] Figure 15A shows the whole body radiance (average radiance p / s / cm2 / sr) at 4 hours postdose of intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1 :1 mRNA FLuc:hEPO, dosed at 10ug / 40uL, in mice. N=3 / group. Positive controls used were LNP 1 and LNP 2 (n=3 / group), and negative control was mice dosed with PBS (n=2). Figures 15B and 15C show the liver (Fig 15B) and spleen (Fig 15C) radiance (average radiance p / s / cm2 / sr) at 4 hours post-dose of intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1 :1 mRNA FLuc:hEPO, dosed at 10ug / 40uL, in mice. N=3 / group. Positive controls used were LNP 1 and LNP 2 (n=3 / group), and negative control was mice dosed with PBS (n=2). Figure 15D shows the right and left side lymph nodes radiance (average radiance p / s / cm2 / sr) at 4 hours post-dose of intramuscular administration of various exemplary modified lipid composition formulations (see Table 13) encapsulating 1 :1 mRNA FLuc:hEPO, dosed at 10ug / 40uL, in mice. N=3 / group. Positive controls used were LNP 1 and LNP 2 (n=3 / group), and negative control was mice dosed with PBS (n=2).DETAILED DESCRIPTION

[0137] Featured herein are modified lipid compositions that contain (a) a structural component comprising one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol; and (b) an ionizable lipid that can increase cell 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 soy lipid extracts or soy-derived lipids, and an ionizable lipid.

[0138] These modified lipid compositions may be formulated with one or more heterologous functional agents, such as polynucleotides, and can be used as delivering vehicles for these heterologous functional agents (e.g., polynucleotides).Complex Lipid Particles

[0139] Complex lipid particles (CLPs) described herein comprise a wide variety of lipids, including structural lipids extracted from one or more natural sources (such as plants or bacteria). In some embodiments, a complex lipid particle is a natural messenger pack (NMP) incorporating natural lipid extracts. In some embodiments, a complex lipid particle is a lipid reconstructed natural messenger pack (LNMP) incorporating natural lipid extracts and at least one exogenous ionizable lipid.

[0140] In some embodiments, the complex lipid particle contains 3 or more lipids extracted from soy or derived from soy.

[0141] The complex lipid particles may also comprise at least exogenous ionizable lipid. Theionizable lipid has two or more of the characteristics listed below:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails; wherein each of the lipid tails 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) an N:P ratio of at least 3.

[0142] The complex lipid particle may comprise between 10% w / w and 99% w / w structural lipids derived from a lipid structure from one or more natural sources, e.g., it may contain at least 10% w / w, at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or about 99% w / w lipids derived from a lipid structure from one or more natural sources.

[0143] In some embodiments, the complex lipid particle comprises about 10-95% w / w of the natural (e.g., plant, bacteria) lipids. For instance, the complex lipid particle comprises 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 of the natural lipids based on the amounts of total lipids in the complex lipid formulation.

[0144] The complex lipid particle may contain 3-1000 lipids extracted from one or more natural (e.g., plant, bacteria) sources. In some embodiments, the natural source is a plant, plant extract, or fragment or part of a plant. In some embodiments, the natural source is a bacteria, bacteria fragment or part of a bacteria. In some embodiments, the natural source is lemon. In some embodiments, the natural source is soy. In other embodiments, the natural source is E. coli.

[0145] In some embodiments, the complex lipid particle contains at least 10 natural lipids belonging to one or more of the classes selected from the group consisting of fatty acyls (FA), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterol. For instance, the complex lipid particle contains 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 natural lipids belonging to one or more of the classes selected from the group consisting of fatty acyls (FA), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterol. In some embodiments, the complex lipid particle contains lipids from at least two or at least three of these different classes.

[0146] In some embodiments, the complex lipid particle contains at least 10 natural lipids belonging to one or more of the classes selected from the group consisting of glycerolipid, sphingolipid, and sterol. For instance, the complex lipid particle contains 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 natural lipids belonging to one or more of the classes selected from the group consisting of glycerolipid, sphingolipid, and sterol. In some embodiments, the complex lipidparticle contains lipids from at least two or at least three of these different classes.

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

[0148] In some embodiments, the complex lipid particle may contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids, triacylglycerols (TG), and sulfolipids (SL). In some embodiments, the CLPs contains one or more glycerophospholipids (GP) selected from the group consisting of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS), and phosphatidylinositols (PI). In some embodiments, the complex lipid particle contains one or more sphingolipids (SP) selected from the group consisting of sulfolipids (SL), glycosyl inositol phosphoryl ceramides (GIPC), glucosylceramides (GCer), ceramides (Cer), and free long-chain bases (LCB). In some embodiments, the complex lipid particle contains one or more phytosterols selected from the group consisting of campesterol, stigmasterol, p-sitosterol, A5-avenasterol, brassicasterol, avenasterol, 4-desmethyl sterol, 4a-monomethyl sterol, A5-sterol, A7-sterol, a-spinasterol, A5,A7-sterol, phytostanol, and sitosterol.

[0149] The CLP may contain one or more natural lipids belonging to one or more classes or subclasses selected from the group consisting of fatty acids, fatty esters, fatty aldehydes, fatty amides, acyclic oxylipins, cyclic oxylipins, glycerolipids, monoradylglycerols, diradylglycerols, trirady Iglycerols, estolides, glycosylmonoacylglycerols, sulfoquinovosylmonoacylglycerols, monogalactosylmonoacylglycerol, digalactosylmonoacylglycerol, sulfoquinovosyldiacylglycerols, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, glycosyldiacylglycerols, glyceropphospholipids, phospholipids, lysophospholipids, phosphatidylinositol phosphates, n-modified phospholipids, oxygenated / oxidized phospholipids, shingolipids, sphingoid bases, ceramides, phosphocereamides, glycophingolipids, sterols, cholesterol, cholesteryl ester, steryl esters, bile acids, sterylglycosides, and acylsterylglycosides. The complex lipid particle may contain one or more natural lipids belonging to one or more of the classes or sub-classes selected from the group consisting of the classes or sub-classes listed above. For instance, the complex lipid particle contains 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 natural lipids belonging to one or more of the classes or sub-classes selected from the group consisting of the classes or sub-classes listed above.

[0150] In some embodiments, the CLP contains one or more natural lipids belonging to one or more of the sub-classes selected from the group consisting of acyl diacylglyceryl glucuronides, acylhexosylceramides, acylsterylglycosides, bile acids, acyl carnitines, cholesteryl esters, ceramides, cardiolipins, coenzyme Qs, diacylglycerols, digalactosyldiacylglycerols, diacylglyceryl glucuronides, dilysocardiolipins, fatty acids, fatty acid esters of hydroxyl fatty acids, hemibismonoacylglycerophosphates, hexosylceramides, lysophosphatidic acids, lysophophatidylcholines, lysophosphatidylethanolamines, N-acyl-lysophosphatidylethanolamines, lysophosphatidylglycerols, lysophosphatidylinositols, lysophosphatidylserines, monogalactosyldiacylglycerols, lysocardiolipins, N-acyl ethanolaminess, N-acyl glycines, N-acyl glycylserines, phosphatidic acids, phosphatidylcholines, phosphatidylethanolamines, phosphatidylethanols, phosphatidylglycerols, phosphatidylinositols, ceramide phosphoinositols, phosphatidylmethanols, phosphatidylserines, steryl esters, stigmasterols, sulfatides, sulfonolipids, sphingomyelins, sulfoquinovosyl diacylglycerosl, sterols, and triacylglycerols. In some embodiments, the complex lipid particle contains at least 10 natural (e.g., plant, bacteria) lipids belonging to one or more of the subclasses selected from the group consisting of the sub-classes listed above. For instance, the complex lipid particle contains 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 natural lipids belonging to one or more of the sub-classes selected from the group consisting of the subclasses listed above.

[0151] The complex lipid particle may contain 10 or more natural lipids belonging to one or more of the sub-classes selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemibismonoacylglycerophosphates, hexosylceramides, lysophophatidylcholines, lysophosphatidylethanolamines, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylethanols, phosphatidylglycerols, phosphatidylinositols, sulfoquinovosyl diacylglycerosl, and sterols. For instance, the complex lipid particle contains 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 natural lipids belonging to one or more of the subclasses selected from the group consisting of the sub-classes listed above.

[0152] The complex lipid particle may contain natural lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or sub-classes of lipids from the natural sources. In some embodiments, the complex lipid particle contains natural lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or sub-classes of lipids from a single natural source (e.g., from only the plant source, or from only the bacteria source). In some embodiments, the CLP may contain natural lipids from only one class or only one sub-class of lipids from the natural sources.

[0153] The identity (and class and subclass) and the amounts of the lipids extracted from the natural source(s) can be analyzed by lipidomics analysis by solubilizing the lipid extracts or complex lipid particles in compatible solvents and analyzing by a 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 reversed-phase high-performance liquid chromatography (RP-HPLC-CAD)), may also be used.

[0154] The complex lipid particle may comprise all or a fraction of the lipid species present in the lipid structure from the particular natural source(s), e.g., it 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 virtually 100% of the lipid species present in the lipid structure from the particular natural source(s).

[0155] The complex lipid particle may comprise reduced or minimized protein matter endogenous tothe one or more natural sources. For instance, the complex lipid particle may contain less than 50% w / w, less than 45% w / w, less than 40% w / w, less than 35% w / w, less than 30% w / w, less than 25% w / w, less than 20% w / w, less than 15% w / w, less than 10% w / w, less than 9% w / w, less than 8% w / w, less than 7% w / w, less than 6% w / w, less than 5% w / w, less than 4% w / w, less than 3% w / w, less than 2% w / w, less than 1 % w / w, less than 0.5% w / w, less than 0.1 % w / w, or essentially free of protein matter endogenous to the one or more natural sources. In some instances, the lipid bilayer of the complex lipid particle does not contain proteins. To calculate %w / w of residual protein matter endogenous to the one or more natural sources, protein concentration is divided by the concentration of the natural lipid extract and then multiplied by 100. Alternatively, %w / w is calculated as the percent of the mass of total protein endogenous to the one or more natural sources based on the mass of the total lipid extract.

[0156] The complex lipid particle may comprise reduced or minimized residual dsDNA matter endogenous to the one or more natural sources. For instance, the complex lipid particle may 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 essentially free of residual dsDNA matter endogenous to the one or more natural sources. In some instances, the lipid bilayer of the complex lipid particle does not contain residual dsDNA. To calculate %w / w of residual dsDNA matter endogenous to the one or more natural sources, total adjusted dsDNA concentration is divided by the concentration of the natural lipid extract and then multiplied by 100. Alternatively, %w / w is calculated as the percent of the mass of total residual dsDNA endogenous to the one or more natural sources based on the mass of the total lipid extract.

[0157] In some embodiments, the complex lipid particle further incorporates a synthetic structural lipid such as a neutral lipid. In some embodiments, the structural lipid component of the complex lipid particle may comprise between 10% w / w and 99% w / w structural lipids derived from a synthetic lipid structure, e.g., it may contain at least 10% w / w, at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or about 99% w / w lipids derived from a synthetic lipid structure.

[0158] In addition to the exogenous ionizable lipid, the complex lipid particle may further comprise at least two other exogenous lipids. The complex lipid particle may include at least 1 % w / w, at least 2% w / w, at least 5% w / w, at least 10% w / w, at least 15% w / w, at least 20% w / w, at least 25% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, or about 95% w / w exogenous lipids. Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and PEG-lipid conjugate. The complex lipid particle may further comprise at least two exogenous lipids. In some embodiments, the complex lipid particle contains an ionizable lipid, a sterol, and PEG-lipid conjugate. Additional exogenous lipids suitable for being included in the complex lipid particle are described herein below.

[0159] In some embodiments, the CLPs contain natural lipids comprising fatty acid-derived tails, said fatty acid-derived tails of the natural lipids being: about 5 to 20% of fatty acid 16:0about 0 to 10% of fatty acid 18:1 (C9) about 0 to 10% of fatty acid 18:1 (C7) about 5 to 30% of fatty acid 18:2 about 2 to 20% of fatty acid 18:3.

[0160] In some embodiments, the CLPs contain phosphatidylcholine (PC) lipids comprising fatty acid-derived tails, said fatty acid-derived tails of the PC lipids being: about 10 to 20% of fatty acid 16:0 about 2 to 5% of fatty acid 18:0 about 7 to 15% of fatty acid 18:1 about 50 to 75% of fatty acid 18:2 about 2 to 10% of fatty acid 18:3.

[0161] In some embodiments, the CLPs contain phosphatidylethanolamines (PE) lipids comprising fatty acid-derived tails, said fatty acid-derived tails of the PE lipids being: about 0.25 to 5% of fatty acid 14:0 about 25 to 45% of fatty acid 16:0 about 5 to 15% of fatty acid 16:1 about 10 to 25% of fatty acid 17:0 about 25 to 45% of fatty acid 18:1 about 2 to 7% of fatty acid 19:0.

[0162] In some embodiments, the CLPs contain natural lipids belonging to the sub-classes of phosphatidylethanolamines, phosphatidylglycerol, and cardiolipin, and comprising: about 50 to 75 wt / wt% of phosphatidylethanolamines (PE) about 15 to 30 wt / wt% of phosphatidylglycerol (PG) about 5 to 15 wt / wt% of cardiolipin (CL).

[0163] In some embodiments, the CLPs contain natural lipids comprising : about 10 to 50 wt / wt% of phosphatidylcholines (PC) about 5 to 50 wt / wt% of phosphatidylethanolamines (PE) about 0 to 15 wt / wt% of triacylglycerol (TG) about 5 to 35 wt / wt% of hexosylceramides (HexCer) about 0 to 5 wt / wt% of phosphatidylglycerol (PG) about 0 to 7 wt / wt% of phosphatidylserines (PS) about 0 to 10 wt / wt% of phosphatidylinositols (PI) about 0 to 5 wt / wt% of cardiolipin (CL).

[0164] In some embodiments, the complex lipid particle contains less than 12% w / w of chloroplast endogenous to the one or more natural sources. In some embodiments, the complex lipid particle contains less than 20% w / w, 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, or less than 0.1 % w / w of chloroplast endogenous to the one or more natural sources.

[0165] In some embodiments, the complex lipid particle contains less than 5% w / w of exogenous antioxidant.

[0166] In some embodiments, the CLPs contain natural lipids comprising about 0 to 20 wt / wt% of cardiolipin (CL).Structural Component

[0167] The structural component comprises one or more lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol. The structural component can 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 plant or animal. In some embodiments, the animal is porcine or bovine. In some embodiments, the plant is soybean. In other embodiments, the structural component is synthetically derived.

[0169] The structural component may have a lipid (e.g., lipid bilayer, unilamellar, or multilamellar structure) structure that includes a natural extracellular vesicle (EV), or segment, portion, or extract (e.g., lipid extract) thereof.

[0170] In some embodiments, the structural component comprises isolated natural extracellular vesicles. Natural EVs refer to an enclosed lipid-bilayer structure that naturally occurs in a source. Natural EVs are derived from a natural source (e.g. plant, bacterium, animal, etc) that comprise structural lipids, and may also comprise natural proteins and / or natural nucleic acids and / or carbohydrate moieties contained in a nanoparticle. The 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, porcine brain, or bovine heart.

[0171] The structural component can include natural EVs, or segments, portions, or extracts, thereof. In some embodiments, the natural EVs are about 5-1000 nm in diameter. For example, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean diameter of about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1 OOOnm, about 1000-1250nm, about 1250-1500nm, about 1500-1750nm, or about 1750-2000nm. In some instances, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean 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 about 5-25 nm. In certain instances, the structural component can include a natural EV, or segment, portion, or extract thereof, having a mean diameter of about 50-200 nm, about 50-300 nm, about 200-500 nm, or about 30-150 nm. A variety of methods (e.g., a dynamic light scattering method) standard in the art can be used to measure the particle diameter of the natural EV, or segment, portion, or extract thereof.

[0172] In some instances, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean surface area of at least 77 nm2, (e.g., at least 77 nm2, at least 100nm2, at least 1000 nm2, at least 1x104nm2, at least 1x105nm2, at least 1x106nm2, or at least 2x106nm2). In some instances, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean surface area of 77 nm2to 3.2 x106nm2(e.g., 77-100 nm2, 100-1000 nm2, 1000-1x104nm2, 1x104- 1x105nm2, 1x105-1x106nm2, or 1x106-3.2x106nm2).

[0173] In some instances, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean volume of at least 65 nm3(e.g., at least 65 nm3, at least 100 nm3, at least 1000 nm3, at least 1x104nm3, at least 1x105nm3, at least 1x106nm3, at least 1x107nm3, at least 1x108nm3, at least 2x108nm3, at least 3x108nm3, at least 4x108nm3, or at least 5x108nm3. In some instances, the structural component can include a natural EV, or segment, portion, or extract thereof, that has a mean volume of 65 nm3to 5.3x108nm3(e.g., 65-100 nm3, 100-1000 nm3, 1000-1x104nm3, 1x104- 1x105nm3, 1x105-1x106nm3, 1x106-1x107nm3, 1x107-1x108nm3, 1x108-5.3x108nm3).

