RNA compositions comprising lipid nanoparticles or lipid reconstituted native messenger packages

Abstract

Disclosed herein are RNA compositions comprising one or more polynucleotides encoding one or more gene editing systems, the one or more polynucleotides being formulated within a lipid reconstituted native messenger packet (LNMP) comprising a native lipid and an ionizable lipid. The disclosure also includes a method for preparing an RNA composition comprising reconstituting a membrane comprising a purified NMP lipid in the presence of an ionizable lipid to produce an LNMP comprising the ionizable lipid, and loading one or more polynucleotides encoding one or more gene editing systems into the LNMP. The present disclosure also includes RNA compositions that can be repeatedly administered.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 424,208, filed on November 10, 2022; U.S. Provisional Application No. 63 / 447,494, filed on February 22, 2023; and U.S. Provisional Application No. 63 / 527,780, filed on July 19, 2023, all of which U.S. Provisional Applications are incorporated herein by reference in their entirety. Background of the Invention

[0003] mRNA therapeutics in commerce or development typically are based on whole microorganisms, protein antigens, peptides, polysaccharides, or deoxyribonucleic acid (DNA) vaccines and combinations thereof. The use of RNA polynucleotides as therapeutics is an emerging field.

[0004] Accordingly, there is a need to develop an enhanced RNA delivery system to enable more effective, more easily scalable, and more stable delivery of RNA therapeutics. Summary of the Invention

[0005] In one aspect, the present disclosure provides an RNA composition for gene editing, the RNA composition comprising one or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems. The one or more polynucleotides are formulated within a composite lipid particle (CLP), such as a lipid - reconstituted native messenger package (LNMP) comprising natural lipids and ionizable lipids. The ionizable lipid has two or more of the following listed properties:

[0006] (i) At least 2 ionizable amines;

[0007] (ii) At least 3 lipid tails, wherein each lipid tail has a length of at least 6 carbon atoms;

[0008] (iii) A pKa of about 4.5 to about 7.5;

[0009] (iv) An ionizable amine and a hetero - organic group separated by a chain having at least two atoms; and

[0010] (v) An N:P ratio of at least 3 (or at least 4).

[0011] On the other hand, the present disclosure provides a method for preparing an RNA composition. The method includes reconstructing a membrane comprising purified NMP lipid in the presence of an ionizable lipid to produce a lipid-reconstituted native messenger particle (LNMP) comprising the ionizable lipid described herein. The method further comprises loading one or more polynucleotides encoding one or more components of a gene editing system or one or more gene editing systems into the LNMP.

[0012] On the other hand, the present disclosure provides an RNA composition for gene editing, the RNA composition comprising:

[0013] one or more polynucleotides encoding one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides being formulated within a plurality of lipid nanoparticles (LNPs) comprising a synthetic structural lipid and an ionizable lipid.

[0014] In all these aspects of the present invention, the ionizable lipid can be selected from one of the following groups of compounds:

[0015] i) a compound of formula its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0016] wherein:

[0017] each A is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, the alkyl or alkenyl being optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0018] each B is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, the alkyl or alkenyl being optionally substituted with a heteroatom or substituted with OH, SH or a halogen;

[0019] each X is independently a biodegradable moiety; and

[0020] W is wherein:

[0021] R5 is OH, SH, NR 10 R 11 ;

[0022] each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl or a cycloalkyl;

[0023] Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, NR 10 R 11 , where each R 10 and R 11 are independently H, a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[0024] Each s is independently 1, 2, 3, 4, or 5;

[0025] Each u is independently 1, 2, 3, 4, or 5;

[0026] t is 1, 2, 3, 4, or 5;

[0027] Each Z independently does not exist, is O, S, or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl group, or a C2-C7 branched or unbranched alkenyl group, provided that when Z does not not exist, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH; and

[0028] Q is O, S, or NR 13 , where each R 13 is H, a C1-C5 alkyl group;

[0029] ii) A compound of formula , a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:

[0030] is a cyclic or heterocyclic moiety;

[0031] Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group, or

[0032] A does not exist, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S-, or a divalent heterocycle;

[0033] Each of X and Z independently does not exist, is -O-, -CO-, -N(R 7)-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0034] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl or aminoalkyl;

[0035] Each M is independently a biodegradable moiety;

[0036] R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 and R 120 each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interrupted by a heteroatom or being substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0037] Each of l and m is an integer from 1 to 10;

[0038] t1 is an integer from 0 to 10; and

[0039] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic or heteroaryl; and

[0040] iii) A compound of formula

[0041] , its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein:

[0042] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl or C2-C5 branched or unbranched alkenyl, or R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, said ring optionally being substituted by R a ;

[0043] R a is H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH or SH;

[0044] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 ; or

[0045] R1 and R2 together form a ring;

[0046] Each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or R 10 and R 11 together form a heterocycle;

[0047] n is 0, 1, 2, 3 or 4;

[0048] Y is O or S;

[0049] Z is absent, is O, S or N(R 12 )(R 12 ), where each R 12 is independently H, a C1-C7 branched or unbranched alkyl group or a C2-C7 branched or unbranched alkenyl group, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH;

[0050] u is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0051] v is 0, 1, 2, 3 or 4;

[0052] y is 0, 1, 2, 3 or 4;

[0053] Each A is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or a halogen;

[0054] Each B is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or a halogen; and

[0055] Each X is independently a biodegradable moiety; and

[0056] iv) A lipid, said lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'):

[0057] Its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0058] Wherein:

[0059] E is each independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl;

[0060] R 13 is branched or unbranched C3-C 10 alkyl;

[0061] r is 1, 2, 3, 4 or 5;

[0062] R a are each independently C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0063] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0064] R t are each independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interrupted by heteroatoms or being substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0065] represents the bond connecting the tail group to the head group; and

[0066] wherein the pKa of the lipid is from about 4 to about 8.

[0067] In some embodiments, the ionizable lipid is a compound of group i) represented by the formula its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein:

[0068] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl group, OH, a halogen, SH, or NR 10 R 11 , or

[0069] each R1 and each R2 together with the carbon atom to which they are attached independently form a ring;

[0070] each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl group, or R 10 and R 11 together form a heterocycle;

[0071] Each R3 and each R4 are independently H, a C2-C 14 branched or unbranched alkyl group (e.g., a C3-C 10 branched or unbranched alkyl group) or a C3-C 10 branched or unbranched alkenyl group, provided that at least one of R3 and R4 is not H;

[0072] Each X is independently a biodegradable moiety;

[0073] Each q is independently 2, 3, 4, or 5;

[0074] V is a branched or unbranched C2-C 10 alkylene group, a C2-C 10 alkenylene group, a C2-C 10 alkynylene group, or a C2-C 10 heteroalkylene group, wherein the alkylene group, alkenylene group, alkynylene group, or heteroalkylene group is optionally substituted by one or more OH, SH, and / or halogen groups;

[0075] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0076] Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 , wherein each v is independently 0, 1, 2, 3, 4, or 5, and R 17 is OH, SH, or N(CH3)2; and

[0077] 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 group, and each R6 is independently H or methyl.

[0078] In some embodiments, the ionizable lipid is a compound of Group (i) represented by the formula , a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing, wherein:

[0079] Each R1 and each R2 is independently H, a C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 , or

[0080] Each R1 and each R2 independently together with the carbon atom to which it is attached forms a ring;

[0081] Each R 10 and R 11 is independently H, a C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle;

[0082] Each R3 and each R4 is independently H, a C2-C 14 branched or unbranched alkyl (e.g., a C3-C 10 branched or unbranched alkyl) or a C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0083] Each X is independently a biodegradable moiety;

[0084] Each s is independently 1, 2, 3, 4, or 5;

[0085] T is –NHC(O)O–, –OC(O)NH–, or a divalent heterocycle optionally substituted with one or more –(CH2) v OH, –(CH2) v SH, –(CH2) v -halo groups;

[0086] Each R7 and each R8 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 , wherein R 17 is OH, SH, or N(CH3)2;

[0087] Each v is independently 0, 1, 2, 3, 4, or 5; and

[0088] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, T is a divalent piperazine or divalent dioxopiperazine.

[0089] In some embodiments, in the above formula of group i), X is -OCO-, -COO-, -NHCO-, or -CONH-.

[0090] In some embodiments, the ionizable lipid is a compound of group ii) represented by one of the following formulas:

[0091]

[0092] Wherein:

[0093] Each m1 is independently an integer from 3 to 6,

[0094] Each l1 is independently an integer from 4 to 8,

[0095] m2 and l2 are each independently an integer from 0 to 3,

[0096] R 80 and R 90 are each independently an unsubstituted C5-C8 alkyl or alkenyl; or R 80 is H or an unsubstituted C1-C4 alkyl or alkenyl, and R 90 is an unsubstituted C5-C 11 alkyl or alkenyl; and

[0097] R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl or alkenyl; or R 110 is H or an unsubstituted C1-C4 alkyl or alkenyl, and R 120 is an unsubstituted C5-C 11 alkyl or alkenyl. In some embodiments, M is -OC(O)- or -C(O)O-;

[0098] is: OH,

[0099] Each R c is independently H or a C1-C3 alkyl;

[0100] Each t1 is independently 1, 2, 3, or 4;

[0101] R 80 and R 90 each independently is H or a C1-C 12 branched or unbranched alkyl; and

[0102] R 110 and R 120 each independently is H or C1-C 12 branched or unbranched alkyl, provided that R 80 and R 90 at least one of which is not H, and R 110 and R 120 at least one of which is not H.

[0103] In some embodiments, the ionizable lipid is a compound of Group (iii), wherein R1 and R2 are each H, or each R1 is H and one of the R2 variables is OH; and X is –OC(O)- or –C(O)O-. In some embodiments, the ionizable lipid is a compound of Group (iii) represented by formula (III), wherein R 20 and R 30 each independently is H or C1-C3 branched or unbranched alkyl; or R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, which ring is optionally substituted by R a substituted; R a is H or OH; Z is absent, is 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).

