Delivery of polynucleotides from lipid nanoparticles comprising RNA and ionizable lipids

Abstract

Provided herein is a method for delivering an RNA composition to a subject for in vivo production of a protein or a peptide in the subject, comprising administering to the subject an RNA composition comprising a polynucleotide that encodes a protein or a peptide, formulated within (a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid, or (b) a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid. Also provided herein are methods for treating diseases or disorders associated with the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph node, pancreas, colon or rectum, caecum, and / or spleen, comprising orally or enterally administering the RNA composition described herein.

Description

Delivery of Polynucleotides CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No.63 / 460,586, filed April 19, 2023; U.S. Provisional Application No.63 / 502,522, filed May 16, 2023; and U.S. Provisional Application No.63 / 547,630, filed November 7, 2023, all of which are herein incorporated by reference in their entirety. BACKGROUND

[0002] The use of polynucleotides as therapeutics is a new and emerging field. A need therefore exists for developing an oral and enteral polynucleotide delivery system for a more effective, easily scalable, and stable delivery of RNA therapeutics that will help with patient’s adherence to the treatment. Delivery methods targeting the gastrointestinal tract and surrounding immune systems are also needed. SUMMARY OF THE INVENTION

[0003] In one aspect, provided herein is a method for delivering an RNA composition to a subject for in vivo production of a protein or a peptide in the subject. The method comprises administering to the subject (e.g., by oral, enteral or intravenous delivery) an RNA composition comprising one or more polynucleotides (e.g., an mRNA or circRNA) that encodes a protein or a peptide that is formulated within (a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid or within (b) a complex lipid particle (CLP), such as a lipid reconstructed natural messenger packs (LNMPs), comprising natural lipids and an ionizable lipid.

[0004] The ionizable lipid has two or more of the characteristics listed below: (i) at least one ionizable amine; (ii) at least three lipid tails, wherein each of the lipid tails is at least six 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.

[0005] In some embodiments, the RNA composition is administered orally or enterally. In some embodiments, the RNA composition is administered systemically (e.g., intravenously).

[0006] In another aspect, provided herein is a method for delivering an RNA composition to the lymphatic transport system and bypassing the liver. The method comprises administering to the subject the RNA composition comprising one or more polynucleotides (e.g., an mRNA or circRNA) encoding one or more polypeptides formulated within a LNP comprising synthetic structural lipids and an ionizable lipid, or a CLP, such as a LNMP, comprising natural lipids and an ionizable lipid. The ionizable lipid has two or more of the characteristics listed below: (i) at least 1 ionizable amine; (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.

[0007] In another aspect, provided herein is a method of treating diseases or disorders associated with the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph node, pancreas, colon or rectum, caecum, and / or spleen. The method comprises orally administering an RNA composition having one or more polynucleotides encoding one or more polypeptides. The one or more polynucleotides are formulated within a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid, or a CLP such as lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid. The ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable amine;(ii) at least three lipid tails, wherein each of the lipid tails is at least six 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.

[0008] In some embodiments, the disease or disorder is pancreatitis, inflammatory bowel disease (IBD), Crohn’s disease, colorectal cancer, or ulcerative colitis.

[0009] In another aspect of the invention, provided herein is a method for in vivo delivery of an antibody to a subject, comprising: administering systemically to a subject an RNA composition comprising one or more polynucleotides (e.g., an mRNA or circRNA) encoding an antibody, formulated within a complex lipid particle (CLP), such as a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid, wherein 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.

[0010] In an alternative aspect of the invention, provided herein is a method for in vivo delivery of an antibody to a subject, comprising: administering systemically to a subject an RNA composition comprising one or more polynucleotides (e.g., an mRNA or circRNA) encoding an antibody, formulated within a plurality of lipid nanoparticles (LNPs) comprising synthetic structural lipids and an ionizable lipid, wherein 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.In another aspect, provided herein is an oral vaccination composition (e.g., for viral or bacterial infection or for anti-tumor), comprising an RNA composition having one or more polynucleotides (e.g., an mRNA or circRNA) encoding one or more polypeptides formulated within (a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and comprising an ionizable lipid, or(b) a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid. The ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable functional group amine;(ii) at least three lipid tails, wherein each of the lipid tails is at least six carbon atoms in length;(iii) a pKa of about 4.5 to about 7.5;(iv) an ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms; and(v) an N:P ratio of at least 3, wherein the mRNA composition is formulated in an oral dosage form. In some embodiments, the polypeptide is an antigenic polypeptide derived from an infectious agent that causes a viral infection or bacterial infection. In some embodiments, the antigenic polypeptide is a corona virus.

[0011] All the embodiments discussed below are applicable to all the above aspects.

[0012] In some embodiments, the polynucleotide is an mRNA or circRNA. In some embodiments, the mRNA or circRNA is derived from (a) a DNA molecule; or (b) an RNA molecule, wherein T is substituted with U.

[0013] In some embodiment, the polynucleotide encodes a protein, peptide, or polypeptide comprising an antibody. In some embodiments, the antibody is a therapeutic. In one embodiment, the antibody is TNF inhibitor or PCSK9 inhibitor. In one embodiment, the RNA composition comprising one or more polynucleotides (e.g., an mRNA or circRNA) encoding the antibody is administered at least one time, optionally two or more times.

[0014] In some embodiments, the in vivo production of the protein or the peptide (or polypeptide) occurs in the subject’s stomach, small intestine, mesenteric lymph node, pancreas, colon, caecum, and / or spleen. In some embodiments, administering the RNA composition results in expression of the protein or the peptide (or polypeptide) detectable in one or more organs in the subject at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In some embodiments, the organs are present along the transit route of the digestive track, and the RNA composition's access to the organs occurs through the lymphatic transport system.

[0015] In some embodiments, the protein or the peptide (or polypeptide) encoded by the polynucleotide (e.g., polyribonucleotide, mRNA, or circRNA) is detectable in the subject’s mesentericlymph node, pancreas, stomach, colon, spleen, and / or small intestine (e.g., villi, Peyer’s patches) at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In some embodiments, the protein or the peptide (or polypeptide) encoded by the polynucleotide is not detectable in the subject’s liver at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

[0016] In some embodiments, the RNA composition is formulated within a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and the ionizable lipid.

[0017] In some embodiments, the RNA composition is formulated within a LNMP comprising natural lipids and the ionizable lipid.

[0018] In some embodiments, the RNA composition is formulated within a CLP. In some embodiments, the CLP is LNMP. Thus, all the embodiments below describing the features relating to LNMP and LNMP formulation are applicable to CLP and CLP formulation.

[0019] In some embodiments, the polynucleotide is encapsulated by the LNPs or LNMPs. In some embodiments, the polynucleotide is embedded on the surface of the LNPs or LNMPs. In some embodiments, the polynucleotide is conjugated to the surface of the LNPs or LNMPs.

[0020] In some embodiments, the RNA composition is administered to the subject in a delayed- release pharmaceutical dosage form comprising (a) a therapeutically effective amount of a polynucleotide (e.g., a mRNA or circRNA); (b) a bile salt or bile acid; and (c) at least one surfactant selected from hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.

[0021] In some embodiments, the RNA composition is administered in the form of a capsule (e.g., a starch capsule, a cellulosic capsule, a hard gelatin capsule, or a soft gelatin capsule). In some embodiments, the RNA composition is administered in the form of a tablet or caplet. In some embodiments, the capsule, tablet, or caplet contains an enteric coating. In some embodiments, the RNA composition is administered in the form of a plurality of particles, granules, beads, pellets, or mixtures thereof.

[0022] In some embodiments, the RNA composition comprises: one or more polynucleotides (e.g., an mRNA or circRNA) encoding one or more polypeptides, formulated within a CLP, such as a lipid reconstructed natural messenger pack (LNMP) comprising natural lipids and an ionizable lipid, wherein the ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable amine functional groups;(ii) at least three lipid tails, wherein each of the lipid tails is at least six carbon atoms in length;(iii) a pKa of about 4.5 to about 7.5;(iv) an ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms; and(v) an N:P ratio of at least 3.

[0023] In some embodiments, the ionizable lipid may be 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), or ALC-315. In one embodiment, the ionizable lipid included isC12-200.

[0024] In some embodiments, the ionizable lipid included is, wherein R is C8-C14 alkyl group.

[0025] In all these aspects, 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-C16branched or unbranched alkyl or C1-C16branched or unbranched alkenyl, optionally substituted with heteroatom or substituted with OH, SH, or halogen; each X is independently a biodegradable moiety; and, R5is OH, SH, or NR10R11; each R6is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; each R7and each R8is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR10R11, wherein each R10and R11is independently H, C1-C3 alkyl, or R10and R11are taken together to form a heterocyclic ring; R7and R8are 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 R12is H, C1-C7 branched orunbranched alkyl, or C2-C7 branched or unbranched alkenyl, and Q is O, S, or NR13, wherein each R13is H, or C1-C5alkyl; 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(O)-, -C(O)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, R60, R70, R80, 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 l and m is an integer from 1 to 10; t1 is an integer from 0 to 10; and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl; and iii) a compound of formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: R20and R30are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, or R20and R30together 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 R1and each R2is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, or R1and R2are taken together to form a cyclic ring; each R10and R11is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R10and R11are 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(R12), wherein each R12is independently H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, the adjacent R1and R2cannot 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 (TI)(TI’), a 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-R13)-O-, - C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, -S-S-, or -C(O-R13)-O-(CH2)r-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R13is branched or unbranched C3-C10alkyl; 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; Rtis each independently H, C1-C16branched or unbranched alkyl or C1-C16branched 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.

[0026] In some embodiments, the ionizable lipid is a compound of group i), represented by a formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: each R1and each R2is independently H, C1-C3branched 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 R10and R11is independently H, C1-C3branched or unbranched alkyl, or R10and R11are taken together to form a heterocyclic ring; each R3 and each R4 is 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 R3and 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 R6is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, or cycloalkyl; each R7and each R8is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vR17, or NR10R11, wherein each v is independently 0, 1, 2, 3, 4, or 5, and R17is OH, SH, or N(CH3)2; and each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, V is a branched or unbranched C2-C3 alkylene, and each R6is independently H or methyl.

[0027] In some embodiments, the ionizable lipid is a compound of group i), represented by a formulapharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: each R1and each R2is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10R11, or each R1and each R2are independently taken together with the carbon atom(s) to which they are attached to form a cyclic ring; each R10and R11is independently H, C1-C3 branched or unbranched alkyl, or R10and R11are taken together to form a heterocyclic ring;each R3and each R4is independently H, C2-C14 branched or unbranched alkyl (e.g., C3-C10 branched or unbranched alkyl), or C3-C10branched or unbranched alkenyl, provided that at least one of R3and R4is 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 R7and each R8is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vR17, or NR10R11, wherein R17is 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. In some embodiments, T is a divalent piperazine or a divalent dioxopiperazine.

[0028] In some embodiments, in the above formulas for group i), X is -OCO-, -COO-, -NHCO-, or -CONH-.

[0029] In some embodiments, the ionizable lipid is a compound of group ii), represented by one of the following formulas:wherein: 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 heterocyclyl or heteroaryl; each M is independently a biodegradable moiety; each m1 is independently an integer from 3 to 6, each l1 is independently an integer from 4 to 8, m2 and l2 are each independently an integer from 0 to 3, R80 and R90 are each independently unsubstituted C5-C8 alkyl or alkenyl; or R80 is H or unsubstituted C1-C4alkyl or alkenyl, and R90is unsubstituted C5-C11alkyl or alkenyl; and R110and R120are each independently unsubstituted C5-C8alkyl or alkenyl; or R110is H orunsubstituted C1-C4 alkyl or alkenyl, and R120 is unsubstituted C5-C11 alkyl or alkenyl. In someeach Rcis independently H or C1-C3alkyl; and each t1 is independently 1, 2, 3, or 4.

[0030] In some embodiments, the ionizable lipid is a compound of group iii), wherein R1and R2are each H, or each R1is H, and one of the R2variables is OH; and X is –OC(O)- or –C(O)O-. In some embodiments, the ionizable lipid is a compound of group iii), represented by formula III, wherein R20and R30are each independently H or C1-C3branched or unbranched alkyl; or R20and R30together 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. In some embodiments, the ionizable lipid is a compound of group iii), represented by formula V.

[0031] In some embodiments, the ionizable lipid is a compound of group iv), wherein the lipid comprises at least one head group and at least one tail group, wherein: the tail group has a structure of formula (TI) or formula TI’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; or R20and R30, together with the adjacent N atom, form a 3 to 7 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups; each of R1and R2is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, OH, halogen, SH, or NR10R11; or R1and R2together form a cyclic ring; each of R10 and R11 is independently H, C1-C3 branched or unbranched alkyl, C2-C3branched or unbranched alkenyl; or R10and R11together form a heterocyclic ring; n is 0, 1, 2, 3 or 4; and 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 R1and R2cannot be OH,wherein: R1is H, C1-C3alkyl, OH, halogen, SH, or NR10R11; R2is OH, halogen, SH, or NR10R11; or R1and R2can be taken together to form a cyclic ring; R10 and R11 are each independently H or C1-C3 alkyl; or R10 and R11 can be taken together to form a heterocyclic ring; R20and R30are each independently H, C1-C5branched 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;R5is OH, SH, (CH2)sOH, or NR10R11; each R6is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, or cycloalkyl; each R7 and R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, halogen, (CH2)vOH, (CH2)vSH, (CH2)sN(CH3)2, or NR10R11, wherein each R10and R11is independently H or C1-C3alkyl, or R10 and R11 are taken together to form a heterocyclic ring; or R7 and R8 are taken together to form a ring; each R20is independently H, or C1-C3branched or unbranched alkyl; R14is a heterocyclic, NR10R11, C(O)NR10R11, NR10C(O)NR10R11, or NR10C(S)NR10R11, wherein each R10 and R11 is independently H, C1-C3 alkyl, C3-C7cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R10and R11are taken together to form a heterocyclic ring; R16is H, =O, =S, or CN; each of s, u, and t is independently 1, 2, 3, 4, or 5; each v is independently 0, 1, 2, 3, 4, or 5; each Y is a divalent heterocyclic; each Z is independently absent, O, S, or NR12, wherein R12is H, C1-C7branched 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-C10alkylene, C2-C10alkenylene, C2-C10alkynylene, or C2-C10heteroalkylene, optionally substituted with one or more OH, SH, and / or halogen groups; and T is –NHC(O)O-, –OC(O)NH-, or a divalent heterocyclic; and iv)wherein:cyclic or heterocyclic moiety;t1 WY is alkyl, hydroxy, hydroxyalkyl, , or; 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; and W 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.

[0032] In some embodiments, 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-C4alkyl; 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:

[0033] In some embodiments, at least one tail group has the structure of formula (TII), (TIII), (TIV), (TV), (TII’), and / or (TIII’), wherein u1 is 3-5, u2 is 0-3, u3 and u4 are each independently 1-7, and Rais each independently methyl.

[0034] In some embodiments, the tail group has the structure of formula (TII) or formula (TIII), wherein each Rais methyl; u1 is 3-5, u2 is 0-3; and u3 and u4 are each independently 1-4.

[0035] In some embodiments, the head group has the structure of one of the following formulasalkyl.each R6, R7, and R8are independently H or methyl; and each of u and t is independently 1, 2, or 3; orR14is a nitrogen-containing 5- or 6- membered heterocyclic, NR10R11, C(O)NR10R11, NR10C(O)NR10R11, or NR10C(S)NR10R11, wherein each R10 and R11 is independently H or C1-C3 alkyl; andeach of u and v is independently 1, 2, or 3; or, wherein: each R6is independently H or methyl; each u is independently 1, 2, or 3; and V is C2-C6 alkylene or C2-C6 alkenylene; oreach R6is independently H or methyl; each R7is independently H; each R8 is methyl; each u is independently 1, 2, or 3; and V is C2-C6alkylene or C2-C6alkenylene; oreach u is independently 1, 2, or 3; and T is a divalent nitrogen-containing 5- or 6- membered heterocyclic; orwherein: each u is independently 1, 2, or 3; Q is O; each Z is independently NR12;and R12is H or C1-C3alkyl; and iv)W is hydroxyl, substituted or unsubstituted hydroxyalkyl, one of the following moieties:wherein each Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -(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, thiolalkyl, heterocyclyl, heteroaryl; or two R8together with the nitrogen atom form a ring, optionally substituted with one or more alkyl, hydroxy,hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, or aminoalkyl; q is 0, 1, 2, 3, 4, or 5; and p is 0, 1, 2, 3, 4, or 5.

[0036] In some embodiments, the ionizable lipid is a compound in Table I, Table II, Table III, or Table IV.

[0037] In some embodiments, the ionizable lipid is Lipid No.2272 or 2243.

[0038] In the CLP or LNMP formulations or the lipid nanoparticle composition, 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).

[0039] In some embodiments, the polynucleotides are polynucleotide constructs, which encode one or more wild type or engineered antigens (or an antibody to an antigen). The antigen may be derived from a tumor, e.g., a tumor specific antigen, a tumor associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the polynucleotide construct encodes anti-TNF.

[0040] In some embodiments, the antigenic polypeptide encoded by the polynucleotide is a tumor antigenic polypeptide comprising p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP- 8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE- A (e.g., MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12), MAGE-B, MAGE-C, MART- 1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or a combination thereof.