[0174] In some instances, the structural component may include an intact natural EV. Alternatively, the structural component may include a segment, portion, or extract of the full surface area of the vesicle (e.g., a segment, portion, or extract including less than 100% (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 10%, less than 5%, or less than 1 %) of the full surface area of the vesicle) of a natural EV. The segment, portion, or extract may be any shape, such as a circumferential segment, spherical segment (e.g., hemisphere), curvilinear segment, linear segment, or flat segment. In instances where the segment is a spherical segment of the vesicle, the spherical segment may represent one that arises from the splitting of a spherical vesicle along a pair of parallel lines, or one that arises from the splitting of a spherical vesicle along a pair of non-parallel lines. Accordingly, the structural component can include a plurality of intact natural EVs, a plurality of natural EV segments, portions, or extracts, or a mixture of intact and segments of EVs. One skilled in the art will appreciate that the ratio of intact to segmented natural EVs will depend on the particular isolation method used. For example, grinding or blending a natural, or part thereof, may produce the structural component that contain a higher percentage of natural EV segments, portions, or extracts than a non-destructive extraction method, such as vacuum-infiltration.

[0175] In instances where, the structural component includes a segment, portion, or extract of a natural EV, the EV segment, portion, or extract may have a mean surface area less than that of an intact vesicle, e.g., a mean surface area less than 77 nm2, 100 nm2, 1000 nm2, 1x104nm2, 1x105nm2, 1x106nm2, or 3.2x106nm2). In some instances, the structural component may include a natural EV, or segment, portion, or extract thereof, that has a mean volume less than that of an intact vesicle, e.g., a mean volume of less than 65 nm3, 100 nm3, 1000 nm3, 1x104nm3, 1x105nm3, 1x106nm3, 1x107nm3, 1x108nm3, or 5.3x108nm3). The structural component may include natural EV segments and / or extracted lipids or a mixture thereof.

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

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

[0178] The structural component may be nature-derived lipids produced from natural EVs, or a segment, portion or extract (e.g., lipid extract) thereof. An exemplary method for producing a structural component (containing a nature-derived lipid) includes (a) providing an initial sample from a natural source; and (b) isolating a crude natural fraction from the initial sample, wherein the crude natural fraction has a decreased level of at least one contaminant or undesired component from the source relative to the level in the initial sample. The method can further include an additional step (c) comprising purifying the crude natural fraction, thereby producing a pure structural component, having a decreased level of at least one contaminant or undesired component from the natural source relative to the level in the crude EV fraction.

[0179] In some instances, the structural component (containing a nature-derived lipid) may be isolated from a natural source by a process which includes the steps of: (a) providing an initial sample from a natural source; (b) isolating a crude natural fraction from the initial sample, wherein the crude natural fraction has a decreased level of at least one contaminant or undesired component from the natural source relative to the level in the initial sample (e.g., a level that is decreased by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%); and (c) purifying the crude natural fraction, thereby producing a pure structural component having a decreased level of at least one contaminant or undesired component from the natural source relative to the level in the crude EV fraction (e.g., a level that is decreased by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%).

[0180] The structural component (containing a nature-derived lipid) may be produced by whole cell extraction. For instance, the cells are first disrupted, and then intracellular and cell membrane / cell wall-associated lipids as well as extracellular hydrocarbons can be separated from the cell mass, such as by use of centrifugation. Intracellular lipids produced in the natural source (e.g. bacteria, plant, animal, etc) are, in some embodiments, extracted after lysing the cells of the source. Additional methods of extracting structural lipids (containing a nature-derived lipid) may be found in U.S. Patent No. 8,592,188, which is incorporated herein by reference in its entirety. The structural component may be produced through synthetic means.

[0181] The structural component (containing a nature-derived lipid) can be produced from a natural source by a variety of methods. For instance, natural EVs can be separated from the source by either destructive (e.g., grinding or blending of a natural source) or non-destructive (washing or vacuum infiltration of a natural source) methods. For instance, the source can be vacuum-infiltrated, ground, blended, or a combination thereof to isolate EVs from the source. For instance, the isolating step may involve vacuum infiltrating the source (e.g., with a vesicle isolation buffer). Alternatively, the isolating step may involve grinding or blending the source to release the EVs.

[0182] Upon isolating the natural EVs, the structural component can be separated or collected into a crude natural fraction (e.g., an apoplastic fraction). For instance, the separating step may involve separating the structural component into a crude natural fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the natural-containing fraction fromlarge contaminants, including natural tissue debris or cells. As such, the crude natural fraction will have a decreased number of large contaminants, as compared to the initial sample from the source. Depending on the method used, the crude natural fraction may additionally comprise a decreased level of natural cell organelles, as compared to the initial sample from the source.

[0183] In some instances, the isolating step may involve centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration.

[0184] The crude natural fraction can be further purified by additional purification methods. For example, the crude natural fraction can be purified by ultracentrifugation, e.g., using a density gradient (iodixanol or sucrose) and / or use of other approaches to remove aggregated components (e.g., precipitation or size-exclusion chromatography). The resulting pure structural component may have a decreased level of contaminants or other undesired components from the natural source (e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipido- proteic structures, nuclei, cell wall components, cell organelles, or a combination thereof) relative to one or more fractions generated during the earlier separation steps, or relative to a pre-established threshold level, e.g., a commercial release specification. For example, the pure structural component may have a decreased level (e.g., by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%), or by about 2x fold, 4x fold, 5x fold, 10x fold, 20x fold, 25x fold, 50x fold, 75x fold, 100x fold, or more than 100x fold), or is substantially free of contaminants or other undesired components (e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipido-proteic structures, nuclei, cell wall components, cell organelles, or a combination thereof) relative to the level in the initial sample.

[0185] For example, protein aggregates may be removed from the structural component. For example, the structural component can be taken through a range of pHs (e.g., as measured using a pH probe) to precipitate out protein aggregates in solution. The pH can be adjusted to, e.g., pH 3, pH 5, pH 7, pH 9, or pH 11 with the addition of, e.g., sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it can be filtered to remove particulates. Alternatively, the structural component can be flocculated using the addition of charged polymers, 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 particulates. Alternatively, aggregates can be solubilized by increasing salt concentration. For example, NaCI can be added to the structural component until it is at, e.g., 1 mol / L. The solution can then be filtered to isolate the structural component. Alternatively, aggregates are solubilized by increasing the temperature. For example, the structural component can be heated under mixing until the solution has reached a uniform temperature of, e.g., 50°C for 5 minutes. The structural component mixture can then be filtered. Alternatively, soluble contaminants from the structural component solutions can be separated by size-exclusion chromatography column according to standard procedures, where the structural lipids elute in the first fractions, whereas proteins and ribonucleoproteins and some lipoproteins are eluted later. The efficiency of protein aggregate removal can be determined by measuring and comparing the protein concentration before and after removal of protein aggregates via BCA / Bradford protein quantification.

[0186] Alternatively, the structural lipids (e.g., soy-derived lipids, cardiolipin, sphingolipid, ceramide,glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol) may be obtained from a commercially available source.

[0187] Any of the production methods described herein can be supplemented with any quantitative or qualitative methods known in the art to characterize or identify the structural component (e.g., the structural lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol) at any step of the production process. For instance, the structural component (e.g., the structural lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol) may be characterized by a variety of analysis methods to estimate yield, concentration, purity, composition, or sizes, by a number of methods known in the art that enable visualization, quantitation, or qualitative characterization (e.g., identification of the composition), such as 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 production process, the structural component (e.g., the structural lipids selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol) can optionally be prepared such that the structural component is at an increased concentration (e.g., by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%; or by about 2x fold, 4x fold, 5x fold, 10x fold, 20x fold, 25x fold, 50x fold, 75x fold, 100x fold, or more than 100x fold) relative to the level in a control or initial sample. The structural component may make up about 0.1% to about 100% of the modified lipid composition, for instance, at about 0.01% to about 100%, about 1 % to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 75% to about 100%.The modified lipid composition — lipid modification of the structural component

[0189] 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] The modification refers to modifying a structural component containing a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure), which may be derived from (e.g., enriched, isolated or purified from) a natural source, and the lipid structure is disrupted (e.g., disrupted by lipid extraction) and reassembled or reconstituted in a liquid phase (e.g., a liquid phase containing a cargo) using standard methods, e.g., reconstituted by a method comprising lipid film hydration and / or solvent injection, to produce the modified lipid composition, as is described herein. In some embodiments, the structural component is modified by reconstructing a film comprising the structural component in the presence of the ionizable lipid.

[0191] In some embodiments, the structural component comprises soy-derived lipids, and the structural lipid is modified by reconstructing a film comprising the purified soy-derived lipids of the structural component with the ionizable lipid.

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

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

[0194] In some instances, processing the structural component to produce a lipid film includes extracting lipids using the Bligh-Dyer method (Bligh and Dyer, J Biolchem Physiol, 37: 911-917, 1959), which is incorporated herein by reference in its entirety. The extracted lipids may be provided as a stock solution, e.g., a solution in chloroform:methanol. Producing the lipid film may comprise, e.g., evaporation of the solvent with a stream of inert gas (e.g., nitrogen).

[0195] The method may, if desired, further comprise sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted modified lipid composition.Structural lipids

[0196] In some embodiments, the structural component comprises structural lipids derived from a natural source. A modified lipid composition may comprise between 10% and 100% lipids derived from the lipid structure from the natural source, e.g., it 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%, at least 95%, or 100% lipids derived from the lipid structure from the natural source. A modified lipid composition may comprise all or a fraction of the lipid species present in the lipid structure from the modified lipid composition, e.g., it 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 100% of the lipid species present in the lipid structure from the natural source. A modified lipid composition may comprise none, a fraction, or all of the protein species present in the lipid structure from the natural source, e.g., it 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 the natural source. In some instances, the lipid bilayer of the modified lipid composition does not contain proteins. In some instances, the lipid structure of the modified lipid composition contains a reduced amount of proteins relative to the lipid structure from the natural source.

[0197] In some embodiments, the structural lipids of the modified lipid composition are selected from the group consisting of soy-derived lipids, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, glucosyl cholesterol In some embodiments, the structural lipids of the modified lipid composition comprise one or more of those structural lipids listed in Tables 1-7 and 9-13.

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

[0199] The structural component may be modified to contain a heterologous agent (e.g., a cellpenetrating agent) that is capable of increasing cell 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) relative to an unmodified structural component. For example, the modified structural component may include (e.g., be loaded with, e.g., encapsulate or be conjugated to) or be formulated with (e.g., be suspended or resuspended in a solution comprising) a cell-penetrating agent, such as an ionizable lipid. Each of the modified structural component 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 may include one or more exogenous lipids, e.g., lipids that are exogenous to the lipid source (e.g., originating from a source that is not the source from which the structural component is produced). The total lipids of the modified lipid composition may 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 examples, the exogenous lipid (e.g., ionizable lipid) is added to amount to 25% or 40% (w / w) of total lipids in the preparation. In some examples, the exogenous lipid is added to the preparation prior to step (b), e.g., mixed with structural lipids prior to step (b).

[0201] Exemplary exogenous lipids include ionizable lipids.

[0202] Exogenous lipids may also include cationic lipids.

[0203] In some instances, the exogenous lipid may be an ionizable lipid or cationic lipid chosen from 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-Ep10, TT3, LP01 , 5A2-SC8, Lipid 5 (Moderna), a cationic sulfonamide amino lipid, an amphiphilic zwitterionic amino lipid, 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 a combination thereof.

[0204] In some embodiments, the exogenous lipid may be an ionizable lipid or cationic lipid chosen from C12-200, MC3, DODAP, DC-cholesterol, DOTAP, Ethyl PC, GL67, KC2, MD1 , OF2, EPC, ZA3- Ep10, TT3, LP01 , 5A2-SC8, Lipid 5 (Moderna), a cationic sulfonamide amino lipid, and an amphiphilic zwitterionic amino lipid and a combination thereof. In some embodiments, the ionizable lipid is chosen from C12-200, MC3, DODAP, and DC-cholesterol or combinations thereof. In some instances, 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 instances, 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 instances, the modified lipid composition comprises at least 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

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

[0207] The agent may increase uptake of the modified lipid composition as a whole or may increase uptake of a portion or component of the modified lipid composition (e.g., the heterologous functional agent) carried by the modified lipid composition. The degree to which cell uptake is increased may vary depending on the source to 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 an increased cell uptake (e.g., animal cell uptake, plant cell uptake, natural cell uptake, or fungal cell uptake) of at least 1 %, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to an unmodified structural component. In some instances, the increased cell uptake is an increased cell uptake of at least 2x-fold, 4x-fold, 5x-fold, 10x -fold, 100x-fold, or 1000x-fold relative to an unmodified structural component.

[0208] In some embodiments, a modified lipid composition that has been modified with a ionizable lipid more efficiently encapsulates a negatively charged a polynucleotide than a modified lipid composition that has not been modified with an ionizable lipid. In some aspects, a modified lipid composition that has been modified with an ionizable lipid has altered biodistribution relative to a modified lipid composition that has not been modified with an ionizable lipid. In some aspects, a modified lipid composition that has been modified with an ionizable lipid has altered (e.g., increased) fusion with an endosomal membrane of a target cell relative to a modified lipid composition that has not been modified with an ionizable lipid.Ionizable lipids

[0209] In some embodiments, the ionizable lipid has at least one (e.g., one, two, three, four or all five) of the characteristics listed below:(i) at least 2 ionizable amines (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, or more than 6 ionizable amines, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more than 12 ionizable amines);(ii) at least 3 lipid tails (e.g., at least 3, at least 4, at least 5, at least 6, or more than 6 lipid tails, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or more than 12 lipid tails), wherein each of the lipid tails is independently at least 6 carbon atoms in length (e.g., at least 6, at least 7, at least 8, at least 9, atleast 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 from about 4.5 to about 7.5 (e.g., a pKa of 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 from about 6.5 and 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) an ionizable amine and a heteroorganic group; and(v) an N:P (amines of ionizable lipid: phosphates of a nucleic acid, such as mRNA) ratio of at least 10.

[0210] In some embodiments, the modified lipid composition has an N / P ratio of about 12 to about17, for example the N / P ratio is about 15 ± 1 , or the N / P ratio is 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, for example the N / P ratio is about 6 ± 1 , or the N / P ratio is 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, LP01 , 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, LP01 , 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, - CO2H, -NH2, -CONH2, optionally 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 include one of the compounds from group i) to group iv) as discussed below.Ionizable lipid compounds I)

[0216] In some embodiments, the ionizable lipid is represented by the following formula I:a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, whereineach A is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each B is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each X is independently a biodegradable moiety; andRs is OH, SH, or NR10R11; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR10R11, wherein each R10 and Rn is independently H, C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic 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 NR12, wherein R12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, andQ is O, S, or NR13, wherein each R13 is H, or C1-C5 alkyl.

[0217] In some embodiments, B is C3-C20 alkyl.

[0218] In some embodiments, W in formula (I) may alternativelywherein:V is branched or unbranched C2-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein each R10 and Rn is independently H, C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring; each v is independently 0, 1 , 2, 3, 4, or 5;R17 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) may alternatively be, wherein:V is C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene; each Re 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) may alternatively, wherein: R14 is a heterocyclic; 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) may alternatively be, wherein:Z is O, S, -C((CH2)VN(RI5)2)-, or N(Ris), wherein R15 is H, C1-C4 branched or unbranched alkyl, and v is 0, 1 , 2, 3, 4, or 5; each R10 is independently H, or C1-C3 alkyl; and each u is independently 0, 1 , 2, 3, 4, or 5.

[0222] In some embodiments, W in formula (I) may alternatively beeach Y is a divalent heterocyclic;Q is O, S, or NH; and each u is independently 1 , 2, 3, 4, or 5.

[0223] In some embodiments, W in formula (I) may alternatively be, wherein:Ri4 is a heterocyclic, NR10R11, C(0)NRioRii, or C(S)NRioRii, wherein each R10 and Rn is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R10 and Rn are taken together to form a heterocyclic ring;R16 is H, =0, =S, or ON; 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) may alternatively be. wherein:T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic optionally substituted with one or more -(CH2)VOH, -(CH2)VSH, and / or -(CH2)v-halogen groups; each R? and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein each R10 and Rn is independently H, C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring;R17 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) may alternatively be. wherein:T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic; and each u is independently 1 , 2, 3, 4, or 5.

[0226] In some embodiments, when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH.

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

[0228] In some embodiments, the ionizable lipid is represented by formula (IX):pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10R11, or each R1 and each R2 are independently taken together with the carbon atom(s) to which they are attached to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, or R10 and Rn are taken together to form a heterocyclic ring; each R3 and each R4is independently H, C2-C14 branched or unbranched alkyl (e.g., C3-C10 branched or unbranched alkyl), or C3-C10 branched or unbranched alkenyl, provided that at least one of Rs and R4is not H; each X is independently a biodegradable moiety;each q is independently 2, 3, 4, or 5;V is branched or unbranched C2-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein each v is independently 0, 1 , 2, 3, 4, or 5, and R17 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 Re is independently H or methyl.

[0230] In some embodiments, the ionizable lipid is represented by one of the following formulas, wherein the definition for the variables are the same as those in formula (X).

[0231] In some embodiments, the disclosure relates to ionizable lipids of Formula (XI):pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10R11, or each Ri and each R2 are independently taken together with the carbon atom(s) to which they are attached to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, or R10 and Rn are taken together to form a heterocyclic ring; each R3 and each R4 is independently H, C2-C1 branched or unbranched alkyl (e.g., C3-C10 branched or unbranched alkyl), or C3-C10 branched or unbranched alkenyl, provided that at least one of Rs 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 heterocyclic optionally substituted with one or more -(CH2)VOH, -(CH2)VSH, -(CH2)v-halogen groups, each R? and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein R17 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 a divalent heterocyclic (e.g., a divalent piperazine, or a divalent dioxopiperazine) optionally substituted with -(CH2)VOH, wherein v is independently 0, 1 , or 2.