[0104] 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:

[0105] the tail group has a structure of formula (TI) or formula (TI'):

[0106] and

[0107] the head group has a structure of one of the following formulas:

[0108] i)

[0109] wherein:

[0110] R 20 and R 30 each independently is H, C1-C5 branched or unbranched alkyl or C2-C5 branched or unbranched alkenyl, the alkyl or alkenyl optionally being interrupted by one or more heteroatoms or being substituted by OH, SH, halogen or cycloalkyl; or

[0111] R 20 and R 30Together with an adjacent N atom, form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl;

[0112] Each of R1 and R2 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0113] R 10 and R 11 Each of which is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl; or R 10 and R 11 together form a heterocyclic ring;

[0114] n is 0, 1, 2, 3 or 4; and

[0115] Z is absent, O, S or NR 12 wherein R 12 is H or C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH;

[0116] ii)

[0117] wherein:

[0118] R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ;

[0119] R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring;

[0120] R 10 and R 11 are each independently H or C1-C3 alkyl; or R 10 and R 11 can combine together to form a heterocyclic ring;

[0121] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl, C2-C5 branched or unbranched alkenyl; or R 20 and R 30 can combine together to form a ring; and

[0122] Each of v and y is independently 1, 2, 3, or 4;

[0123] iii)

[0124] wherein W is

[0125]

[0126]

[0127] wherein

[0128] R5 is OH, SH, (CH2) s OH or NR 10 R 11 ;

[0129] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0130] Each of R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[0131] Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group;

[0132] R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H, a C1-C3 alkyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, where the alkyl, cycloalkyl, and cycloalkenyl groups are optionally substituted with one or more NH and / or oxo groups, or R10 and R 11 together form a heterocycle;

[0133] R 16 is H, =O, =S or CN;

[0134] Each of s, u, and t is independently 1, 2, 3, 4, or 5;

[0135] Each v is independently 0, 1, 2, 3, 4, or 5;

[0136] Each Y is a divalent heterocycle;

[0137] Each Z independently does not exist, is O, S or NR 12 , where R 12 is H, C1-C7 branched or unbranched alkyl or C2-C7 branched or unbranched alkenyl;

[0138] Q is O, S, CH2 or NR 13 , where each R 13 is H, C1-C5 alkyl;

[0139] V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, and the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and

[0140] T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle; and

[0141] iv) where:

[0142] is a cyclic or heterocyclic moiety;

[0143] Y is alkyl, hydroxy, hydroxyalkyl,

[0144] A does not exist, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S- or -S-S-;

[0145] Each of X and Z is independently absent, or is -O-, -C(O)-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0146] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl;

[0147] t1 is an integer from 0 to 10; and

[0148] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic or heteroaryl; and

[0149] wherein the pKa of the lipid is from about 4 to about 8.

[0150] 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:

[0151] Wherein:

[0152] R 7 are each independently H or methyl;

[0153] R b is independently H or C1-C4 alkyl in each case; and

[0154] u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and

[0155] the head group has the structure of one of the following formulas:

[0156] i) where m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0157] ii)

[0158] iii) and

[0159] iv)

[0160] In some embodiments, at least one tail group has a structure of formula (TII), (TIII), (TIV), (TV), (TII') and / or (TIII'), where u1 is 3 - 5, u2 is 0 - 3; u3 and u4 are each independently 1 - 7, and R a are each independently methyl.

[0161] In some embodiments, the tail group has a structure of formula (TII) or formula (TIII), where each R a is methyl; u1 is 3 - 5, u2 is 0 - 3; and u3 and u4 are each independently 1 - 4.

[0162] In some embodiments, the head group has a structure of one of the following formulas:

[0163] i)

[0164] ii) where each R 20 and R 30 are independently C1 - C3 alkyl;

[0165] iii) where:

[0166] W is where:

[0167] each R6, R7 and R8 are independently H or methyl; and

[0168] each of u and t is independently 1, 2 or 3; or

[0169] W is where:

[0170] R 16 is H or =O;

[0171] R 14 is a 5 - or 6 - membered nitrogen - containing heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 are independently H or C1 - C3 alkyl; and

[0172] each of u and v is independently 1, 2 or 3; or

[0173] W is wherein:

[0174] each R6 is independently H or methyl;

[0175] each u is independently 1, 2 or 3; and

[0176] V is C2-C6 alkylene or C2-C6 alkenylene; or

[0177] W is wherein:

[0178] each R6 is independently H or methyl;

[0179] each R7 is independently H;

[0180] each R8 is methyl;

[0181] each u is independently 1, 2 or 3; and

[0182] V is C2-C6 alkylene or C2-C6 alkenylene; or

[0183] W is wherein:

[0184] each u is independently 1, 2 or 3; and

[0185] T is a divalent nitrogen-containing 5- or 6-membered heterocycle; or

[0186] W is wherein:

[0187] each u is independently 1, 2 or 3;

[0188] Q is O;

[0189] each Z is independently NR 12 ; and

[0190] R 12 is H or C1-C3 alkyl; and

[0191] iv) wherein:

[0192] W is hydroxy, substituted or unsubstituted hydroxyalkyl, one of the following moieties: wherein

[0193] each Q independently does not exist, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q C(R 7)2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0194] R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thioalkyl or N + (R 7 )3–alkylene-Q-;

[0195] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol, thioalkyl, heterocyclic group, heteroaryl; or two R 8 together with the nitrogen atom form a ring, which ring is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl;

[0196] q is 0, 1, 2, 3, 4 or 5; and

[0197] p is 0, 1, 2, 3, 4 or 5.

[0198] In some embodiments, the ionizable lipid is a compound in Table I, Table II, Table III or Table IV.

[0199] In some embodiments, the ionizable lipid is a lipid numbered 2252, 2272, 2320, 2439, 2356, 2243, 2431, 2455, 2454, 2424, 2425, 2433, 2275, 2220, 2335 or 2282.

[0200] In a CLP (or LNP) formulation or an LNP formulation, more than one ionizable lipid can be used for the ionizable lipid component: one or more ionizable lipids from the ionizable lipids of the formula compounds in groups i)-iv) can be used alone or in combination with different ionizable lipids from the formula compounds in groups i)-iv).

[0201] In all these aspects of the present invention, in some embodiments, the CLP is LNMP, and the CLP formulation encapsulating one or more polynucleotides is LNMP. Accordingly, all embodiments described below regarding the features related to LNMP and LNMP formulations are applicable to CLP and CLP formulations.

[0202] In some embodiments, the polynucleotide is encapsulated by the lipid-reconstituted native messenger package (LNMP). In some embodiments, the polynucleotide is encapsulated by the lipid-reconstituted plant messenger package (LPMP). In some embodiments, the polynucleotide is embedded on the surface of the LNMP. In some embodiments, the polynucleotide is conjugated to the surface of the LNMP.

[0203] In some embodiments, the LNMP is produced by a method comprising lipid extrusion. In some embodiments, the LNMP is produced by a method comprising treating a solution of a lipid extract comprising the NMP in a microfluidic device comprising an aqueous phase, thereby producing the LNMP. In some embodiments, the aqueous phase comprises the polynucleotide.

[0204] In some embodiments, the native lipid of the LPMP is extracted from a plant source (such as lemon or algae).

[0205] In a CLP formulation (e.g., LNP) or an LNP formulation, for the ionizable lipid component, the ionizable lipid from the compounds of formulae in groups i)-iv) can be used in combination with one or more other ionizable lipids.

[0206] In some embodiments, the ionizable lipid of the LNMP is selected from the group consisting of: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315. In one embodiment, the ionizable lipid is C12-200. In some embodiments, the ionizable lipid is wherein R is a C8-C14 alkyl group.

[0207] In some embodiments, the reconstitution is carried out in the presence of a sterol, thereby producing an LNMP comprising native lipid, ionizable lipid, and sterol.

[0208] In some embodiments, the reconstitution is carried out in the presence of a PEGylated lipid (or PEG-lipid conjugate), thereby producing an LNMP comprising native lipid, ionizable lipid, and PEG-lipid conjugate.

[0209] In some embodiments, the LNMP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the LNP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0210] In some embodiments, the sterol is cholesterol or sitosterol.

[0211] In some embodiments, the PEG-lipid conjugate comprises PEG-2k. 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-lipid conjugate is PEG2000-DMG or PEG2000-PE.

[0212] In some embodiments, the amount of the PEG-lipid conjugate is about 1.5 - 2.5 mol%.

[0213] In some embodiments, the amount of the ionizable lipid is about 30 - 50 mol%, about 30 - 40 mol%, or about 45 - 55%. In one embodiment, the amount of the ionizable lipid is about 50 mol% or 35 mol%.

[0214] In some embodiments, the LNMP comprises:

[0215] about 20 mol% to about 50 mol% of the ionizable lipid,

[0216] about 20 mol% to about 60 mol% of the natural lipid,

[0217] about 7 mol% to about 50 mol% of the sterol, and

[0218] about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0219] In one embodiment, the molar ratio of ionizable lipid:natural lipid:sterol:PEG-lipid in the LNMP is about 35:50:12.5:2.5. In one embodiment, the molar ratio of ionizable lipid:natural lipid:sterol:PEG-lipid in the LNMP is about 35:20:42.5:2.5. In one embodiment, the molar ratio of ionizable lipid:natural lipid:sterol:PEG-lipid in the LNMP is about 35:16:46.5:2.5. In one embodiment, the molar ratio of ionizable lipid:natural lipid:sterol:PEG-lipid in the LNMP is about 50:10:38.5:1.5. In one embodiment, the molar ratio of ionizable lipid:natural lipid:sterol:PEG-lipid in the LNMP is about 50:20:28.5:1.5.

[0220] In some embodiments, the LNMP comprises:

[0221] natural lipids extracted from lemon or algae,

[0222] ionizable lipids (e.g., ionizable lipids from Table I, Table II, Table III, or Table IV), cholesterol, and

[0223] DMPE-PEG2k.

[0224] In one embodiment, the LNMP comprises:

[0225] natural lipids extracted from lemon,

[0226] ionizable lipids from Table I, Table II, Table III, or Table IV,

[0227] cholesterol, and

[0228] DMPE-PEG2k. The LNMP may have a molar ratio of ionizable lipid:lemon lipid:cholesterol:DMPE-PEG2k of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:16:46.5:2.5, about 50:10:38.5:1.5, or about 50:20:28.5:1.5.

[0229] In one embodiment, the LNMP comprises:

[0230] natural lipids extracted from algae,

[0231] ionizable lipids (e.g., ionizable lipids from Table I, Table II, Table III, or Table IV),

[0232] cholesterol, and

[0233] DMPE-PEG2k. The ionizable lipid:algal lipid:cholesterol:DMPE-PEG2k molar ratio that the LNMP can include is about 35:20:42.5:2.5, about 35:50:12.5:2.5, about 35:16:46.5:2.5, about 50:10:38.5:1.5 or about 50:20:28.5:1.5.

[0234] In some embodiments, the LNP comprises:

[0235] about 20 mol% to about 50 mol% of the ionizable lipid,

[0236] about 5 mol% to about 40 mol% of the synthetic structural lipid,

[0237] about 20 mol% to about 50 mol% of the sterol, and

[0238] about 0.5 mol% to about 3 mol% of the polyethylene glycol (PEG)-lipid conjugate.