[0041] In some embodiments, the antigenic polypeptide encoded by the polynucleotide is a tumor antigenic polypeptide comprising CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4- IBB, 5T4, AGS-5 , AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Foxp3, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, Integrin av, Integrin ανβ , LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, anti-PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1 , TRAILR2,VEGFR- 1 , VEGFR-2, VEGFR-3, and variants thereof.

[0042] In some embodiments, the tumor antigenic polypeptide is IL2 peptide, IL-2-Ra, Anti-CD19 antibody, anti-CD20 antibody, chimeric antigen receptor T cell (CAR-T) antibody, anti-HER2 antibody, etanercept (e.g., Enbrel), adalimumab (e.g., Humira), epoetin alfa (e.g., Epogen), filgrastim (e.g., Neupogen), pembrolizumab (e.g., Keytruda), rituximab (e.g., Rituxan), romiplostim (e.g., Nplate), sargramostim (e.g., Leukine), or a fragment or subunit thereof. In one embodiment, the tumorantigenic polypeptide is IL2 peptide, or a fragment or subunit thereof. In one embodiment, tumor antigenic polypeptide is epoetin alfa (e.g., Epogen), or a fragment or subunit thereof.

[0043] In some embodiments, the tumor antigenic polypeptide comprises a tumor antigen selected from the group consisting of a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, and a combination thereof. In one embodiment, the tumor antigenic polypeptide comprises a lung cancer antigen.

[0044] In some embodiments, the polynucleotide may be a mRNA, an siRNA or siRNA 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 circular RNA (circRNA), a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is an mRNA. In one embodiment, the polynucleotide is a circRNA.

[0045] In one embodiment, the polynucleotide is an mRNA which encodes a molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence provided in any one of Table 3.

[0046] In one embodiment, the polynucleotide is an mRNA which encodes an IL-2 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of an IL-2 molecule provided in any one of Tables V-VII and Table 3.

[0047] In some embodiments, the mRNA is (a) a DNA molecule, or (b) an RNA molecule. In the mRNA, T is optionally substituted with U.

[0048] In some embodiments, the mRNA is a DNA molecule. The DNA molecule can further comprise a promoter. In some embodiments, the promoter is a T7 promoter, a T3 promoter, or an SP6 promoter. In some embodiments, the promoter is located at the 5’ UTR.

[0049] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may be a self- replicating RNA molecule.

[0050] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may further comprise a 5’ cap. The 5’ cap can have a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, a Cap 3 structure, a Cap 0 structure, or any combination thereof.

[0051] In some embodiments, the polynucleotide is an mRNA which encodes an IL-2 molecule. In one embodiment, the IL-2 molecule comprises a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof. In one embodiment, the IL-2 molecule comprises a variant of a naturally occurring IL-2 molecule (e.g., an IL-2 variant, e.g., as described herein), or a fragment thereof.

[0052] In some embodiments, the mRNA comprises a 5' untranslated region (UTR) and / or a 3' UTR.

[0053] In some embodiments, the mRNA comprises a 5' UTR. The 5' UTR may comprise a Kozak sequence.

[0054] In some embodiments, the mRNA comprises a 3' UTR. In some embodiments, the 3’ UTR comprises one or more sequences derived from an amino-terminal enhancer of split (AES). In someembodiments, the 3’ UTR comprises a sequence derived from mitochondrially encoded 12S mRNA (mtRNRl).

[0055] In some embodiments, the mRNA comprises a poly(A) sequence. In one embodiment, the poly(A) sequence is a 110-nucleotide sequence consisting of a sequence of 30 adenosine residues, a 10-nucleotide linker sequence, and a sequence of 70 adenosine residues.

[0056] In some embodiments, the natural lipids of the LNMPs are extracted from a plant source, such as lemon or algae. In some embodiments, the natural lipids are extracted from lemon. In some embodiments, the natural lipids are extracted from a bacteria source, such as E. coli or Salmonella typhimurium.

[0057] In some embodiments, for the ionizable lipid component, the ionizable lipids from the compounds of formulas in groups i)-iv) can be used in combination with one or more other ionizable lipids. For instance, one or more other ionizable lipids can include 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 additional ionizable lipid included is C12-200.

[0058] In some embodiments, the LNMP is produced by a method comprising lipid extrusion. In some embodiments, the LNMP is produced by a method comprising processing a solution comprising a lipid extract of the PMPs in a microfluidics device comprising an aqueous phase, thereby producing the LNMPs. In some embodiments, the aqueous phase comprises the polynucleotides. In some embodiments, the reconstitution is performed in the presence of a sterol, thereby producing a LNMP that comprises natural lipids, an ionizable lipid, and a sterol. In some embodiments, the sterol is cholesterol or sitosterol.

[0059] In some embodiments, the reconstitution is performed in the presence of a PEGylated lipid (or a PEG-lipid conjugate), thereby producing a LNMP that comprises natural lipids, the ionizable lipid, and a PEG-lipid conjugate. In some embodiments, the LNMPs further comprise a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0060] In some embodiments, the LNP composition comprises one or more ionizable lipids, one or more synthetic structural lipids, a sterol, and one or more PEG-modified lipids.

[0061] In some embodiments, the LNMPs or the LNP composition further comprise a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0062] 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-DMG is PEG2000-DMG or PEG2000-PE.

[0063] In some embodiments, the synthetic structural lipid of the LNP composition is a phospholipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl- phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol- phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl- phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0064] In some embodiments, the LNP composition comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 5 mol% to about 60 mol% of the synthetic structural lipids, about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0065] In some embodiments, the LNP composition comprises: about 20 mol% to about 50 mol% of the ionizable lipid from Table I, Table II, Table III, or Table IV, about 5 mol% to about 60 mol% of the synthetic structural lipids, about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0066] In some embodiments, the LNP composition comprises ionizable lipid: synthetic structural lipids:sterol:PEG-lipid at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, 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.

[0067] In some embodiments, the LNMP comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 5 mol% to about 60 mol% of the natural lipids, and optionally a neutral lipid, about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0068] In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:50:12.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:20:42.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:30:32.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:16:46.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:25:37.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:40:22.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 45:10:43.5:1.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 35:16:46.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio ofabout 50:20:28.5:1.5. In one embodiment, the LNMPs comprise the ionizable lipid:natural lipids:sterol:PEG-lipid at a molar ratio of about 50:10:38.5:1.5.

[0069] In some embodiments, the LNMP may further comprise a neutral lipid as a helper lipid. In some embodiments, the natural lipids may be used in combination with a neutral lipid as a structural lipid component. The neutral lipid may be used in a molar ratio of neutral lipid:natural lipid of 10:1 to 1:10, or 3:1 to 1:3, e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. Non-limiting examples of neutral lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl- phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl- phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In one embodiment, the structural lipid component in the LNMP may comprise a natural lipid + DOPE, or a natural lipid + DSPC.

[0070] In these embodiments, the LNMP thus comprises the ionizable lipids described herein, a structural lipid comprising natural lipids and a neutral lipid, a sterol and / or a PEG-lipid.

[0071] In some embodiments, the LNMP comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 5 mol% to about 60 mol% of a structural lipid component (i.e., the natural lipids and the neutral lipid), about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0072] In one embodiment, the LNMPs comprise the ionizable lipid:(natural lipids+ neutral lipid):sterol:PEG-lipid at a molar ratio of about 35:50:12.5:2.5. In one embodiment, the LNMPs comprise the ionizable lipid:(natural lipids+ neutral lipid):sterol:PEG-lipid at a molar ratio of about 35:20:42.5:2.5. For instance, the LNMPs may comprise the ionizable lipid:(natural lipids+ neutral lipid):sterol:PEG-lipid at a molar ratio of about 35:(10+10):42.5:2.5. In one embodiment, the LNMPs comprise ionizable lipid: (natural lipids+ neutral lipid):sterol:PEG-lipid at a molar ratio of about 50:20:28.5:1.5. For instance, the LNMPs may comprise the ionizable lipid:(natural lipids+ neutral lipid):sterol:PEG-lipid at a molar ratio of about 50:(10+10):28.5:1.5.

[0073] In some embodiments, the LNMPs comprise: natural lipids extracted from lemon or algae, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV,cholesterol, and DMG-PEG.

[0074] In some embodiments, the LNMPs comprise: natural lipids extracted from lemon or algae, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMPE-PEG2k.

[0075] In one embodiment, the LNMPs comprise: natural lipids extracted from lemon, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMG-PEG or DMPE-PEG2k. The LNMPs may comprise the ionizable lipid:lemon lipids:cholesterol: DMPE-PEG2k at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (lemon lipids + neutral lipid) :cholesterol: DMPE-PEG2k at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid:lemon lipids:cholesterol: DMG-PEG at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (lemon lipids + neutral lipid) :cholesterol: DMG-PEG at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0076] In one embodiment, the LNMPs comprise: natural lipids extracted from algae, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMPE-PEG2k. The LNMPs may comprise the ionizable lipid:algae lipids:cholesterol: DMPE- PEG2k at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (algae lipids + neutral lipid) :cholesterol: DMPE-PEG2k at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0077] In one embodiment, the LNMPs comprise: natural lipids extracted from algae, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMG-PEG. The LNMPs may comprise the ionizable lipid:algae lipids:cholesterol: DMG-PEG at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (algae lipids + neutral lipid) :cholesterol: DMG-PEG at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0078] In some embodiments, the LNMPs comprise: natural lipids extracted from E. coli or Salmonella typhimurium, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, andDMPE-PEG2k.

[0079] In one embodiment, the LNMPs comprise: natural lipids extracted from E. coli, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMPE-PEG2k. The LNMPs may comprise the ionizable lipid: E. coli lipids:cholesterol: DMPE-PEG2k at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (E. coli lipids + neutral lipid) :cholesterol: DMPE-PEG2k at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0080] In one embodiment, the LNMPs comprise: natural lipids extracted from Salmonella typhimurium, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMPE-PEG2k. The LNMPs may comprise the ionizable lipid: Salmonella typhimurium lipids:cholesterol: DMPE-PEG2k at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (Salmonella typhimurium lipids + neutral lipid):cholesterol: DMPE-PEG2k at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0081] In some embodiments, the LNMPs comprise: natural lipids extracted from E. coli or Salmonella typhimurium, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMG-PEG.

[0082] In one embodiment, the LNMPs comprise: natural lipids extracted from E. coli, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMG-PEG. The LNMPs may comprise the ionizable lipid: E. coli lipids:cholesterol: DMG- PEG at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (E. coli lipids + neutral lipid) :cholesterol: DMG-PEG at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0083] In one embodiment, the LNMPs comprise: natural lipids extracted from Salmonella typhimurium, and optionally a neutral lipid, the ionizable lipid from Table I, Table II, Table III, or Table IV, cholesterol, and DMG-PEG. The LNMPs may comprise the ionizable lipid: Salmonella typhimurium lipids:cholesterol: DMG-PEG at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMPs may comprise the ionizable lipid: (Salmonella typhimurium lipids + neutral lipid):cholesterol: DMG-PEG at a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

[0084] In some embodiments, the LNMP 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 LNMP 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. In one embodiment, the LNMP is a lipid nanoparticle.

[0085] In some embodiments, the LNMP has a size of less than about 200 nm. In one embodiment, the LNMP has a size of less than about 150 nm. In one embodiment, the LNMP has a size of less than about 100 nm. In one embodiment, the LNMP has a size of about 80 nm to about 100 nm. In one embodiment, the LNMP has a size of about 55 nm to about 80 nm.

[0086] In some embodiments, the LNMP has an N:P ratio of at least 3, for instance, an N:P ratio of 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 6 to 30, 6 to 20, 6 to 15, or 6 to 12.

[0087] In some embodiments, the RNA composition has a total lipid:polynucleotide weight ratio of about 50:1 to about 10:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 44:1 to about 24:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 40:1 to about 28:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 38:1 to about 30:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 37:1 to about 33:1.

[0088] In some embodiments, the RNA 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 NaCl.

[0089] In some embodiments, the RNA 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.

[0090] In some embodiments, the aqueous phase and the lipid solution are mixed at a 3:1 volumetric ratio.

[0091] In some embodiments, the RNA composition further comprises one or more cryoprotectants. The one or more cryoprotectants may be sucrose, glycerol, or a combination thereof. In one embodiment, the RNA composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0092] In some embodiments, the RNA composition is a lyophilized composition. The lyophilized RNA composition may comprise one or more lyoprotectants. The lyophilized RNA composition may comprise a poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized RNA composition comprises a poloxamer, e.g., poloxamer 188.

[0093] In some embodiments, the RNA composition is a lyophilized composition. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1.0 % w / w of the polynucleotides. In one embodiment, the lyophilized RNA composition comprises about 1.0 to about 5.0 % w / w lipids. In one embodiment, the lyophilized RNA composition comprises about 0.5 to about2.5 % w / w of TRIS buffer. In one embodiment, the lyophilized RNA composition comprises about 0.75 to about 2.75 % w / w of NaCl. In one embodiment, the lyophilized RNA composition comprises about 85 to about 95 % w / w of a sugar, e.g., sucrose. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1.0 % w / w of a poloxamer, e.g., poloxamer 188. In one embodiment, the lyophilized RNA composition comprises about 1.0 to about 5.0 % w / w of potassium sorbate.

[0094] In another aspect, provided herein is a method of delivering an RNA composition in a subject, comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

[0095] In another aspect, provided herein is a method of inducing an immune response in a subject, comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

[0096] In another aspect, provided herein is a method of treating or preventing a cancer in a subject, comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

[0097] In some embodiments, the LNMP 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 LNMP 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. In one embodiment, the LNMP is a lipid nanoparticle.

[0098] In some embodiments, the LNMP has a size of less than about 200 nm. In one embodiment, the LNMP has a size of less than about 150 nm. In one embodiment, the LNMP has a size of less than about 100 nm. In one embodiment, the LNMP has a size of about 55 nm to about 80 nm.

[0099] In some embodiments, the RNA composition has a total lipid:polynucleotide weight ratio of about 50:1 to about 10:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 44: 1 to about 24: 1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 40: 1 to about 28: 1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 38: 1 to about 30:1. In one embodiment, the RNA composition has a total lipid:polynucleotide weight ratio of about 37: 1 to about 33:1.

[0100] In some embodiments, the methods provided herein comprising a lipid nanoparticle having a bacteria-derived lipid composition, comprising (a) a natural component comprising one or more lipids extracted from a natural source; and (b) an ionizable lipid.

[0101] In some embodiments, the natural source is selected from bacteria, animal, insect, archaea, and fungi.

[0102] In some embodiments, the methods provided herein comprising a lipid nanoparticle having a bacteria-derived lipid composition, comprising (a) a bacterial component comprising one or more lipids extracted from a bacterial source; and (b) an ionizable lipid.

[0103] one or more lipids extracted from a bacterial source; and (b) an ionizable lipid to the targetcell.

[0104] In some embodiments, the lipid nanoparticle comprises purified bacterial lipid of the bacteria component (a) in the presence of the ionizable lipid (b) to produce the bacteria-derived lipid composition.

[0105] In some embodiments, the bacterial source is selected from Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, and Thermoanaerobacterium. In one embodiment, the bacterial source is Escherichia (e.g., E. coli). In one embodiment, the bacterial source is Salmonella (e.g., Salmonella typhimurium).

[0106] In some embodiments, the bacterial component comprises isolated bacterial extracellular vesicles.

[0107] In some embodiments, the bacterial component is modified by reconstructing a film comprising the bacterial component in the presence of the ionizable lipid.

[0108] In some embodiments, the bacterial component is modified by reconstructing a film comprising the purified bacteria lipids of the bacterial component with the ionizable lipid.

[0109] 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 3.

[0110] The present invention provides LNP composition or LNMPs comprising one or more active agents or therapeutic agents, methods of making the lipid particles, and methods of delivering and / or administering the lipid particles (e.g., for the treatment of a disease or disorder).

[0111] In preferred embodiments, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent or therapeutic agent in the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease or protease. In other preferred embodiments, the lipid particles are substantially non-toxic to mammals such as humans.

[0112] In one aspect, provided herein is a particle comprising: (a) a nucleic acid;(b) an ionizable lipid comprising less than 50 mol% of the total lipid present in the particle; (c) a composition of natural source lipids comprising from 10 mol % to 90 mol % of the total lipid present in the particle; (d) a sterol (e.g. cholesterol or its derivative) comprising from and 5 mol% to 40 mol% of the total lipid present in the particle; and (e) a conjugated lipid (e.g. PEG-lipid) that inhibits aggregation of particles comprising from 0.5 mol % to 3 mol % of the total lipid present in the particle.

[0113] In another aspect, provided herein is a particle comprising: (a) a nucleic acid; (b) an ionizable lipid comprising about 10 to about 45 mol% of the total lipid present in the particle; (c) a composition of natural source lipids comprising from about 10 mol % to about 90 mol % of the total lipid present in the particle; (d) a sterol (e.g. cholesterol or its derivative) comprising from and 10 mol% to about 50 mol% of the total lipid present in the particle; and (e) a conjugated lipid (e.g. PEG-lipid) that inhibits aggregation of particles comprising from 0 mol % to about 10 mol % of the total lipid present in the particle.