[0233] In some embodiments, in each of the above formulas, X is -OC(O)-, -C(O)O-, -SS-, - N(R18)C(O)-, -C(O)N(R18)-, -C(O-RI3)-O-, -C(O)O(CH2)a-, -OC(O)(CH2)a-, -C(O)N(R18)(CH2)a-, - N(R18)C(O)(CH2)a-,-C(O-Ri3)-O-(CH2)a-, wherein each R18is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R13 is independently C3-C10 alkyl, and each a is independently 0-16. In one embodiment, each X is independently -OCO-, -COO-, -NHCO-, or -CONH-. In one embodiment, at least one X is -SS-.

[0234] More embodiments of the ionizable lipid of formula (I), in the Ionizable lipid compounds group i), may be found in PCT Application No. PCT / US22 / 50725, filed on November 22, 2022, the content of which is incorporated herein by reference in its entirety. In particular, all the ionizable lipids of formulas (l)-(XII) of PCT Application No. PCT / US22 / 50725 are suitable for use as the ionizable lipidsin this disclosure, and are incorporated herein by reference in its entirety.

[0235] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table I below.Table I. Exemplary ionizable lipid compounds.Ionizable lipid compounds ii)

[0236] In some embodiments, the ionizable lipid is represented by the following formula II:pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:cyclic or heterocyclic moiety;Y is alkyl, hydroxy, hydroxyalkylA is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -S-S-, or a bivalent heterocycle; each of X and Z is independently absent, -O-, -CO-, -N(R7)-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(0)-, -C(0)N(R7)-, or -S-; each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; each of R30, R40, R50, Reo, R70, Rso, R90, R100, R110, and R120 is independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of I and m is an integer from 1 to 10; t1 is an integer from 0 to 10; andW is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocylyl or heteroaryl.

[0237] In some embodiments, Y is hydroxyl or

[0238] In some embodiments,is selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetra hydrofuran, tetrahydropyran, morpholine, and dioxane. In some

[0239] In some embodiments, the ionizable lipid is represented by formula:All the variables in this formula have been defined and exemplified as those described in the above embodiments.

[0240] In some embodiments, the ionizable lipid is represented by formula:A is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -S-S-, or a bivalent heterocycle; each R7is 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 heterocylyl or heteroaryl; each M is independently a biodegradable moiety; each ml is independently an integer from 3 to 6, each 11 is independently an integer from 4 to 8, m2 and I2 are each independently an integer from 0 to 3,Rao and R90 are each independently unsubstituted Cs-Ca alkyl; or Rao is H or unsubstituted Ci- 04 alkyl, and R90 is unsubstituted C5-C11 alkyl; andR110 and R120 are each independently unsubstituted Cs-Ca alkyl; or Ruo is H or unsubstituted C1-C4 alkyl, and R120 is unsubstituted C5-C11 alkyl. All the other variables in these formulas have been defined and exemplified as those described in the above embodiments. In some embodiments, in these formulas, Rao is H or unsubstituted C1-C2 alkyl, and R90 is unsubstituted Ca-Cio alkyl; and Ruoand R120 are each independently unsubstituted C5-C8 alkyl. In some embodiments, Rso, R90, R110, and R120 are each independently unsubstituted C5-C8 alkyl.

[0241] In some embodiments, in the above formulas, A is absent, -O-, -N(R7)-, N(R7)C(O)-,-C(O)O-, wherein R6is independently H, alkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, thiol, thiolalkyl, or N+(R7)s-alkylene-Q-; and R7is H or C1-C3 alkyl.

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

[0243] In some embodiments, in the above formulas, W is hydroxyl, hydroxyalkyl, or one of the following:each Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R7)2-, -C(O)N(R7)-, -C(S)N(R7)-, or -N(R7)-; each R6is independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol, thiolalkyl, or N+(R7)a-alkylene-Q-; each R8is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol, or thiolalkyl, or two R8together with the nitrogen atom may form a ring; each 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 formulas,X is absent, -O-, or -C(O)-;each Rcis independently H or C1-C3 alkyl; each t1 is independently 1 , 2, 3, or 4; each of R30, R40, R50, and Reo is H or C1-C4 branched or unbranched alkyl;R70 is H; and each of Rao and R90 is independently H or C1-C12 branched or unbranched alkyl;R100 is H; and each of Rno and R120 is independently H or C1-C12 branched or unbranchedalkyl, provided that at least one of Rao and R90 is not H, and at least one of Rno and R120 is not H;I is from 3 to 7; and m is from 1 to 5.

[0246] More embodiments of the ionizable lipid of formula (II), in the Ionizable lipid compounds group ii), may be found in PCT Application No. PCT / US23 / 16300, filed on March 24, 20223, the content of which is incorporated herein by reference in its entirety. In particular, all 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 suitable for use as the ionizable lipids in this disclosure, and are incorporated herein by reference in its entirety.

[0247] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table II below. Table II. Exemplary ionizable lipid compounds.Ionizable lipid compounds Hi)

[0248] In some embodiments, the ionizable lipid is represented by formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein:R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, orR20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra;Rais H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, orR1 and R2 are taken together to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, orR10 and R11 are taken together to form a heterocyclic ring; n is 0, 1 , 2, 3 or 4;Y is O or S;Z is absent, O, S, or N(Ri2>(Ri2), wherein each R12 is independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH; each A is each independently C1-C16 branched or unbranched alkyl, or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; each B is each independently C1-C16 branched or unbranched alkyl, or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; each X is independently a biodegradable moiety.

[0249] In some embodiments, R20 and R30 are each independently H or C1-C3 branched or unbranched alkyl. In some embodiments, R20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra. In some embodiments, Rais H, C1-C3 branched or unbranched alkyl or OH. In one embodiment, Rais 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 is represented by formula (V):(V), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, whereinR1 is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, andR2 is H, OH, halogen, SH, or NR10R11, orR1 and R2 are taken together to form a cyclic ring;R10 and R11 are each independently H or C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring;Q is OH or -(OCH2CH2)uNR2oR3o,R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, orR20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring optionally substituted with Ra;Rais H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; u is O, 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; each A is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; each B is each independently C1-C16 branched or unbranched alkyl or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; and each X is independently a biodegradable moiety.

[0252] In some embodiments, the disclosure relates to ionizable lipids of one of the following formulas:( ), 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. Other variables are defined as in formulas III) and V) above.

[0253] In some embodiments, in the above formulas, X is -OC(O)-, -C(O)O-, -N(R7)C(O)-, - C(O)N(R7)-,-C(O-Ri3)-O-, -C(O)O(CH2)S-, -OC(O)(CH2)S-, -C(O)N(R7)(CH2)S-, -N(R7)C(O)(CH2)S-, -C(O-RI3)-O-(CH2)S-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R13 is independently C3-C10 alkyl, and each s is independently 0-16. 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] More embodiments of the ionizable lipid of formula (III) or (V), in the Ionizable lipid compounds group iii), may be found in PCT Application No. PCT / US22 / 50111 , filed on November 16, 2022, the content of which is incorporated herein by reference in its entirety. In particular, all the ionizable lipids of formulas (lO)-(VIIO) and formulas (l)-(VIID) of PCT Application No.PCT / US22 / 50111 are suitable for use as the ionizable lipids in this disclosure, and are incorporated herein by reference in its entirety.

[0255] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table III below. Table III. Exemplary ionizable lipid compounds.Ionizable lipid compounds iv)

[0256] In some embodiments, the ionizable lipid is a lipid comprising at least one head group and at least onpharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:E is each independently a biodegradable group;Rais each independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; u1 and u2 are each independently 0, 1 , 2, 3, 4, 5, 6, or 7;R‘ is each independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; represents the bond connecting the tail group to the head group; and wherein the lipid has a pKa from about 4 to about 8.

[0257] In some embodiments, E is each independently -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -C(O-RI3)-O-, -C(O)O(CH2)r, -C(O)N(R7) (CH2)r-, -S-S-, or -C(O-Ri3)-O-(CH2)r-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R13 is branched or unbranched C3-C10 alkyl; and r is 1 , 2, 3, 4, or 5. In some embodiments, E is each independently -OC(O)-, -C(O)O-,-N(R7)C(O)-, or -C(O)N(R7)-, wherein R7is 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 (TH):

[0259] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (Till):alkyl. The definitions of other variables in (Till) are the same as those defined above in (Tl).

[0260] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV):or 4. The definitions of other variables in (TIV) are the same as those defined above in (Tl).

[0261] In some embodiments, the lipid comprises at least one head group and at least one tail groupindependently H or methyl; and Rbis in each occasion independently H or C1-C4 alkyl. The definitions of other variables in (TV) are the same as those defined above in (Tl).

[0262] In some embodiments, the lipid comprises at least one head group and at least one tail groupis in each occasion independently H or C1-C4 alkyl. The definitions of other variables in (TH’) are the same as those defined above in (Tl’).

[0263] In some embodiments, the lipid comprises at least one head group and at least one tail groupeach independently H or methyl; and Rbis in each occasion independently H or C1-C4 alkyl. The definitions of other variables in (Till’) are the same as those defined above in (TH).

[0264] In some embodiments, the lipid comprises at least one tail group of the formulas (TH), (Till), (TIV), (TV), (TH’), and (Till’), whereinR7is each independently H or methyl;Rbis in each occasion independently 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 wherein the lipid has a pKa from about 4 to about 8.

[0265] In some embodiments, the lipid comprises tw, three, four, 0 or more tail groups that have a formula of (T), (Tl), (Til), (Till), (TIV), (TV), (TH’), and / or (Till’), and each tail group may be the same or different.

[0266] In some embodiments, , in any of the above formulas (T), (Tl), (Til), and (Till), (TIV), (TV), (Tl’), (TH’), and / or (Till’), Rais each independently C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl, or C2-C5 branched or unbranched alkynyl. In some embodiments, Rais each independently C1-C3 branched or unbranched alkyl. In one embodiment, each Rais methyl.

[0267] In some embodiments, in any of the above formulas (T), (Tl), (Til), and (Till), (TIV), (TV), (Tl’), (TH’), and / or (Till’), u1 is 3, 4, or 5. In some embodiments, in any of the above formulas (T), (Tl), (Til), and (Till), (TIV), (TV), (TH), (TH’), and / or (Till’), u2 is 0, 1 , 2, or 3. In some embodiments, in any of the above formulas (T), (Tl), (TH), and (Till), (TIV), (TV), (Tl’), (Til’), and / or (Till’), u3 and u4 are each independently 1-7, for instance, u3 and u4 are each independently 1 , 2, 3, or 4.

[0268] In some embodiments, the lipid comprises at least one tail of formula (Till), wherein each Rais methyl; Rbis in each occasion independently 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 (Till), wherein the two tails of formula (Till) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (Till), wherein each tail may be the same or different. In some embodiments, the lipid has four tails of formula (Till), wherein each tail may be the same or different. In some embodiments, in each tail of formula (Till), each Rais methyl, and u1 is 3, u2 is 2, and u3 is 4.

[0269] In some embodiments, the lipid comprises at least one tail of formula (Til), wherein each Rais 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 (Til), wherein the two tails of formula (Til) are the same. In some embodiments, the lipid has at least two tails of formula (TH), wherein the two tails of formula (Til) are or different. In some embodiments, the lipid comprises at least three tails of formula (Til), wherein each tail may be the same or different. In some embodiments, the lipid has four tails of formula (TH), wherein each tail may be the same or different.

[0270] In some embodiments, the lipid comprises at least one tail of formula (TIV), wherein each Rais 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), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), wherein each tail may be the same or different.

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

[0272] In some embodiments, the lipid has at least two tails of formula (TH’), wherein each tail may be the same or different. In some embodiments, the lipid has at least three tails of formula (TH’), wherein each tail may be the same or different. In some embodiments, the lipid has at least four tails of formula (TH’), wherein each tail may be the same or different.

[0273] In some embodiments, the lipid has at least two tails of formula (Till’), wherein each tail may be the same or different. In some embodiments, the lipid has at least three tails of formula (Till’), wherein each tail may be the same or different. In some embodiments, the lipid has at least four tails of formula (Till’), wherein each tail may be the same or different.In some embodiments, the lipid has at least one tail of formula (Til) and / or at least one tail of formula (Till); the lipid further comprises at least one tail that does not have a formula (T), (Tl), (Til), (Till), (TIV), (TV), (TIT), and / or (Till’). That is to say, the lipid further comprises at least one tail that does not contain a gem-di functional groups bonded to the same carbon next to E (e.g., -C(O)O-).

[0274] In some embodiments, the lipid further comprises at least one tail that does not have a formula (T), (Tl), (TH), (Till), (TIV), (TV), (Tl ), (TH’), and / or (Till’). That is to say, the lipid further comprises at least one tail that does not contain a gem-di functional groups bonded to the samecarbon next to E.

[0275] In some embodiments, the lipid further comprises at least one tail that does not have a formula (T), (Tl), (TH), (Till), (TIV), (TV), (Tl’), (TH’), and / or (Till’). That is to say, the lipid further comprises at least one tail that does not contain a gem-di functional groups bonded to the same carbon next to E.

[0276] In some embodiments, the lipid further comprises at least one tail of formula (TNG-I):whereinE is each independently a biodegradable group as described herein (e.g., -OC(O)-, -C(O)O-, -N(R7)C(O)-, -S-S-, or -C(O)N(R7)-); u1 and u2 are each independently 0, 1 , 2, 3, 4, 5, 6, or 7; andR7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0277] In some embodiments, the at least one tail of formula (TNG-I) can be represented byRbis in each occasion independently H or C1-C4 alkyl.

[0278] All the embodiments above regarding the definitions of E, Rb, R‘, u1 , u2, u3 and u4, as described above relating to the tail group containing a gem-di functional group bonded to the same carbon next to E, having a formula (T), (Tl), (TH), (Till), (TIV), (TV), (TH’), or (Till’), are also applicable to the tail group that does not contain a gem-di functional groups bonded to the same carbon next to E, having a formula (TNG-I), (TNG-II), or (TNG-III).

[0279] In some embodiments, the lipid further comprises at least two tails that do not have a formula (T), (Tl), (Til), (Till), (TIV), (TV), (TH), (TH’), and / or (Till’). In some embodiments, the lipid comprises two tail groups of formula (TNG-II) or (TNG-III), and wherein 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 a formula (T), (Tl), (TH), (Till), (TIV), (TV), (Tl’), (TH’), and / or (Till’). In some embodiments, the lipid comprises three tail groups of formula (TNG-II) or (TNG-III), and wherein each tail group may be the same or different,

[0281] In some embodiments, the head group of the lipid has a structure of formula (HA-I):R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or substituted with OH, SH, halogen, or cycloalkyl groups; orR20 and R30, together with the adjacent N atom, form a 3 to 7 membered heterocylic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; each of R1 and R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11 ; or R1 and R2 together form a cyclic ring; each of R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl; or R10 and Rn together form a heterocyclic ring; n is 0, 1 , 2, 3 or 4;Z is absent, O, S, or NR12, wherein R12 is H or C1-C7 branched or unbranched alkyl; provided that when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, SH.

[0282] In some embodiments, R20 and R30 together with the adjacent N atom form a 3 to 7 membered heterocylic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups.

[0283] In some embodiments, the head group of the ionizable lipid has a structure of formula (HA-wherein: each of R1 and R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11 ; or R1 and R2 are taken together to form a cyclic ring; each of R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl; or R10 and Rn are taken together to form a heterocyclic ring; 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 NR12, wherein R12 is H or C1-C7 branched or unbranched alkyl; provided that when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH; and represents the bond connecting the head group to the tail group.

[0284] In some embodiments, the head group of the ionizable lipid has a structure of formula (HA-wherein Z is absent, O, S, or NR12; and R12 is H or C1-C7 branched or unbranched alkyl. The definitions of other variables in (HA-III) are the same as those defined above in (HA-IA).

[0285] In some embodiments, the head group has a structure of:or, wherein:Rc is H or alkyl, optionally substituted with OH; and ml is 1 , 2, or 3.

[0286] In some embodiments, the head group of the ionizable lipid has a structure of formula (HA-V):wherein:Ri is H, C1-C3 alkyl, OH, halogen, SH, or NR10R11 ;R2 is OH, halogen, SH, or NR10R11 ; or R1 and R2 can be taken together to form a cyclic ring;R10 and R11 are each independently H or C1-C3 alkyl; or R10 and Rn can be taken together to form a heterocyclic ring;R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R20 and R30 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 a structure of formula (HA-VI):(HA-VI). The definitions of all variables in (HA-VI) are the same as those defined above in (HA-V).

[0288] In some embodiments, in any of the above formulas (HA-V) or (HA-VI), each of R20 and R30 are independently C1-C3 alkyl. In one embodiment, each of R20 and R30 are independently methyl.

[0289] In some embodiments, the head group of the ionizable lipid has a structure of formula (HB-I):whereinR5is OH, SH, (CH2)SOH, or NR10R11; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and Re are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2)vOH, (CH2)vSH, (CH2)sN(CH3)2, or NR10R11, wherein each R10 and R11 is independently H or C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring; or R7 and Re are taken together to form a ring; each R20 is independently H, or C1-C3 branched or unbranched alkyl;Ri s a heterocyclic, NR10R11, C(0)NRioRn, NRioC(0)NRioRii, or NRioC(S)NRioRii, wherein each R10 and Rn is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R10 and Rn are taken together to form a heterocyclic ring;R16 is H, =0, =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 heterocyclic; each Z is independently absent, O, S, or NR12, wherein R12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl;Q is O, S, CH2, or NR13, wherein each R13 is H, or C1-C5 alkyl;V is branched or unbranched C2-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; andT is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic.