[0239] In one embodiment, the ionizable lipid:synthetic structural lipid:sterol:PEG-lipid molar ratio that the LNP comprises is about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:16:46.5:2.5, about 50:10:38.5:1.5 or about 50:20:28.5:1.5..

[0240] In some embodiments, the LNP comprises:

[0241] synthetic structural lipid,

[0242] ionizable lipid (e.g., ionizable lipids from Table I, Table II, Table III or Table IV),

[0243] cholesterol, and

[0244] PEG-lipid.

[0245] In some embodiments, the (e.g., LNP) comprises:

[0246] synthetic structural lipid,

[0247] ionizable lipid (e.g., 2252, 2272, 2320, 2439, 2356, 2243, 2431, 2455, 2454, 2424, 2425, 2433, 2275, 2220, 2335 or 2282),

[0248] cholesterol, and

[0249] DMG-PEG2000.

[0250] In some embodiments, the LNMP is a lipophilic moiety selected from the group consisting of lipid complexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellipods, micelles, and emulsions. In one embodiment, the LNMP is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In one embodiment, the LNMP is a lipid nanoparticle.

[0251] In some embodiments, the size of the LNMP is less than about 200 nm. In one embodiment, the size of the LNMP is less than about 150 nm. In one embodiment, the size of the LNMP is less than about 100 nm. In one embodiment, the size of the LNMP is from about 55 nm to about 80 nm. In one embodiment, the size of the LNMP is from about 80 nm to about 100 nm.

[0252] In some embodiments, the N:P ratio of the LNMP or LNP is at least 3, for example, the N:P ratio is 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. In one embodiment, the N / P ratio is 6 ± 1. In one embodiment, the N / P ratio is 3 ± 1. In one embodiment, the N / P ratio is 15 ± 1.

[0253] In one embodiment, the polypeptide is erythropoietin or epoetin alfa (Epogen) or a fragment or subunit thereof.

[0254] In some embodiments, one or more polynucleotides encode one or more components of a gene editing system. In some embodiments, one or more polynucleotides encode one or more gene editing systems. In some embodiments, the gene editing system comprises an RNA-guided DNA binder.

[0255] In some embodiments, the polynucleotide can be mRNA, siRNA or siRNA precursor, microRNA (miRNA) or miRNA precursor, plasmid, Dicer-substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), piwi-interacting RNA (piRNA), ribozyme, deoxyribozyme (DNAzyme), aptamer, circular RNA (circRNA), guide RNA (gRNA), single guide RNA (sgRNA), or a DNA molecule encoding any of these RNAs.

[0256] In one embodiment, the one or more polynucleotides comprise mRNA or modified mRNA.

[0257] In some embodiments, the mRNA is derived from (a) a DNA molecule or (b) an RNA molecule. In the mRNA, T is optionally replaced by U.

[0258] In some embodiments, the mRNA is derived from a DNA molecule. The DNA molecule may 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.

[0259] In some embodiments, the mRNA is derived from an RNA molecule. The RNA molecule may be a self-replicating RNA molecule.

[0260] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may further comprise a 5' cap. The 5' cap may have a cap1 structure, a cap1 (m6A) structure, a cap2 structure, a cap3 structure, a cap0 structure, or any combination thereof.

[0261] In some embodiments, the mRNA comprises a 5' untranslated region (UTR) and / or a 3'UTR.

[0262] In some embodiments, the mRNA comprises a 5'UTR. The 5'UTR may comprise a Kozak sequence.

[0263] In some embodiments, the mRNA comprises a 3'UTR. In some embodiments, the 3'UTR comprises one or more sequences derived from the split amino-terminal enhancer (AES). In some embodiments, the 3'UTR comprises a sequence derived from mitochondrially encoded 12S rRNA (mtRNRl).

[0264] In some embodiments, the mRNA comprises a poly(A) sequence. In one embodiment, the poly(A) sequence is an 110-nucleotide sequence consisting of a sequence of 30 adenosine residues, a 10-nucleotide linker sequence, and a sequence of 70 adenosine residues.

[0265] In some embodiments, the DNA-binding agent of the RNA guide of the gene editing system is a Cas nuclease mRNA. In some embodiments, the Cas nuclease mRNA is a class II Cas nuclease mRNA. In one embodiment, the class II Cas nuclease is a Cas9 nuclease mRNA.

[0266] In some embodiments, the one or more polynucleotides comprise a gRNA or a modified gRNA. In one embodiment, the gRNA is a dual guide RNA (dgRNA) or a sgRNA. In some embodiments, the gRNA is a modified gRNA, and the modified gRNA comprises a modification selected from the group consisting of: 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, and 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the gRNA is a modified gRNA, and the modified gRNA comprises a modification at one or more nucleotides among the first five nucleotides at the 5' end or the 3' end. In some embodiments, the gRNA is a modified gRNA, and the modified gRNA comprises a PS bond between the first four nucleotides or the last four nucleotides. In some embodiments, the modified gRNA further comprises a 2'-O-Me modified nucleotide at the first three nucleotides at the 5' end or the 3' end.

[0267] In some embodiments, the one or more polynucleotides comprise a gRNA and a class II Cas nuclease mRNA. In some embodiments, the gRNA and the class II Cas nuclease mRNA are present in a ratio in the range of about 10:1 to about 1:10 by weight. In some embodiments, the gRNA and the class II Cas nuclease mRNA are present in a ratio in the range of about 5:1 to about 1:5 by weight. In some embodiments, the gRNA and the class II Cas nuclease mRNA are present in a ratio in the range of about 2:1 to about 1:2 by weight. In some embodiments, the gRNA and the class II Cas nuclease mRNA are present in a ratio of about 2:1 or about 1:1 by weight.

[0268] In some embodiments, the one or more polynucleotides comprise an RNA, and the RNA comprises an open reading frame encoding an RNA-guided DNA binder, wherein the uridine content of the open reading frame is in the range from its minimum uridine content to 150% of the minimum uridine content. In some embodiments, the one or more polynucleotides comprise an mRNA, and the mRNA comprises an open reading frame encoding an RNA-guided DNA binder, wherein the uridine dinucleotide content of the open reading frame is in the range from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

[0269] In some embodiments, the RNA composition further comprises at least one template nucleic acid.

[0270] In some embodiments, the total lipid:polynucleotide weight ratio of the RNA composition is from about 50:1 to about 10:1. In one embodiment, the total lipid:polynucleotide weight ratio of the RNA composition is from about 44:1 to about 24:1. In one embodiment, the total lipid:polynucleotide weight ratio of the RNA composition is from about 40:1 to about 28:1. In one embodiment, the total lipid:polynucleotide weight ratio of the RNA composition is from about 38:1 to about 30:1. In one embodiment, the total lipid:polynucleotide weight ratio of the RNA composition is from about 37:1 to about 33:1.

[0271] In some embodiments, the RNA composition (e.g., the aqueous phase) further comprises a HEPES or TRIS buffer. The pH of the HEPES or TRIS buffer can be from about 7.0 to about 8.5. The concentration of the HEPES or TRIS buffer can be from about 7 mg / mL to about 15 mg / mL. The aqueous phase can further comprise from about 2.0 mg / mL to about 4.0 mg / mL NaCl.

[0272] 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.

[0273] In some embodiments, the aqueous phase and the lipid solution are mixed at a volume ratio of 3:1.

[0274] In some embodiments, the RNA composition further comprises one or more cryoprotectants. The one or more cryoprotectants can be sucrose, glycerol or a combination thereof. In one embodiment, the RNA composition comprises a combination of sucrose at a concentration of from about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of from about 50 mg / mL to about 70 mg / mL.

[0275] In some embodiments, the RNA composition is a lyophilized composition. The lyophilized RNA composition can comprise one or more lyoprotectants. The lyophilized RNA composition can comprise poloxamer, potassium sorbate, sucrose or any combination thereof. In one embodiment, the lyophilized RNA composition comprises poloxamer, such as poloxamer 188.

[0276] In some embodiments, the RNA composition is a lyophilized composition. In one embodiment, the lyophilized RNA composition comprises from about 0.01% w / w to about 1.0% w / w polynucleotide. In one embodiment, the lyophilized RNA composition comprises from about 1.0% w / w to about 5.0% w / w lipid. In one embodiment, the lyophilized RNA composition comprises from about 0.5% w / w to about 2.5% w / w TRIS buffer. In one embodiment, the lyophilized RNA composition comprises from about 0.75% w / w to about 2.75% w / w NaCl. In one embodiment, the lyophilized RNA composition comprises from about 85% w / w to about 95% w / w sugar, such as sucrose. In one embodiment, the lyophilized RNA composition comprises from about 0.01% w / w to about 1.0% w / w poloxamer, such as poloxamer 188. In one embodiment, the lyophilized RNA composition comprises from about 1.0% w / w to about 5.0% w / w potassium sorbate.

[0277] On the other hand, provided herein is a method of delivering a gene editing system to a subject in need thereof, the method comprising administering to the subject an RNA composition comprising:

[0278] One or more polynucleotides encoding one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides being formulated within:

[0279] (a) A plurality of lipid nanoparticles (LNPs), the plurality of LNPs comprising a synthetic structural lipid and an ionizable lipid; or

[0280] (b) A lipid-reconstituted native messenger package (LNMP), the LNMP comprising a native lipid and an ionizable lipid,

[0281] Wherein the ionizable lipid has two or more of the following listed properties:

[0282] (i) At least 2 ionizable amines;

[0283] (ii) At least 3 lipid tails; wherein each lipid tail of the lipid tails has a length of at least 6 carbon atoms;

[0284] (iii) A pKa of about 4.5 to about 7.5;

[0285] (iv) An ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and

[0286] (v) An N:P ratio of at least 3.

[0287] On the other hand, the present disclosure provides a method for gene editing in a cell or a subject, the method comprising contacting the cell or administering to the subject:

[0288] one or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides being formulated within:

[0289] (a) a plurality of lipid nanoparticles (LNPs), the plurality of LNPs comprising a synthetic structural lipid and an ionizable lipid; or

[0290] (b) a lipid-reconstituted natural messenger package (LNMP), the LNMP comprising a natural lipid and an ionizable lipid,

[0291] wherein the ionizable lipid has two or more of the following listed properties:

[0292] (i) at least 2 ionizable amines;

[0293] (ii) at least 3 lipid tails; wherein each of the lipid tails has a length of at least 6 carbon atoms;

[0294] (iii) a pKa of about 4.5 to about 7.5;

[0295] (iv) an ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and

[0296] (v) an N:P ratio of at least 3,

[0297] wherein the gene editing system or the one or more components of the one or more gene editing systems are delivered to the cell or the subject to modify the genome of the cell or the subject.