[0114] In certain embodiments, the LNP or LNMP particle comprises: (a) a nucleic acid (e.g., mRNA or circRNA); (b) a ionizable lipid comprising from about 20 mol % to about 45 mol % of the total lipid present in the particle; (c) a non-ionizable lipid comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and (d) a conjugated lipid that inhibits aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.

[0115] In some embodiments, the LNP or LNMP particle comprises: (a) an mRNA or circRNA; (b) a ionizable lipid comprising from about 25 mol % to about 35 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.

[0116] In some embodiments, the LNP or LNMP particle comprises: (a) an mRNA or circRNA; (b) a ionizable lipid comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.

[0117] In some embodiments, provided herein are methods for treating a disease or disorder in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a therapeutically effective amount of a LNP or LNMP described herein orally.

[0118] In some embodiments, provided herein is a composition in the form of enterically coated capsules, tablets, caplets, or multiparticulate carriers such as particles, pellets, granules and beads.

[0119] In some embodiments, provided herein are a method and delivery system for the administration of a polynucleotide, wherein the drug, a bile salt or bile acid, and at least one surfactant are present in a single dosage form.

[0120] In some embodiments, provided herein is a dosage form comprised of an osmotically activated device in which a semipermeable membrane encapsulates a bile salt or bile acid, at least one surfactant as provided herein, and a hydrophilic drug.

[0121] In some embodiments, provided herein is a delayed release pharmaceutical dosage form for oral administration of low molecular weight heparin, wherein the dosage form comprises a composition of: (a) a therapeutically effective amount of low molecular weight heparin; (b) a bile salt or bile acid; (c) at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof; and (d) a means for delaying release of the composition from the dosage form following oral administration. In a preferred embodiment, the composition further includes a solubilizer to ensure good solubilization and / or dissolution of one or more components in the composition.

[0122] In some embodiments, the dosage form is not limited with respect to size, shape or general configuration, and may comprise, for example, a capsule, a tablet or a caplet, or a plurality of particles, granules, beads, or pellets that may or may not be encapsulated. Furthermore, either the heparin or the bile salt or bile acid may be present as a coating. In addition, the dosage form or components of the dosage form may be enterically coated; for example, a capsule or tablet may be enterically coated, and multiparticulate dosage forms such as drug-containing particles, pellets, granules and beads may be enterically coated as well. The enteric coating will generally comprise a bioerodible, gradually hydrolyzable and / or gradually water-soluble material, suitable for providing a desired delayed release profile.

[0123] In some embodiments, any bile salt or acid may be employed, so long as the selected compound is at least partially solubilized or suspended in the composition.

[0124] To ensure good solubilization and / or dissolution of the bile salt or acid, and to minimize precipitation thereof, additional formulation-aiding excipients may be incorporated into the aforementioned dosage form. Such excipients include, for example, bufferants, cosolvents, complexing agents, and crystal growth inhibitors. Additionally, processing techniques such as size reduction, co-precipitation, coacervation, lyophilizing, spray drying, eutectic mixing, solid solutioning or other appropriate techniques may be used to make the bile salt or acid more amenable to rapid dissolution. If suspended, the bile salt or acid can be in any of a number of forms, e.g., crystalline, amorphous, nanosized, micronized, or milled.

[0125] In some embodiments, a suitable hydrophilic surfactant will generally have an HLB value of at least 10, while suitable lipophilic surfactants will generally have an HLB value of or less than about 10. The co-administration of low molecular weight heparin with a bile salt or acid and at least one surfactant as provided herein substantially enhances the transmembrane absorption of the drug.

[0126] While not wishing to be bound by theory, it is proposed that the substantially homogeneous, optically clear aqueous dispersion that results immediately upon contact with an aqueous medium such as gastrointestinal fluid makes the drug immediately available for bioabsorption, i.e., the drug is rapidly and effectively "presented" to a target absorption site within the body. The optically clear aqueous dispersion that is formed is generally characterized as having an absorbance of less than about 0.3 at 400 nm measured at 100X dilution. In another embodiment, a method is provided foradministering low molecular weight heparin to a patient, the method comprising administering a therapeutically effective amount of the polynucleotide along with a bile salt or acid and at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. Typical dosages for orally administered low molecular weight heparin using the dosage forms of the invention are on the order of 700 to 400,000 IU / day, generally in the range of about 2500 to 10,000 IU / day, while typical dosages for orally administered unfractionated heparin are on the order of 2,500 to 800,000 Units / day. Generally, the drug will be given for the treatment or prevention of thrombosis.

[0127] Some embodiments, drug delivery systems are provided that comprise an osmotically activated device, i.e., an osmotically activated tablet or capsule, which houses a therapeutically effective amount of a hydrophilic drug, a bile salt or bile acid, and at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. In this embodiment, the drug-containing composition is encapsulated in a semipermeable membrane or barrier containing a small orifice. As known in the art with respect to so-called "osmotic pump" drug delivery devices, the semipermeable membrane allows passage of water in either direction, but not drug or other components of the drug-containing composition. Therefore, when the device is exposed to aqueous fluids, water will flow into the device due to the osmotic pressure differential between the interior and exterior of the device, and as water flows into the device, the drug-containing formulation in the interior will be "pumped" out through the orifice. The rate of drug release dD / dt, will be equivalent to the inflow rate of water times the drug concentration. In a preferred embodiment, the osmotically activated device is enterically coated with a coating material effective to provide the desired delayed release profile.

[0128] In some embodiments, a drug delivery system is provided for oral administration of a polysaccharide drug, the system comprised of a first dosage form and a second dosage form, wherein the first dosage form contains a therapeutically effective amount of the polysaccharide drug, and the second dosage form contains a bile salt or bile acid in combination with at least one surfactant selected from hydrophilic surfactants, lipophilic surfactants, and mixtures thereof, wherein at least one of the dosage forms is a delayed release dosage form, e.g., coated with an enteric coating. The polysaccharide drug may be, for example, glucosamine, a glycosaminoglycan, dextran, xylan, pentasaccharide, polygalacturonic acid, polymannuronic acid, chitin, pharmaceutically acceptable salts, esters or other derivatives thereof, and combinations of any of the foregoing. The dosage forms may be administered simultaneously or sequentially; in the latter case, either the first dosage form may be administered first, followed by administration of the second dosage form, or the second dosage form may be administered first, followed by administration of the first dosage form.

[0129] In some embodiments, the RNA 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 NaCl.

[0130] In some embodiments, the RNA 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 ofabout 3.2.

[0131] In some embodiments, the aqueous phase and the lipid solution are mixed at a 3:1 volumetric ratio.

[0132] In some embodiments, the RNA composition further comprises one or more cryoprotectants. The one or more cryoprotectants may be sucrose, glycerol, or a combination thereof. In one embodiment, the RNA composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0133] In some embodiments, the RNA composition is a lyophilized composition. The lyophilized mRNA composition may comprise one or more lyoprotectants. The lyophilized RNA composition may comprise a poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized RNA composition comprises a poloxamer, e.g., poloxamer 188.

[0134] In some embodiments, the RNA composition is a lyophilized composition. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1 .0 % w / w of the polynucleotides. In one embodiment, the lyophilized RNA composition comprises about 1.0 to about 5.0 % w / w lipids. In one embodiment, the lyophilized RNA composition comprises about 0.5 to about 2.5 % w / w of TRIS buffer. In one embodiment, the lyophilized RNA composition comprises about 0.75 to about 2.75 % w / w of NaCI. In one embodiment, the lyophilized RNA composition comprises about 85 to about 95 % w / w of a sugar, e.g., sucrose. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1.0 % w / w of a poloxamer, e.g., poloxamer 188. In one embodiment, the lyophilized RNA composition comprises about 1 .0 to about 5.0 % w / w of potassium sorbate.

[0135] In another aspect, provided herein is a method of delivering an RNA in a subject, comprising administering to a subject the RNA composition discussed in the above aspects of the invention.

[0136] In another aspect, provided herein is a method of inducing an immune response in a subject, comprising administering to a subject the RNA composition discussed in the above aspects of the invention.

[0137] In another aspect, provided herein is a method of treating or preventing a cancer in a subject, comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

[0138] In these aspects of the invention, the RNA composition may be administered by oral, intravenous, intradermal, intramuscular, intranasal, intraocular, or rectal, and / or subcutaneous administration. In certain embodiments, the RNA composition is administered by oral, enteral, intravenous, intramuscular, and / or subcutaneous administration.

[0139] In some embodiments, the RNA composition is administered at a dosage level sufficient to deliver about 0.01 mg / kg to about 0.2 mg / kg of the RNA to the subject. In some embodiments, the RNA composition is administered at a dosage level sufficient to deliver 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg of the RNA to the subject.

[0140] In some embodiments, the RNA composition is administered to the subject once, twice, three times, four times, or more. In some embodiments, the RNA composition is administered to the subject once or twice.

[0141] In some embodiments, the method further comprises administering an additional therapeutic agent to the subject.

[0142] In some embodiments, the additional therapeutic agent is an anti-cancer therapeutic agent.

[0143] In some embodiments, the additional therapeutic agent is a therapeutic agent that treats and / or prevents chronic pain. In one embodiment, the additional therapeutic agent is an opioid analgesics such as buprenorphine, a non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam SR, or combinations thereof.

[0144] In some embodiments, the additional therapeutic agent is administered prior to, concurrent with, or after the administration of the RNA composition.Definitions

[0145] As used herein, the term “effective amount,” “effective concentration,” or “concentration effective to” refers to an amount of a LNMP, or nucleic acid composition, sufficient to affect the recited result or to reach a target level (e.g., a predetermined or threshold level) in or on a target organism.

[0146] 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. As defined herein, the term “nucleic acid” and “polynucleotide” are interchangeable and refer to RNA or DNA that is linear or branched, 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).

[0147] As used herein, the terms “circRNA,” “circular polyribonucleotide,” “circular RNA,” and “circular polyribonucleotide molecule” 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.

[0148] As used herein, the term “expression sequence” is a nucleic acid sequence that encodes a product, e.g., a polypeptide or a regulatory nucleic acid. An exemplary expression sequence thatcodes for a polypeptide can comprise a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon”.

[0149] As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are used interchangeably and mean a monoribonucleotide molecule or 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. In some embodiments, the linear RNA has a 5’ end or 3’ end that is modified or protected from degradation (e.g., by a 5’ end protectant or a 3’ end protectant). In some embodiments, the linear RNA has non-covalently linked 5’ or 3’ ends. A linear RNA can be used as a starting material for circularization through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing-catalyzed circularization methods.

[0150] As used herein, the term “polyribonucleotide cargo” herein includes any sequence including at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo includes one or multiple expression sequences, wherein each expression sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo includes one or multiple noncoding sequences, such as a polyribonucleotide having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo includes a combination of expression and noncoding sequences. In embodiments, the polyribonucleotide cargo includes one or more polyribonucleotide sequence described herein, such as one or multiple regulatory elements, internal ribosomal entry site (IRES) elements, or spacer sequences.

[0151] As used herein, the elements of a nucleic acid are “operably connected” or “operably linked” if they are positioned on the vector such that they can be transcribed to form a linear RNA that can then be circularized into a circular RNA using the methods provided herein.

[0152] As used herein, a “spacer” or “spacer sequence” refers to any contiguous, non-coding nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Exemplary spacer sequences include, but are not limited to, poly(X) sequences as described herein, repetitive or random non-coding DNA or RNA sequences located 3’ or 5’ to open reading frames, or 3’ or 5’ untranslated regions. Any spacer sequence deemed appropriate by the skilled artisan for the polyribonucleotides described herein are contemplated by this disclosure.

[0153] As used interchangeably herein, the terms “poly(X)” and “poly(X) sequence” refer to an untranslated, contiguous region of any nucleic acid molecule of at least 5 nucleotides in length and consisting of individual adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) residues, or some combination thereof. For example, in some embodiments, a poly(A) sequence may be sequence of adenine residues. In other embodiments, a poly(A-T) sequence is a combination of adenine and thymine residues, In other embodiments, a poly(A-U) sequence may be a combination of adenine and uracil residues. In some embodiments, a poly(A-G) sequence is a combination of adenine and guanine residues. In some embodiments, a poly(G-C) sequence is a combination of guanine and cytosine residues. In some embodiments, a poly(X) sequence may be at least about 50 nucleotides to about 700 nucleotides in length, at least about 60 nucleotides to about 600 nucleotides in length, at least about 70 nucleotides to about 500 nucleotides in length, at least about 80nucleotides to about 400 nucleotides in length, at least about 90 nucleotides to about 300 nucleotides in length, at least about 100 nucleotides to about 200 nucleotides in length. In some embodiments, the poly(X) sequence may be at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 nucleotides in length. In some embodiments, a poly(X) sequence may be located 3’ to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the poly(X) sequence may be 3’ to a termination element (e.g., a stop codon) such that the poly(X) sequence is not translated. In some embodiments, a poly(X) sequence may be located 3’ to a termination element and a 3’ spacer sequence.

[0154] As used herein, the terms “nicked RNA,” “nicked linear polyribonucleotide,” and “nicked linear polyribonucleotide molecule” are used interchangeably and mean a polyribonucleotide molecule having a 5’ and 3’ end that results from nicking or degradation of a circular RNA.

[0155] 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, at least 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.

[0156] 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).

[0157] 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.

[0158] 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.

[0159] As used herein, the term “antibody” encompasses an immunoglobulin, whether natural orpartly 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.

[0160] As used herein, the term “heterologous” refers to an agent (e.g., a polypeptide) that is either (1) exogenous to the plant (e.g., originating from a source that is not the plant or plant part from which the PMP is produced) (e.g., an agent which is added to the PMP using loading approaches described herein) or (2) endogenous to the plant cell or tissue from which the PMP is produced, but present in the PMP (e.g., added to the PMP using loading approaches described herein, genetic engineering, as well as in vitro or in vivo approaches) at a concentration that is higher than that found in nature (e.g., higher than a concentration found in a naturally-occurring plant extracellular vesicle).

[0161] 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.

[0162] As used herein, the term “modified NMPs” or “modified LNMPs” refers to a composition including a plurality of NMPs or LNMPs that include one or more heterologous agents (e.g., one or more exogenous lipids, such as a ionizable lipids, e.g., a NMP or LNMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) capable of increasing cell uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the NMP or LNMP, or a portion or component thereof, relative to an unmodified NMP or LNMP; capable of enabling or increasing delivery of a heterologous functional agent (e.g., an agricultural or therapeutic agent) by the NMP or LNMP 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 NMPs or LNMPs may be modified in vitro or in vivo.

[0163] As used herein, the term “unmodified NMPs” or “unmodified LNMPs” refers to a composition including a plurality of NMPs or LNMPs that lack a heterologous cell uptake agent capable of increasing cell uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the NMP.

[0164] As used herein, the term “modified PMPs” or “modified LPMPs” refers to a composition including a plurality of PMPs or LPMPs that include one or more heterologous agents (e.g., one or more exogenous lipids, such as a ionizable lipids, e.g., a PMP or LPMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) capable of increasing cell uptake (e.g., animal cell uptake, plantcell uptake, bacterial cell uptake, or fungal cell uptake) of the PMP or LPMP, or a portion or component thereof, relative to an unmodified PMP or LPMP; capable of enabling or increasing delivery of a heterologous functional agent (e.g., an agricultural or therapeutic agent) by the PMP or LPMP 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 PMPs or LPMPs may be modified in vitro or in vivo.

[0165] As used herein, the term “unmodified PMPs” or “unmodified LPMPs” refers to a composition including a plurality of PMPs or LPMPs that lack a heterologous cell uptake agent capable of increasing cell uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the PMP.

[0166] As used herein, the term “cell uptake” refers to uptake of a NMP or LNMP or a portion or component thereof (e.g., a polynucleotide carried by the NMP or LNMP) by a cell, such as an animal cell, a plant cell, bacterial cell, or fungal cell. For example, uptake can involve transfer of the NMP (e.g., LNMP) 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 NMPs (e.g., LNMPs) may occur via active or passive cellular mechanisms. Cell uptake includes aspects in which the entire NMP (e.g., LNMP) 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 LNMP (e.g., a LNMP comprising an ionizable lipid, e.g., a LNMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) has an increased rate of endosomal escape relative to an unmodified LNMP. Cell uptake also includes aspects in which the NMP (e.g., LNMP) 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 LNMPs has an increased rate of fusion with the membrane of the target cell (e.g., is more fusogenic) relative to an unmodified LNMP.

[0167] 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.

[0168] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, fruit, harvested produce, tumor tissue, sap (e.g., xylem sap and phloem sap), and various forms of cells and culture (e.g., single cells, protoplasts, embryos, and callus tissue). The plant tissue may be in a plant or in a plant organ, tissue, or cell culture. In addition, a plant may be genetically engineered to produce a heterologous protein or RNA.

[0169] As used herein, the term “Bacteria” refers to whole bacteria or parts of bacteria. Further divisions of bacteria can be classified as coccals, bacillus, spirillum, or vibrio, and varying phylumsinclude but are not limited to Proteobacteria, Firmicutes, Bacteroids, sphingobacteria, Flavobacteria, Fusobacteria, Spirochaetes, Chlorobia, Cyanobacteria, Thermomicrobia, Xenobacteria, or Aquificae. Example of specific bacteria species include Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Parts of bacteria include cellular components such as peptidoglycan, outer membranes, inner membranes, cell walls, RNA polymerase, metabolic products, polypeptides, proteins. Flagella, pili, ribosomes, mesosome, cytoplasm, or chromosome. A bacteria may be genetically engineered to produce a heterologous protein or RNA, or may be genetically engineered to not produce an endogenous protein or RNA.