[0290] In some embodiments, in formulawherein: each Re, 7, and Re are independently H or methyl; and each of u and t is independently 1 , 2, or 3.

[0291] In some embodiments, in formulawherein:R16 is H or =0;R14 is a nitrogen-containing 5- or 6- membered heterocyclic, NR10R11, C(0)NRioRn, NRioC(0)NRioRn, or NRioC(S)NRioRn, wherein each R10 and Rn is independently H or C1-C3 alkyl; and each of u and v is independently 1 , 2, or 3.

[0292] In some embodiments, in formula (HB-I), W is, wherein: each Re 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 formulaeach Re is independently H or methyl; each R7 is independently H; each Re is methyl; each u is independently 1 , 2, or 3; andV is C2-C6 alkylene or C2-C6 alkenylene.

[0294] In some embodiments, in formula (HB-I), W is, wherein: each u is independently 1 , 2, or 3; andT is a divalent nitrogen-containing 5- or 6- membered heterocyclic.

[0295] In some embodiments, in formulawherein: each u is independently 1 , 2, or 3;Q is O;each Z is independently NR12; and R12 is H or C1-C3 alkyl.

[0296] In some embodiments, the head group has the structure of:independently 1 or 2.

[0297] In some embodiments, the head group of the ionizable lipid has a structure of formula (HC-I):Y is alkyl, hydroxy, hydroxyalkyl,A is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, - C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, or -S-S-; each of X and Z is independently absent, -O-, -C(O)-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, or -S-; each R7is 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; andW 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 a structure of formula

[0299] In some embodiments, in the above formulas,A is absent, -O-, -N(R7)-, -OC(O)-, or -C(O)O-;X is absent, -O-, or -C(O)-; andZ is -O-, -C(O)O-, or -OC(O)-.

[0300] In some embodiments, the head group has a structure of formula

[0301] In some embodiments, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:wherein each Q is independently absent, -0-, -C(0)-, -C(S)-, -C(0)0-, -(CH2)q-C(R7)2-, - C(O)N(R7)-, -C(S)N(R7)-, or -N(R7);R6is independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O- alkylene-N(R7)2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl, or N+(R7)3-alkylene-Q-; each R8is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, or thiolalkyl, heterocyclyl, heteroaryl, or two R8together with the nitrogen atom may form a ring, optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl; q is 0, 1 , 2, 3, 4, or 5; and p is 0, 1 , 2, 3, 4, or 5.

[0302] In some embodiments, W is one of the following:acceptable salts thereof, and stereoisomers of any of the foregoing, whereinRi is each independently H, C1-C3 alkyl, OH, halogen, SH, or NR10R11; R1 and R2 can be taken together to form a cyclic ring; R10 and Rn are each independently H, C1-C3 alkyl, and R10 and R11 can be taken together to form a heterocyclic ring;R2 is each independently H, C1-C3 alkyl, OH, halogen, SH, or NR10R11; R1 and R2 can be taken together to form a cyclic ring; R10 and Rn are each independently H, C1-C3 alkyl, and R10 and R11 can be taken together to form a heterocyclic ring; m is 1 , 2, 3, 4, 5, 6, 7 or 8; n is 0, 1 , 2, 3 or 4; r is each independently 0, 1 , 2, 3, 4, 5, 6, 7 or 8;R3 is each independently H, or C3-C10 alkyl;R4 is each independently H, or C3-C10 alkyl; provided that at least one of R3 and R4 is not H;Z is absent, O, S, or NRI2; wherein RI2is C1-C7 alkyl;X’ is a biodegradable moiety.

[0304] some embodiments, X’ is-OCO-, -COO-, -NR7CO-, -CONR7-, -C(O-Ri3)-O-(acetal), -COO(CH2)S-, -CONH(CH2)S-, or-C(O-Ri3)-O-(CH2)s-; wherein R7is H or C1-C3 alkyl; and R13 is C3-C10 alkyl.

[0305] In some embodiments, at least one X in the formulamethyl.

[0306] In some embodiments, m =3. In some embodiments, n = 0 or 1 . In some embodiments, each R R1 and R2is 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] More embodiments of the above ionizable lipids comprising at least one head group (e.g., 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 (Tl) or (T1 ’) (e.g., tail group of formula (Til), (Till), TIV, TV, TH’, or Till’), in the Ionizable lipid compounds group iv), may be found in PCT Application No. PCT / US23 / 31669, filed on August 31 , 2023,, the content of which is incorporated herein by reference in its entirety. Moreover, all the ionizable lipids of formulas (LA-I)-(LA-VII), (LB-I)-(LB-VII), (LC-IA)-(LC-IC), (LC-IIA)-(LC-IIC), and (LC-IIIA)-(LC-IIIE) of PCT Application No. PCT / US23 / 31669, filed on August 31 , 2023 are suitable for use as the ionizable lipids in this disclosure, and are incorporated herein by reference in its entirety.

[0309] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table IV below. Table IV. Exemplary ionizable lipid compounds.Other ionizable lipids

[0310] In the modified lipid compositions, more than one ionizable lipid can be used for the ionizable lipid component: one or more of the ionizable lipids from the compounds of formulas in groups i)-iv) can be used alone or in combination with a different ionizable lipid from the compounds of formulas in groups i)-iv).

[0311] In some embodiments, the ionizable lipid do 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, LP01 , 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, LP01 , 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0313] In some embodiments, the modified lipid composition comprises an ionizable lipid being represented by the following formula III:wherein R is Cs-Ci4 alkyl group.

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

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

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

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

[0318] In some instances, 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 more than 90% ionizable lipid, e.g., 1 %-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%- 70%, 70%-80%, or 80%-90% ionizable lipid, e.g., about 25%-75% ionizable lipid (e.g., about 25%- 75% ionizable lipid), all in mol%.

[0319] The ionizable lipid described herein may include 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 lipid described herein include at least 3 lipid tails. A lipid tail may be a Cs-Cis hydrocarbon (e.g., Ce-Cis alkyl or Ce-Cis alkanoyl). An amine core may be substituted with one or more lipid tails at a nitrogen atom (e.g., one hydrogen atom attached to the nitrogen atom may be replaced with a lipid tail).

[0320] In some embodiments, the amine core has a structure of:

[0321] In some embodiments, the amine core has a structure of:

[0322] In some embodiments, the amine core has a structure of:

[0323] In some embodiments, the amine core has a structure of:

[0324] In some embodiments, the amine core has a structure of:

[0325] In some embodiments, the amine core has a structure of:

[0326] In some embodiments, the amine core has a structure of:

[0327] In some embodiments, the amine core has a structure of:

[0328] The modified lipid composition may further contain cationic lipids.

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

[0330] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

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

[0332] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

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

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

[0335] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salts thereof, wherein each instance of RLis independently optionally substituted C6-C40 alkenyl.

[0336] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0337] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:, and pharmaceutically acceptable salts thereof.

[0338] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0339] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0340] Other suitable lipids for use in the modified lipid composition and methods for making and using thereof include the lipids as described in International Patent Publication WO 2015 / 184256, which is incorporated herein by reference in its entirety. In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salt thereof, wherein each X independently is O or S; each Y independently is O or S; each m independently is 0 to 20; each n independently is 1 to 6; each RA 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; and each RB 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 composition and methods for making and usingthereof include a lipid, “Target 23”, having a compound structure of:, (Target 23) and pharmaceutically acceptable salts thereof.

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

[0343] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salt thereof.

[0344] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salt thereof.

[0345] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:salt thereof.

[0346] Other suitable lipids for use in the modified lipid composition and methods for making and using thereof include lipids as described in United States Provisional Patent Application Serial Number 62 / 758,179, which is incorporated herein by reference in its entirety.

[0347] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:, or a pharmaceutically acceptable salt thereof, wherein each R1and R2is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or arylene; each L1is independently an ester, thioester, disulfide, or anhydride group; each L2is independently C2-C10 aliphatic; each X1is independently H or OH; and each R3is independently C6-C20 aliphatic.

[0348] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salt thereof.

[0349] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salt thereof.

[0350] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salt thereof.

[0351] Other suitable lipids for use in the modified lipid composition and methods for making and using thereof include the lipids as described in J. McClellan, M. C. King, Cell 2010, 141 , 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which is incorporated herein by reference in its entirety.

[0352] In certain embodiments, the lipids of the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

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

[0354] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:thereof.

[0355] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:acceptable salts thereof.

[0356] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:thereof.

[0357] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:

[0358] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:thereof.

[0359] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0360] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0361] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0362] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0363] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0364] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0365] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0366] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

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

[0368] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0369] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:

[0370] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0371] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0372] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0373] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0374] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:acceptable salts thereof.

[0375] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0376] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0377] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:acceptable salts thereof.

[0378] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0379] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:salts thereof.

[0380] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0381] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:thereof.

[0382] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0383] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:thereof.

[0384] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:salts thereof.

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

[0386] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid of the following formula:or a pharmaceutically acceptable salt thereof, wherein one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, or - NRaC(=O)O-; and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, ,NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3is Ci- 024 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Rais H or C1-C12 alkyl;R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl; R3is H, OR5, ON, -C(=O)OR4, - OC(=O)R4or -NR5C(=O)R4; R4is C1-C12 alkyl; R5is H or Ci-C6alkyl; and x is 0, 1 or 2.

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

[0388] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:acceptable salts thereof.

[0389] In some embodiments, the modified lipid composition and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

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

[0391] In some embodiments, the lipids of the modified lipid composition and methods for making and using thereof include a compound of one of the following formulas:pharmaceutically acceptable salts thereof.For any one of these four formulas, R4 is independently selected from -(CH2)nQ and -(CH2)nCHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -ON, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2I-N(H)C(S)N(H)(R), and a heterocycle; and n is 1 , 2, or 3.

[0392] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0393] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0394] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0395] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0396] Other suitable lipids for use in the modified lipid composition and methods for making and using thereof include the lipids as described in International Patent Publication WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference in its entirety. In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0397] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0398] In certain embodiments, the modified lipid composition and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0399] In certain embodiments, the modified lipid composition and methods for making and usingthereof include a lipid having a compound structure ofpharmaceutically acceptable salts thereof.

[0400] In some embodiments, the modified lipid compositions described herein may include a ionizable lipid as described in, may be formulated as described in, or may comprise or be comprised by a composition as described in WO2016118724, WO2016118725, WO2016187531 ,WO2017176974, WO2018078053, WO2019027999, WO2019036030, WO2019089828,WO2019099501 , W02020072605, W02020081938, W02020118041 , W02020146805, or WO2020219876, each of which is incorporated by reference herein in its entirety.Other lipids and other agents

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

[0402] The modified lipid compositions 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 compositions can further include stabilizing molecules that increase the stability of the modified lipid compositions (e.g., for at least one day at room temperature, and / or stable for at least one week at 4°C).

[0403] In some embodiments, the modified lipid composition further includes a sterol, e.g., sitosterol, sitostanol, B-sitosterol, 7a-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or an analog (e.g., a glycoside, ester, or peptide) of any sterol. In some examples, the exogenous sterol is added to the preparation prior to step (b), e.g., mixed with structural lipids prior to step (b). The exogenous sterol may be added to amount to, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% (w / w) of total lipids and sterols in the preparation.

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

[0405] In some embodiments, a modified lipid composition that has been modified with a sterol has altered stability (e.g., increased stability) relative to a modified lipid composition that has not been modified with a sterol. In some aspects, a modified lipid composition that has been modified with a sterol has a greater rate of fusion with a membrane of a target cell relative to a modified lipid composition that has not been modified with a sterol.

[0406] In some instances, the modified lipid compositions comprise an exogenous lipid and an exogenous sterol.

[0407] In some embodiments, the modified lipid composition further includes a PEGylated lipid. Polyethylene glycol (PEG) length can vary from 1 kDa to 10kDa; in some aspects, PEG having a length of 2kDa is used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some instances, the modified lipid compositions comprise 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%, 20%, 30%, 40%, 50%, or more 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 in mol%. In some embodiments, the modified lipid composition comprises about 0.1 %-10% PEGylated lipid (e.g., C14-PEG2k, C18- PEG2k, or DMPE-PEG2k), e.g., about 1 %-3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid, all in mol%.

[0408] In some embodiments, a modified lipid composition that has been modified with a PEGylated lipid has altered stability (e.g., increased stability) relative to a modified lipid composition that has not been modified with a PEGylated lipid. In some embodiments, a modified lipid composition that has been modified with a PEGylated lipid has altered particle size relative to a modified lipid composition that has not been modified with a PEGylated lipid. In some embodiments, a modified lipid composition that has been modified with a PEGylated lipid is less likely to be phagocytosed than a modified lipid composition that has not been modified with a PEGylated lipid. The addition of PEGylated lipids can also affect stability in Gl tract and enhance particle migration through mucus. PEG may be used as a method to attach targeting moieties.

[0409] In some embodiments, the modified lipid composition comprises 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%-50% natural-derived lipids (e.g., about 10%-20% natural-derived lipids, e.g., about 10%, 12.5%, 16%, or 20% natural-derived lipids); about 30%-75% ionizable lipids (e.g., about 35% or about 50% ionizable lipids); about 35%-50% sterol (e.g., about 36%, 38.5%, 42.5%, or 46.5% sterol); and about 0.1 %-10% PEGylated lipid (e.g., about 1 %-3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid), all in mol%.

[0410] In some embodiments, the modified lipid composition comprise a molar ratio of about 5%- 60% natural-derived lipids (e.g., about 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60% natural-derived lipids, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60% natural-derivedlipids); about 25%-75% ionizable lipids (e.g., about 35% or about 50% ionizable lipids); about 10%- 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 %-10% PEGylated lipid (e.g., about 0.5%-5% PEGylated lipid, e.g., about 1 %-3% PEGylated lipid, or about 1 .5% or about 2.5% PEGylated lipid).

[0411] In some embodiments, the ionizable lipids, structural lipids, sterol, and PEGylated lipid 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, structural lipids, sterol, and PEGylated lipid 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, structural lipids, sterol, and PEGylated lipid 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 lipids, structural lipids, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:12.5:2.5.

[0415] In some embodiments, the ionizable lipids, structural lipids, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:1 1 .5:3.5.

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

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

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

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

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

[0421] In some embodiments, a modified lipid composition that has been modified with an ionizable lipid (and / or cationic lipid) and a sterol and / or a PEGylated lipid more efficiently encapsulates a negatively charged cargo (e.g., a nucleic acid) than a modified lipid composition that has not been modified with an ionizable lipid (and / or cationic lipid) and a sterol and / or a PEGylated lipid. The modified lipid composition may have an encapsulation efficiency for a cargo (e.g., a heterologous functional agent such as nucleic acid, e.g., RNA or DNA) that is at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more than 99%, e.g., it may have an encapsulation efficiency of 5%-30%, 30%-50%, 50%-70%, 70%-80%, 80%-90%, 90%-95%, or 95%- 100%.

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

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

[0424] In some instances, the organic solvent in which the lipid film is dissolved is dimethylformamide:methanol (DMF:MeOH). Alternatively, the organic solvent or solvent combination may be, e.g., acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1- buthanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofurammethanol, dimethyl sulfoxide:methanol, or dimethylformamide:methanol.

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

[0426] The lipid solution and the aqueous phase may be mixed in the microfluidics device at any suitable ratio. In some examples, aqueous phase and the lipid solution are mixed at a 3:1 volumetric ratio.

[0427] The modified lipid composition may optionally include additional agents, e.g., cell-penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. The modified lipid composition can carry or associate with additional agents in a variety of ways to enable delivery of the heterologous functional agent to a target cell, e.g., by encapsulating the heterologous functional agent, incorporation of the heterologous functional agent in the lipid bilayer structure, or association of the heterologous functional agent (e.g., by conjugation) with the surface of the lipid bilayer structure. The heterologous functional agent can be incorporated into the modified lipid composition either in vivo or in vitro (e.g., in tissue culture, in cell culture, or synthetically incorporated).Zeta Potential

[0428] The 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, e.g., a zeta potential of greater than -30 mV when in the absence of cargo, greater than -20 mV, greater than -5mV, greater than 0 mV, or about 30 mv when in the absence of cargo. In some examples, the modified lipid composition has a negative zeta potential, e.g., 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 when in the absence of cargo. In some examples, the modified lipid composition has a positive zeta potential, e.g., 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 when in the absence of cargo. In some examples, the modified lipid composition has a zeta potential of about 0.