[0298] In some embodiments, the RNA composition is administered at least once.

[0299] In some embodiments, the RNA composition is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, at least twenty times or more. In some embodiments, the RNA composition is administered 2 - 8 times. In some embodiments, the delivery of the gene editing system or the result of gene editing is improved after multiple administrations.

[0300] In some embodiments, the RNA composition is administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. In some embodiments, the RNA composition is administered to the subject eight times. In some embodiments, the RNA composition is administered to the subject five times at one-week intervals. In some embodiments, the RNA composition is administered to the subject two times, three times, four times, five times, six times, seven times, eight times, ten times or more, wherein the RNA composition is administered to the subject at one-week, two-week, three-week, four-week, five-week or more-week intervals.

[0301] In some embodiments, the RNA composition further comprises at least one template nucleic acid.

[0302] In some embodiments, at least two RNA compositions are administered to the subject or contacted with the cell: a first RNA composition comprising mRNA; and a second RNA composition comprising a guide RNA nucleic acid. In some embodiments, the first RNA composition and the second RNA composition are administered simultaneously. In some embodiments, the first RNA composition and the second RNA composition are administered sequentially.

[0303] In some embodiments, a single RNA composition is contacted with the cell or administered to the subject, wherein the single RNA composition comprises mRNA and a guide RNA nucleic acid.

[0304] In these aspects of the invention, the RNA composition can 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, intravenous, intramuscular and / or subcutaneous administration.

[0305] In some embodiments, the RNA composition is administered at a dose level sufficient to deliver from about 0.01 mg / kg to about 4 mg / kg of mRNA to the subject. In some embodiments, the RNA composition is administered at a dose level sufficient to deliver 0.125 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg or 3 mg / kg of mRNA to the subject.

[0306] In some embodiments, the method further comprises administering an additional therapeutic agent to the subject.

[0307] In some embodiments, the additional therapeutic agent is administered before, simultaneously with or after the administration of the RNA composition.

[0308] In these aspects of the invention related to methods for delivering gene editing systems or gene editing methods, the RNA composition can be formulated with b) a CLP such as an LNMP, as described herein.

[0309] In some embodiments, the ionizable lipid of the LNMP is selected from the group consisting of: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315. In one embodiment, the ionizable lipid is C12-200. In some embodiments, the ionizable lipid is wherein R is a C8-C14 alkyl group.

[0310] In these aspects of the invention related to methods for delivering gene editing systems or gene editing methods, the RNA composition can be formulated with a) an LNP, as described herein.

[0311] In these aspects of the invention related to methods for delivering gene editing systems or gene editing methods, the ionizable lipid in the CLP (such as an LNMP) or LNP can be selected from one of the following groups of compounds:

[0312] i) a compound of the formula or a pharmaceutically acceptable salt thereof or a stereoisomer of any of the foregoing,

[0313] wherein:

[0314] each A is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, the alkyl or alkenyl optionally being substituted with a heteroatom or being substituted with OH, SH, or a halogen;

[0315] each B is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, the alkyl or alkenyl optionally being substituted with a heteroatom or being substituted with OH, SH, or a halogen;

[0316] each X is independently a biodegradable moiety; and

[0317] W is

[0318]

[0319]

[0320] wherein

[0321] R5 is OH, SH, NR 10 R 11 ;

[0322] each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group or a cycloalkyl group;

[0323] each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, NR 10 R 11 , where each R 10 and R 11 are independently H, a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle;

[0324] each s is independently 1, 2, 3, 4 or 5;

[0325] each u is independently 1, 2, 3, 4 or 5;

[0326] t is 1, 2, 3, 4 or 5;

[0327] each Z is independently absent, O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl group or a C2-C7 branched or unbranched alkenyl group; and

[0328] Q is O, S or NR 13 , where each R 13 is H, a C1-C5 alkyl group;

[0329] ii) a compound of formula its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0330] wherein:

[0331] is a cyclic or heterocyclic moiety;

[0332] Y is an alkyl group, a hydroxy group, a hydroxyalkyl group or

[0333] A is absent, -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle;

[0334] Each of X and Z independently is absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0335] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl or aminoalkyl;

[0336] Each M is independently a biodegradable moiety;

[0337] R 30 , R 40 , R 50 , R 60 , R 70 , R 80 , R 90 , R 100 , R 110 and R 120 is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl optionally being interrupted by a heteroatom or being substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0338] Each of l and m is an integer from 1 to 10;

[0339] t1 is an integer from 0 to 10; and

[0340] W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl or substituted or unsubstituted heterocyclic group or heteroaryl; and

[0341] iii) A compound of formula , its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein:

[0342] R 20 and R 30 are each independently H, C1-C5 branched or unbranched alkyl or C2-C5 branched or unbranched alkenyl, or R 20 and R 30Forms a 3- to 7-membered ring together with an adjacent N atom, and the ring is optionally substituted by R a substituted;

[0343] R a is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH or SH;

[0344] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 or

[0345] R1 and R2 together form a ring;

[0346] Each R 10 and R 11 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or R 10 and R 11 together form a heterocyclic ring;

[0347] n is 0, 1, 2, 3 or 4;

[0348] Y is O or S;

[0349] Z is absent, is O, S or N(R 12 )(R 12 ), where each R 12 is independently H, a C1-C7 branched or unbranched alkyl group or a C2-C7 branched or unbranched alkenyl group, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH;

[0350] u is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0351] v is 0, 1, 2, 3 or 4;

[0352] y is 0, 1, 2, 3 or 4;

[0353] Each A is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or is optionally substituted by OH, SH or a halogen;

[0354] Each B is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16A branched or unbranched alkenyl group, wherein the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or is optionally substituted by OH, SH, or halogen; and

[0355] Each X is independently a biodegradable moiety; and

[0356] iv) a lipid, said lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'):

[0357] Its pharmaceutically acceptable salts or stereoisomers of any of the foregoing,

[0358] wherein:

[0359] E is each independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl;

[0360] R 13 is a branched or unbranched C3-C 10 alkyl;

[0361] r is 1, 2, 3, 4 or 5;

[0362] R a are each independently C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0363] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0364] R t are each independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl group is optionally interrupted by a heteroatom or is substituted by OH, SH, or halogen or cycloalkyl or substituted cycloalkyl;

[0365] represents the bond connecting the tail group to the head group; and

[0366] wherein the pKa of the lipid is from about 4 to about 8.

[0367] In some embodiments, the ionizable lipid is a compound in Table I, Table II, Table III, or Table IV. In some embodiments, the ionizable lipid is 2272, 2320, 2439, 2356, 2243, 2431, 2455, 2454, 2424, 2433, 2425, 2275, 2220, or 2335.

[0368] On the other hand, provided herein is a method capable of repeated administration to a subject in need thereof, the method comprising administering to the subject an RNA composition comprising:

[0369] one or more polynucleotides (e.g., one or more components of a gene editing system or one or more polynucleotides of one or more gene editing systems), the one or more polynucleotides formulated within:

[0370] (a) a plurality of lipid nanoparticles (LNPs), the plurality of LNPs comprising a synthetic structural lipid and an ionizable lipid; or

[0371] (b) a lipid-reconstituted native messenger package (LNMP), the LNMP comprising a native lipid and an ionizable lipid,

[0372] wherein the ionizable lipid has two or more of the following listed properties:

[0373] (i) at least 2 ionizable amines;

[0374] (ii) at least 3 lipid tails; wherein each lipid tail has a length of at least 6 carbon atoms;

[0375] (iii) a pKa from about 4.5 to about 7.5;

[0376] (iv) an ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and

[0377] (v) an N:P ratio of at least 3.

[0378] In some embodiments, the RNA composition is administered at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or more times. In some embodiments, the RNA composition is administered 2 - 8 times. In some embodiments, the delivery or administration of the RNA composition results in improved outcomes after multiple administrations.

[0379] In some embodiments, the RNA composition is administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. In some embodiments, the RNA composition is administered to the subject eight times. In some embodiments, the RNA composition is administered to the subject five times at one-week intervals. In some embodiments, the RNA composition is administered to the subject two times, three times, four times, five times, six times, seven times, eight times, ten times, or more, wherein the RNA composition is administered to the subject at one-week intervals, two-week intervals, three-week intervals, four-week intervals, five-week intervals, or more-week intervals.

[0380] Definitions

[0381] As used herein, the term "effective amount", "effective concentration", or "concentration effective to" refers to the amount of the LNMP or nucleic acid composition sufficient to achieve the result or reach the target level (e.g., a predetermined or threshold level) in or on the target organism.

[0382] As used herein, the term "therapeutic agent" refers to an agent that can act on an animal, such as 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 antiviral agent, an insecticide, a nematicide, an antiparasitic agent, or a vermifuge.

[0383] As defined herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to linear or branched, single-stranded or double-stranded RNA or DNA or hybrids 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 generally linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs having other types of bonds or backbones (e.g., phosphoramidate, thiophosphate, dithiophosphate, O-methylphosphoramidite, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA) and peptide nucleic acid (PNA) bonds or backbones, etc.). Nucleic acids can be single-stranded, double-stranded or contain portions of both single-stranded and double-stranded sequences. Nucleic acids 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 for example inosine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine).

[0384] As used herein, the terms "peptide", "protein" or "polypeptide" encompass any chain of natural or non-natural occurring amino acids (D-amino acids 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), whether there are post-translational modifications (e.g., glycosylation or phosphorylation) or whether there are one or more non-aminoacyls (e.g., sugars, lipids, etc.) covalently linked to the peptide, and include for example natural proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptidomimetics or peptidomimetics. The size of the polypeptide can be for example at least 0.1 kD, at least 1 kD, at least 5 kD, at least 10 kD, at least 15 kD, at least 20 kD, at least 30 kD, at least 40 kD, at least 50 kD or greater than 50 kD. The polypeptide can be a full-length protein. Alternatively, the polypeptide can contain one or more domains of a protein.

[0385] As used herein, the term "animal" refers to human and non-human animals (including for example dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, chickens and non-human primates).

[0386] As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes disease or symptoms of disease in an animal by, for example: (i) directly infecting the animal, (ii) producing an agent that causes disease or symptoms of disease in the animal (e.g., a bacterium that produces a virulent toxin, etc.), and / or (iii) triggering an immune response (e.g., an inflammatory response) in the animal (e.g., a biting insect, e.g., a bedbug). 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 of causing a disease or symptom in a human, either alone or in concert with another pathogen.