[0170] As used herein, the term “Arthropod” refers to any animal within the phylum Arthropoda, or any animal section, part, organ, tissue, egg, cell, or progeny of the same. Example animals include insects, spiders, and crustaceans. Arthropod cells include, without limitation, cells from eggs, suspension cultures, embryos, tissue, organs, exoskeletons, segments, and appendages. Arthropod parts include body segments, appendages, exoskeleton, eggs, organs, embryos, and various forms of cells and culture. Arthropod tissue may be in an arthropod or in an organ, tissue, or cell culture. An arthropod may be genetically engineered to produce a heterologous protein or RNA. An arthropod may be genetically engineered to not produce an endogenous protein or RNA.

[0171] As used herein, the term “Fungi” refers to whole fungi, fungi organs, fungi tissue, spores, fungi cells, and progeny of the same. Example fungi include yeasts, mushrooms, molds, and mildews. Fungi cells include without limitation cells from spores, suspension cultures, mycelium, hyphae, thallus, cell walls, tissue, gametophytes, sporophytes, and organs. Fungal tissue may be in a fungus or in an organ, tissue, or cell culture. A fungus may be genetically engineered to produce a heterologous protein or RNA. A fungus may be genetically engineered to not produce an endogenous protein or RNA.

[0172] As used herein, the term “Archaea” refers to whole archaea or parts of archaea. Example archaea include euryarchaeota, crenarchaeota, and koraarchaeota. Parts of archaea include cellular components such as RNA polymerases, glycerol-ether lipids, membranes, cell walls, polypeptides, proteins, and metabolic products. Archaea may be genetically engineered to produce a heterologous protein or RNA, or may be genetically engineered to not produce an endogenous protein or RNA.

[0173] As used herein, the term “plant extracellular vesicle”, “plant EV”, or “EV” refers to an enclosed lipid-bilayer structure naturally occurring in a plant. Optionally, the plant EV includes one or more plant EV markers. As used herein, the term “plant EV marker” refers to a component that is naturally associated with a plant, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof, including but not limited to any of the plant EV markers listed in the Appendix. In some instances, the plant EV marker is an identifying marker of a plant EV but is not a pesticidal agent. In some instances, the plant EV marker is an identifying marker of a plant EV and also a pesticidal agent (e.g., either associated with or encapsulated by the plurality of PMPs or LPMPs, or not directly associated with or encapsulated by the plurality of PMPs or LPMPs).

[0174] As used herein, the term “natural messenger pack” or “NMP” refers to a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure), that is about 5-2000 nm (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50-150 nm, or at least 70-120 nm) in diameter that is derived from (e.g., enriched, isolated or purified from) a natural source or segment, portion, or extract thereof, including lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated therewith and that has been enriched, isolated or purified from a natural source, a part of a natural source, or a cell of a natural source, the enrichment or isolation removing one or more contaminants or undesired components from the source. NMPs may be highly purified preparations of naturally occurring EVs. Preferably, at least 1% of contaminants or undesired components from the natural source are removed (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%) of one or more contaminants or undesired components from the source, e.g., source cell wall components; pectin; organelles (e.g., mitochondria; plastids such as chloroplasts, leucoplasts or amyloplasts; and nuclei); chromatin (e.g., a chromosome); or molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipido-proteic structures). Preferably, a NMP is at least 30% pure (e.g., at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure, or 100% pure) relative to the one or more contaminants or undesired components from the natural source as measured by weight (w / w), spectral imaging (% transmittance), or conductivity (S / m).

[0175] As used herein, the term “plant messenger pack” or “PMP” refers to a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure), that is about 5-2000 nm (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50- 150 nm, or at least 70-120 nm) in diameter that is derived from (e.g., enriched, isolated or purified from) a plant source or segment, portion, or extract thereof, including lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated therewith and that has been enriched, isolated or purified from a plant, a plant part, or a plant cell, the enrichment or isolation removing one or more contaminants or undesired components from the source plant. PMPs may be highly purified preparations of naturally occurring EVs. Preferably, at least 1% of contaminants or undesired components from the source plant are removed (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%) of one or more contaminants or undesired components from the source plant, e.g., plant cell wall components; pectin; plant organelles (e.g., mitochondria; plastids such as chloroplasts, leucoplasts or amyloplasts; and nuclei); plant chromatin (e.g., a plant chromosome); or plant molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipido-proteic structures). Preferably, a PMP is at least 30% pure (e.g., at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure, or 100% pure) relative to the one or more contaminants or undesired components from the source plant as measured by weight (w / w), spectral imaging (% transmittance), or conductivity (S / m).

[0176] A lipid reconstructed NMP (LNMP) is used herein. For instance, a lipid reconstructed PMP (LPMP) is used herein. The terms “lipid reconstructed NMP” and “LNMP” refer to a NMP that has been derived from a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure) derived from (e.g., enriched, isolated or purified from) a natural source, wherein the lipid structure is disrupted (e.g., disrupted by lipid extraction) and reassembled orreconstituted 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 LNMP, 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 NMPs. Alternatively, LNMPs may be produced using a microfluidic device (such as a NanoAssemblr® IGNITE™ microfluidic instrument (Precision NanoSystems)). The terms “lipid reconstructed PMP” and “LPMP” are defined in the same manner as “lipid reconstructed NMP” and “LNMP,” when the natural source is a plant source.

[0177] As used herein, the term “pure” refers to a PMP preparation in which at least a portion (e.g., at least 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%) of plant cell wall components, plant organelles (e.g., mitochondria, chloroplasts, and nuclei), or plant molecule aggregates (protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipido-proteic structures) have been removed relative to the initial sample isolated from a plant, or part thereof.

[0178] 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 (such as plants or bacteria), 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). Thus, any disclosure herein describing the features relating to LNMP and LNMP formulation are applicable to CLP and CLP formulation.

[0179] The complex lipid particle may contain 3-1000 lipids extracted from one or more natural (e.g., plant, bacteria) sources. The complex lipid particle may contain natural (e.g., plant, bacteria) 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 sub-classes of lipids from the natural (e.g., plant, bacteria) source. The complex lipid particle may comprise all or a fraction of the lipid species present in the lipid structure from the natural (e.g., plant, bacteria) 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%, atleast 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 plant source or from a bacteria source.

[0180] The complex lipid particle may comprise reduced or minimized protein matter endogenous to the one or more natural (i.e. plant, bacteria) 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, or less than 50% w / w of the protein matter endogenous to the one or more natural (e.g., plant, bacteria) sources. In some instances, the lipid bilayer of the complex lipid particle does not contain proteins.

[0181] 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.

[0182] 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.

[0183] As used herein, the term “exogenous lipid” refers to a lipid that is exogenous to the natural source (e.g., plant, bacteria), 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 cell-penetrating 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.

[0184] As used herein, the term “cationic lipid” refers to an amphiphilic molecule (e.g., a lipid or alipidoid) that is positively charged, containing a cationic group (e.g., a cationic head group).

[0185] 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).

[0186] It has been surprisingly found that ionizable lipids comprising alkyl chains with multiple sites of 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.

[0187] 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 nanoparticle comprising the ionizable lipid under different pH conditions. The surface charge of the lipid nanoparticle in 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.

[0188] 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.

[0189] 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).

[0190] As used herein, the term “lipidoid” refers to a molecule having one or more characteristics of a lipid.

[0191] As used herein, the term “stable LNMP formulation” or “stable CLP formulation” refers to a CLP formulation or a LNMP 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 the initial number of CLPs or LNMPs (e.g., CLPs or LNMPs per mL of solution) relative to the number of CLPs or LNMPs in the CLP formulation or LNMP 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 RNA formulated within the CLP or LNMP) relative to the initial activity of the CLP or LNMP (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)).

[0192] Alternatively, the expression refers to a CLP formulation or LNMP 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 LNMP 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)).

[0193] Alternatively, the expression refers to a CLP formulation or a LNMP 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 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 size of the CLPs or LNMPs (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)).

[0194] In some embodiments, the stable CLP or LNMP formulation continues to encapsulate or remains associated with an exogenous peptide, polypeptide, or protein with which the CLP or LNMP formulation 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.

[0195] 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1 is a photograph showing the level of nanoluciferase in mice treated orally (PO) with reconstructed LPMPs (recPMPs) derived from lemon, formulated with C12-200 as the ionizable lipid, and comprising nanoluciferase (nLuc) mRNA - nLuc-Flag and nLuc secreted.

[0197] Figure 2 is a photograph showing the level of nanoluciferase in mice treated orally (PO) with reconstructed LPMPs (recPMPs) derived from lemon, formulated with C12-200 as the ionizable lipid, and comprising nanoluciferase (nLuc) mRNA - nLuc-Flag and nLuc secreted in liver, stomach, colon, spleen, small intestine, mesenteric lymph nodes, pancreas, and caecum with 5 minutes exposure time.

[0198] Figure 3 is a photograph showing the level of nanoluciferase in mice treated orally (PO) with reconstructed LPMPs (recPMPs) derived from lemon, formulated with C12-200 as the ionizable lipid, and comprising nanoluciferase (nLuc) mRNA - nLuc-Flag and nLuc secreted in liver, stomach, colon, spleen, small intestine, mesenteric lymph nodes, pancreas, and caecum with 5 minutes exposure time.

[0199] Figure 4 is an illustration of organs showing the level of nanoluciferase in mice treated orally (PO) with reconstructed LPMPs (recPMPs) derived from lemon, formulated with C12-200 as the ionizable lipid, and comprising nanoluciferase (nLuc) mRNA - nLuc-Flag and nLuc secreted in liver, stomach, colon, spleen, small intestine, mesenteric lymph nodes, pancreas, and caecum.

[0200] Figures 5A-5F show the average radiance in the liver (Figure 5A), spleen (Figure 5B), pancreas (Figure 5C), MLNs (Figure 5D), gastro-intestine (Gl) (Figure 5E), and inguinal lymph nodes (Figure 5F) of mice 24 hours after either oral (PO) or intrajejunal (IJ) delivery of a mRNA-LNP formulation employing ionizable lipid 2243 (nLuc-Flag mRNA, I J-1 Oug, P0-200ug). N= 5 / group as compared to naive mice (n=2).

[0201] Figure 6A shows a representative section of the small intestine (SI) from a naive tdTomato mouse as a negative control. Figure 6B shows a representative section of the SI 48-hours post- intrajejunal administration of 2243 a mRNA-LNP formulation employing the ionizable lipid 2243 (nLuc- Flag : CRE mRNA, 15ug; N= 2), wherein the light gray indicates transfected cells. Figure 6C highlights a Peyer’s Patch shown in Figure 6B. Figure 6D highlights villi shown in Figure 6B.

[0202] Figure 7 depicts the concentration of anti-TNFa antibody levels 24h post-dose in the plasma of the mice given 0% DSS and a single intravenous dose of a LNP / mRNA formulation employing the ionizable lipid 2243 (LNP 2243 I anti-TNFa mRNA, 0.6mg / kg) and in the plasma of the mice given 2% DSS and a single intravenous dose of a LNP / mRNA formulation employing the ionizable lipid 2243 (LNP 2243 I anti-TNFa mRNA, 0.6mg / kg). The control was the plasma from naive mice (0% DSS and no administration of LNP 2243 I anti-TNFa mRNA). N=5 / group.

[0203] Figure 8A shows the concentration of TNFa in feces three-days post dose in the mice given 2% DSS and in the mice given 2% DSS and a single intravenous dose of a LNP / mRNA formulation employing the ionizable lipid 2243 (LNP 2243 I anti-TNFa mRNA, 0.6mg / kg). The control was feces from naive mice (0% DSS and no administration of LNP 22431 anti-TNFa mRNA). N=5 / group. Figure 8B shows the concentration of calprotectin in feces pre-dose (3 days after the start of DSS), 24h post-dose (5 days after start of DSS) in the mice given 2% DSS and in the mice given 2% DSS and a single intravenous dose of LNP / mRNA formulation employing the ionizable lipid 2243 (2243 LNP / anti-TNFa mRNA, 0.6mg / kg), and 3 days post-dose (7 days after the start of DSS) in the mice given 2% DSS and in the mice given 2% DSS and a single intravenous dose of LNP / mRNA formulation employing the ionizable lipid 2243 (2243 LNP I anti-TNFa mRNA, 0.6mg / kg). The control was feces from naive mice (0% DSS and no administration of LNP 22431 anti-TNFa mRNA). N=5 / group.

[0204] Figure 9 shows the measured antibody concentration (huIgG, ng / mL) in the plasma of mice at Days 0, 1 , 3, 7, and 14, respectively, after a single intravenous dose of an exemplary LPMP / mRNA formulation employing the ionizable lipid 2272 (recLemon LPMP 2272 I anti-PCSK9 mRNA, 0.3mg / kg) N=5.

[0205] Figure 10A shows the measured antibody concentration (huIgG, ng / mL) in the plasma of mice 24h after a single intravenous dose of an exemplary LPMP / mRNA formulation the ionizable lipid 2272 (recLemon LPMP 2272 I anti-PCSK9 mRNA, 0.3mg / kg) N=10. PBS was used as a control (N=1). Figure 10B shows the measured antibody concentration (huIgG, ng / mL) in MLNs and colon / caecum of mice 24h after a single intravenous dose of an exemplary LPMP / mRNA formulation employing the ionizable lipid 2272 (recLemon LPMP 22721 anti-PCSK9 mRNA, 0.3mg / kg) N=10. PBS was used as a control (N=1).

[0206] Figures 11 A-11 D show the antibody concentration (huIgG, ng / mL) in the colon / caecum contents, mesenteric lymph nodes (MLNs), small intestine (SI) content, and plasma of mice 24 hours after intrajejunal delivery of an exemplary LPMP / mRNA formulation employing the ionizable lipid 2272 (anti-PCSK9 mRNA, 65-75ug). N = 19 / 3 independent experiments as compared to PBS (n = 16 13 independent experiments). Statistically identified outliers were removed.DETAILED DESCRIPTION

[0207] The inventors have surprising found that when administering a nano-luciferase via oral gavage intracellularly (nluc-Flag) and via secretion (nLuc-secreted), resulting in broad transfection of the digestive tract (extra-hepatic tissues) and lymphoid organs, no activity was observed in the liver. nLuc-Flag and nLuc-secreted were not detected in the liver; they were only detected in the Gl tract and associated lymphoid tissues. In addition, a higher expression of nLuc-secreted in the colon was observed, as compared to nluc-Flag.

[0208] Featured herein are RNA therapeutics that can safely direct to the lymphatic transport system without delivering to the liver. Such selectivity would be useful for treating pancreatitis, IBD, ulcerative colitis, Crohn’s disease, colorectal cancer, and also oral vaccination (norovirus, RSV, flu, shingle, COVID, and the like).

[0209] In some embodiments, the potentially unlocking RNA therapies include, but not limited to Gl cancers (i.e. stomach, pancreas, colon); mRNA for cancer antigens, diptheria toxin, anti-PD1 , chemokines, Cytokines (II2, 12, 27, IFNg, IL15), receptors, pancreatitis (mRNA for anti-inflammatory drugs), IBD (mRNA for IL10, anti-TNF, siRNA for TNFa, mRNA for inhibitory receptors), ulcerative colitis, Crohn’s disease, infectious diseases (viral, bacterial & fungal), and microbiome modulation.

[0210] The RNA compositions may be used to induce a balanced immune response against cancers,comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example. These RNA compositions include one or more polynucleotides (e.g., RNA such as mRNA or circRNA) encoding one or more tumor antigenic polypeptides, formulated within a complex lipid particle (CLP). In some embodiments, the CLP is a lipid reconstructed natural messenger packs (LNMPs) comprising lipids extracted from one or more natural sources (i.e. , natural lipids) and an ionizable lipid. NMPs are lipid assemblies produced wholly or in part from natural source extracellular vesicles (EVs), or segments, portions, or extracts thereof. PMPs are lipid assemblies produced wholly or in part from plant extracellular vesicles (EVs), or segments, portions, or extracts thereof. LNMPs are NMPs derived from a lipid structure wherein the lipid structure is disrupted and reassembled or reconstituted in a liquid phase. LPMPs are PMPs derived from a lipid structure wherein the lipid structure is disrupted and reassembled or reconstituted in a liquid phase.

[0211] The disclosure also includes a method for making a RNA composition, comprising reconstituting a film comprising purified NMP lipids in the presence of an ionizable lipid to produce a LNMP comprising the ionizable lipid, and loading into the LNMPs with one or more polynucleotides encoding one or more polypeptides.Complex Lipid Particles and Lipid Reconstructed Natural Messenger Packs (LNMPs)Complex Lipid Particles

[0212] 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.

[0213] The complex lipid particles may also comprise at least exogenous ionizable lipid. 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.

[0214] 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.

[0215] 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 (e.g., plant, bacteria) lipids based on the amounts of total lipids in the complex lipid formulation.

[0216] 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.

[0217] 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.

[0218] 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 lipid particle contains lipids from at least two or at least three of these different classes.

[0219] In some embodiments, the complex lipid particle may contain one or more glycerolipids (GL) or glycerophospholipids (GP), which may also include glycolipids.

[0220] 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.