[0429] The zeta potential of the modified lipid composition may be measured using any method known in the art. Zeta potentials are generally measured indirectly, e.g., calculated using theoretical models from the data obtained using methods and techniques known in the art, e.g., electrophoretic mobility or dynamic electrophoretic mobility. Electrophoretic mobility is typically measured using microelectrophoresis, electrophoretic light scattering, or tunable resistive pulse sensing. Electrophoretic light scattering is based on dynamic light scattering. Typically, zeta potentials are accessible from dynamic light scattering (DLS) measurements, also known as photon correlation spectroscopy or quasi-elastic light scattering.Natural EV-Markers

[0430] The structural components (e.g., structural lipids) in the modified lipid composition and methods of making and using thereof may have a range of markers that identify the structural component as being produced. In some embodiments, the structural component is natural. As used herein, the term “natural EV-marker” refers to a component that is naturally associated with the natural source and incorporated into or onto the natural EV, such as a natural protein, a natural nucleic acid, a natural small molecule, a natural lipid, or a combination thereof.Loading of Agents

[0431] The modified lipid composition can include a heterologous functional agent, e.g., a cellpenetrating agent and / or a heterologous agricultural agent (e.g., pesticidal agent, fertilizing agent, herbicidal agent, plant-modifying agent), a heterologous therapeutic agent (e.g., an antifungal agent, an antibacterial agent, a virucidal agent, an anti-viral agent, an insecticidal agent, a nematicidal agent, an antiparasitic agent, or an insect repellent)), such as those described herein,

[0432] The modified lipid composition can carry or associate with 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), e.g., by encapsulating the agent, incorporation of the agent in the lipid bilayer structure, or association of the agent (e.g., by conjugation) with the surface of the lipid bilayer structure of the modified lipid composition. In some instances, the heterologous functional agent (e.g., cell-penetrating agent) is included in the formulation made of the modified lipid composition formulation, as described herein.

[0433] The heterologous functional agent can be incorporated or loaded into or onto the modified lipid composition by any methods known in the art that allow association, directly or indirectly, between the modified lipid composition and agent. The agent can be incorporated into the modified lipid composition by an in vivo or in vitro (e.g., in tissue culture, or in cell culture), or both in vivo and in vitro methods.

[0434] In some instances, the modified lipid composition is loaded in vitro. The heterologous functional agent may be loaded onto or into (e.g., may be encapsulated by) the modified lipid composition using, but not limited to, physical, chemical, and / or biological methods (e.g., in tissue culture or in cell culture). For example, the agent may be introduced into the modified lipid composition by one or more of electroporation, sonication, passive diffusion, stirring, lipid extraction, or extrusion. In some instances, the agent is incorporated into the modified lipid composition using a microfluidic device, e.g., using a method in which lipids are provided in an organic phase, the agent is provided in an aqueous phase, and the organic and aqueous phases are combined in themicrofluidics device to produce a modified lipid composition comprising the heterologous functional agent. Loaded modified lipid composition can be assessed to confirm the presence or level of the loaded agent using a variety of methods, such as HPLC (e.g., to assess small molecules), immunoblotting (e.g., to assess proteins); and / or quantitative PCR (e.g., to assess nucleotides). However, it should be appreciated by those skilled in the art that the loading of a heterologous functional agent of interest into modified lipid composition is not limited to the above-illustrated methods.

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

[0436] In some instances, the modified lipid composition is stably associated with the heterologous functional agent prior to and following delivery of the modified lipid composition. In other instances, the modified lipid composition is associated with the agent such that the agent becomes dissociated from the modified lipid composition following delivery of the modified lipid composition.

[0437] The modified lipid composition can be loaded or the modified lipid composition can be formulated with various concentrations of the heterologous functional agent, depending on the particular agent or use. For example, in some instances, the modified lipid composition is loaded or formulated to include 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 an agent. In some instances, the modified lipid composition is loaded or formulated to include 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) or less wt% of an agent. For example, the modified lipid composition can include 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%, about 10 to about 20 wt% of the agent. In some instances, the modified lipid composition can be loaded or formulated with about 1 , 5, 10, 50, 100, 200, or 500, 1 ,000, 2,000 (or any range between about 1 and 2,000) or more pg / ml of an agent. A modified lipid composition can be loaded or formulated with about 2,000, 1 ,000, 500, 200, 100, 50, 10, 5, 1 (or any rangebetween about 2,000 and 1) or less pg / ml of an agent.

[0438] In some instances, the modified lipid composition is loaded or formulated to include 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% of the heterologous functional agent. In some instances, the modified lipid composition can be loaded or formulated with at least 1 pg / ml, at least 5 pg / ml, at least 10 pg / ml, at least 50 pg / ml, at least 100 pg / ml, at least 200 pg / ml, at least 500 pg / ml, at least 1 ,000 pg / ml, at least 2,000 pg / ml of the agent.

[0439] In some instances, the modified lipid composition is formulated with the heterologous functional agent by suspending the modified lipid compositions in a solution comprising or consisting of the agent, e.g., by vigorous mixing. The agent (e.g., cell-penetrating agent, e.g., nucleic acids, enzyme, detergent, ionic, fluorous, or zwitterionic liquid, or ionizable lipid may comprise, e.g., less than 1% or at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the solution.FormulationsAgricultural Formulations

[0440] The modified lipid composition described herein can be formulated into an agricultural composition.

[0441] To allow ease of application, handling, transportation, storage, and effective activity, the modified lipid composition can be formulated with other substances. The modified lipid composition can be formulated into, for example, baits, concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, microencapsulations, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions. For further information on formulation types see “Catalogue of Pesticide Formulation Types and International Coding System” Technical Monograph n° 2, 5th Edition by CropLife International (2002).

[0442] The modified lipid composition can be applied as aqueous suspensions or emulsions prepared from concentrated formulations of such agents. Such water-soluble, water-suspendable, or emulsifiable formulations are either solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which may be compacted to form water dispersible granules, comprise an intimate mixture of the modified lipid composition, a carrier, and surfactants. The carrier is usually selected from among the attapulgite clays, the montmorillonite clays, the diatomaceous earths, or the purified silicates. Effective surfactants, including from about 0.5% to about 10% of the wettable powder, are found among sulfonated lignins, condensed naphthalenesulfonates, naphthalenesulfonates, alkylbenzenesulfonates, alkyl sulfates, and non-ionic surfactants such as ethylene oxide adducts of alkyl phenols.

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

[0444] Aqueous suspensions comprise suspensions of water-insoluble modified lipid composition dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Suspensions are prepared by finely grinding the composition and vigorously mixing it into a carrier comprised of water and surfactants. 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 granular compositions that are particularly useful for applications to the soil. Granular compositions usually contain from about 0.5% to about 10% by weight of the modified lipid composition, dispersed in a carrier that comprises clay or a similar substance. Such compositions are usually prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier which has been pre-formed to the appropriate particle size, in the range of 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 and crushing and drying to obtain the desired granular particle size.

[0446] Dusts containing the modified lipid composition are prepared by intimately mixing a modified lipid composition in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts can suitably contain from about 1% to about 10% of the packets. They can be applied as a seed dressing or as a foliage application with a dust blower machine.

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

[0448] The modified lipid composition can also be applied in the form of an aerosol composition. In such compositions the packets are dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve.

[0449] Another embodiment is an oil-in-water emulsion, wherein the emulsion comprises oily globules which are each provided with a lamellar liquid crystal coating and are dispersed in an aqueous phase, wherein each oily globule comprises at least one compound which is agriculturally active, and is individually coated with a monolamellar or oligolamellar layer including: (1) at least one non-ionic lipophilic surface-active agent, (2) at least one non-ionic hydrophilic surface-active agent and (3) at least one ionic surface-active agent, wherein the globules having a mean particle diameter of less than 800 nanometers. Further information on the embodiment is disclosed in U.S. patent publication 20070027034 published Feb. 1 , 2007. For ease of use, this embodiment will be referred to as “OIWE.”

[0450] Additionally, generally, when the molecules disclosed above are used in a formulation, such formulation can also contain other components. These components include, but are not limited to, (this is a non-exhaustive and non-mutually exclusive list) wetters, spreaders, stickers, penetrants, buffers, sequestering agents, drift reduction agents, compatibility agents, anti-foam agents, cleaning agents, and emulsifiers. A few components are described forthwith.

[0451] A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank to reduce the wetting time of wettable powders and to improve the penetration of water 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; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

[0452] A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. Dispersing agents are added to agrochemical formulations to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. They are widely used in wettable powders, suspension concentrates and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to reaggregation of particles. The most commonly used surfactants are anionic, non-ionic, or mixtures of the two types. For wettable powder formulations, the most common dispersing agents are sodium lignosulfonates. For suspension concentrates, very good adsorption and stabilization are obtained using polyelectrolytes, such as sodium naphthalene sulfonate formaldehyde condensates. Tristyrylphenol ethoxylate phosphate esters are also used. Non-ionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates. In recent years, new types of very high molecular weight polymeric surfactants have been developed as dispersing agents. These have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. These high molecular weight polymers can give very good long-term stability to suspension concentrates because the hydrophobic backbones have many anchoring points onto the particle surfaces. Examples of dispersing agents used in agrochemical formulations are: sodium lignosulfonates; sodium naphthalene sulfonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alkyl ethoxylates; EO-PO (ethylene oxide - propylene oxide) block copolymers; and graft copolymers.

[0453] An emulsifying agent is a substance which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent, the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain alkylphenol or aliphatic alcohol with twelve or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzenesulfonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 willnormally provide good stable emulsions. Emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

[0454] A solubilizing agent is a surfactant which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics, sorbitan monooleates, sorbitan monooleate ethoxylates, and methyl oleate esters.

[0455] Surfactants are sometimes used, either alone or with other additives such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the modified lipid composition on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the modified lipid composition. However, they are often nonionics such as: alkyl ethoxylates; linear aliphatic alcohol ethoxylates; aliphatic amine ethoxylates.

[0456] A carrier or diluent in an agricultural formulation is a material added to the modified lipid composition to give a product of the required strength. Carriers are usually materials with high absorptive capacities, while diluents are usually materials with low absorptive capacities. Carriers and diluents are used in the formulation of dusts, wettable powders, granules, and water-dispersible granules.

[0457] Organic solvents are used mainly in the formulation of emulsifiable concentrates, oil-in-water emulsions, suspoemulsions, and ultra low volume formulations, and to a lesser extent, granular formulations. Sometimes mixtures of solvents are used. The first main groups of solvents are aliphatic paraffinic oils such as kerosene or refined paraffins. The second main group (and the most common) comprises the aromatic solvents such as xylene and higher 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 into water. Alcohols are sometimes used as cosolvents to increase solvent power. Other solvents may include vegetable oils, seed oils, and esters of vegetable and seed oils.

[0458] Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. 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-gelling agents for many years. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or are synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum; locust bean gum; carrageenan; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). Other types of anti-settling agents are based on modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0459] Microorganisms can cause spoilage of formulated products. Therefore preservation agentsare used to eliminate or reduce their effect. Examples of such agents include, but are not limited to: propionic acid and its sodium salt; sorbic acid and its sodium or potassium salts; 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 water-based formulations to foam during mixing operations in production and in application through a spray tank. In order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles. Generally, there are two types of anti-foam agents, namely silicones and non-silicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the non-silicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the antifoam agent is to displace 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, e.g., plant and animal sources. Specific examples are: vegetable oils, seed oils, and esters thereof, also alkoxylated alkyl polyglucosides.

[0462] In some instances, the modified lipid composition can be freeze-dried or lyophilized. See U.S. Pat. No. 4,311 ,712. The modified lipid composition can later be reconstituted on contact with water or another liquid. Other components can be added to the lyophilized or reconstituted modified lipid composition, for example, other heterologous functional agents, agriculturally acceptable carriers, or other materials in accordance with the formulations described herein.

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

[0464] For further 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.Pharmaceutical Formulations

[0465] The modified lipid compositions are formulated into pharmaceutical compositions (i.e. , a modified lipid composition composition), e.g., for administration to an animal (e.g., a human). The pharmaceutical composition may be administered to an animal (e.g., human) with a pharmaceutically acceptable diluent, carrier, and / or excipient. Depending on the mode of administration and the dosage, the pharmaceutical composition of the methods described herein will be formulated into suitable pharmaceutical compositions to permit facile delivery. The single dose may be in a unit dose form as needed.

[0466] The modified lipid composition may be formulated for e.g., oral administration, intravenous administration (e.g., injection or infusion), intramuscular, or subcutaneous administration to an animal.For injectable formulations, various effective pharmaceutical carriers are known in the art (See, e.g., Remington: The Science and Practice of Pharmacy, 22nded., (2012) and ASHP Handbook on Injectable Drugs, 18thed., (2014)).

[0467] Suitable pharmaceutically acceptable carriers and excipients are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol. The modified lipid composition may be formulated according to conventional pharmaceutical practice. The concentration of the compound in the formulation will vary depending upon a number of factors, including the dosage of the active agent (e.g., modified lipid compositions and nucleic acids) to be administered, and the route of administration.

[0468] For oral administration to an animal, 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(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, 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); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like. Formulations for oral use may also be provided in unit dosage form as chewable tablets, non-chewable tablets, caplets, capsules (e.g., as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium). The compositions disclosed herein may also further include an immediate-release, extended release or delayed-release formulation.

[0469] For parenteral administration to an animal, the modified lipid composition may be formulated in the form of liquid solutions or suspensions and administered by a parenteral route (e.g., subcutaneous, intravenous, or intramuscular). The pharmaceutical composition can be formulated for injection or infusion. Pharmaceutical compositions for parenteral administration can be formulated using a 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 media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEM), andF-12 medium). Formulation methods are known in the art, see e.g., Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor & Francis Group, CRC Press (2009).Heterologous Functional Agents

[0470] The modified lipid composition can further include a heterologous functional agent, such as a heterologous functional agent (e.g., a heterologous agricultural agent (e.g., pesticidal agent, fertilizing agent, herbicidal agent, plant-modifying agent) or a heterologous therapeutic agent (e.g., an antifungal agent, an antibacterial agent, a virucidal agent, an anti-viral agent, an insecticidal agent, a nematicidal agent, an antiparasitic agent, or an insect repellent)). For example, the modified lipid composition may encapsulate the heterologous functional agent. Alternatively, the heterologous functional agent can be embedded on or conjugated to the surface of the modified lipid composition. In some instances, the modified lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different heterologous functional agents. Heterologous functional agents may be added at any step during the manufacturing process effective to introduce the agent into the modified lipid composition.

[0471] In certain instances, the heterologous functional agent (e.g., a heterologous agricultural agent (e.g., pesticidal agent, fertilizing agent, herbicidal agent, plant-modifying agent, 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 virucidal agent, an anti-viral agent, a nematicidal agent, an antiparasitic agent, or an insect repellent)) can be modified. For example, the modification can be a chemical modification, e.g., conjugation to a marker, e.g., fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation or operational linkage to 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 cation moiety.

[0472] Examples of heterologous functional agents are outlined below.Heterologous agricultural agents

[0473] The modified lipid composition can include a heterologous agricultural agent (e.g., an agent that effects a plant or an organism that associates with a plant and can be loaded into a modified lipid composition), such as a pesticidal agent, herbicidal agent, fertilizing agent, or a plant-modifying agent.

[0474] For example, in some instances, the modified lipid composition may include a pesticidal agent. The pesticidal agent can be an antifungal agent, an antibacterial agent, an insecticidal agent, a molluscicidal agent, a nematicidal agent, a virucidal agent, or a combination thereof. The pesticidal agent can be a chemical agent, such as those well known in the art. Alternatively or additionally, the pesticidal agent can be a peptide, a polypeptide, a nucleic acid, a polynucleotide, or a small molecule. The pesticidal agent may be an agent that can decrease the fitness of a variety of plant pests or can be one that targets one or more specific target plant pests (e.g., a specific species or genus of plant pests).

[0475] In some instances, the modified lipid composition may include one or more heterologous fertilizing agents. Examples of heterologous fertilizing agents include plant nutrients or plant growth regulators, such as those well known in the art. Alternatively, or additionally, the fertilizing agent canbe a peptide, a polypeptide, a nucleic acid, or a polynucleotide that can increase the fitness of a plant symbiont. The fertilizing agent may be an agent that can increase the fitness of a variety of plants or plant symbionts or can be one that targets one or more specific target plants or plant symbionts (e.g., a specific species or genera of plants or plant symbionts).

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

[0477] The modified lipid composition described herein can further include an antibacterial agent. In some instances, the modified lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antibacterial agents. For example, the antibacterial agent can decrease the fitness of (e.g., decrease growth or kill) a natural plant pest (e.g., a natural plant pathogen). A modified lipid composition including an antibiotic can be contacted with a target pest, or plant infested thereof, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside or on the target pest; and (b) decrease fitness of the target pest. The antibacterials may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0478] As used herein, the term “antibacterial agent” refers to a material that kills or inhibits the growth, proliferation, division, reproduction, or spread of bacteria, such as phytopathogenic bacteria, and includes bactericidal (e.g., disinfectant compounds, antiseptic compounds, or antibiotics) or bacteriostatic agents (e.g., compounds or antibiotics). Bactericidal antibiotics kill bacteria, while bacteriostatic antibiotics only slow their growth or reproduction.

[0479] Bactericides can include disinfectants, antiseptics, or antibiotics. The most used disinfectants can comprise: active chlorine (i.e., hypochlorites (e.g., sodium hypochlorite), chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide etc.), active oxygen (peroxides, such as peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate and urea perhydrate), iodine (iodpovidone (povidone-iodine, Betadine), Lugol’s solution, iodine tincture, iodinated nonionic surfactants), concentrated alcohols (mainly ethanol, 1 -propanol, called also n-propanol and 2-propanol, called isopropanol and mixtures thereof; further, 2-phenoxyethanol and 1- and 2-phenoxypropanols are used), phenolic substances (such as phenol (also called carbolic acid), cresols (called Lysole in combination with liquid potassium soaps), halogenated (chlorinated, brominated) phenols, such as hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, Dibromol and salts thereof), cationic surfactants, such as some quaternary ammonium cations (such as benzalkonium chloride, cetyl trimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride) and others, nonquaternary compounds, such as chlorhexidine, glucoprotamine, octenidine dihydrochloride etc.), strong oxidizers, such as ozone and permanganate solutions; heavy metals and their salts, such as colloidal silver, silver nitrate, mercury chloride, phenylmercury salts, copper sulfate, copper oxide-chloride, copper hydroxide, copper octanoate, copper oxychloride sulfate, copper sulfate, copper sulfate pentahydrate, etc. Heavy metals and their salts are the most toxic, and environment- hazardous bactericides and therefore, their use is strongly oppressed or canceled; further, also properly concentrated strong acids (phosphoric, nitric, sulfuric, amidosulfuric, toluenesulfonic acids) and alkalis (sodium, potassium, calcium hydroxides).