[0387] As used herein, the term "heterologous" refers to an agent (e.g., a polypeptide) that is (1) exogenous to a plant (e.g., from a source that is not the plant or plant part that produces the PMP) (e.g., an agent added to the PMP using the loading methods described herein) or (2) endogenous to a plant cell or tissue that produces the PMP, but is present in the PMP at a higher concentration than found in nature (e.g., higher than the concentration found in naturally occurring plant extracellular vesicles) (e.g., added to the PMP using the loading methods, genetic engineering, and in vitro or in vivo methods described herein).

[0388] As used herein, the "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. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0389] As used herein, the term "modified NMP" or "modified LNMP" refers to a composition comprising a plurality of NMPs or LNMPS (e.g., one or more exogenous lipids such as ionizable lipids, e.g., an NMP or LNMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) that includes one or more heterologous agents, wherein the one or more heterologous agents are capable of increasing the cellular 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; are capable of effecting or increasing the delivery of a heterologous functional agent (e.g., an agricultural agent or a therapeutic agent) to a cell by the NMP or LNMP; and / or are capable of effecting or increasing the loading (e.g., loading efficiency or loading capacity) of an exogenous functional agent (e.g., an agricultural agent or a therapeutic agent). The NMP or LNMP can be modified in vitro or in vivo.

[0390] As used herein, the term "unmodified NMP" or "unmodified LNMP" refers to a composition comprising a plurality of NMPs or LNMPS that lack a heterologous cellular uptake agent capable of increasing the cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the NMP.

[0391] As used herein, the term "modified PMP" or "modified LPMP" refers to a composition comprising a plurality of PMPs or LPMPs (e.g., one or more exogenous lipids such as ionizable lipids, e.g., a PMP or LPMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) that includes one or more heterologous agents, wherein the one or more heterologous agents are capable of increasing the cellular uptake (e.g., animal cell uptake, plant cell 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; are capable of effecting or increasing the delivery of a heterologous functional agent (e.g., an agricultural agent or a therapeutic agent) to a cell by the PMP or LPMP; and / or are capable of effecting or increasing the loading (e.g., loading efficiency or loading capacity) of an exogenous functional agent (e.g., an agricultural agent or a therapeutic agent). The PMP or LPMP can be modified in vitro or in vivo.

[0392] As used herein, the term "unmodified PMP" or "unmodified LPMP" refers to a composition comprising a plurality of PMPs or LPMPs that lack a heterologous cellular uptake agent capable of increasing the cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the PMP.

[0393] As used herein, the term "cellular uptake" refers to the uptake of NMP or LNMP or a portion or component thereof (e.g., a polynucleotide carried by NMP or LNMP) by a cell such as an animal cell, a plant cell, a bacterial cell, or a fungal cell. For example, uptake can involve the transfer of NMP (e.g., LNMP) or a component thereof from the extracellular environment to or across the cell membrane, cell wall, extracellular matrix, or into the intracellular environment of the cell. Cellular uptake of NMP (e.g., LNMP) can occur via active or passive cellular mechanisms. Cellular uptake includes aspects in which the entire NMP (e.g., LNMP) is taken up by the cell, such as by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of the target cell after endocytosis and endosomal escape. In some embodiments, the endosomal escape rate of modified LNMP (e.g., LNMP comprising an ionizable lipid, e.g., LNMP comprising an ionizable lipid and a sterol and / or a PEGylated lipid) is increased relative to unmodified LNMP. Cellular uptake also includes aspects in which 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 after membrane fusion. In some embodiments, the fusion rate of LNMP with the membrane of the target cell is increased (e.g., more fusogenic) relative to unmodified LNMP.

[0394] As used herein, the term "cell permeant" refers to an agent that modifies the 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 cellular uptake relative to a cell that has not been contacted with the agent.

[0395] As used herein, the term "plant" refers to an entire plant, plant organs, plant tissues, seeds, plant cells, and progeny of the seeds. Plant cells include, but are not limited to, 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, fruits, harvestables, tumor tissues, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, and callus tissue). Plant tissues can be in a plant or in a plant organ, tissue, or cell culture. Additionally, plants can be genetically engineered to produce heterologous proteins or RNAs.

[0396] As used herein, the term "bacterium" refers to a whole bacterium or a part of a bacterium. Additional classifications of bacteria can be classified as cocci, bacilli, spirilla or vibrios, and different phyla include, but are not limited to, Proteobacteria, Firmicutes, Bacteroids, Sphingobacteria, Flavobacteria, Fusobacteria, Spirochaetes, Chlorobia, Cyanobacteria, Thermomicrobia, Xenobacteria or Aquificae. Examples of specific bacterial species include Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae and Pseudomonas aeruginosa. Parts of bacteria include cellular components such as peptidoglycan, outer membrane, inner membrane, cell wall, RNA polymerase, metabolites, polypeptides, proteins, flagella, pili, ribosomes, mesosomes, cytoplasm or chromosomes. Bacteria can be genetically engineered to produce heterologous proteins or RNAs, or can be genetically engineered to not produce endogenous proteins or RNAs.

[0397] As used herein, the term "arthropod" refers to any animal within the phylum Arthropoda or any animal segment, part, organ, tissue, egg, cell or progeny thereof. Example animals include insects, spiders and crustaceans. Arthropod cells include, but are not limited to, cells from eggs, suspension cultures, embryos, tissues, organs, exoskeletons, segments and appendages. Arthropod parts include segments, appendages, exoskeletons, eggs, organs, embryos and various forms of cells and cultures. Arthropod tissues can be located within an arthropod or within an organ, tissue or cell culture. Arthropods can be genetically engineered to produce heterologous proteins or RNAs. Arthropods can be genetically engineered to not produce endogenous proteins or RNAs.

[0398] As used herein, the term "fungus" refers to the whole fungus, fungal organs, fungal tissues, spores, fungal cells, and their progeny. Exemplary fungi include yeast, mushrooms, molds, and mildews. Fungal cells include, but are not limited to, cells from spores, suspension cultures, mycelia, hyphae, thalli, cell walls, tissues, gametophytes, spores, and organs. Fungal tissues may be located within the fungus or within an organ, tissue, or cell culture. Fungi can be genetically engineered to produce heterologous proteins or RNAs. Fungi can be genetically engineered to not produce endogenous proteins or RNAs.

[0399] As used herein, the term "archaea" refers to the whole archaea or parts of archaea. Exemplary archaea include Euryarchaeota, Crenarchaeota, and Korarchaeota. Parts of archaea include cellular components such as RNA polymerase, glycerol-ether lipids, membranes, cell walls, polypeptides, proteins, and metabolites. Archaea can be genetically engineered to produce heterologous proteins or RNAs, or can be genetically engineered to not produce endogenous proteins or RNAs.

[0400] As used herein, the term "extracellular vesicle" or "EV" refers to a closed lipid bilayer structure that naturally occurs in an organism or cell. Optionally, the EV includes one or more EV markers. As used herein, the term "EV marker" refers to a component that is naturally associated with a particular organism, such as a protein, nucleic acid, small molecule, lipid, or a combination thereof. In some cases, the EV marker is an identification marker for the EV but not an insecticide. In some cases, the EV marker is an identification marker for the EV and is also an insecticide (e.g., associated with or encapsulated by multiple NMPs, or not directly associated with or encapsulated by multiple NMPs).

[0401] As used herein, the term "natural messenger package" or "NMP" refers to a lipid structure (e.g., lipid bilayer, monolayer, multi-layered structure; e.g., vesicular lipid structure) having a diameter of 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) that is derived from a natural source or a segment, portion or extract thereof (e.g., enriched, isolated or purified therefrom), which includes lipid or non-lipid components (e.g., peptides, nucleic acids or small molecules) that are associated therewith and have been enriched, isolated or purified from a natural source, such as a plant, arthropod, fungus, archaea or bacterium; an arthropod, plant, fungus, archaea or bacterium part; or an arthropod, plant, fungus, archaea or bacterium cell, and the enrichment or isolation removes one or more contaminants or undesired components from the source. NMP can also be separated from other natural sources, such as algae or animal organs. NMP can be a highly purified preparation of naturally occurring EVs. Preferably, at least 1% (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 the contaminants or undesired components from the source are removed from one or more contaminants or undesired components from the source, such as cell wall components; membrane components; chitin; organelles (e.g., mitochondria; nucleoli; Golgi complexes; ribosomes, endoplasmic reticulum and nuclei); chromatin; or molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates or lipid-protein structures). Preferably, 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 source, as measured by weight (w / w), spectral imaging (transmittance %) or conductivity (S / m).

[0402] As used herein, the terms "plant extracellular vesicle", "plant EV", or "EV" refer to a naturally occurring, enclosed lipid bilayer structure in plants. Optionally, the plant EVs include one or more plant EV markers. As used herein, the term "plant EV marker" refers to a component that is naturally associated with plants, such as a plant protein, plant nucleic acid, plant small molecule, plant lipid, or a combination thereof, including but not limited to any plant EV marker listed in the appendix. In some cases, the plant EV marker is an identification marker for plant EVs but not an insecticide. In some cases, the plant EV marker is an identification marker for plant EVs and is also an insecticide (e.g., associated with or encapsulated by multiple PMPs or LPMPs, or not directly associated with or encapsulated by multiple PMPs or LPMPs).

[0403] As used herein, the term "plant messenger package" or "PMP" refers to a lipid structure (e.g., lipid bilayer, monolayer, multi-layer structure; e.g., vesicular lipid structure) having a diameter of 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) that is derived from a plant source or a segment, portion, or extract thereof (e.g., enriched, isolated, or purified therefrom), and includes lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) that are associated therewith and have been enriched, isolated, or purified from a plant, plant part, or plant cell, wherein the enrichment or isolation removes one or more contaminants or undesired components from the source plant. The PMP can be a highly purified preparation of naturally occurring EVs. Preferably, at least 1% (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 the contaminants or undesired components from the source plant are removed from one or more contaminants or undesired components from the source plant, such as plant cell wall components; pectin; plant organelles (e.g., mitochondria; plastids, such as chloroplasts, leucoplasts, or amyloplasts; and nuclei); plant chromatin (e.g., plant chromosomes); or plant molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipid-protein structures). Preferably, the 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).

[0404] Lipid-reconstituted NMP (LNMP) is used herein. For example, lipid-reconstituted PMP (LPMP) is used herein. The terms "lipid-reconstituted NMP" and "LNMP" refer to NMP that has been derived from a lipid structure (e.g., lipid bilayer, monolayer, multi-layer structure; e.g., vesicular lipid structure) sourced from an arthropod, plant, fungus, archaea, or bacterial source (e.g., enriched, isolated, or purified therefrom), 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 cargo) using standard methods, e.g., reconstituted by methods including lipid membrane hydration, multi-fluid, and / or solvent injection, to produce LNMP as described herein. If desired, the method can further include sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted NMP. Alternatively, a microfluidic device (such as IGNITE TM a microfluidic instrument (Precision NanoSystems, Inc.)) can be used to produce LNMP.