[0221] 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.

[0222] 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 glycyl serines, 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.

[0223] 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,-M-phosphatidy lethanols, 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.

[0224] 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.

[0225] The identity (and class and subclass) and the amounts of the lipids extracted from the natural source(s) can be analyzed by lipidomic 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.

[0226] 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).

[0227] The complex lipid particle may comprise reduced or minimized protein matter endogenous to the 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.

[0228] 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 naturalsources. 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.

[0229] 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.

[0230] 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.

[0231] 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 16:0 about 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.

[0232] 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.

[0233] 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 16:1about 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.

[0234] 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).

[0235] 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).

[0236] 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.

[0237] In some embodiments, the complex lipid particle contains less than 5% w / w of exogenous antioxidant.

[0238] In some embodiments, the CLPs contain natural lipids comprising about 0 to 20 wt / wt% of cardiolipin (CL).Natural messenger packs (NMPs)

[0239] A plurality of NMPs in a modified NMP formulation may be loaded with the exogenous peptide, polypeptide, or protein such that at least 5%, at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 95% of NMPs in the plurality of NMPs encapsulate the exogenous peptide, polypeptide, or protein. In some embodiments the NMP is derived from an arthropod, fungi, archaea, plant, or bacteria. For instance, one example of NMP derived from a plant source is a plant NMP, which may be referred to as a PMP, which is a lipid (e.g., lipid bilayer, unilamellar, or multilamellar structure) structure that includes a plant EV, or segment, portion, or extract (e.g., lipid extract) thereof. Additional descriptions about PMPs can be found in the section “Plant Messenger Pack (PMPs),” in PCT Application No. PCT / US22 / 47107, filed on October 19, 2022, which is incorporated herein by reference in its entirety.

[0240] NMPs can include Arthropod, Plant, Fungi, Archaea, or Bacteria EVs, or segments, portions, or extracts, thereof, in which the EVs are about 5-2000 nm in diameter. For example, the NMP caninclude an 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 NMP includes a Arthropod, Plant, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, that has a mean diameter of about 5-1400 nm, 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 Arthropod, Plant, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, has a mean diameter of about 50-200 nm. In certain instances, the EV, or segment, portion, or extract thereof, has a mean diameter of about 50-300 nm. In certain instances, the EV, or segment, portion, or extract thereof, has a mean diameter of about 200-500 nm. In certain instances, the EV, or segment, portion, or extract thereof, has a mean diameter of about 30-150 nm.

[0241] In some instances, the NMP may include a Arthropod, Plant, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, that has a mean diameter of at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, or at least 1300. In some instances, the NMP includes a Arthropod, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, that has a mean diameter less than 1400 nm, less than 1000 nm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, or less than 50 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 EVs, or segment, portion, or extract thereof.

[0242] In some instances, the NMP may include an Arthropod, Plant, Fungi, Archaea, or Bacteria 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). In some instances, the NMP may include a Arthropod, Fungi, Archaea, or Bacteria 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). In some instances, the NMP may include a Arthropod, Plant, Fungi, Archaea, or Bacteria 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 100 nm2, at least 1000 nm2, at least 1x104nm2, at least 1x105nm2, at least 1x106nm2, or at least 2x106nm2). In some instances, the NMP may include a Arthropod, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, that has a meanvolume 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.

[0243] In some instances, the NMP can have the same size as the Arthropod, Plant, Fungi, Archaea, or Bacteria EV or segment, extract, or portion thereof. Alternatively, the NMP may have a different size than the initial EV from which the NMP is produced. For example, the NMP may have a diameter of about 5-2000 nm in diameter. For example, the NMP can have 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 1000-1200 nm, about 1200-1400 nm, about 1400-1600 nm, about 1600 - 1800 nm, or about 1800 - 2000 nm. In some instances, the NMP may have a mean diameter of at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm, or about 2000 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 NMPs. In some instances, the size of the NMP is determined following loading of heterologous functional agents, or following other modifications to the NMPs.

[0244] In some instances, the NMP may have a mean surface area of 77 nm2to 1 .3 x107nm2(e.g., 77-100 nm2, 100-1000 nm2, 1000-1x104nm2, 1x104- 1x105nm2, 1x105-1x106nm2, or 1x106-1 ,3x107nm2). In some instances, the NMP may have a mean volume of 65 nm3to 4.2 x109nm3(e.g., 65-100 nm3, 100-1000 nm3, 1000-1x104nm3, 1x104- 1x105nm3, 1x105-1x106nm3, 1x106-1x107nm3, 1x107- 1x108nm3, 1x108-1x109nm3, or 1x109- 4.2 x109nm3). In some instances, the NMP has a mean surface area of at least 77 nm2, (e.g., at least 77 nm2, at least 100 nm2, at least 1000 nm2, at least 1x104nm2, at least 1x105nm2, at least 1x106nm2, or at least 1x107nm2). In some instances, the NMP 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 1x109nm3, at least 2x109nm3, at least 3x109nm3, or at least 4x109nm3).

[0245] In some instances, the NMP may include an intact Arthropod, Plant, Fungi, Archaea, or Bacteria EV. In some embodiments, the NMP may include a non-plant natural source such as algae or animal-derived organs EV, or segment, portion, or extract thereof. Alternatively, the NMP 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 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 thesplitting 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 plurality of NMPs can include a plurality of intact EVs, a plurality of 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 EVs will depend on the particular isolation method used. For example, grinding or blending an Arthropod, Fungi, Plant, Archaea, or Bacteria, or part thereof, may produce NMPs that contain a higher percentage of EV segments, portions, or extracts than a non-destructive extraction method, such as vacuum-infiltration.

[0246] In instances where, the NMP includes a segment, portion, or extract of a Arthropod, Fungi, Archaea, or Bacteria 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 EV segment, portion, or extract has a surface area of less than 70 nm2, 60 nm2, 50 nm2, 40 nm2, 30 nm2, 20 nm2, or 10 nm2). In some instances, the NMP may include a Arthropod, Fungi, Archaea, or Bacteria 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).

[0247] In instances where the NMP includes an extract of a Arthropod, Plant, Fungi, Archaea, or Bacteria EV, e.g., in instances where the NMP includes lipids extracted (e.g., with chloroform or ethanol) from a EV, the NMP may include at least 1 %, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% of lipids extracted (e.g., with chloroform or with ethanol) from a Arthropod, Fungi, Archaea, or Bacteria EV. The NMPs in the plurality may include Arthropod, Plant, Fungi, Archaea, or Bacteria EV segments and / or EV-extracted lipids or a mixture thereof.Production of NMPs

[0248] NMPs may be produced from Arthropod, Fungi, Plant, Archaea, or Bacteria EVs, or a segment, portion or extract (e.g., lipid extract) thereof, that occur naturally in Arthropod, Fungi, Plant, Archaea, or Bacteria, or parts thereof, including tissues or cells. In some embodiments, the NMP may include a non-plant natural source such as algae or animal-derived organs EV, or segment, portion, or extract thereof. An exemplary method for producing NMPs includes (a) providing an initial sample from a source, or a part thereof, wherein the source or part thereof comprises EVs; and (b) isolating a crude NMP fraction from the initial sample, wherein the crude NMP fraction has a decreased level of at least one contaminant or undesired component from the source or part thereof relative to the level in the initial sample. The method can further include an additional step (c) comprising purifying the crude NMP fraction, thereby producing a plurality of pure NMPs, wherein the plurality of pure NMPs have a decreased level of at least one contaminant or undesired component from the Arthropod, Fungi, Archaea, or Bacteria or part thereof relative to the level in the crude EV fraction. Each production step is discussed in further detail, below.

[0249] For instance, PMPs may be produced from plant EVs, or a segment, portion or extract (e.g.,lipid extract) thereof, that occur naturally in plants, or parts thereof, including plant tissues or plant cells. An exemplary method for producing PMPs includes (a) providing an initial sample from a plant, or a part thereof, wherein the plant or part thereof comprises EVs; and (b) isolating a crude PMP fraction from the initial sample, wherein the crude PMP fraction has a decreased level of at least one contaminant or undesired component from the plant or part thereof relative to the level in the initial sample. The method can further include an additional step (c) comprising purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs have a decreased level of at least one contaminant or undesired component from the plant or part thereof relative to the level in the crude EV fraction. Each production step is discussed in further detail, below. Exemplary methods regarding the isolation and purification of NMPs (e.g., plant NMPs, PMPs) is found, for example, in Rutter and Innes, Plant Physiol. 173(1): 728-741 , 2017; Rutter et al, Bio. Protoc. 7(17): e2533, 2017; Regente et al, J of Exp. Biol. 68(20): 5485-5496, 2017; Mu et al, Mol. Nutr. Food Res., 58, 1561-1573, 2014, and Regente et al, FEBS Letters. 583: 3363-3366, 2009, each of which is herein incorporated by reference. Additional descriptions about the production of PMPs can be found in the section “Production of PMPs,” in PCT Application No. PCT / US22 / 47107, filed on October 19, 2022, which is incorporated herein by reference in its entirety.

[0250] For example, a plurality of NMPs may be isolated from a Arthropod, Fungi, Plant, Archaea, or Bacteria by a process which includes the steps of: (a) providing an initial sample from a source, or a part thereof, wherein the source or part thereof comprises EVs; (b) isolating a crude NMP fraction from the initial sample, wherein the crude NMP fraction has a decreased level of at least one contaminant or undesired component from the Arthropod, Fungi, Plant, Archaea, or Bacteria or part thereof 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 NMP fraction, thereby producing a plurality of pure NMPs, wherein the plurality of pure NMPs have a decreased level of at least one contaminant or undesired component from the source or part thereof 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%).

[0251] The NMPs provided herein can include an Arthropod, Fungi, Archaea, or Bacteria EV, or segment, portion, or extract thereof, isolated from a variety of sources.

[0252] For instance, the plant NMPs, PMPs, can include a plant EV, or segment, portion, or extract thereof, produced from a variety of plants. PMPs may be produced from any genera of plants (vascular or nonvascular), including but not limited to angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, selaginellas, horsetails, psilophytes, lycophytes, algae (e.g., unicellular or multicellular, e.g., archaeplastida), or bryophytes. In certain instances, PMPs can be produced using a vascular plant, for example monocotyledons or dicotyledons or gymnosperms. For example, PMPs can be produced using alfalfa, apple, Arabidopsis, banana, barley, a Brassica species (e.g., Arabidopsis thaliana or Brassica napus), canola, castor bean, chicory, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, dioscorea, eucalyptus, fescue, flax, gladiolus, liliacea, linseed, millet, muskmelon, mustard, oat, oil palm, oilseedrape, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugarbeet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass, wheat or vegetable crops such as lettuce, celery, broccoli, cauliflower, cucurbits; fruit and nut trees, such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; vines, such as grapes, kiwi, hops; fruit shrubs and brambles, such as raspberry, blackberry, gooseberry; forest trees, such as ash, pine, fir, maple, oak, chestnut, popular; with alfalfa, canola, castor bean, corn, cotton, crambe, flax, linseed, mustard, oil palm, oilseed rape, peanut, potato, rice, safflower, sesame, soybean, sugarbeet, sunflower, tobacco, tomato, or wheat.

[0253] PMPs (i.e., plant NMPs) may be produced using a whole plant (e.g., a whole rosettes or seedlings) or alternatively from one or more plant parts (e.g., leaf, seed, root, fruit, vegetable, pollen, phloem sap, or xylem sap). For example, PMPs can be produced using shoot vegetative organs / structures (e.g., leaves, stems, or tubers), roots, flowers and floral organs / structures (e.g., pollen, bracts, sepals, petals, stamens, carpels, anthers, or ovules), seed (including embryo, endosperm, or seed coat), fruit (the mature ovary), sap (e.g., phloem or xylem sap), plant tissue (e.g., vascular tissue, ground tissue, tumor tissue, or the like), and cells (e.g., single cells, protoplasts, embryos, callus tissue, guard cells, egg cells, or the like), or progeny of same. For instance, the isolation step may involve (a) providing a plant, or a part thereof. In some examples, the plant part is an Arabidopsis leaf. The plant may be at any stage of development. For example, the PMPs can be produced using seedlings, e.g., 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, or 8-week old seedlings (e.g., Arabidopsis seedlings). Other exemplary PMPs can include PMPs produced using roots (e.g., ginger roots), fruit juice (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., Arabidopsis phloem sap), or xylem sap (e.g., tomato plant xylem sap). In some embodiments, the PMPs are produced from algae or lemon.

[0254] NMPs may be isolated from any genera of Arthropod, Fungi, Archaea, or Bacteria, including but not limited to crabs, crawfish, shrimp, spiders, scorpions, crickets, grasshoppers, beetles, millipedes, ticks, mites, centipedes, ants, wasps, dragonflies, flies, gnats, other insects and crustaceans, yeast, mushrooms, puffballs, stinkhorns, boletes, smuts, bunts, bracket fungi, jelly fungi, toadstools, molds, rusts, earth stars, chanterelles, ergot, pyroIobus, picrophilus, methanogens, crenarchaeota, nanoarchaeota, ignicoccus, cenarchaeum, halophiles, Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia,Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0255] NMPs may be produced from a whole Arthropod, Fungi, Archaea, or Bacteria (e.g., a whole insect, spider, crustacean, fungi, or single cell of archaea or bacteria) or alternatively from one or more source parts (e.g., segments, organs, eggs, spores, mycelium, tissue, membrane or cell wall). For example, NMPs can be produced from organs / structures / tissues / cell cultures (e.g., body segments, appendages, organs, eggs, exoskeleton, embryos, spores, mycelium, hyphae, thallus, suspension cultures, cell walls, inner or outer membranes, gametophytes, sporophytes, polymerases, glycerol-ether lipids, metabolic products, flagella, pili, ribosomes or organelles) or progeny of same. The source may be at any stage of development. In some embodiments, the NMP is produced from an insect or fungi, (e.g. cricket, yeast, or mushroom). In some embodiments, the NMP is produced from a bacteria or archaea (e.g. E. coli). In some embodiments, the NMP is produced from algae (e.g. kelp or chlorella). In some embodiments, the NMP is produced from an animal organ (e.g. brain or blood).

[0256] NMPs can be produced from a plant, Arthropod, Fungi, Archaea, or Bacteria, or part thereof, by a variety of methods. Any method that allows release of the EV-containing fraction of a source, or an otherwise extracellular fraction that contains NMPs comprising secreted EVs (e.g., cell culture media) is suitable in the present methods. EVs can be separated from the source or source part by either destructive (e.g., grinding or blending) or non-destructive (washing or vacuum infiltration) methods. For instance, the plant, Arthropod, Fungi, Archaea, or Bacteria, or part thereof, can be vacuum-infiltrated, ground, blended, or a combination thereof to isolate EVs from the source or source part, thereby producing NMPs. For instance, the isolating step may involve (b) isolating a crude NMP fraction from the initial sample (e.g., a plant, Arthropod, Fungi, Archaea, or Bacteria or part, or a sample derived from a plant, Arthropod, Fungi, Archaea, or Bacteria or part), wherein the crude NMP fraction has a decreased level of at least one contaminant or undesired component from the source or part thereof relative to the level in the initial sample; wherein the isolating step involves vacuum infiltrating the plant, Arthropod, Fungi, Archaea, or Bacteria (e.g., with a vesicle isolation buffer) to release and collect the desired fraction. Alternatively, the isolating step may involve (b) grinding or blending the source to release the EVs, thereby producing NMPs.

[0257] Upon isolating the plant, Arthropod, Fungi, Archaea, or Bacteria EVs, thereby producing NMPs, the NMPs can be separated or collected into a crude NMP fraction. For instance, the separating step may involve separating the plurality of NMPs into a crude NMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the NMP-containing fraction from large contaminants, including tissue debris, cells, or cell organelles. As such, the crude NMP fraction will have a decreased number of large contaminants, including, for example, tissue debris, cells, or cell organelles (e.g., nuclei, mitochondria, etc.), as compared to the initial sample from the source or source part.

[0258] The crude NMP fraction can be further purified by additional purification methods to produce a plurality of pure NMPs. For example, the crude NMP fraction can be separated from other source components by ultracentrifugation, e.g., using a density gradient (iodixanol or sucrose), sizeexclusion, and / or use of other approaches to remove aggregated components (e.g., precipitation orsize-exclusion chromatography). The resulting pure NMPs may have a decreased level of contaminants or undesired components from the source (e.g., one or more non-NMP components, such as 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 NMPs 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) of source organelles or cell wall components relative to the level in the initial sample. In some instances, the pure NMPs are substantially free (e.g., have undetectable levels) of one or more non-NMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipido-proteic structures), nuclei, cell wall components, cell organelles, or a combination thereof. Further examples of the releasing and separation steps can be found in WO 2021 / 041301. The NMPs may be at a concentration of, e.g., 1x109, 5x109, 1x1010, 5x1010, 5x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, or more than 1x1013NMPs / mL.

[0259] For example, protein aggregates may be removed from isolated NMPs. For example, the isolated NMP solution 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 isolated NMP solution 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 isolated NMP solution until it is at, e.g., 1 mol / L. The solution can then be filtered to isolate the NMPs. Alternatively, aggregates are solubilized by increasing the temperature. For example, the isolated NMPs can be heated under mixing until the solution has reached a uniform temperature of, e.g., 50°C for 5 minutes. The NMP mixture can then be filtered to isolate the NMPs. Alternatively, soluble contaminants from NMP solutions can be separated by size-exclusion chromatography column according to standard procedures, where NMPs 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. In some embodiments, protein aggregates are removed before the exogenous peptide, polypeptide, or protein is encapsulated by the NMP. In other embodiments, protein aggregates are removed after the exogenous peptide, polypeptide, or protein is encapsulated by the NMP.