[0480] As antiseptics (i.e., germicide agents that can be used on human or animal body, skin, mucoses, wounds and the like), few of the above mentioned disinfectants can be used, under proper conditions (mainly concentration, pH, temperature and toxicity toward man / animal). Among them, important are: properly diluted chlorine preparations (i.e., Daquin’s solution, 0.5% sodium or potassium hypochlorite solution, pH-adjusted to pH 7-8, or 0.5-1% solution of sodium benzenesulfochloramide (chloramine B)), some iodine preparations, such as iodopovidone in various galenics (ointment, solutions, wound plasters), in the past also Lugol’s solution, peroxides as urea perhydrate solutions and pH-buffered 0.1-0.25% peracetic acid solutions, alcohols with or without antiseptic additives, used mainly for skin antisepsis, weak organic acids such as sorbic acid, benzoic acid, lactic acid and salicylic acid some phenolic compounds, such as hexachlorophene, triclosan and Dibromol, and cation-active compounds, such as 0.05-0.5% benzalkonium, 0.5-4% chlorhexidine, 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 commonly classified based on their mechanism of action, chemical structure, or spectrum of activity.

[0482] The antibiotic described herein may target any natural function or growth processes and may be either bacteriostatic (e.g., slow or prevent natural growth) or bactericidal (e.g., kill bacteria). In some instances, the antibiotic is a bactericidal antibiotic. In some instances, the bactericidal antibiotic is one that targets the natural cell wall (e.g., penicillins and cephalosporins); one that targets the cell membrane (e.g., polymyxins); or one that inhibits essential natural enzymes (e.g., rifamycins, lipiarmycins, quinolones, and sulfonamides). In some instances, the bactericidal antibiotic is an aminoglycoside (e.g., kasugamycin). In some instances, the antibiotic is a bacteriostatic antibiotic. In some instances 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 antibiotic described herein may have any level of target specificity (e.g., narrow- or broad-spectrum). In some instances, the antibiotic is a narrow-spectrum antibiotic, and thus targets specific types 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 are found in Table 1 of WO 2021 / 041301 , which is incorporated herein by reference in its entirety. One skilled in the art will appreciate that a suitable concentration of each antibiotic in the composition depends on factors such as efficacy, stability of the antibiotic, number of distinct antibiotics, the formulation, and methods of application of thecomposition.Antifungal agents

[0484] The modified lipid composition can further include an antifungal agent. In some instances, the modified lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antifungal agents. For example, the antifungal agent can decrease the fitness of (e.g., decrease growth or kill) a fungal plant pest. A modified lipid composition including an antifungal can be contacted with a target fungal pest, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside or on the target fungus; and (b) decrease fitness of the target fungus. The antifungals may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0485] As used herein, the term "fungicide" or “antifungal agent” refers to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of fungi, such as phytopathogenic fungi. Many different types of antifungal agent have been produced commercially. Non limiting examples of antifungal agents include: azoxystrobin, mancozeb, prothioconazole, folpet, tebuconazole, difenoconazole, captan, bupirimate, or fosetyl-AI. Further exemplary fungicides include, but are not limited to, strobilurins, azoxystrobin, dimoxystrobin, enestroburin, 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), methfuroxam, metsulfovax, ofurace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbolid, salicylanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamides, bixafen, boscalid, carboxylic acid morpholides, dimethomorph, flumorph, benzamides, flumetover, fluopicolid (picobenzamid), zoxamid, carboxamides, carpropamid, diclocymet, mandipropamid, silthiofam, azoles, triazoles, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, enilconazole, epoxiconazole, fenbuconazole, flusilazol, fluquinconazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimenol, triadimefon, triticonazole, Imidazoles, cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, benzimidazoles, benomyl, carbendazim, fuberidazole, thiabendazole, ethaboxam, etridiazole, hymexazol, nitrogen-containing heterocyclyl compounds, pyridines, fuazinam, pyrifenox, pyrimidines, bupirimate, cyprodinil, ferimzone, fenarimol, mepanipyrim, nuarimol, pyrimethanil, piperazines, triforine, pyrroles, fludioxonil, fenpiclonil, morpholines, aldimorph, dodemorph, fenpropimorph, tridemorph, dicarboximides, iprodione, procymidone, vinclozolin, acibenzolar-S-methyl, anilazine, captan, captafol, dazomet, diclomezin, fenoxanil, folpet, fenpropidin, famoxadon, fenamidon, octhilinone, probenazole, proquinazid, pyroquilon, quinoxyfen, tricyclazole, carbamates, dithiocarbamates, ferbam, mancozeb, maneb, metiram, metam, propineb, thiram, zineb, ziram, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, guanidines, dodine, iminoctadine, guazatine, kasugamycin, polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing heterocyclyl compounds, isoprothiolane, dithianone, organophosphorouscompounds, edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, Organochlorine compounds, thiophanate-methyl, chlorothalonil, dichlofluanid, toly Ifluanid , flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton, spiroxamine, cyflufenamid, cymoxanil, metrafenon, N-2-cyanophenyl- 3,4-dichloroisothiazol-5-carboxamide (isotianil), 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-methylpyrazol-e-4-carboxamide, 5-chloro-7-(4- methylpi peridin-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]triazo-le-1- sulfonamide, methyl-(2-chloro-5-[1-(3-methylbenzyloxyimino)-ethyl]benzyl)carbamate, 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-ynyloxy]-3- methoxyphenyl)ethyl)-2-metha-nesulfonylamino-3-methylbutyramide, N-(2-(4-[3-(4-chlorophenyl)prop- 2-ynyloxy]-3-methoxyphenyl)ethyl)-2-ethan-esulfonylamino-3-methylbutyramide, N-(4'-bromobiphenyl- 2-yl)-4-difluoromethyl-2-methylthiazol-5-carboxamide, N-(4'-trifluoromethylbiphenyl-2-yl)-4- difluoromethyl-2-methylthiazol-5-carboxamide, N-(4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2- methylt-hiazol-5-carboxamide, or methyl 2-(ortho-((2,5-dimethylphenyloxy-methylene)phenyl)-3- methoxyacrylate. One skilled in the art will appreciate that a suitable concentration of each antifungal in the composition depends on factors such as efficacy, stability of the antifungal, number of distinct antifungals, the formulation, and methods of application of the composition.Insecticides

[0486] The modified lipid composition can further include an insecticide. In some instances, the modified lipid compositions include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different insecticide agents. For example, the insecticide can decrease the fitness of (e.g., decrease growth or kill) an insect plant pest. A modified lipid composition including an insecticide can be contacted with a target insect pest, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of insecticide concentration inside or on the target insect; and (b) decrease fitness of the target insect. The insecticides may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0487] As used herein, the term "insecticide" or “insecticidal agent” refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of insects, such as agricultural insect pests. Non limiting examples of insecticides are shown in Table 2 of WO 2021 / 041301 , which is incorporated herein by reference in its entirety. Additional non-limiting examples of suitable insecticides include biologies, hormones or pheromones such as azadirachtin, Bacillus species, Beauveria species, codlemone, Metarrhizium species, Paecilomyces species, thuringiensis, and Verticillium species, and active compounds having unknown or non-specified mechanisms of action such as fumigants (such as aluminum phosphide, methyl bromide and sulphuryl fluoride) and selective feeding inhibitors (suchas cryolite, flonicamid and pymetrozine). One skilled in the art will appreciate that a suitable concentration of each insecticide in the composition depends on factors such as efficacy, stability of the insecticide, number of distinct insecticides, the formulation, and methods of application of the composition.Nematicide

[0488] The modified lipid composition can further include a nematicide. In some instances, the modified lipid composition includes 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 decrease the fitness of (e.g., decrease growth or kill) a nematode plant pest. A modified lipid composition including a nematicide can be contacted with a target nematode pest, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of nematicide concentration inside or on the target nematode; and (b) decrease fitness of the target nematode. The nematicides may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0489] As used herein, the term "nematicide" or “nematicidal agent” refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of nematodes, such as agricultural nematode pests. Non limiting examples of nematicides are shown in Table 3 of WO 2021 / 041301 , which is incorporated herein by reference in its entirety. One skilled in the art will appreciate that a suitable concentration of each nematicide in the composition depends on factors such as efficacy, stability of the nematicide, number of distinct nematicides, the formulation, and methods of application of the composition.Molluscicide

[0490] The modified lipid composition can further include a molluscicide. In some instances, the modified lipid composition includes 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 decrease the fitness of (e.g., decrease growth or kill) a mollusk plant pest. A modified lipid composition including a molluscicide can be contacted with a target mollusk pest, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of molluscicide concentration inside or on the target mollusk; and (b) decrease fitness of the target mollusk. The molluscicides may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0491] As used herein, the term "molluscicide" or “molluscicidal agent” refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of mollusks, such as agricultural mollusk pests. A number of chemicals can be employed as a molluscicide, including metal salts such as iron(lll) phosphate, aluminium sulfate, and ferric sodium EDTA,[3][4], metaldehyde, methiocarb, or acetylcholinesterase inhibitors. One skilled in the art will appreciate that a suitable concentration of each molluscicide in the composition depends on factors such as efficacy, stability of the molluscicide, number of distinct molluscicides, the formulation, and methods of application of the composition.Virucides

[0492] The modified lipid composition can further include a virucide. In some instances, the modified lipid composition includes 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 decrease the fitness of (e.g., decrease or eliminate) a viral plant pathogen. A modified lipid composition including a virucide as described herein can be contacted with a target virus, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of virucide concentration; and (b) decrease or eliminate the target virus. The virucides described herein may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0493] As used herein, the term "virucide" or “antiviral” refers to a substance that kills or inhibits the growth, proliferation, reproduction, development, or spread of viruses, such as agricultural virus pathogens. A number of agents can be employed as a virucide, including chemicals or biological agents (e.g., nucleic acids, e.g., dsRNA). One skilled in the art will appreciate that a suitable concentration of each virucide in the composition depends on factors such as efficacy, stability of the virucide, number of distinct virucides, the formulation, and methods of application of the composition.Herbicides

[0494] The modified lipid composition can further include one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) herbicide. For example, the herbicide can decrease the fitness of (e.g., decrease or eliminate) a weed. A modified lipid composition including an herbicide can be contacted with a target weed in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of herbicide concentration on the plant and (b) decrease the fitness of the weed. The herbicides may be formulated in a 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. A number of chemicals can be employed as a 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, haloxyfop, and quizalofop esters, pyridinecarboxylic acid herbicides, such as aminopyralid, picloram, and clopyralid esters, pyrimidinecarboxylic acid herbicides, such as aminocyclopyrachlor esters, pyridyloxyalkanoic acid herbicides, such as fluoroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides, such as bromoxynil and ioxynil esters, esters of the arylpyridine carboxylic acids, and arylpyrimidine carboxylic acids of the generic structures disclosed in U.S. Pat. No. 7,314,849, U.S. Pat. No. 7,300,907, and U.S. Pat. No. 7,642,220, each of which is incorporated by reference herein in its entirety. In certain embodiments, the herbicide can be selected from thegroup consisting of 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butylate, carfentrazone, chloramben, chlorimuron, chlorproham, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cyanazine, cycloate, DCPA, desmedipham, dichlobenil, diclofop, diclosulam, diethatyl, difenzoquat, diflufenzopyr, dimethenamid-p, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethametsulfuron, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluthiacet, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, haloxyfop, 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, oxyfluorfen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propachlor, propanil, prosulfuron, pyrazon, pyridate, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, vernolate. One skilled in the art will appreciate that a suitable concentration of each herbicide in the composition depends on factors such as efficacy, stability of the herbicide, number of distinct herbicides, the formulation, and methods of application of the composition.Repellents

[0496] The modified lipid composition can further include a repellent. In some instances, the modified lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different repellents. For example, the repellent can repel any of the pests described herein (e.g., insects, nematodes, or mollusks); microorganisms (e.g., phytopathogens or endophytes, such as bacteria, fungi, or viruses); or weeds. A modified lipid composition including a repellent can be contacted with a target plant, or plant infested therewith, in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of repellent concentration; and (b) decrease the levels of the pest on the plant relative to an untreated plant. The repellent may be formulated in a modified lipid composition for any of the methods described herein, and in certain instances, may be associated with the modified lipid composition.

[0497] In some instances, the repellent is an insect repellent. Some examples of well-known insect repellents include: benzil; 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 (Indalone); dibutyl adipate; dibutyl phthalate; di-normal-butyl succinate (Tabatrex); N,N-diethyl-meta-toluamide (DEET); dimethyl carbate (endo,endo)-dimethyl bicyclo[2.2.1] hept-5-ene-2,3-dicarboxylate); dimethyl phthalate; 2-ethyl-2-butyl-1 ,3-propanediol; 2-ethyl-1 ,3- hexanediol (Rutgers 612); di-normal-propyl isocinchomeronate (MGK Repellent 326); 2- phenylcyclohexanol; p-methane-3,8-diol, and normal-propyl N,N-diethylsuccinamate. Other repellents include citronella oil, dimethyl phthalate, normal-butylmesityl oxide oxalate and 2-ethyl hexanediol-1 ,3 (See, Kirk-Othmer Encyclopedia of Chemical Technology, 2nd Ed., Vol. 11 : 724-728; and TheCondensed Chemical Dictionary, 8th Ed., p 756).

[0498] An insect repellent may be a synthetic or nonsynthetic insect repellent. Examples of synthetic insect repellents include methyl anthranilate and other anthranilate-based insect repellents, benzaldehyde, DEET (N,N-diethyl-m-toluamide), dimethyl carbate, dimethyl phthalate, icaridin (i.e., picaridin, Bayrepel, and KBR 3023), indalone (e.g., as used in a "6-2-2" mixture (60% Dimethyl phthalate, 20% Indalone, 20% Ethylhexanediol), IR3535 (3-[N-Butyl-N-acetyl]-aminopropionic acid, ethyl ester), metofluthrin, permethrin, SS220, or tricyclodecenyl allyl ether. Examples of natural insect repellents include beautyberry (Callicarpa) leaves, birch tree bark, bog myrtle (Myrica Gale), catnip oil (e.g., nepetalactone), citronella oil, essential oil of the 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.Fertilizing agents

[0499] The modified lipid composition can further include a heterologous fertilizing agent. In some instances, the heterologous fertilizing agent is associated with the modified lipid composition. For example, a modified lipid composition may encapsulate the heterologous fertilizing agent.Additionally, or alternatively, the heterologous fertilizing agent can be embedded on or conjugated to the surface of the modified lipid composition.

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

[0501] In some instances, the heterologous fertilizing agent can be modified. For example, the modification can be a chemical modification, e.g., conjugation to a marker, e.g., fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation or operational linkage to 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), a cation moiety.

[0502] In some instances, the heterologous fertilizing agent includes any material of natural or synthetic origin that is applied to soils or to plant tissues to supply one or more plant nutrients essential to the growth of plants. The plant nutrient may include a macronutrient, micronutrient, or a combination 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 a nitrogen fertilizer including, but not limited to urea, ammonium nitrate, ammonium sulfate, non-pressure nitrogen solutions, aqua ammonia, anhydrous ammonia, ammonium thiosulfate, sulfur-coated urea, urea-formaldehydes, IBDU, polymer-coated urea, calcium nitrate, ureaform, or methylene urea, phosphorous fertilizers such as diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, concentrated superphosphate and triple superphosphate, or potassium fertilizers suchas potassium chloride, potassium sulfate, potassium-magnesium sulfate, potassium nitrate. Such compositions can exist as free salts or ions within the composition. Fertilizers may be designated by the content of one or more of its components, such as nitrogen, phosphorous, or potassium. The content of these elements in a fertilizer may be indicated by the N — P — K value (where N=nitrogen content by weight percentage, P=phosphorous content by weight percentage, and K=potassium content by weight percentage).

[0503] Inorganic fertilizers, on the other hand, are manufactured from non-living materials and include, for example, ammonium nitrate, ammonium sulfate, urea, potassium chloride, potash, ammonium phosphate, anhydrous ammonia, and other phosphate salts. Inorganic fertilizers are readily commercially available and contain nutrients in soluble form that are immediately available to the plant. Inorganic fertilizers are generally inexpensive, having a low unit cost for the desired element. One skilled in the art will appreciate that the exact amount of a given element in a fertilizing agent may be calculated and administered to the plant or soil.

[0504] Fertilizers may be further classified as either organic fertilizers or inorganic fertilizers. Organic fertilizers include fertilizers having a molecular skeleton with a carbon backbone, such as in compositions derived from living matter. Organic fertilizers are made from materials derived from living things. Animal manures, compost, bonemeal, feather meal, and blood meal are examples of common organic fertilizers. Organic fertilizers, on the other hand, are typically not immediately available to plants and require soil microorganisms to break the fertilizer components down into simpler structures prior to use by the plants. In addition, organic fertilizers may not only elicit a plant growth response as observed with common inorganic fertilizers, but natural organic fertilizers may also stimulate soil microbial population growth and activities. Increased soil microbial population (e.g., plant symbionts) may have significant beneficial effects on the physical and chemical properties of the soil, as well as increasing disease and pest resistance.