[0405] Lipid-reconstituted PMP (LPMP) is used herein as a subcategory of lipid-reconstituted NMP. The terms "lipid-reconstituted PMP" and "LPMP" refer to PMP that has been derived from a lipid structure (e.g., lipid bilayer, monolayer, multi-layer structure; e.g., vesicular lipid structure) sourced from a plant source (e.g., enriched, isolated, or purified therefrom), 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 cargo) using standard methods, e.g., reconstituted by methods including lipid membrane hydration and / or solvent injection, to produce LPMP as described herein. If desired, the method can further include sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted LPMP. Alternatively, a microfluidic device (such as IGNITE TM a microfluidic instrument (Precision NanoSystems)) can be used to produce LPMP.

[0406] 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 molecular aggregates (protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates or lipid-protein structures) have been removed relative to the initial sample isolated from a plant or a part thereof.

[0407] As used herein, the term "complex lipid particle" refers to a lipid particle characterized by complexity and comprising various lipids, said 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 from 10% w / w to 99% w / w of structural lipids derived from lipid structures from one or more natural sources. For example, the complex lipid particle 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 of lipids derived from lipid structures from one or more natural sources. In some cases, a complex lipid particle incorporating a natural lipid extract may also be referred to as a natural messenger package (NMP). For example, a complex lipid particle incorporating a plant lipid extract may also be referred to as a plant messenger package (PMP). In some cases, a complex lipid particle incorporating a natural lipid extract and at least one exogenous ionizable lipid may also be referred to as a lipid-reconstituted natural messenger package (LNMP). For example, a complex lipid particle incorporating a plant lipid extract and at least one exogenous ionizable lipid may also be referred to as a lipid-reconstituted plant messenger package (LPMP). Thus, any disclosure herein describing features related to LNMPs and LNMP formulations applies to CLPs and CLP formulations.

[0408] The composite lipid particles can contain 3 to 1000 lipids extracted from one or more natural (e.g., plant, bacterial) sources. The composite lipid particles can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of natural (e.g., plant, bacterial) lipids from natural (e.g., plant, bacterial) sources. The composite lipid particles can contain all or a portion of the lipid species present in the lipid structures from natural (e.g., plant, bacterial) sources. For example, the composite lipid particles can 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 nearly 100% of the lipid species present in the lipid structures from natural sources. The composite lipid particles can contain all or a portion of the lipid species present in the lipid structures from a specific natural source. For example, the composite lipid particles can 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 nearly 100% of the lipid species present in the lipid structures from a plant source or from a bacterial source.

[0409] The composite lipid particles can contain reduced or minimized proteinaceous material endogenous to the one or more natural (i.e., plant, bacterial) sources. For example, the composite lipid particles can 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 proteinaceous material endogenous to the one or more natural (e.g., plant, bacterial) sources. In some cases, the lipid bilayer of the composite lipid particles does not contain protein.

[0410] The composite lipid particles can also include synthetic structural lipids, such as neutral lipids, as a structural lipid component. The structural lipid component of the composite lipid particles can contain 10% w / w to 99% w / w of structural lipids derived from synthetic lipid structures (as opposed to lipids extracted from natural sources). For example, the composite lipid particles can 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 approximately 99% w / w of lipids derived from synthetic lipid structures.

[0411] The composite lipid particles can further comprise at least two exogenous lipids. The composite lipid particles can comprise 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 of exogenous lipids. Exemplary exogenous lipids include sterols and PEG-lipid conjugates. The composite lipid particles can be used to encapsulate one or more exogenous nucleic acids or polynucleotides encoding one or more peptides, polypeptides or proteins such that the exogenous nucleic acids or polynucleotides can be delivered to target cells or tissues.

[0412] As used herein, the term "exogenous lipid" refers to a lipid that is exogenous to a natural source (e.g., plant, bacterium), i.e., the lipid is from a source that is not the natural source from which the lipid is extracted (e.g., a lipid added to a composite lipid particle formulation using the methods described herein). The term "exogenous lipid" does not exclude lipids from natural sources (such as sterols from plant sources). That is, an exogenous lipid can be a lipid from a natural source (such as a sterol from a plant source that is exogenous to the plant source from which the lipid is extracted, e.g., the exogenous lipid can be a sterol from a plant source added to a composite lipid particle formulation). As another example, an exogenous lipid can be a lipid from a natural source that is exogenous to the particular natural source from which the lipid is extracted (e.g., a lipid from a bacterial source that is exogenous to the plant source from which the lipid is extracted, or vice versa). An exogenous lipid can be a cell permeant, can be capable of increasing the delivery of one or more polynucleotides to cells by a composite lipid formulation, and / or can be capable of increasing the loading of polynucleotides (e.g., loading efficiency or loading capacity). In some embodiments, the exogenous lipid can be a stabilizing lipid. In some embodiments, the exogenous lipid can be a structural lipid (e.g., a synthetic structural lipid). Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols and PEGylated lipids.

[0413] As used herein, the term "cationic lipid" refers to a positively charged amphiphile (e.g., a lipid or lipidoid) containing a cationic group (e.g., a cationic head group).

[0414] As used herein, the term "ionizable lipid" refers to an amphiphile (e.g., a lipid or 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 charged species) under a given condition (e.g., pH). It has surprisingly been found that ionizable lipids comprising an alkyl chain having multiple unsaturated sites, e.g., at least two or three unsaturated sites, are particularly useful for forming lipid particles having increased membrane fluidity. Many ionizable lipids and related analogs suitable for use herein have been described in the following: U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patents Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, the ionizable lipid is ionizable such that it can exist in a positively charged form upon dissociation according to pH. The ionization of the ionizable lipid affects the surface charge of lipid nanoparticles comprising the ionizable lipid under different pH conditions. The surface charge of the lipid nanoparticles can in turn affect their plasma protein uptake, blood clearance, and tissue distribution (Semple, S.C. et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and their ability to form non-bilayer structures that may affect endosomal lysis for intracellular delivery of nucleic acids (Hafez, I.M. et al., Gene Ther 8:1188-1196 (2001)).

[0415] In some embodiments, the ionizable lipid is a lipid that is generally neutral, e.g., at physiological pH (e.g., pH of about 7), but can carry a net charge at acidic pH or basic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH of about 7, but can carry a net charge at acidic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH of about 7, but can carry a net charge at basic pH. In some embodiments, the ionizable lipid does not include those cationic lipids or anionic lipids that generally carry a net charge at physiological pH (e.g., pH of about 7).

[0416] As used herein, the term "lipidoid" refers to a molecule having one or more lipidic properties.

[0417] As used herein, the term "stable LNMP formulation" or "stable CLP formulation" refers to, relative to the amount of CLP or LNMP in the CLP formulation or LNMP formulation (e.g., at the time of production or formulation), optionally within a defined temperature range (e.g., 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)), for a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days or at least 90 days), retain 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 amount of CLP or LNMP (e.g., CLP or LNMP per mL of solution); or relative to the initial activity of CLP or LNMP (e.g., at the time of production or formulation), optionally within a defined temperature range (e.g., 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)), retain 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 permeability activity and / or the activity of mRNA formulated within CLP or LNMP).

[0418] Alternatively, the expression means that, relative to the initial activity (e.g., at the time of production or formulation) of the CLP formulation or LNMP formulation optionally at a defined temperature range (e.g., 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)), the CLP formulation or LNMP composition 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 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).

[0419] Alternatively, the expression means that, relative to the initial size (e.g., at the time of production or formulation) of the CLP or LNMP optionally at a defined temperature range (e.g., 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)), the CLP formulation or LNMP formulation retains its particle size 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), i.e., the particle size does not increase, or increases by 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).

[0420] In some embodiments, the stable CLP or LNMP formulation continues to encapsulate or remain associated with an exogenous peptide, polypeptide, or protein that the CLP or LNMP formulation has been loaded with, e.g., continues to encapsulate or remain associated with the 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 days or more.

[0421] As used herein, the term "treatment" refers to the administration of a pharmaceutical composition to an animal for prophylactic and / or therapeutic purposes.

[0422] As used herein, the term "gene editing system" or "genome editing system" refers to any genome editing technology, including CRISPR-Cas nuclease systems, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or other known means of inducing gene editing.

[0423] As used herein, "RNA-guided DNA binder" means a polypeptide or polypeptide complex having RNA and DNA binding activities, or the DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders include Cas endonucleases / nickases and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nuclease" encompasses Cas endonucleases, Cas nickases, and dCas DNA binders. Cas endonucleases / nickases and dCas DNA binders include the Csm or Cmr complexes of type III CRISPR systems, their Cas 10, Csm1 or Cmr2 subunits, the Cascade complex of type I CRISPR systems, their Cas3 subunit, and class 2 Cas nucleases. As used herein, "class 2 Cas nuclease" is a single-chain polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include class 2 Cas endonucleases / nickases (e.g., H840A, D10A, or N863A variants) that further have RNA-guided DNA endonuclease / nickase activity, and class 2 dCas DNA binders in which the endonuclease / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2e3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H69SA variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0424] Figure 1A is a graph depicting the TTR concentration (μg / mL) in the blood of mice intravenously administered 3 mg / kg recLemon LPMP (1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS on day 0, day 2, and day 7. N = 5 / group. Figure 1B Shows the amount (%) of gene editing of the target gene (TTR) in the bone marrow, spleen, and target organ liver in the case of intravenous administration of LPMP (3 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components or an equal volume of PBS. N = 5 / group.

[0425] Figure 2ADepicts the TTR concentration (μg / mL) in the blood of mice intravenously administered various exemplary recLemon LPMP (1 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS on days 0, 3, 7, 14, 21, 28, and 35 after dosing. N = 3 / group. Figure 2B Depicts the TTR concentration (μg / mL) in the blood of mice intravenously administered various exemplary LNPs (1 mg / kg LNP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS on days 0, 3, 7, 14, 21, 28, and 35 after dosing. N = 3 / group.

[0426] Figure 3A Shows the percentage of TTR protein in the liver relative to baseline 7 days after intravenous administration of various exemplary recLemon LPMP formulations (Figure 5) (1 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS that deliver CRISPR / Cas gene editing components. N = 3 / group. Figure 3B Shows the percentage of TTR protein in the liver relative to baseline 7 days after intravenous administration of various LNP formulations (Figure 5) (1 mg / kg LNP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS that deliver CRISPR / Cas gene editing components. N = 3 / group.