[0260] In some embodiments, the preparation of NMPs from natural sources is through an ethanol extraction method. In some aspects, a 3:2 ethyl acetate:ethanol solvent aids in extraction. In someembodiments, the preparation of NMPs from natural sources is through a modified Matyash extraction method. In some aspects, a 1 :2 MeOH:MTBE solvent aids in extraction.

[0261] 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 NMPs at any step of the production process. NMPs may be characterized by a variety of analysis methods to estimate NMP yield, NMP concentration, NMP purity, NMP composition, or NMP sizes. NMPs can be evaluated by a number of methods known in the art that enable visualization, quantitation, or qualitative characterization (e.g., identification of the composition) of the NMPs, 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). In certain instances, methods (e.g., mass spectroscopy) may be used to identify EV markers present on the NMP. To aid in analysis and characterization, of the NMP fraction, the NMPs can additionally be labelled or stained. For example, the NMPs can be stained with 3,3’-dihexyloxacarbocyanine iodide (DIOCe), a fluorescent lipophilic dye, PKH67 (Sigma Aldrich); Alexa Fluor® 488 (Thermo Fisher Scientific), or DyLight™ 800 (Thermo Fisher). In the absence of sophisticated forms of nanoparticle tracking, this relatively simple approach quantifies the total membrane content and can be used to indirectly measure the concentration of NMPs (Rutter and Innes, Plant Physiol. 173(1): 728-741 , 2017; Rutter et al, Bio. Protoc. 7(17): e2533, 2017). For more precise measurements, and to assess the size distributions of NMPs, nanoparticle tracking, nano flow cytometry, or Tunable Resistive Pulse Sensing can be used.

[0262] During the production process, the NMPs can optionally be prepared such that the NMPs are 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 EV level in a control or initial sample. The isolated NMPs may make up about 0.1% to about 100% of the NMP composition, such as any one of 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%. In some instances, the composition described herein includes at least any of 0.1%, 0.5%, 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more NMPs, e.g., as measured by wt / vol, percent NMP protein composition, and / or percent lipid composition (e.g., by measuring fluorescently labelled lipids)). In some instances, the concentrated agents are used as commercial products, e.g., the final user may use diluted agents, which have a substantially lower concentration of active ingredient. In some embodiments, the composition described herein is formulated as a NMP concentrate formulation, e.g., an ultra-low- volume concentrate formulation. In some embodiments, the NMPs in the composition are at a concentration effective to increase the fitness of an organism, e.g., a plant, an animal, an insect, a bacterium, or a fungus. In other aspects, the NMPs in the composition are at a concentration effective to decrease the fitness of an organism, e.g., a plant, an animal, an insect, a bacterium, or a fungus.

[0263] NMPs can be produced from a variety of Arthropod, Fungi, Plant, Archaea, or Bacteria, or one or more parts thereof (e.g., segments, organs, eggs, spores, mycelium, tissue, membrane or cellwall). For example, NMPs can be produced from organs / structures / tissues / cell cultures (e.g., body segments, appendages, organs, eggs, exoskeleton, embryos, spores, mycelium, hyphae, thallus, suspension cultures, cell walls, inner or outer membranes, gametophytes, sporophytes, polymerases, glycerol-ether lipids, metabolic products, flagella, pili, ribosomes or organelles) or progeny of same. The source may be at any stage of development. In some embodiments, the NMP is produced from an insect or fungi, (e.g. cricket, yeast, or mushroom). In some embodiments, the NMP is produced from a bacteria or archaea (e.g. E. coli). In some embodiments, the NMP is produced from an algae (e.g. kelp or chlorella). In some embodiments, the NMP is produced from an animal organ (e.g. brain or blood).

[0264] NMPs can be produced and purified by a variety of methods, for example, by using a density gradient (iodixanol or sucrose) in conjunction with ultracentrifugation and / or methods to remove aggregated contaminants, e.g., precipitation or size-exclusion chromatography.

[0265] In some instances, the NMPs of the present compositions and methods can be isolated from an Arthropod, Fungi, Archaea, or Bacteria, or part thereof, and used without further modification to the NMP. In other instances, the NMP can be modified prior to use, as outlined further herein. In some instances, the NMPs are PMPs. In some instances, the NMPs of the present compositions and methods can be isolated from a plant, or part thereof, and used without further modification to the NMP. In other instances, the NMP can be modified prior to use, as outlined further herein.Lipid Reconstructed Natural Messenger Packs (LNMPs)

[0266] A lipid reconstructed NMP (LNMP) is used herein. LNMP refers to a NMP that has been derived from a lipid structure (e.g., a lipid bilayer, unilamellar, multilamellar structure; e.g., a vesicular lipid structure) derived from (e.g., enriched, isolated or purified from) a natural source, wherein 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 LNMP, as is described herein. For instance, a plant LNMP can be referred to as a LPMP, a lipid reconstructed plant messenger pack, which is derived from a plant source.

[0267] The method for a lipid reconstructing NMP (e.g., LPMP) may, if desired, further comprise sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted LNMPs. Alternatively, LNMPs (e.g., LPMPs) may be produced using a microfluidic device (such as a NanoAssemblr® IGNITE™ microfluidic instrument (Precision NanoSystems)).

[0268] In some embodiments, the LNMPs (e.g., LPMPs) are produced by a process which comprises the steps of (a) providing a plurality of purified NMPs (e.g., purified PMPs); (b) processing the plurality of NMPs (e.g., PMPs) 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, thereby producing the LNMPs (e.g., LPMPs).

[0269] In some instances, processing the plurality of NMPs (e.g., PMPs) to produce a lipid film includes extracting lipids from the plurality of NMPs, e.g., extracting lipids using the Bligh-Dyer method (Bligh and Dyer, J Biolchem Physiol, 37: 911-917, 1959). The extracted lipids may beprovided 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).Natural lipids

[0270] A LNMP may comprise between 10% and 100% lipids derived from the lipid structure from the natural source (e.g., lemon or algae), e.g., 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 LNMP may comprise all or a fraction of the lipid species present in the lipid structure from the natural source (e.g., lemon or algae), 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 LNMP may comprise none, a fraction, or all of the protein species present in the lipid structure from the natural source (e.g., lemon or algae), e.g., 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 (e.g., lemon or algae). In some instances, the lipid bilayer of the LNMP does not contain proteins. In some instances, the lipid structure of the LNMP contains a reduced number of proteins relative to the lipid structure from the natural source.

[0271] In some embodiments, the natural lipids of the LNMPs are extracted from a plant source, such as lemon or algae. In some embodiments, the natural lipids of the LNMPs are extracted from a bacteria source, such as Escherichia or Salmonella.

[0272] In some embodiments, the natural lipids of the LNMPs are extracted from lemon or algae.Exogenous lipids

[0273] The LNMPs may be modified to contain a heterologous agent (e.g., a cell-penetrating 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, bacterial cell uptake, or fungal cell uptake) relative to an unmodified LNMP. For example, the modified LNMPs 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 LNMPs may comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0274] LNMPs may include one or more exogenous lipids, e.g., lipids that are exogenous to the natural source (e.g., originating from a source that is not the source or source part from which the LNMP is produced). The lipid composition of the LNMP 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 extracted NMP lipids prior to step (b).

[0275] Exemplary exogenous lipids include ionizable lipids. The ionizable lipids in the LNMP compositions herein include one or more from the compounds of groups i)-iv) as described herein.

[0276] Exogenous lipids may also include cationic lipids.

[0277] In some instances, the exogenous lipid may also include 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 or a combination thereof.

[0278] In some embodiments, the exogenous lipid may also include 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 or 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).

[0279] In some instances, the LNMPs comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0280] In some instances, the LNMPs comprise a molar ratio of 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 some embodiments, the LNMP comprises 25% C12-200. In some embodiments, the LNMP comprises a molar ratio of 35% C12-200. In some embodiments, the LNMP comprises a molar ratio of 50% C12-200. In some embodiments, the LNMP comprises 40% MC3. In some embodiments, the LNMP comprises a molar ratio of 50% C12-200. In some embodiments, the LNMP comprises 20% or 40% DC-cholesterol. In some embodiments, the LNMP comprises 25% or 40% DOTAP.

[0281] The agent may increase uptake of the LNMP as a whole or may increase uptake of a portion or component of the LNMP (e.g., the mRNA or circRNA therapeutic) carried by the LNMP. The degree to which cell uptake is increased may vary depending on the natural source or source part to which the composition is delivered, the LNMP formulation, and other modifications made to the LNMP, For example, the modified LNMPs may have an increased cell uptake (e.g., animal celluptake, plant cell uptake, bacterial 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 LNMP. 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 LNMP.

[0282] In some embodiments, a LNMP that has been modified with an ionizable lipid more efficiently encapsulates a negatively charged a polynucleotide than a LNMP that has not been modified with an ionizable lipid. In some aspects, a LNMP that has been modified with an ionizable lipid has altered biodistribution relative to a LNMP that has not been modified with an ionizable lipid. In some aspects, a LNMP 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 LNMP that has not been modified with an ionizable lipid.Ionizable lipids

[0283] 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, 11 , 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, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or more than 18 carbon atoms in length, e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more than 25 carbon atoms in length);(iii) an acid dissociation constant (pKa) of 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 mRNA or circRNA) ratio of at least 3 (or at least 4);

[0284] 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.

[0285] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

[0286] The ionizable lipid in the LNMP compositions included one of the compounds from group i) to group iv) as discussed below.Ionizable lipid compounds i)

[0287] 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, 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 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.

[0288] In some embodiments, B is C3-C20 alkyl.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] In some embodiments, W in formula (I) may alternatively be, wherein: each Y is a divalent heterocyclic;Q is O, S, or NH; and each u is independently 1 , 2, 3, 4, or 5.

[0294] 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 CN; each v is independently 0, 1 , 2, 3, 4, or 5; and each u is independently 1 , 2, 3, 4, or 5.

[0295] 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 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;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.

[0296] 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.

[0297] In some embodiments, when Z is not absent, the adjacent R1 and R2 cannot be OH, NR10R11, or SH.

[0298] In some embodiments, the heterocyclic is a piperazine, piperazine dione, piperazine-2,5- dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic or heteroaromatic.

[0299] 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, oreach 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-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 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 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.

[0300] 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.

[0301] 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).

[0302] 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-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 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.

[0303] In some embodiments, T is a divalent heterocyclic (e.g., a divalent piperazine, or a divalentdioxopiperazine) optionally substituted with -(CH2)VOH, wherein v is independently 0, 1 , or 2.

[0304] 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-.

[2100] 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 lipids in this disclosure, and are incorporated herein by reference in its entirety.

[0305] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table I below.Table I. Exemplary ionizable lipid compounds.lonizable lipid compounds ii)

[0306] 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:is cyclic or heterocyclic moiety;Y is alkyl, hydroxy, hydroxyalkyl or;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 of X and Z is independently absent, -O-, -CO-, -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, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; each of R30, R40, R50, R60, R70, R80, 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 l and m is an integer from 1 to 10; t1 is an integer from 0 to 10; and W is hydroxyl, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl. A

[0307] In some embodiments, Y is hydroxyl ort1 W.

[0308] In some embodiments,selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane. In some ,

[0309] In some embodiments, the ionizable lipid is represented by formula:All the variables in this formula have been defined and exemplified asthose described in the above embodiments.

[0310] 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 heterocyclyl or heteroaryl; each M is independently a biodegradable moiety; each m1 is independently an integer from 3 to 6, each l1 is independently an integer from 4 to 8, m2 and l2 are each independently an integer from 0 to 3, R80 and R90 are each independently unsubstituted C5-C8 alkyl; or R80 is H or unsubstituted C1- C4 alkyl, and R90 is unsubstituted C5-C11 alkyl; and R110and R120are each independently unsubstituted C5-C8alkyl; or R110is H or unsubstituted C1-C4alkyl, and R120is unsubstituted C5-C11alkyl. 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, R80 is H or unsubstituted C1-C2 alkyl, and R90 is unsubstituted C6-C10 alkyl; and R110 and R120are each independently unsubstituted C5-C8alkyl. In some embodiments, R80, R90, R110, and R120are each independently unsubstituted C5-C8alkyl.

[0311] In some embodiments, in the above formulas, A is absent, -O-, -N(R7)-, N(R7)C(O)-,independently H, alkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, thiol, thiolalkyl, or N+(R7)3-alkylene- Q-; and R7is H or C1-C3 alkyl.

[0312] In some embodiments, in the above formulas, t1 is 0, 1 , 2, 3 or 4; and t is 0, 1 , or 2.

[0313] 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)3-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.

[0314] 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 R60is H or C1-C4branched or unbranched alkyl; R70is H; and each of R80and R90is independently H or C1-C12branched or unbranched alkyl; R100 is H; and each of R110 and R120 is independently H or C1-C12 branched or unbranched alkyl, provided that at least one of R80 and R90 is not H, and at least one of R110 and R120 is not H; l is from 3 to 7; and m is from 1 to 5.

[0315] In some embodiments, in the above formulas, Y oris: OH,,,

[0316] 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, 2023, 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.

[0317] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table II below.Ionizable lipid compounds iii)

[0318] 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, or R20and R30together 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 R1and each R2is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, or R1 and R2 are taken together to form a cyclic ring; each R10and R11is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or R10and R11are 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(R12), wherein each R12is independently H, C1-C7branched or unbranched alkyl, or C2-C7branched 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.

[0319] In some embodiments, R20and R30are each independently H or C1-C3 branched or unbranched alkyl. In some embodiments, R20and R30together with the adjacent N atom form a 3 to 7 membered cyclic ring, optionally substituted with Ra. In some embodiments, Rais H, C1-C3branched or unbranched alkyl or OH. In one embodiment, Rais H or OH.

[0320] In some embodiments, Z is absent, S, O, or NH. In some embodiments, n is 0, 1, or 2.

[0321] In some embodiments, the ionizable lipid is represented by formula (V):(V), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: R1is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR10R11,and R2is H, OH, halogen, SH, or NR10R11,or R1 and R2 are taken together to form a cyclic ring; R10and R11are each independently H or C1-C3 alkyl, or R10and R11are taken together to form a heterocyclic ring; Q is OH or -(OCH2CH2)uNR20R30, R20 and R30 are each independently H, C1-C5 branched or unbranched alkyl, or C2-C5 branched or unbranched alkenyl, or R20and R30together 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; 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-C16branched or unbranched alkyl or C2-C16branched 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.

[0322] 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.

[0323] In some embodiments, in the above formulas, X is –OC(O)-, -C(O)O-, -N(R7)C(O)-, - C(O)N(R7)-, -C(O-R13)-O-, -C(O)O(CH2)s-, -OC(O)(CH2)s-, -C(O)N(R7)(CH2)s-, -N(R7)C(O)(CH2)s-, -C(O-R13)-O-(CH2)s-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R13is independently C3-C10alkyl, 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.

[0324] 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 (IO)-(VIIO) and formulas (I)-(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.

[0325] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table III below. Table III. Exemplary ionizable lipid compounds.Ionizable lipid compounds iv)

[0326] In some embodiments, the ionizable lipid is a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI’):pharmaceutically 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; Rtis each independently H, C1-C16branched or unbranched alkyl or C1-C16branched 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.

[0327] In some embodiments, E is -OC(O)-, -C(O)O-, -N(R7)C(O)-, -C(O)N(R7)-, -C(O-R13)-O-, -C(O)O(CH2)r-, -C(O)N(R7) (CH2)r-, or -S-S-, or -C(O-R13)-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.

[0328] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII):

[0329] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (Till):(Tl II a), wherein u3 is 0, 1 , 2, 3, 4, 5, 6, or 7; 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 (Tl).

[0330] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV):

[0331] In some embodiments, the lipid comprises at least one head group and at least one tail group, 2, 3, 4, 5, 6, or 7; R7is each independently 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).

[0332] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (wherein u3 is 0, 1 , 2, 3, 4, 5, 6, or 7; and Rbis 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’).

[0333] 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).

[0334] 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.

[0335] In some embodiments, the lipid comprises two, 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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-).

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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).

[0349] 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,

[0350] 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,

[0351] In some embodiments, the head group of the lipid has a structure of formula (HA-I):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 heterocyclic 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.

[0352] In some embodiments, R20 and R30 together with the adjacent N atom form a 3 to 7 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl groups.

[0353] 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; andrepresents the bond connecting the head group to the tail group.

[0354] 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).

[0355] In some embodiments, the head group has a structure of:, wherein:Rc is H or alkyl, optionally substituted with OH; and ml is 1 , 2, or 3.

[0356] 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.

[0357] 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).

[0358] In some embodiments, in any of the above formulas (HA-V) or (HA-VI), each R20 and R30 are independently C1-C3 alkyl. In one embodiment, each R20 and R30 are independently methyl.

[0359] In some embodiments, the head group of the ionizable lipid has a structure of formula (HB-I):whereinRs is 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 R11 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;Rids 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.