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

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

[0507] The heterologous fertilizing agent may include a plant growth regulator. Exemplary plant growth regulators include auxins, cytokinins, gibberellins, and abscisic acid. In some instances, the plant growth regulator is abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6- dimethylpuridine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3 -acetic acid , maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmon, thidiazuron, triapenthenol, tributyl phosphorotrithioate, 2,3,5-tri- iodobenzoic acid, trinexapac-ethyl and uniconazole. Other plant growth regulators that can be incorporated seed coating compositions are described in US 2012 / 0108431 , which is incorporated by reference in its entirety.Plant-modifying agents

[0508] The modified lipid composition described herein include one or more heterologous plantmodifying agents. For example, the modified lipid composition may encapsulate the heterologous plant-modifying agent. Alternatively or additionally, the heterologous plant-modifying agent can be embedded on or conjugated to the surface of the modified lipid composition.

[0509] In some instances, the plant-modifying agent can include a peptide or a nucleic acid. The plant-modifying agent may be an agent that increases the fitness of a variety of plants or can be one that targets one or more specific plants (e.g., a specific species or genera of plants). Additionally, in some instances, 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-modifying agents.

[0510] Further, in some instances, the heterologous plant-modifying agent (e.g., an agent including a nucleic acid molecule or peptide) can be modified. For example, the modification can be a chemical modification, e.g., conjugation to a marker, e.g., fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation or operational linkage to 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), a cation moiety.Polypeptides

[0511] The modified lipid composition may include a polypeptide. In some instances, the modified lipid composition includes a polypeptide or functional fragments or derivative thereof.

[0512] Examples of polypeptides include an enzyme (e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, a DNAse, or an ubiquitination protein), a pore-forming protein, a signaling ligand, a cell penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene editing protein (e.g., CRISPR-Cas system, TALEN, or zinc finger), riboprotein, a protein aptamer, or a chaperone.

[0513] Polypeptides may include naturally occurring polypeptides or recombinantly produced variants. In some instances, the polypeptide may be a functional fragments or variants 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 with 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, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide. In some instances, the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identity to a protein of interest.

[0514] The polypeptides may be formulated in a modified lipid composition. The compositionsdisclosed herein may include any number or type (e.g., classes) of polypeptides, such as at least about any one of 1 polypeptide, 2, 3, 4, 5, 10, 15, 20, or more polypeptides. A suitable concentration of each polypeptide in the composition depends on factors such as efficacy, stability of the polypeptide, number of distinct polypeptides in the composition, the formulation, and methods of application of the composition. In some instances, each polypeptide in a liquid composition is from about 0.1 ng / mL to about 100 mg / mL. In some instances, each polypeptide in a solid composition is from about 0.1 ng / g to about 100 mg / g.

[0515] Methods of making a polypeptide are routine in the art. See, in general, 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] Methods for producing a polypeptide involve expression in plant cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, mammalian cells, or other cells under the control of appropriate promoters. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5’ or 3’ flanking nontranscribed sequences, and 5’ or 3’ nontranslated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with natural, fungal, yeast, and mammalian cellular 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 employed to express and manufacture a recombinant polypeptide agent. Examples of mammalian expression systems include CHO cells, COS cells, HeLA and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in, e.g., Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Purification of proteins 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). Formulation of protein therapeutics 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 instances, the modified lipid composition includes an antibody or antigen binding fragment thereof. For example, an agent described herein may be an antibody that blocks or potentiates activity and / or function of a component. The antibody may act as an antagonist or agonist of a polypeptide (e.g., enzyme or cell receptor). The making and use of antibodies against a target antigen is known in the art. See, for example, Zhiqiang An (Ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1st Edition, Wiley, 2009 and also Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 2013, for methods of making recombinantantibodies, including antibody engineering, use of degenerate oligonucleotides, 5’-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques.Nucleic acids

[0519] In some instances, the modified lipid composition includes a nucleic acid (a polynucleotide or a polyribonucleotide). Numerous nucleic acids are useful in the modified lipid composition and methods described herein. The modified lipid composition may include any number or type (e.g., classes) of heterologous nucleic acids (e.g., DNA molecule (e.g., plasmid) or RNA molecule, e.g., mRNA, circRNA, guide RNA (gRNA), or inhibitory RNA molecule or precursor thereof (e.g., siRNA, shRNA, or miRNA or a precursor of any of these), or a hybrid DNA-RNA molecule), such as at least about 1 class or variant of a nucleic acid, or 2, 3, 4, 5, 10, 15, 20, or more classes or variants of nucleic acids. A suitable concentration of each nucleic acid in the composition depends on factors such as efficacy, stability of the nucleic acid, number of distinct nucleic acids, the formulation, and methods of application of the composition. Examples of nucleic acids useful herein include a DNA molecule (e.g., a plasmid), an mRNA, a circRNA, an siRNA, a Dicer substrate small interfering RNA (dsiRNA), an antisense RNA, a short interfering RNA (siRNA) or siRNA precursor (e.g., one or more strands of RNA that hybridize inter- or intra-molecularly to form at least partially double-stranded RNA having at least about 20 contiguous base-pairs), a short hairpin (shRNA), a microRNA (miRNA) or miRNA precursor, an asymmetric interfering RNA (aiRNA), a peptide nucleic acid (PNA), a morpholino, a locked nucleic acid (LNA), a piwi-interacting RNA (piRNA), a ribozyme, a deoxyribozymes (DNAzyme), an aptamer (DNA, RNA), a circular RNA (circRNA), a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs.Nucleic Acids Encoding Peptides

[0520] In some instances, the modified lipid composition includes a nucleic acid encoding a polypeptide. Nucleic acids encoding a polypeptide may have a length from 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 5000 to about 6000 nts, about 6000 to about 7000 nts, about 7000 to about 8000 nts, about 8000 to about 9000 nts, about 9000 to about 10,000 nts, about 10,000 to about 15,000 nts, about 10,000 to about 20,000 nts, about 10,000 to about 25,000 nts, about 10,000 to about 30,000 nts, about 10,000 to about 40,000 nts, about 10,000 to about 45,000 nts, about 10,000 to about 50,000 nts, or any range there between.

[0521] The modified lipid composition may also include active variants of a nucleic acid sequence of interest. In some instances, the variant of the nucleic acids 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, e.g., over a specified region or over theentire sequence, to a sequence of a nucleic acid of interest. In some instances, the modified lipid composition includes an active polypeptide encoded by a nucleic acid variant. In some instances, 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, e.g., over a specified region or over the entire amino acid sequence, to a sequence of a polypeptide of interest or the naturally derived polypeptide sequence.

[0522] Certain methods for expressing a nucleic acid encoding a protein may involve expression in cells, including insect, yeast, plant, bacteria, or other cells under the control of appropriate promoters. Expression vectors may include nontranscribed elements, such as an origin of replication, a suitable promoter and enhancer, and other 5’ or 3’ flanking nontranscribed sequences, and 5’ or 3’ nontranslated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with natural, fungal, yeast, and mammalian cellular 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. A nucleic acid sequence coding for a desired gene can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, a gene of interest can be produced synthetically, rather than cloned.

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

[0525] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 basepairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is 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, it appears that individual elements can 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 drivinghigh levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1 a (EF-1a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as 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 may be an inducible promoter. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0528] The expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibioticresistance genes, such as neo and the like.

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

[0530] In some instances, an organism may be genetically modified to alter expression of one or more proteins. Expression of the one or more proteins may be modified for a specific time, e.g., development or differentiation state of the organism. In one instances, the invention includes a composition to alter expression of one or more proteins, e.g., proteins that affect activity, structure, or function. Expression of the one or more proteins may be restricted to a specific location(s) or widespread throughout the organism.mRNA

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

[0532] In some instances, the modified RNA agent of interest described herein has modified nucleosides or nucleotides. Such modifications are known and are described, e.g., in WO 2012 / 019168. Additional modifications are described, e.g., in WO 2015 / 038892; WO 2015 / 038892; WO 2015 / 089511 ; WO 2015 / 196130; WO 2015 / 196118 and WO 2015 / 196128 A2, which are herein incorporated by reference in their entirety.

[0533] In some instances, the modified RNA encoding a polypeptide of interest has one or more terminal modification, e.g., a 5’ cap structure and / or a poly-A tail (e.g., of between 100-200 nucleotides in length). The 5’ cap structure may be selected from the group consisting of CapO, Capl, 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 cases, the modified RNAs also contain a 5‘ UTR including at least one Kozak sequence, and a 3‘ UTR. Such modifications are known and are described, e.g., in WO 2012 / 135805 and WO 2013 / 052523, which are incorporated herein by reference in their entirety. Additional terminal modifications are described, e.g., in WO 2014 / 164253 and WO 2016 / 011306, WO 2012 / 045075, and WO 2014 / 093924, which are incorporated herein by reference in their entirety. Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNA) which may include at least one chemical modification are described in WO 2014 / 028429, which is incorporated herein by reference in its entirety.

[0534] In some instances, a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 ‘-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5’- / 3’- linkage may be intramolecular or intermolecular. Such modifications are described, e.g., in WO 2013 / 151736.

[0535] Methods of making and purifying modified RNAs are known and disclosed in the art. For example, modified RNAs are made using only in vitro transcription (IVT) enzymatic synthesis. Methods of making IVT polynucleotides are known in the art and are described in WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151671 , WO 2013 / 151672, WO 2013 / 151667 and WO 2013 / 151736. Methods of purification include purifying an RNA transcript including a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and / or a plurality of uracils or derivatives thereof (polyT / U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO 2014 / 152031); using ion (e.g., anion) exchange chromatography that allows for separation of longer RNAs up to 10,000 nucleotidesin length via a scalable method (WO 2014 / 144767); and subjecting a modified mRNA sample to DNAse treatment (WO 2014 / 152030).

[0536] Formulations of modified RNAs are known and are described, e.g., in WO 2013 / 090648. For example, the formulation may be, but is not limited to, nanoparticles, poly(lactic-co-glycolic acid)(PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles and combinations thereof.

[0537] Modified RNAs encoding polypeptides in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151736; Tables 6 and 7 International Publication No. WO 2013 / 151672; Tables 6, 178 and 179 of International Publication No. WO 2013 / 151671 ; Tables 6, 185 and 186 of International Publication No WO 2013 / 151667. Any of the foregoing may be synthesized as an IVT polynucleotide, chimeric polynucleotide or a circular polynucleotide, and each may include one or more modified nucleotides or terminal modifications.Circular Polyribonucleotides

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

[0539] The polyribonucleotide may include an IRES (internal ribosome entry site) operably linked to an expression sequence encoding a polypeptide. The circular polyribonucleotide may include a splice junction, e.g., 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 as disclosed in International Patent Publication Nos. WO2019 / 118919, WO 2020 / 023655, WO 2020 / 180751 , WO 2020 / 180752, WO 2020 / 181013, WO 2020 / 198403, WO 2020 / 257730, WO 2020 / 257727, WO 2020 / 252436, each of which is hereby incorporated by reference in its entirety.

[0540] In some embodiments, the circular polynucleotide further includes a polyribonucleotide cargo. In some embodiments, the polyribonucleotide cargo includes an expression (or coding) sequence, a non-coding sequence, or a combination of an expression (coding) sequence and a non-coding sequence. In some embodiments, the polyribonucleotide cargo includes an expression (coding) sequence encoding a polypeptide. In some embodiments, the polyribonucleotide includes an IRES operably linked to an expression sequence encoding a polypeptide. In some embodiments, the IRES is located upstream of the expression sequence. In some embodiments, the IRES is located downstream of the expression sequence. In some embodiments, the circular polyribonucleotide further includes a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment. The spacer region may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length ribonucleotides in length. The spacer region may be, e.g., from 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 includes a polyA sequence. In some embodiments, the spacerregion includes a polyA-C sequence. In some embodiments, the spacer region includes a polyA-G sequence. In some embodiments, the spacer region includes a polyA-T sequence. In some embodiments, the spacer region includes a random sequence. In some embodiments, the first annealing region and the second annealing region are joined, thereby forming a circular polyribonucleotide.

[0541] In some embodiments, the circular RNA is a produced by a deoxyribonucleotide template or a linear RNA described herein. In some embodiments, the circular RNA is produced by any of the methods described herein.

[0542] In some embodiments, the circular polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1 ,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0543] In some embodiments, the circular polyribonucleotide is between 500 nucleotides and 20,000 nucleotides, between 1 ,000 and 20,000 nucleotides, between 2,000 and 20,000 nucleotides, or between 5,000 and 20,000 nucleotides. In some embodiments, the circular polyribonucleotide is between 500 nucleotides and 10,000 nucleotides, between 1 ,000 and 10,000 nucleotides, between 2,000 and 10,000 nucleotides, or between 5,000 and 10,000 nucleotides.

[0544] As a result of its circularization, the circular polyribonucleotide may include certain characteristics that distinguish it from linear RNA. For example, the circular polyribonucleotide may be less susceptible to degradation by exonuclease as compared to linear RNA. As such, the circular polyribonucleotide can be more stable than a linear RNA, especially when incubated in the presence of an exonuclease. The increased stability of the circular polyribonucleotide compared with linear RNA makes circular polyribonucleotide more useful as a cell transforming reagent to produce polypeptides and can be stored more easily and for longer than linear RNA. The stability of the circular polyribonucleotide treated with exonuclease can be tested using methods standard in art which determine whether RNA degradation has occurred (e.g., by gel electrophoresis). Moreover, unlike linear RNA, the circular polyribonucleotide can be less susceptible to dephosphorylation when the circular polyribonucleotide is incubated with phosphatase, such as calf intestine phosphatase.

[0545] The circular polyribonucleotides described herein and compositions or pharmaceutical compositions thereof may be used in therapeutic and veterinary methods of dosing to produce a level of circular polyribonucleotide, a level of binding to a target, or a level of protein in a plurality of cells after providing the plurality with at least two doses of circular polyribonucleotide. In some embodiments, the circular polyribonucleotide is non-immunogenic in a mammal, e.g., a human. In some embodiments, the circular polyribonucleotide is capable of replicating or replicates in a cell froman aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non- tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, the disclosure includes a cell that includes the circular polyribonucleotide described herein, in which the cell is a cell from an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (horses, cows, pigs, chickens etc.), a human cell, a cultured cell, a primary cell or a cell line, a stem cell, a progenitor cell, a differentiated cell, a germ cell, a cancer cell (e.g., tumorigenic, metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, or any combination thereof. In some embodiments, the cell is modified to include the circular polyribonucleotide.

[0546] In some embodiments, the circular polyribonucleotide includes sequences for expression products. In some embodiments, the circular polyribonucleotide includes a binding site for binding to a target. In some embodiments, the circular polyribonucleotide is provided to a plurality of cells via any a dosing regimen described herein. In some embodiments, the circular polyribonucleotide as described herein induces a response or response level in a subject. In some embodiments, the expression products encoded by the sequences included in the circular polyribonucleotide are expressed in one or more of cells in the plurality of cells.

[0547] In some embodiments, the circular polyribonucleotide has a half-life of at least that of a linear counterpart, e.g., linear expression sequence, or linear polyribonucleotide. In some embodiments, the circular polyribonucleotide has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell for from about 1 hour to about 60 days, e.g., about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the circular polyribonucleotide has a half-life or persistence in a cell post division. In certain embodiments, the circular polyribonucleotide has a half-life or persistence in a dividing cell for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain embodiments, the circularpolyribonucleotide has a half-life or persistence in a dividing cell of from about 10 minutes to about 60 days, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days.

[0548] In some embodiments, the circular polyribonucleotide modulates a cellular function, e.g., transiently, or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 10 minutes, e.g., at least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for from about 1 hour to about 60 days, e.g., from about 1 hour to about 30 days, e.g., for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 60 days.Inhibitory RNA

[0549] In some instances, the modified lipid composition includes an inhibitory RNA molecule, e.g., that acts via the RNA interference (RNAi) pathway. In some instances, the inhibitory RNA molecule decreases the level of gene expression and / or decreases the level of a protein. In some instances, the inhibitory RNA molecule inhibits expression of a gene. For example, an inhibitory RNA molecule may include a short interfering RNA or its precursor, short hairpin RNA, and / or a microRNA or its precursor that targets a gene. Certain RNA molecules can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules include RNA or RNA-like structures typically containing 15-50 base pairs (such as about 18-25 base pairs) and having a nucleobase sequence identical (or complementary) or nearly identical (or substantially complementary) to a coding sequence in an expressed target gene within the cell. RNAi molecules include, but are not limited to: short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), short hairpin RNAs (shRNA), meroduplexes, dicer substrates, and multivalent RNA interference (U.S. Pat. Nos.8,084,599 8,349,809, 8,513,207 and 9,200,276). A shRNA is a RNA molecule including a hairpin turn that decreases expression of target genes via RNAi. shRNAs can be delivered to cells in the form of plasmids, e.g., viral or natural vectors, e.g., by transfection, electroporation, or transduction). A microRNA is a non-coding RNA molecule that typically has a length of about 21 or 22 nucleotides. MiRNAs bind to target sites on mRNA molecules and silence the mRNA, e.g., by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. In some instances, the inhibitory RNA molecule decreases the level and / or activity of a negative regulator of function. In other instances, the inhibitor RNA molecule decreases the level and / or activity of an inhibitor of a positive regulator of function. The inhibitory RNA molecule can be chemically synthesized ortranscribed in vitro.