[0427] Figure 4A Shows the percentage of TTR protein in the liver relative to baseline 21 days after intravenous administration of various exemplary recLemon LPMP formulations (1 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS that deliver CRISPR / Cas gene editing components. N = 3 / group. Figure 4B Shows the percentage of TTR protein in the liver relative to baseline 21 days after intravenous administration of various LNP formulations (1 mg / kg LNP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) or an equal volume of PBS that deliver CRISPR / Cas gene editing components. N = 3 / group.

[0428] Figure 5AShows the percentage of TTR protein in the liver relative to baseline 28 days after intravenous administration of various exemplary recLemonLPMP formulations (1 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components or an equal volume of PBS. N = 3 / group. Figure 5B Shows the percentage of TTR protein in the liver relative to baseline 28 days after intravenous administration of various LNP formulations (1 mg / kg LNP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components or an equal volume of PBS. N = 3 / group.

[0429] Figure 6A Shows the percentage of TTR protein in the liver relative to baseline 35 days after intravenous administration of various exemplary recLemonLPMP formulations (1 mg / kg recLemon LPMP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components or an equal volume of PBS. N = 3 / group. Figure 6B Shows the percentage of TTR protein in the liver relative to baseline 35 days after intravenous administration of various LNP formulations (1 mg / kg LNP; 1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components or an equal volume of PBS. N = 3 / group.

[0430] Figure 7A Depicts the TTR concentration (ug / mL) in the blood of mice that received intravenous administration of various exemplary recLemon LPMP (1:1 Cas9mRNA:sgRNA mTTR_G211) at a dose of 1.5 mg / kg or 0.3 mg / kg recLemon LPMP or an equal volume of PBS on day 0, day 3, and day 7 after dosing. N = 6 - 7 / group. Figure 7B Shows the percentage of TTR protein in the liver relative to baseline 7 days after intravenous administration of various exemplary recLemonLPMP formulations (1:1 Cas9 mRNA:sgRNA mTTR_G211) delivering CRISPR / Cas gene editing components at a dose of 1.5 mg / kg or 0.3 mg / kg recLemon LPMP or an equal volume of PBS. N = 6 - 7 / group.

[0431] Figure 8AShows the whole-body radiation rate (average radiation rate p / s / cm 2 / sr) at 4 hours after administration of various exemplary recLemon LPMP and LNP formulations (LNP2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE or recLemon LPMP_CM) encapsulating 1:1 mRNA FLuc:hEPO administered intravenously at 0.5 mg / kg to mice given 8 doses (each dose separated by one to two weeks). LNP Lipid 5 was measured starting from dose 3. N = 5 / group. Figure 8B Shows the whole-body hEPO concentration (pg / mL) at 0 hours (before administration), 4 hours after administration, and 24 hours after administration of various exemplary recLemon LPMP and LNP formulations (LNP 2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE or recLemon LPMP_CM) encapsulating 1:1 mRNA FLuc:hEPO administered intravenously at 0.5 mg / kg to mice given 5 doses (each dose separated by one week). Pre-administration measurements were not performed before doses 4 and 5. LNP Lipid 5 was measured starting from dose 3. N = 5 / group.

[0432] Figure 9A Shows the whole-body radiation rate (average radiation rate p / s / cm 2 / sr) at 4 hours after administration of various exemplary recLemon LPMP and LNP formulations (LNP 2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE or recLemon LPMP_CM) (0.125 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) administered intravenously to mice given 5 doses (each dose separated by one week). N = 5 / group. Figure 9B Shows the whole-body hEPO concentration (pg / mL) at 0 hours (before administration), 4 hours after administration, and 24 hours after administration of various exemplary recLemon LPMP and LNP formulations (LNP 2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE or recLemon LPMP_CM) (0.125 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) administered intravenously to mice given 5 doses (each dose separated by one week). N = 5 / group.

[0433] Figure 10A Shows the anti-PEG IgM antibody titers at 0 hours before administration in mice administered various exemplary recLemon LPMP and LNP formulations (LNP 2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE, or recLemon LPMP_CM) intravenously at 5 doses (each dose 1 week apart) (0.5 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO). N = 5 / group. Figure 10B Shows the anti-PEG IgG antibody titers at 0 hours before administration in mice administered various exemplary recLemon LPMP and LNP formulations (LNP 2272, recLemon LPMP 2272, LNP Lipid 5, LNP C12, recLemon LPMP C12_EE, or recLemon LPMP_CM) intravenously at 5 doses (each dose 1 week apart) (0.5 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO). N = 5 / group.

[0434] Figure 11A Shows the systemic hEPO concentration (pg / mL) after administration in mice administered various exemplary recLemon LPMP and LNP formulations (recLemon LPMP 2272, recLemon LPMP 2320, or 2320LNP) intravenously at 5 doses (each dose 1 week apart) (1 mg / kg or 0.5 mg / kg per dose; encapsulating 3:1 mRNA FLuc:hEPO). N = 5 / group. Figure 11B Shows the systemic radiation rate (average radiation rate p / s / cm 2 / sr) at 4 hours after administration in mice administered various exemplary recLemon LPMP and LNP formulations (recLemon LPMP 2272, recLemon LPMP 2320, or 2320LNP) intravenously at 5 doses (each dose 1 week apart) (1 mg / kg or 0.5 mg / kg per dose; encapsulating 3:1 mRNA FLuc:hEPO). N = 5 / group. Figure 11C and 11DShows anti-PEG IgG ( Figure 11C ) and anti-PEG IgM ( Figure 11D ) antibody titers at 0 hours before dosing in mice given 5 doses (each dose 1 week apart) of various exemplary recLemon LPMP and LNP formulations (recLemon LPMP 2272, recLemon LPMP 2320 or 2320LNP) (1 mg / kg or 0.5 mg / kg per dose; encapsulating 3:1 mRNA Fluc:hEPO). N = 5 / group.

[0435] Figure 12A Shows the whole body radiation rate (average radiation rate p / s / cm 2 / sr) at 4 hours after dosing in mice given 5 doses (each dose 1 week apart) of various exemplary recLemon LPMP and LNP formulations (recLemon LPMP 2272, recLemon LPMP 2320, recLemon LPMP2439, 2272LNP, 2320LNP or 2439LNP) (0.2 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) administered subcutaneously. N = 5 / group. Figure 12B and 12C Shows anti-PEG IgG (12B) and anti-PEG IgM (12C) antibody titers at 0 hours before dosing in mice given 5 doses (each dose 1 week apart) of various exemplary recLemon LPMP and LNP formulations (recLemon LPMP2272, recLemon LPMP 2320, recLemon LPMP 2439, 2272LNP, 2320LNP or 2439LNP) (0.2 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO). N = 5 / group.

[0436] Figure 13A Shows the whole body radiation rate (average radiation rate p / s / cm 2 / sr) at 4 hours after dosing in mice given 5 doses (each dose 2 weeks apart) of recLemon LPMP 2272 or 2272LNP (0.5 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) administered subcutaneously (SQ) or intravenously (IV). N = 5 / group. Figure 13BShows the systemic hEPO concentration (pg / mL) after administration of recLemon LPMP 2272 or 2272LNP (0.5 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) subcutaneously or intravenously in mice given 5 doses (each dose 2 weeks apart). N = 5 / group. Figure 13C and 13D Shows anti-PEG IgG ( Figure 13C ) and anti-PEG IgM ( Figure 13D ) antibody titers at 0 hours before administration of recLemon LPMP 2272 or 2272LNP (0.5 mg / kg per dose; encapsulating 1:1 mRNA FLuc:hEPO) in mice given 5 doses (each dose 2 weeks apart). N = 5 / group. 2272LNP and PBS are controls. DETAILED DESCRIPTION

[0437] The present disclosure features RNA compositions (e.g., mRNA / gRNA encoding gene editing systems) that can safely direct the body's cellular machinery to edit the genome, and methods of using these RNA compositions for delivering gene editing systems or for gene editing. The RNA compositions can be administered repeatedly (e.g., 5 times, 6 times, 7 times, 8 times). These RNA compositions include one or more polynucleotides (e.g., RNA, such as messenger RNA (mRNA or gRNA)), the one or more polynucleotides encoding one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides formulated within a lipid nanoparticle (LNP) or a complex lipid particle (CLP). In some embodiments, the CLP is a lipid-reconstituted native messenger package (LNMP), the LNMP comprising lipids extracted from one or more natural sources (i.e., natural lipids) and an ionizable lipid. NMP is a lipid assembly produced wholly or in part from natural extracellular vesicles (EVs) or segments, portions, or extracts thereof. PMP is a lipid assembly produced wholly or in part from plant extracellular vesicles (EVs) or segments, portions, or extracts thereof. LNMP is an NMP derived from a lipid structure, wherein the lipid structure is disrupted and reassembled or reconstituted in the liquid phase. For example, LPMP is a PMP derived from a lipid structure, wherein the lipid structure is disrupted and reassembled or reconstituted in the liquid phase.

[0438] On the other hand, the present disclosure also includes a method of repeatedly administering to a subject in need thereof, the method comprising administering to the subject an RNA composition comprising one or more polynucleotides, the one or more polynucleotides formulated within an LNP or LNMP as described herein.

[0439] The present disclosure also includes a method for preparing an RNA composition, the method comprising reconstructing a membrane comprising purified NMP lipids in the presence of an ionizable lipid to produce LNMP comprising the ionizable lipid, and loading one or more components of a gene editing system or one or more polynucleotides of one or more gene editing systems into the LNMP.

[0440] Composite lipid particles and lipid-reconstituted natural messenger packages (LNMP)

[0441] Complex lipid particles

[0442] The composite lipid particles (CLP) described herein comprise various lipids, including structural lipids extracted from one or more natural sources such as plants or bacteria. In some embodiments, the composite lipid particles are natural messenger packages (NMP) incorporating natural lipid extracts. In some embodiments, the composite lipid particles are lipid-reconstituted natural messenger packages (LNMP) incorporating natural lipid extracts and at least one exogenous ionizable lipid.

[0443] The composite lipid particles may also comprise at least an exogenous ionizable lipid. The ionizable lipid has two or more of the following listed properties:

[0444] (i) At least 2 ionizable amines;

[0445] (ii) At least 3 lipid tails; wherein each lipid tail has a length of at least 6 carbon atoms;

[0446] (iii) A pKa of about 4.5 to about 7.5;

[0447] (iv) An ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and

[0448] (v) An N:P ratio of at least 3.

[0449] The composite lipid particles may comprise 10% w / w to 99% w / w structural lipids derived from lipid structures from one or more natural sources. For example, the composite lipid particles 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 lipid structures from one or more natural sources.