[0360] In some embodiments, in formulawherein: each Re, R7, and Re are independently H or methyl; and each of u and t is independently 1 , 2, or 3.

[0361] 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; andeach of u and v is independently 1 , 2, or 3.

[0362] 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; andV is C2-C6 alkylene or C2-C6 alkenylene.each Re is independently H or methyl; each Rz is independently H; each Re is methyl; each u is independently 1 , 2, or 3; andV is C2-C6 alkylene or C2-C6 alkenylene., ,T is a divalent nitrogen-containing 5- or 6- membered heterocyclic.

[0365] In some embodiments, in formulawherein: each u is independently 1 , 2, or 3;Q is O; each Z is independently NR12; andR12 is H or C1-C3 alkyl.

[0366] In some embodiments, the head group has the structure of:independently 1 or 2.

[0367] In some embodiments, the head group of the ionizable lipid has a structure of formula (HC-I):Y( Cycle j(HC-I), wherein( Cycle )V— ' is 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.

[0368] In some embodiments, the head group has a structure of formula

[0369] 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)-.

[0200] In some embodiments, the head group has a structure of formula

[0370] In some embodiments, W is hydroxyl, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:wherein each Q is independently absent, -O-, -C(O)-, -C(S)-, -C(O)O-, -(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.

[0371] In some embodiments, W is one of the following:

[0372] In some embodiments, the ionizable lipid is represented by formula ofpharmaceutically 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 NR12; wherein R12 is C1-C7 alkyl;X’ is a biodegradable moiety.

[0373] In some embodiments, each X is. In some embodiments, X’ is-OCO-, -COO-, -NR7CO-, -CONR7-, -C(O-Ri3)-O-(acetal), -COO(CH2)S-, -CONH(CH2)S-, -C(O-Ri3)-O-(CH2)s-; wherein R7is H or C1-C3 alkyl; and R13 is C3-C10 alkyl.In some embodiments, at least one X in the formula isor o, wherein R7is H or methyl. In one embodiment, eachor, , wherein R7is H or methyl.

[0374] In some embodiments, m =3. In some embodiments, n = 0 or 1 . In some embodiments, each R Ri and R2 is H. In some embodiments, Z is absent.

[0375] In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z isNH.In some embodiments, r is 3. In some embodiments, r is 4.

[0376] 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.

[0377] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table IV below.Table IV. Exemplary ionizable lipid compounds.

[0378] In some embodiments, a lipid membrane of the LNMPs comprises at least 35% of the lipid compound from group i), e.g., at least 40%, 45%, 50% , 55%, 60%, 65%, 70% , 75% 80%, 85%, 90%, or more than 90% of the lipid compound from group i), e.g., 35%-40%, 40%-50%, 50%-60% , 60%- 70%, 70%-80% , or 80%-90% of the lipid compound from group i).

[0379] In some embodiments, a lipid membrane of the LNMPs comprises at least 35% of the lipid compound from group ii), e.g., at least 40%, 45%, 50% , 55%, 60%, 65%, 70% , 75% 80%, 85%, 90%, or more than 90% of the lipid compound from group ii), e.g., 35%-40%, 40%-50%, 50%-60% , 60%- 70%, 70%-80% , or 80%-90% of the lipid compound from group ii).

[0380] In some embodiments, a lipid membrane of the LNMPs comprises at least 35% of the lipid compound from group iii), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or more than 90% of the lipid compound from group iii), e.g., 35%-40%, 40%-50% , 50%-60%, 60%- 70%, 70%-80% , or 80%-90% of the lipid compound from group iii).

[0381] In some embodiments, a lipid membrane of the LNMPs comprises at least 35% of the lipid compound from group iii), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80% , 85%, 90%, or more than 90% of the lipid compound from group iii), e.g., 35%-40%, 40%-50% , 50%-60%, 60%- 70%, 70%-80% , or 80%-90% of the lipid compound from group iv).

[0382] In some instances, the LNMPs comprise at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0383] In some instances, the LNMPs comprise a molar ratio of 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). Other ionizable lipids

[0384] In the LNMP formulations, 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).

[0385] 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.

[0386] In some embodiments, the additional 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.

[0387] In some embodiments, the additional ionizable lipid is represented by the following formula III:wherein R is C8-C14alkyl group.

[0388] 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 C8-C18hydrocarbon (e.g., C6-C18alkyl or C6-C18alkanoyl). 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).

[0389] In some embodiments, the amine core has a structure of:.

[0390] In some embodiments, the amine core has a structure of:.

[0391] In some embodiments, the amine core has a structure of:

[0392] In some embodiments, the amine core has a structure of:

[0393] In some embodiments, the amine core has a structure of:

[0394] In some embodiments, the amine core has a structure of:

[0395] In some embodiments, the amine core has a structure of:

[0396] In some embodiments, the amine core has a structure of:

[0397] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0398] In certain embodiments, the RNA composition (e.g., the mRNA therapeutic composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0399] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0400] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0401] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0402] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0403] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0404] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0405] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0406] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structurepharmaceutically acceptable salts thereof.

[0407] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0408] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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 RNA composition (e.g., the mRNA 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.

[0409] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid, “Target 23”, having acompound structure of:, (Target 23) and pharmaceutically acceptable salts thereof.

[0410] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0411] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salt thereof.

[0412] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salt thereof.

[0413] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salt thereof.

[0414] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0415] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0416] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid of the following formula:pharmaceutically acceptable salt thereof.

[0417] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid of the following formula:(Compound 2), or a pharmaceutically acceptable salt thereof.

[0418] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid of the following formula:(Compound 3), or a pharmaceutically acceptable salt thereof.

[0419] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0420] In certain embodiments, the lipids of the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0421] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0422] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0423] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:salts thereof.

[0424] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0425] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0426] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:

[0427] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:

[0428] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compoundstructure:pharmaceutically acceptable salts thereof.

[0429] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound

[0430] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:thereof.

[0431] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:salts thereof.

[0432] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0433] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0434] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0435] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0436] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0437] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0438] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0439] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0440] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0441] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0442] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0443] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0444] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0445] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0446] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0447] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:thereof.

[0448] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0449] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:

[0450] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0451] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0452] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0453] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0454] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid of the following formula:-NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-Cs cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24 alkyl or Ce- C24 alkenyl; R3is H, OR5, ON, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4; R4is C1-C12 alkyl; R5is H or Ci-Ce alkyl; and x is 0, 1 or 2.

[0455] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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 RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0456] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0457] In some embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having the compound structure:pharmaceutically acceptable salts thereof.

[0458] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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.

[0459] In some embodiments, the lipids of the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a compound 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)2, -N(H)C(S)N(H)(R), and a heterocycle; and n is 1 , 2, or 3.

[0460] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0461] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0462] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0463] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0464] Other suitable lipids for use in the RNA composition (e.g., the mRNA composition or the circRNA 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 RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0465] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structurepharmaceutically acceptable salts thereof.

[0466] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNAcomposition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0467] In certain embodiments, the RNA composition (e.g., the mRNA composition or the circRNA composition) and methods for making and using thereof include a lipid having a compound structure of:pharmaceutically acceptable salts thereof.

[0468] In some embodiments, the LNMPs 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 W02020219876, each of which is incorporated by reference herein in its entirety.

[0469] The ionizable lipids disclosed herein may be used to form the LNMP composition together with one or more natural lipids disclosed herein. In some embodiments, the LNMP composition is formulated to further comprise one or more therapeutic agents. In some embodiments, the LNMP composition is a lipid nanoparticle that encapsulates or is associated with the one or more mRNA compositions.

[0470] In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) disclosed herein has an N / P ratio of at least 3, for instance, an N / P ratio of 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 6 to 30, 6 to 20, 6 to 15, or 6 to 12. For example, the N / P ratio may be 6 ± 1 , or the N / P ratio may be 6 ± 0.5. In some embodiments, the N / P ratio is about 6. In some embodiments, the N / P ratio is about 3 (e.g., 3 ± 1 or 3 ± 0.5). In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) has an N / P ratio of about 12 toabout 17, 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. In some embodiments, the N / P ratio is about 12 (e.g., 12 ± 1 or 12 ± 0.5).

[0471] In some embodiments, the disclosure relates to a composition comprising (i) one or more compounds chosen from the ionizable lipids of Formula (l)-(lll), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing and (ii) a lipid component. In some embodiments, the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the one or more compounds.

[0472] In some embodiments, the disclosure relates to a composition comprising (i) one or more lipid nanoparticles and (ii) one or more lipid components.

[0473] In some embodiments, one or more lipid components comprise one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component(s) comprise(s) one or more helper lipids, one or more PEG lipids, and one or more neutral lipids.

[0474] Non-limiting examples of neutral lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids may be acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0475] In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) comprises a phytosterol or a combination of a phytosterol and cholesterol. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol, stigmasterol, b- sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151 , Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175 and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of Compound S-140, Compound S-151 , Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In someembodiments, the phytosterol is a combination of Compound S-141 , Compound S-140, Compound S- 143 and Compound S-148. In some embodiments, the phytosterol comprises a sitosterol or a salt or an ester thereof. In some embodiments, the phytosterol comprises a stigmasterol or a salt or an ester thereof.Other lipids and other agents

[0476] The exogenous lipid may be a cell-penetrating agent, may be capable of increasing delivery of a polypeptide by the LNMP 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.

[0477] The LNMPs can be modified with other components (e.g., lipids, e.g., sterols, e.g., cholesterol; or small molecules) to further alter the functional and structural characteristics of the LNMP. For example, the LNMPs can be further modified with stabilizing molecules that increase the stability of the LNMPs (e.g., for at least one day at room temperature, and / or stable for at least one week at 4°C).

[0478] In some embodiments, the LNMP is modified with 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 extracted NMP 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.

[0479] In some embodiments, the sterol is cholesterol or sitosterol. In some instances, the LNMPs 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 LNMP 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 LNMP comprises a molar ratio of about 20%-40% sterol.

[0480] In some embodiments, a LNMP that has been modified with a sterol has altered stability (e.g., increased stability) relative to a LNMP that has not been modified with a sterol. In some aspects, a LNMP that has been modified with a sterol has a greater rate of fusion with a membrane of a target cell relative to a LNMP that has not been modified with a sterol.

[0481] In some instances, the LNMPs comprise an exogenous lipid and an exogenous sterol.

[0482] In some embodiments, the LNMP is modified with 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 LNMPs comprise a molar ratio of 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. In some embodiments, the LNMP comprises a molar ratio of 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. In some embodiments, a LNMP that has been modified with a PEGylated lipid has altered stability (e.g., increased stability) relative to a LNMP that has not been modified with a PEGylated lipid. In some embodiments, a LNMP that has been modified with a PEGylated lipid has altered particle size relative to a LNMP that has not been modified with a PEGylated lipid. In some embodiments, a LNMP that has been modified with a PEGylated lipid is less likely to be phagocytosed than a LNMP 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.

[0483] In some embodiments, the LNMPs are modified with 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).

[0484] In some embodiments, the modified LNMPs comprise a molar ratio of about 5%-50% LNMP lipids (e.g., about 10%-20% LNMP lipids, e.g., about 10%, 12.5%, 16%, or 20% LNMP 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).

[0485] In some embodiments, the modified LNMPs comprise a molar ratio of about 5%-60% LNMP lipids (e.g., about 10%-20%, 20%-30%, 30%40%, 40%-50%, or 50%-60% LNMP lipids, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60% LNMP lipids); 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).

[0486] In some embodiments, the ionizable lipids, LNMP 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 NMP.

[0487] In some embodiments, the ionizable lipids, natural 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 NMP.

[0200] In some embodiments, the ionizable lipids, natural 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 NMP.

[0488] In some embodiments, the ionizable lipids, natural lipids, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:12.5:2.5.

[0489] In some embodiments, the ionizable lipids, natural lipids, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:1 1 .5:3.5.

[0490] In some embodiments, the ionizable lipids, natural lipids, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:20:42.5:2.5.

[0491] In some embodiments, a LNMP 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 LNMP that has not been modified with an ionizable lipid (and / or cationic lipid) and a sterol and / or a PEGylated lipid. The modified LNMP may have an encapsulation efficiency for the cargo (e.g., 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., may have an encapsulation efficiency of 5%-30%, 30%-50%, 50%-70%, 70%-80%, 80%-90%, 90%-95%, or 95%- 100%.

[0492] Cell uptake of the modified LNMPs can be measured by a variety of methods known in the art. For example, the LNMPs, 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.

[0493] In some embodiments, a LNMP formulation provided herein comprises two or more different modified LNMPs, e.g., comprises modified LNMPs derived from different unmodified LNMPs (e.g., unmodified LNMPs from two or more different natural sources) and / or comprises modified LNMPs comprising different species and / or different ratios of ionizable lipids, sterols, and / or PEGylated lipids.

[0494] 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.

[0495] 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 nucleic acid (e.g., an siRNA or siRNA precursor (e.g., dsRNA), miRNA or miRNA precursor, mRNA, circRNA, or plasmid (pDNA)) or a small molecule.

[0496] 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.LNMPs may optionally include additional agents, e.g., cell-penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. The LNMPs can carry or associate with additional agents in a variety of ways to enable delivery of the agent to a target plant or animal, 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. Nucleic acid molecules can be incorporated into the LNMPs either in vivo or in vitro (e.g., in tissue culture, in cell culture, or synthetically incorporated).Zeta Potential

[0497] The LNMPs comprising an ionizable lipid 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 LNMP 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 LNMP has a positive zeta potential, e.g., azeta 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 LNMP has a zeta potential of about 0.

[0498] The zeta potential of the LNMP 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.Plant EV-Markers

[0499] The LNMPs in the RNA composition (e.g., mRNA composition or circRNA composition) and methods of making and using thereof may have a range of markers that identify the LNMPs (e.g. LPMPs) as being produced using a plant EV, and / or including a segment, portion, or extract thereof. As used herein, the term “plant EV-marker” refers to a component that is naturally associated with a plant and incorporated into or onto the plant EV in planta, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof. Examples of plant EV-markers can be found, for example, in Rutter and Innes, Plant Physiol. 173(1): 728-741 , 2017; Raimondo et al., Oncotarget. 6(23): 19514, 2015; Ju et al., Mol. Therapy. 21 (7):1345-1357, 2013; Wang et al., Molecular Therapy. 22(3): 522-534, 2014; and Regente et al, J of Exp. Biol. 68(20): 5485-5496, 2017; each of which is incorporated herein by reference.

[0500] Additional examples of the suitable plant EV-markers include those described and listed in International Patent Application Publication No. WO 2021 / 041301 , which is incorporated herein by reference in its entirety.Bacterial EV-Markers

[0501] The bacterial components (e.g., bacterial lipids) in the bacteria-derived lipid composition and methods of making and using thereof may have a range of markers that identify the bacterial component as being produced. As used herein, the term “bacterial EV-marker” refers to a component that is naturally associated with a bacterium and incorporated into or onto the bacterial EV, such as a bacterial protein, a bacterial nucleic acid, a bacterial small molecule, a bacterial lipid, or a combination thereof.Natural Source EV-Markers

[0502] The NMPs may have a range of markers that identify the NMP as being produced from a specific source EV, and / or including a segment, portion, or extract thereof. As used herein, the term “EV-marker” refers to a component that is naturally associated with a specific source and incorporated into or onto the EV in vivo, such as a protein, a nucleic acid, a small molecule, a lipid, or a combination thereof. Examples of source EV-markers include but are not limited to peptidoglycan, lipopolysaccharide, ester-linked lipids, ether-linked lipids, circular DNA, chitin, beta-glucan, pekilo,mycoprotein, cerato-platanins, exotoxins, diacylglycerol, triglycerides, phosphatidylcholine, phosphatidylinositol, ornithine lipids, glycolipids, sphingolipids, hopanoids, or ergosterol.

[0503] The source EV marker can include a lipid. Examples of lipid markers that may be found in the NMP include lipid A, lipopolysaccharide, ergosterol, ornithine lipids (OLs), sulfolipids, diacylglyceryl- N,N,N-trimethylhomoserine (DGTS), glycolipids (GLs), diacylglycerol (DAG), hopanoids (HOPs), glucosyleramide, sterylglycosides, ether-linked lipids, or a combination thereof.

[0504] Other EV markers may include lipids that accumulate in sources in response to abiotic or biotic stressors.

[0505] Alternatively, the source EV marker may include a protein. In some instances, the protein EV marker may be an antimicrobial or antiviral protein naturally produced by the source, including proteins that are secreted in response to abiotic or biotic stressors. Some examples of protein EV markers include but are not limited to cecropins, moricins, defensins, proline- and glycine-rich peptides, fungal immunomodulatory proteins, flagellin, encapsulin, streptavidin, internalin, pilin, halocin, or archaeocins. In some instances, the EV marker can include a protein involved in lipid metabolism. In some instances, the protein EV marker is a cellular trafficking protein in the source. In certain instances where the EV marker is a protein, the protein marker may lack a signal peptide that is typically associated with secreted proteins. Unconventional secretory proteins seem to share several common features like (i) lack of a leader sequence, (ii) absence of PTMs specific for ER or Golgi apparatus, and / or (iii) secretion not affected by brefeldin A which blocks the classical ER / Golgi- dependent secretion pathway. One skilled in the art can use a variety of tools freely accessible to the public to evaluate a protein for a signal sequence, or lack thereof.

[0506] In instances where the EV marker is a protein, the protein may have an amino acid sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a known EV marker.