[0550] In some instances, the nucleic acid is a DNA, a RNA, or a PNA. In some instances, the RNA is an inhibitory RNA. In some instances, the inhibitory RNA inhibits gene expression. In some instances, the nucleic acid is an mRNA, a modified mRNA, or a DNA molecule that increases expression of an enzyme (e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, a DNAse, or an ubiquitination protein), a pore-forming protein, a signaling ligand, a cell penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene editing protein (e.g., CRISPR-Cas system, TALEN, or zinc finger), riboprotein, a protein aptamer, or a chaperone. In some instances, the nucleic acid is an mRNA, a modified mRNA, or a DNA molecule that increases the expression of an enzyme (e.g., a metabolic enzyme, a recombinase enzyme, a helicase enzyme, an integrase enzyme, a RNAse enzyme, a DNAse enzyme, or an ubiquitination protein), a pore-forming protein, a signaling ligand, a cell penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene editing protein (e.g., a CRISPR-Cas system, a TALEN, or a zinc finger), a riboprotein, a protein aptamer, or a chaperone. In some aspects, the nucleic acid encodes the enzyme, pore-forming protein, signaling ligand, cell penetrating peptide, transcription factor, receptor, antibody, nanobody, gene editing protein, riboprotein, protein aptamer, or chaperone. In some instances, the increase in expression is an increase in expression of about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to a reference level (e.g., the expression in an untreated subject). In some instances, the increase in expression is an increase in expression of about 2x fold, about 4x fold, about 5x fold, about 10x fold, about 20x fold, about 25x fold, about 50x fold, about 75x fold, or about 100x fold or more, relative to a reference level (e.g., the expression in an untreated subject).

[0551] In some instances, the nucleic acid is an antisense RNA, a dsiRNA, a siRNA, a shRNA, a miRNA, an aiRNA, a PNA, a morpholino, a LNA, a piRNA, a ribozyme, a DNAzyme, an aptamer (DNA, RNA), a circRNA, a gRNA, or a DNA molecules (e.g., a plasmid) that acts to reduce expression of, e.g., an enzyme (a metabolic enzyme, a recombinase enzyme, a helicase enzyme, an integrase enzyme, a RNAse enzyme, a DNAse enzyme, a polymerase enzyme, a ubiquitination protein, a superoxide management enzyme, or an energy production enzyme), a transcription factor, a secretory protein, a structural factor (actin, kinesin, or tubulin), a riboprotein, a protein aptamer, a chaperone, a receptor, a signaling ligand, or a transporter. In some instances, the decrease in expression is a decrease in expression of about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to a reference level (e.g., the expression in an untreated subject). In some instances, the decrease in expression is a decrease in expression of about 2x fold, about 4x fold, about 5x fold, about 10x fold, about 20x fold, about 25x fold, about 50x fold, about 75x fold, or about 100x fold or more, relative to a reference level (e.g., the expression in an untreated subject).

[0552] RNAi molecules include a sequence substantially complementary, or fully complementary, to all or a fragment of a target gene. RNAi molecules may complement sequences at the boundary between introns and exons to prevent the maturation of newly generated nuclear RNA transcripts of specific genes into mRNA for transcription. RNAi molecules complementary to specific genes canhybridize with the mRNA for a target gene and prevent its translation. The antisense molecule can be DNA, RNA, or a derivative or hybrid thereof. Examples of such derivative molecules include, but are not limited to, peptide nucleic acid (PNA) and phosphorothioate-based molecules such as deoxyribonucleic guanidine (DNG) or ribonucleic guanidine (RNG).

[0553] RNAi molecules can be provided as ready-to-use RNA synthesized in vitro or as sense and antisense RNA sequences (or DNA encoding sense and antisense RNA sequences) transfected into cells which will yield RNAi molecules upon transcription. Hybridization of the RNA molecule with, e.g., the target mRNA results in degradation of the hybridized complex by RNAse H and / or inhibition of the formation of translation complexes. Both result in a failure to produce the product of the original gene.

[0554] The length of the RNAi molecule that hybridizes to the transcript of interest may be around 10 nucleotides, between about 15 or 30 nucleotides, or about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In embodiments, the RNAi molecule hybridizes to the transcript of interest to form a perfectly or near-perfectly double-stranded region of at least about 17 base pairs; in embodiments the double-stranded region includes at least about 10 contiguous base pairs. The degree of identity of the antisense sequence to the targeted transcript may be at least 75%, at least 80%, at least 85%, at least 90...

Claims

What is claimed is:1 . A modified lipid composition, comprising:(a) a structural component comprising one or more lipids selected from the group consisting of a soy-derived lipid, cardiolipin, sphingolipid, ceramide, glucosyl ceramide, lactosyl ceramide, galactosyl cholesterol, and glucosyl cholesterol; modified by(b) an ionizable lipid; wherein the one or more lipids increase the delivery of the modified lipid composition to spleen, characterized by an increased spleen-to-liver delivery ratio.

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

3. The modified lipid composition of claim 1 , wherein the structural component comprises a sphingolipid and the spingolipid is sphingomyelin lipid derived from animal brain.

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

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

6. 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 modified lipid composition of claim 5 or 6, wherein the soy PL mixture comprises about 30% to about 50% of PC, about 20% to about 40% of PE, about 10% to about 25% of PI, about 1 % to about 15% of PA, about 1 % to about 15% of LPC, and about 0% to about 10% of other lipids.

8. The modified lipid composition of claim 7, wherein the soy PL mixture comprises PC:PE:PI:PA:LPC:other lipids at a percentage ratio of about 38:30:18:7:7:0.

9. The modified lipid composition of claim 5 or 6, wherein the soy polar lipid composition comprise about 40% to about 50% of PC,about 15% to about 30% of PE, about 10% to about 25% of PI, about 1 % to about 15% of PA, about 0% to about 10% of LPC, and about 0% to about 15% of other lipids.

10. The modified lipid composition of claim 9, wherein the soy polar lipid composition comprises PC:PE:PI:PA:LPC:other lipids at a percentage ratio of about 46:22:18:7:0:7.11 . The modified lipid composition of claim 4, wherein the soy-derived lipid increases the delivery of the modified lipid composition to spleen, characterized by an increased spleen-to-liver delivery ratio.

12. The modified lipid composition of claim 1 or 11 , wherein the spleen-to-liver delivery ratio is 1 .0 or greater.

13. The modified lipid composition of claim 1 or 11 , wherein an increase in the amount of the structural lipid component present in the modified lipid composition increases the spleen-to-liver delivery ratio.

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

15. The modified lipid composition of claim 1 or 4, wherein the structural component comprises purified soy-derived lipids, and the structural component is modified by reconstructing the purified soy-derived lipids with the ionizable lipid.

16. The modified lipid composition of claim 1 or 4, wherein the ionizable lipid has one or more characteristics selected from the group consisting of:(i) at least 2 ionizable amines;(ii) at least 3 lipid tails, wherein each of the lipid tails 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) an N:P ratio of at least 3.

17. 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 modified lipid composition of claim 1 or 4, wherein the ionizable lipid is represented by a compound, wherein each R is independently aC8-C14 alkyl group.

19. The modified lipid composition of claim 1 or 4, wherein the ionizable lipid is selected from one of the following groups of compounds: i) a compound of formulapharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: each A is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each B is independently C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each X is independently a biodegradable moiety; andRs is OH, SH, or NR10R11; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR10R11, wherein each R10 and Rn is independently H, C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic 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 NR12, wherein R12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, andQ is O, S, or NR13 ii) a compound of formulapharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:cyclic or heterocyclic moiety;Y is alkyl, hydroxy, hydroxyalkyl orA is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -S-S-, or a bivalent heterocycle; each of X and Z is independently absent, -O-, -CO-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(0)-, -C(0)N(R7)-, or -S-; each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; each of R30, R40, R50, Reo, R70, Rso, R90, R100, R110, and R120 is independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of I and m is an integer from 1 to 10; t1 is an integer from 0 to 10; andW is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocylyl or heteroaryl; iii) a compound of formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein:R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, or R20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra;Rais H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, or SH; each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, orR1 and R2 are taken together to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R10 and Rn are taken together to form a heterocyclic ring; n is 0, 1 , 2, 3 or 4;Y is O or S;Z is absent, O, S, or N(Ri2>, wherein each R12 is independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH; v is 0, 1 , 2, 3, or 4; y is 0, 1 , 2, 3, or 4; each A is each independently C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, optionally interrupted with one or more heteroatoms or optionally substituted with OH, SH, or halogen; each B is each independently C1-C16 branched or unbranched alkyl, or C2-C16 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or optionally substituted with 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 (Tl) or (Tl’)pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:E is each independently -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -C(O-RI3)-O-, -C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, -S-S-, or -C(O-Ri3)-O-(CH2)r-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl;R13 is branched or unbranched C3-C10 alkyl; r is 1 , 2, 3, 4, or 5;Rais each independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; u1 and u2 are each independently 0, 1 , 2, 3, 4, 5, 6, or 7;R‘ is each independently H, C1-C16 branched or unbranched alkyl or C1-C16 branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; andS represents the bond connecting the tail group to the head group; and wherein the lipid has a pKa from about 4 to about 8.

20. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of group i), represented by a formula(IX), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: each Ri and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10R11, or each Ri and each R2 are independently taken together with the carbon atom(s) to which they are attached to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, or R10 and Rn are taken together to form a heterocyclic ring; each R3 and each R4 is independently H, C2-C1 branched or unbranched alkyl (e.g., C3-C10 branched or unbranched alkyl), or C3-C10 branched or unbranched alkenyl, provided that at least one of Rs 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-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R? and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein each v is independently 0, 1 , 2, 3, 4, or 5, and R17 is OH, SH, or N(CHs)2; and each m is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.21 . The modified lipid composition of claim 20, wherein V is a branched or unbranched C2-C3 alkylene, and each Re is independently H or methyl.

22. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of groupi), represented by a formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: each Ri and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10R11, or each R1 and each R2 are independently taken together with the carbon atom(s) to which they are attached to form a cyclic ring; each R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, or R10 and Rn are taken together to form a heterocyclic ring; each R3 and each R4 is independently H, C2-C1 branched or unbranched alkyl (e.g., C3-C10 branched or unbranched alkyl), or C3-C10 branched or unbranched alkenyl, provided that at least one of Rs 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 heterocyclic optionally substituted with one or more -(CH2)VOH, -(CH2)VSH, -(CH2)v-halogen groups, each R? and each Rs is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vRi7, or NR10R11, wherein R17 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.

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 20-23, wherein X is -OCO-, -COO-, -CONH-, or -NHCO-.

25. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of group ii), represented by one of the following formulas:wherein:A is absent, -0-, -N(R7)-, -O-alkylene-, -alkylene-O-, -0C(0)-, -C(0)0-,-N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, -S-S-, or a bivalent heterocycle; each R7is 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 heterocylyl or heteroaryl; each M is independently a biodegradable moiety; each ml is independently an integer from 3 to 6, each 11 is independently an integer from 4 to 8, m2 and I2 are each independently an integer from 0 to 3,Rao and R90 are each independently unsubstituted Cs-Ca alkyl or alkenyl; or Rao is H or unsubstituted C1-C4 alkyl or alkenyl, and R90 is unsubstituted C5-C11 alkyl or alkenyl; andR110 and R120 are each independently unsubstituted Cs-Ca alkyl or alkenyl; or Rno is H or unsubstituted C1-C4 alkyl or alkenyl, and R120 is unsubstituted C5-C11 alkyl or alkenyl.

26. The modified lipid composition of claim 25, wherein:each Rr;is independently H or C1-C3 alkyl; and each t1 is independently 1 , 2, 3, or 4.

27. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of group iii), wherein Ri and R2 are each H, or each R1 is H, and one of the R2 variables is OH; and X is -OC(O)- or -C(O)O-.

28. The modified lipid composition of claim 27, wherein the ionizable lipid is a compound of group iii), represented by formula III), wherein:R20 and R30 are each independently H or C1-C3 branched or unbranched alkyl; or R20 and R30 together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra;Rais H or OH;Z is absent, S, O, or NH; and n is 0, 1 , or 2.

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

30. The modified lipid composition of claim 19, wherein the ionizable lipid is a compound of group iv), comprising at least one head group and at least one tail group, wherein: the tail group has a structure of formula (Tl) (or Tl’); and the head group has a structure of one of the following formulas:wherein:R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, optionally interrupted with one or more heteroatoms or substituted with OH, SH, halogen, or cycloalkyl groups; orR20 and R30, together with the adjacent N atom, form a 3 to 7 membered heterocylic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; each of R1 and R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11 ; or R1 and R2 together form a cyclic ring; each of R10 and Rn is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl; or R10 and Rn together form a heterocyclic ring; n is 0, 1 , 2, 3 or 4; andZ is absent, O, S, or NR12, wherein R12 is H or C1-C7 branched or unbranched alkyl; provided that when Z is not absent, the adjacent R1 and R2 cannot be OH,wherein:R1 is H, C1-C3 alkyl, OH, halogen, SH, or NR10R11;R2 is OH, halogen, SH, or NR10R11; or R1 and R2 can be taken together to form a cyclic ring;R10 and R11 are each independently H or C1-C3 alkyl; or R10 and Rn can be taken together to form a heterocyclic ring;R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R20 and R30 can be taken together to form a cyclic ring; and each of v and y is independently 1 , 2, 3, or 4;whereinR5is OH, SH, (CH2)SOH, or NR10R11; each Re is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7 and Rs are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, (CH2)vOH, (CH2)vSH, (CH2)SN(CH3)2, or NR10R11, wherein each R10 and Rn is independently H or C1-C3 alkyl, or R10 and Rn are taken together to form a heterocyclic ring; or R7 and Rs are taken together to form a ring; each R20 is independently H, or C1-C3 branched or unbranched alkyl;Ri s a heterocyclic, NR10R11, C(0)NRioRn, NRioC(0)NRioRii, or NRioC(S)NRioRii, wherein each R10 and Rn is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R10 and Rn are taken together to form a heterocyclic ring;R16 is H, =0, =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 heterocyclic; each Z is independently absent, O, S, or NR12, wherein R12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl;Q is O, S, CH2, or NR13, wherein each R13 is H, or C1-C5 alkyl;V is branched or unbranched C2-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, or C2-C10 heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; andT is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic; ands cyclic or heterocyclic moiety;Y is alkyl, hydroxy, hydroxyalkyl,;A is absent, -O-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -N(R7)C(O)N(R7)-, -S-, or -S-S-; each of X and Z is independently absent, -O-, -C(O)-, -N(R7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, or -S-; each R7is 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; andW is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; and wherein the lipid has a pKa from about 4 to about 8.

31. The modified lipid composition of claim 30, wherein the ionizable lipid is a compound of group iv), and wherein at least one tail group of the lipid has one of the following formulas:R7is each independently H or methyl;Rbis in each occasion independently H or C1-C4 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 a structure of one of the following formulas:

32. The modified lipid composition of claim 31 , wherein at least one tail group has the structure of formula (Til), (Till), (TIV), (TV), (TH’), and / or (Till’), wherein u1 is 3-5, u2 is 0-3, u3 and u4 are each independently 1-7, and Rais each independently methyl.

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

34. The modified lipid composition of claim 33, wherein the ionizable lipid is35. 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 of claim 35, wherein the modified lipid composition comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 10 mol% to about 75 mol% of the structural component, about 0 mol% to about 45 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

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

40. The modified lipid composition of claim 38, wherein the modified lipid composition comprises ionizable lipid:structural lipid:sterol:PEG-lipid at 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 . The modified lipid composition of claim 35, wherein the structural lipid comprises structural lipid 1 and structural lipid 2, wherein structural lipid 1 and structural lipid 2 are different structural lipids.

42. The modified lipid composition of claim 41 , wherein the modified lipid composition comprises ionizable lipid :(structural lipid 1 +structural lipid 2):sterol:PEG-lipid at 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. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition is a lipophilic moiety selected from the group consisting of a lipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion.

44. The modified lipid composition of claim 1 or 4, wherein the modified lipid composition is a liposome selected from the group consisting of a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide-containing nanoliposome, and a multivesicular liposome.

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

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

47. The modified lipid composition of claim 45, wherein the lipid nanoparticle has a size of less than about 100 nm.

48. The modified lipid composition of claim 45, wherein the average polydispersity index (PDI) of the lipid nanoparticle ranges from about 0.1 to about 0.4.

49. The modified lipid composition of claim 45, wherein the average PDI of the lipid nanoparticle ranges from 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 . The modified lipid composition of claim 50, wherein the heterologous functional agent is encapsulated by, embedded on the surface of, or conjugated to the surface of the modified lipid composition.

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

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

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

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. 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. A method for delivering one or more heterologous functional agents to a cell or a subject, the method comprising contacting the cell with or administering to the subject the one or more heterologous functional agents and the modified lipid composition of any one of claims 1-56, wherein the heterologous functional agent is encapsulated by, embedded on the surface of, or conjugated to the surface of the modified lipid composition,58. The method of claim 57, wherein the one or more heterologous functional agents is delivered to splenic cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or myeloid cells.

59. The method of claim 57, wherein the modified lipid composition increases the transfection of the splenic cells, immune cells, lymphoid cells, hematopoietic stem cells, and / or myeloid cells.

60. The method of claim 59, wherein the splenic lymphoid cells are transfected at a frequency of at least 1 %, at least 2%, or at least 5% of the parent population.61 . The method of claim 59, wherein the splenic myeloid cells are transfected at a frequency of at least 5%, at least 10%, or at least 15% of the parent population.

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

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

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