[0507] In some instances, the EV marker includes a nucleic acid encoded in the source, e.g., an Arthropod, Plant, Fungi, Archaea, or Bacteria RNA, DNA, or PNA. For example, the NMP may include dsRNA, mRNA, a circular RNA (circRNA), a viral RNA, a microRNA (miRNA), or a small interfering RNA (siRNA) encoded by the source. In some instances, the nucleic acid may be one that is associated with a protein that facilitates the long-distance transport of RNA. In some instances, the nucleic acid EV marker may be one involved in host-induced gene silencing (HIGS), which is the process by which a source silences foreign transcripts of pathogens. In some instances, the nucleic acid may be a microRNA.

[0508] In instances where the EV marker is a nucleic acid, the nucleic acid may have a nucleotide sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a known EV marker.

[0509] In some instances, the EV marker includes a compound produced by the source. For example, the compound may a component of the cell wall (e.g. lipopolysaccharide). For example, the compound may be a defense compound produced in response to abiotic or biotic stressors, such as pathogens or extreme environmental stress.

[0510] In some instances, the NMP may also be identified as being produced from a source EVbased on the lack of certain markers (e.g., lipids, polypeptides, or polynucleotides) that are not typically produced by these sources, but are generally associated with other organisms (e.g., markers of animal EVs or plant EVs). For example, in some instances, the NMP lacks lipids typically found in animal EVs or plant EVs.

[0511] EV markers can be identified using any approaches known in the art that enable identification of small molecules (e.g., mass spectroscopy, mass spectrometry), lipids (e.g., mass spectroscopy, mass spectrometry), proteins (e.g., mass spectroscopy, immunoblotting), or nucleic acids (e.g., PCR analysis). In some instances, a NMP composition described herein includes a detectable amount, e.g., a pre-determined threshold amount, of an EV marker described herein.Loading of Agents (e.g., nucleic acids)

[0512] The LNMPs are modified to include a therapeutic agent (e.g., a nucleic acid molecule) to form the RNA composition (e.g., mRNA composition or circRNA composition). The LNMPs 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), e.g., by encapsulating the agent, incorporation of the component in the lipid bilayer structure, or association of the component (e.g., by conjugation) with the surface of the lipid bilayer structure of the LNMP. In some instances, the agent is included in the LNMP formulation, as described herein.

[0513] The agent can be incorporated or loaded into or onto the LNMPs by any methods known in the art that allow association, directly or indirectly, between the LNMPs and agent. The agents can be incorporated into the LNMPs by an in vivo method (e.g., in planta, e.g., through production of LNMPs from a transgenic plant that comprises the agent), or in vitro (e.g., in tissue culture, or in cell culture), or both in vivo and in vitro methods.

[0514] In some instances, the LNMPs are loaded in vitro. The substance may be loaded onto or into (e.g., may be encapsulated by) the LNMPs 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 LNMPs by one or more of electroporation, sonication, passive diffusion, stirring, lipid extraction, or extrusion. In some instances, the agent is incorporated into the LNMP using a microfluidic device, e.g., using a method in which LNMP lipids are provided in an organic phase, the heterologous functional agent is provided in an aqueous phase, and the organic and aqueous phases are combined in the microfluidics device to produce a LNMP comprising the heterologous functional agent. Loaded LNMPs 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 substance of interest into LNMPs is not limited to the above-illustrated methods.

[0515] In some instances, the agent can be conjugated to the LNMP, in which the agent is connected or joined, indirectly or directly, to the LNMP. For instance, one or more agents can be chemically linked to a LNMP, such that the one or more agents are joined (e.g., by covalent or ionic bonds) directly to the lipid bilayer of the LNMP. In some instances, the conjugation of various agentsto the LNMPs can be achieved by first mixing the one or more agents with an appropriate crosslinking 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 LNMPs 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 LNMPs from the agent conjugated to the LNMPs. As part of combining the mixture with a sucrose gradient, and an accompanying centrifugation step, the LNMPs conjugated to the agent are then seen as a band in the sucrose gradient, such that the conjugated LNMPs can then be collected, washed, and dissolved in a suitable solution for use as described herein.

[0516] In some instances, the LNMPs are stably associated with the agent prior to and following delivery of the LNMP, e.g., to an animal. In other instances, the LNMPs are associated with the agent such that the agent becomes dissociated from the LNMPs following delivery of the LNMP, e.g., to an animal.

[0517] The LNMPs can be loaded or the LNMP can be formulated with various concentrations of the agent, depending on the particular agent or use. For example, in some instances, the LNMPs are loaded or the LNMP is formulated such that the LNMP formulation disclosed herein includes 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 LNMPs are loaded or the LNMP is formulated such that the LNMP formulation includes 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 LNMP formulation 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 LNMP can be loaded or the LNMP is 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 LNMP of the invention can be loaded or a LNMP can be formulated with about 2,000, 1 ,000, 500, 200, 100, 50, 10, 5, 1 (or any range between about 2,000 and 1) or less pg / ml of an agent.

[0518] In some instances, the LNMPs are loaded or the LNMP is formulated such that the LNMP formulation disclosed herein includes 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 an agent. In some instances, the LNMP can be loaded or the LNMP can be 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 an agent.

[0519] In some instances, the LNMP is formulated with the agent by suspending the LNMPs in a solution comprising or consisting of the agent, e.g., suspending or resuspending the LNMPs 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.Pharmaceutical Formulations

[0520] The modified LNMPs are formulated into pharmaceutical compositions (i.e. , an RNA composition such as a mRNA composition or a circRNA 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.

[0521] The LNMP / RNA composition (e.g., mRNA composition or circRNA 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)).

[0522] 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 LNMP / RNA composition (e.g., mRNA composition or circRNA 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., LNMPs and nucleic acids) to be administered, and the route of administration.

[0523] For oral administration to an animal, the LNMP / RNA composition (e.g., mRNA composition or circRNA 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.

[0524] For parenteral administration to an animal, the LNMP / RNA composition (e.g., mRNA composition or circRNA 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), and F-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).Polynucleotides

[0525] The LNMP / RNA composition (e.g., mRNA composition or circRNA composition) includes one or more nucleic acid molecules, e.g., polynucleotides, which encode one or more wild type or engineered proteins, peptides, or polypeptides. Exemplary polynucleotides, e.g., polynucleotide constructs, include antigen - encoding RNA polynucleotides, e.g., mRNAs, linear polyribonucleotides, or circRNAs.

[0526] Examples of polypeptides that can be used herein can include an enzyme (e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, a DNAse, or an ubiquitination protein), a poreforming 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.

[0527] Polypeptides included herein 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.

[0528] The polypeptide may have a length from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 aminoacids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1 ,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptide has a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1 ,500 amino acids, less than about 1 ,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.

[0529] The LNMP / RNA composition (e.g., mRNA composition or circRNA composition) 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 LNMP / mRNA composition depends on factors such as efficacy, stability of the polypeptide, number of distinct polypeptides in the formulation, and methods of application of the formulation. In some instances, each polypeptide in a liquid formulation is from about 0.1 ng / mL to about 100 mg / mL. In some instances, each polypeptide in a solid formulation is from about 0.1 ng / g to about 100 mg / g.Nucleic Acids Encoding Peptides

[0530] In some instances, the LNMP / RNA composition (e.g., mRNA composition or circRNA composition) include a heterologous 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 therebetween.

[0531] The LNMP / RNA composition (e.g., mRNA composition or circRNA 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 the entire sequence, to a sequence of a nucleic acid of interest. In some instances, the invention includes an active polypeptide encoded by a nucleic acid variant as described herein. 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.

[0532] 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 bacterial, 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.

[0533] 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.

[0534] 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.

[0535] 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.

[0536] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence 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 virusimmediate 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.

[0537] 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 to 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.

[0538] 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.

[0539] 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.

[0540] 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 instance, provided is 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

[0541] The LNMP / mRNA 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, bacterial 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).

[0542] 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.

[0543] 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.

[0544] 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.

[0545] 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, which are incorporated herein by reference in their entirety. 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 nucleotides in length via a scalable method (WO 2014 / 144767); and subjecting a modified mRNA sample to DNAse treatment (WO 2014 / 152030).

[0546] 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 (reLNPs) and combinations thereof.

[0547] Modified RNAs encoding polypeptides in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are discl...

Claims

What is claimed is:1 . A method for delivering an RNA composition to a subject for in vivo production of a protein or a peptide in the subject, comprising: administering to the subject an RNA composition comprising a polynucleotide that encodes a protein or a peptide, formulated within(a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid, or(b) a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid, wherein the ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable amine functional groups;(ii) at least three lipid tails, wherein each of the lipid tails is at least six carbon atoms in length;(iii) a pKa of about 4.5 to about 7.5;(iv) an ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms; and(v) an N:P ratio of at least 3.

2. The method of claim 1 , wherein the RNA composition is administered orally or enterally.

3. The method of claim 1 , wherein the RNA composition is administered systemically.

4. The method of claim 1 , wherein the polynucleotide is an mRNA or circRNA, and optionally wherein the mRNA or circRNA is derived from(a) a DNA molecule; or(b) an RNA molecule, wherein T is substituted with U.

5. The method of claim 1 , wherein the protein or peptide comprises an antibody.

6. The method of claim 5, wherein the antibody is a therapeutic, a TNF inhibitor, or PCSK9 inhibitor.

7. The method of claim 1 , wherein the in vivo production of the protein or the peptide occurs in the subject’s stomach, small intestine, mesenteric lymph node, pancreas, colon, caecum, and / or spleen.

8. The method of claim 1 , wherein the administering of the RNA composition results in expression of the protein or the peptide encoded by the polynucleotide detectable in one or more organs in the subject at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration, and wherein the organs are present along the transit route of the digestive track, and the RNA composition accesses the organs through the lymphatic transport system.

9. The method of claim 1 , wherein the protein or the peptide encoded by the polynucleotide is detectable in the subject’s mesenteric lymph node, pancreas, stomach, colon, small intestine, spleen, villi, and / or Peyer’s patches at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

10. The method of claim 1 , wherein the protein or the peptide encoded by the polynucleotide is not detectable in the subject’s liver at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

11. A method of treating a disease or disorder associated with the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph node, pancreas, colon or rectum, caecum, and / or spleen, comprising: orally or enterally administering an RNA composition having a polynucleotide encoding one or more polypeptides formulated within(a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid, or(b) a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid, wherein the ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable amine functional group;(ii) at least three lipid tails, wherein each of the lipid tails is at least six carbon atoms in length;(iii) a pKa of about 4.5 to about 7.5;(iv) an ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms; and(v) an N:P ratio of at least 3.

12. The method of claim 11 , wherein the disease or disorder is pancreatitis, IBD, Crohn’s disease, colorectal cancer, or ulcerative colitis.

13. The method of any one of claims 1-12, wherein the RNA composition is formulated within (a) the plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and the ionizable lipid.

14. The method of any one of claims 1-12, wherein the RNA composition is formulated within (b) the lipid reconstructed natural messenger pack (LNMP) comprising natural lipids and the ionizable lipid.

15. The method of any one of the proceeding claims, wherein the RNA composition is administered to the subject in a delayed-release pharmaceutical dosage form comprising (a) a therapeutically effective amount of the polynucleotide; (b) a bile salt or bile acid; and (c) at least one surfactant selected from hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.effective amount of the polynucleotide; (b) a bile salt or bile acid; and (c) at least one surfactant selected from hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.

16. The method of claim 15, wherein the RNA composition is administered in the form of a capsule.

17. The method of claim 16, wherein the capsule is a starch capsule, a cellulosic capsule, a hard gelatin capsule, or a soft gelatin capsule.

18. The method of claim 15, wherein the RNA composition is administered in the form of a tablet or caplet.

19. The method of any one of claims 16-18, wherein the capsule, tablet, or caplet contains an enteric coating.

20. The method of claim 15, wherein the RNA composition is administered in the form of a plurality of particles, granules, beads, pellets, or mixtures thereof.

21. The method of claim any one of the proceeding claims, 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.

22. The method of any one of cla of the following groups of compounds:i) a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: each A is independently C1-C16branched or unbranched alkyl or C1-C16branched 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 SH, or halogen;, ; or , wherein R5is OH, SH, or NR10R11; each R6is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, or cycloalkyl; each R7and each R8is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, or NR10R11, wherein each R10and R11is independently H, C1-C3alkyl, or R10and R11are taken together to form a heterocyclic ring; each s is independently 1, 2, 3, 4, or 5; each u is indepe t is 1, 2, 3, 4 or 5 each Z is indepe R12, wherein R12is H, C1-C7branched or unbranched alkyl, or C2-C7branc , and Q is O, S, or NR1r C1-C5 alkyl;X Z R R 50 30R60M M R40 lmR R12070110ii) a compound of formula(II), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: Cycle is a cyclic or heterocyclic moiety; A t1Y is alkyl, hydroxy, hydroxyalkyl orW; 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 of X and Z is independently absent, -O-, -CO-, -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, hydroxyalkyl, or aminoalkyl; each M is independently a biodegradable moiety; each of R30, R40, R50, R60, R70, R80, R90, R100, R110, and R120 is independently H, C1-C16 branched or unbranched alkyl or C1-C16branched or unbranched alkenyl, optionally interrupted with heteroatom or substituted with OH, SH, or halogen, or cycloalkyl or substituted cycloalkyl; each of l and m is an inte f 1 t 10 t1 is an integer from 0 to W is hydroxyl, substitutetituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl;R1R2A X B Z N R20N n A X B R iii) a compound of formula30R1R2 Y(III) or A X B Q y n vNR1R2A X B (V), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein: R20and R30are each independently H, C1-C5branched or unbranched alkyl, or C2-C5branched or unbranched alkenyl, or R20and R30together 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 R1and each R2is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, OH, halogen, SH, or NR10R11, or R1and R2are taken together to form a cyclic ring; each R10and R11is independently H, C1-C3branched or unbranched alkyl, C2-C3branched or unbranched alkenyl, or R10and R11are 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(R12), wherein each R12is independently H, C1-C7branched or unbranched alkyl, or C2-C7branched or unbranched alkenyl, provided that when Z is not absent, the adjacent R1and R2cannot 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-C16branched or unbranched alkyl, or C2-C16branched 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 unbranc substituted with OH, SHiv) a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI’) Rtu2tE E R u1aau2Rtu1 a (TI) orRaR RR Rt(TI’), a 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-R13)-O-, -C(O)O(CH2)r-, -C(O)N(R7)(CH2)r-, -S-S-, or -C(O-R13)-O-(CH2)r-, wherein each R7is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R13is branched or unbranched C3-C10alkyl; r is 1, 2, 3, 4, or 5; each independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; nd u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; Rtis each independently H, C1-C16branched or unbranched alkyl or C1-C16branched 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.

23. able II, Table III, or T 24.or .

25. The method of claim 14, wherein the natural lipids of the LNMP are extracted from lemon or algae.

26. The method of claim 13 or 14, wherein the LNMP or the LNP composition further comprise a sterol and a polyethylene glycol (PEG)-lipid conjugate.

27. The method of claim 26, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

28. The method of claim 27, wherein the PEG-DMG is PEG2000-DMG or PEG2000-PE.

29. The method of claim 26, wherein the LNMP comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 5 mol% to about 60 mol% of the natural lipids, about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

30. The method of claim 29, wherein the LNMP comprises ionizable lipid : natural lipids:sterol:PEG-lipid at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, 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 .5.31 . The method of claim 13, wherein the synthetic structural lipid of the LNP composition is a phospholipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol- phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

32. The method of claim 26, wherein the LNP composition comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 5 mol% to about 60 mol% of the synthetic structural lipids,about 7 mol% to about 50 mol% of the sterol, and about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

33. The method of claim 32, wherein the LNP composition comprises ionizable lipid: synthetic structural lipids:sterol:PEG-lipid at a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, 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, about45:10:43.5:1 .5, about 50:20:28.5:1 .5, or about 50:10:38.5:1 .5.

34. The method of claim 1 , wherein the RNA composition has a total lipid : polyn ucleotide weight ratio ranging from about 50:1 to about 10:1.

35. The method of claim 34, wherein the RNA composition has a total lipid :polynucleotide weight ratio ranging from about 40:1 to about 28:1 .

36. The method of claim 34, wherein the RNA composition has a total lipid :polynucleotide weight ratio ranging from about 37:1 to about 33:1 .

37. An oral vaccination composition, comprising an RNA composition comprising a polynucleotide encoding one or more polypeptides formulated within(a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and comprising an ionizable lipid, or(b) a lipid reconstructed natural messenger packs (LNMPs) comprising natural lipids and an ionizable lipid, wherein the ionizable lipid has two or more of the characteristics listed below:(i) at least one ionizable functional group amine;(ii) at least three lipid tails, wherein each of the lipid tails is at least six carbon atoms in length;(iii) a pKa of about 4.5 to about 7.5;(iv) an ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms; and(v) an N:P ratio of at least 3, wherein the RNA composition is formulated in an oral dosage form.

38. The oral vaccination composition of claim 37, wherein the polypeptide is an antigenic polypeptide derived from an infectious agent that causes a viral or bacterial infection, optionally wherein the antigenic polypeptide is a corona virus.

39. The oral vaccination composition of claim 37, wherein the polypeptide is a tumor antigenic polypeptide.

Images