[0450] In some embodiments, the composite lipid particles comprise about 10-95% w / w of natural (e.g., plant, bacterial) lipids. For example, based on the amount of total lipids in the composite lipid formulation, the composite lipid particles comprise 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 natural (e.g., plant, bacterial) lipids.

[0451] The composite lipid particles can contain 3-1000 lipids extracted from one or more natural (e.g., plant, bacterial) sources. In some embodiments, the natural source is a plant, a plant extract, or a fragment or part of a plant. In some embodiments, the natural source is a bacterium, a bacterial fragment, or a part of a bacterium. In some embodiments, the natural source is lemon. In some embodiments, the natural source is soybean. In other embodiments, the natural source is Escherichia coli.

[0452] In some embodiments, the composite lipid particles contain at least 10 natural lipids that belong to one or more of the categories selected from the group consisting of: fatty acyls (FAs), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterols. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids that belong to one or more of the categories selected from the group consisting of: fatty acyls (FAs), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterols. In some embodiments, the composite lipid particles contain lipids from at least two or at least three of these different categories.

[0453] In some embodiments, the composite lipid particles contain at least 10 natural lipids, and the natural lipids are one or more of the categories selected from the group consisting of glycerides, sphingolipids, and sterols. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids, and the natural lipids are one or more of the categories selected from the group consisting of glycerides, sphingolipids, and sterols. In some embodiments, the composite lipid particles contain lipids from at least two or at least three of these different categories.

[0454] In some embodiments, the composite lipid particles can contain one or more glycerides (GL) or glycerophospholipids (GP), which may also include glycolipids.

[0455] In some embodiments, the composite lipid particles can contain one or more glycerides selected from the group consisting of: phospholipids (PL), galactolipids, triacylglycerols (TG), and sulfolipids (SL). In some embodiments, the CLP contains one or more glycerophospholipids (GP) selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI). In some embodiments, the composite lipid particles contain one or more sphingolipids (SP) selected from the group consisting of: sphingolipids (SL), glycosylinositolphosphoceramides (GIPC), glucosylceramides (GCer), ceramides (Cer), and free long-chain bases (LCB). In some embodiments, the composite lipid particles contain one or more phytosterols selected from the group consisting of: campesterol, stigmasterol, β-sitosterol, Δ5-avenasterol, brassicasterol, avenasterol, 4-demethylsterols, 4α-monomethylsterols, Δ5-sterols, Δ7-sterols, α-spinasterol, Δ5,Δ7-sterols, phytostanol, and sitostanol.

[0456] The CLP may contain one or more natural lipids, and the natural lipids belong to one or more categories or subcategories selected from the group consisting of: fatty acids, fatty acid esters, fatty aldehydes, fatty acid amides, acyclic oxylipins, cyclic oxylipins, glycerides, monoacylglycerols, diacylglycerols, triacylglycerols, estolides, glycosyl monoacylglycerols, sulfoquinovosyl monoacylglycerols, monogalactosyl monoacylglycerols, digalactosyl monoacylglycerols, sulfoquinovosyl diacylglycerols, monogalactosyl diacylglycerols, digalactosyl diacylglycerols, glycosyl diacylglycerols, glycerophospholipids, phospholipids, lysophospholipids, phosphatidylinositol phosphates, n-modified phospholipids, oxygenated / oxidized phospholipids, sphingolipids, sphingoid bases, ceramides, phosphocereamides, glycosphingolipids, sterols, cholesterol, cholesterol esters, sterol esters, bile acids, sterylglycosides, and acylsterylglycosides. The composite lipid particles may contain one or more natural lipids, and the natural lipids belong to one or more of the categories or subcategories selected from the group consisting of the categories or subcategories listed above. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids, and the natural lipids belong to one or more of the categories or subcategories selected from the group consisting of the categories or subcategories listed above.

[0457] In some embodiments, the CLP contains one or more natural lipids, and the natural lipids are one or more of the subclasses selected from the group consisting of: acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dihydrocardiolipin, fatty acid, fatty acid ester of hydroxy fatty acid, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, cardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol ester, stigmasterol, sulfoglycolipid, sulfatide, sphingomyelin, sulfoquinovosyldiacylglycerol, sterol, and triacylglycerol. In some embodiments, the composite lipid particle contains at least 10 natural (e.g., plant, bacterial) lipids, and the natural lipids are one or more of the subclasses selected from the group consisting of the subclasses listed above. For example, the composite 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, and the natural lipids are one or more of the subclasses selected from the group consisting of the subclasses listed above.

[0458] The composite lipid particles can contain 10 or more natural lipids, which belong to one or more subclasses selected from the group consisting of: acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, lysophosphatidylglycerols, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerols, and sterols. For example, the composite lipid particles contain at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids, which belong to one or more subclasses selected from the group consisting of the subclasses listed above.

[0459] The composite lipid particles can contain at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of natural lipids from natural sources. In some embodiments, the composite lipid particles contain at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of natural lipids from a single natural source (e.g., only from a plant source or only from a bacterial source). In some embodiments, the CLP can contain only one class or only one subclass of natural lipids from natural sources.

[0460] By lipidomic analysis, by dissolving the lipid extract or composite lipid particles in a compatible solvent and analyzing by mass spectrometry (e.g., MS / MS), the identity (as well as class and subclass) and amount of lipids extracted from natural sources can be analyzed. Other known methods can also be used, 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)).

[0461] The composite lipid particles can contain all or a portion of the lipid species present in the lipid structures from a specific natural source. For example, the composite lipid particles can 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 almost 100% of the lipid species present in the lipid structures from a specific natural source.

[0462] The composite lipid particles can comprise reduced or minimized proteinaceous materials that are endogenous to the one or more natural sources. For example, the composite lipid particles can 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 be substantially free of proteinaceous materials that are endogenous to the one or more natural sources. In some cases, the lipid bilayer of the composite lipid particles does not contain proteins. To calculate the % w / w of the residual proteinaceous materials that are endogenous to the one or more natural sources, the protein concentration is divided by the concentration of the native lipid extract and then multiplied by 100. Alternatively, the % w / w is calculated as the percentage of the mass of the total protein that is endogenous to the one or more natural sources based on the mass of the total lipid extract.

[0463] The composite lipid particles can comprise reduced or minimized residual dsDNA materials that are endogenous to the one or more natural sources. For example, the composite lipid particles can contain less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w, less than 0.005% w / w, less than 0.001% w / w or be substantially free of residual dsDNA materials that are endogenous to the one or more natural sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. To calculate the % w / w of the residual dsDNA materials that are endogenous to the one or more natural sources, the total adjusted dsDNA concentration is divided by the concentration of the native lipid extract and then multiplied by 100. Alternatively, the % w / w is calculated as the percentage of the mass of the total residual dsDNA that is endogenous to the one or more natural sources based on the mass of the total lipid extract.

[0464] In some embodiments, the composite lipid particles further incorporate synthetic structural lipids, such as neutral lipids. In some embodiments, the structural lipid component of the composite lipid particles can comprise from 10% w / w to 99% w / w of the structural lipids derived from synthetic lipid structures. For example, the composite lipid particles can 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 of lipids derived from synthetic lipid structures.

[0465] In addition to the exogenous ionizable lipid, the composite lipid particles can further comprise at least two other exogenous lipids. The composite lipid particles can comprise 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 of exogenous lipids. Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and PEG-lipid conjugates. The composite lipid particles can further comprise at least two exogenous lipids. In some embodiments, the composite lipid particles contain an ionizable lipid, a sterol, and a PEG-lipid conjugate. Additional exogenous lipids suitable for inclusion in the composite lipid particles are described below.

[0466] In some embodiments, the CLP contains natural lipids, and the natural lipids comprise fatty acid-derived tails, and the fatty acid-derived tails of the natural lipids are:

[0467] about 5% to 20% of fatty acid 16:0

[0468] about 0% to 10% of fatty acid 18:1(C9)

[0469] about 0% to 10% of fatty acid 18:1(C7)

[0470] about 5% to 30% of fatty acid 18:2

[0471] about 2% to 20% of fatty acid 18:3.

[0472] In some embodiments, the CLP contains phosphatidylcholine (PC) lipids, and the PC lipids comprise fatty acid-derived tails, and the fatty acid-derived tails of the PC lipids are:

[0473] about 10% to 20% of fatty acid 16:0

[0474] about 2% to 5% of fatty acid 18:0

[0475] About 7% to 15% of fatty acid 18:1

[0476] About 50% to 75% of fatty acid 18:2

[0477] About 2% to 10% of fatty acid 18:3.

[0478] In some embodiments, the CLP contains phosphatidylethanolamine (PE) lipids, the PE lipids comprising fatty acid-derived tails, and the fatty acid-derived tails of the PE lipids are:

[0479] About 0.25% to 5% of fatty acid 14:0

[0480] About 25% to 45% of fatty acid 16:0

[0481] About 5% to 15% of fatty acid 16:1

[0482] About 10% to 25% of fatty acid 17:0

[0483] About 25% to 45% of fatty acid 18:1

[0484] About 2% to 7% of fatty acid 19:0.

[0485] In some embodiments, the CLP contains natural lipids, the natural lipids belonging to the subclasses of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin and comprising:

[0486] About 50 wt / wt% to 75 wt / wt% of phosphatidylethanolamine (PE)

[0487] About 15 wt / wt% to 30 wt / wt% of phosphatidylglycerol (PG)

[0488] About 5 wt / wt% to 15 wt / wt% of cardiolipin (CL).

[0489] In some embodiments, the CLP contains natural lipids, the natural lipids comprising:

[0490] About 10 wt / wt% to 50 wt / wt% of phosphatidylcholine (PC)

[0491] About 5 wt / wt% to 50 wt / wt% of phosphatidylethanolamine (PE)

[0492] About 0 wt / wt% to 15 wt / wt% of triacylglycerol (TG)

[0493] About 5 wt / wt% to 35 wt / wt% of hexosylceramide (HexCer)

[0494] Phosphatidylglycerol (PG) from about 0 wt / wt% to 5 wt / wt%

[0495] Phosphatidylserine (PS) from about 0 wt / wt% to 7 wt / wt%

[0496] Phosphatidylinositol (PI) from about 0 wt / wt% to 10 wt / wt%

[0497] Cardiolipin (CL) from about 0 wt / wt% to 5 wt / wt%.

[0498] In some embodiments, the composite lipid particles contain less than 12% w / w of chloroplasts endogenous to the one or more natural sources. In some embodiments, the composite lipid particles contain 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 chloroplasts endogenous to the one or more natural sources.

[0499] In some embodiments, the composite lipid particles contain less than 5% w / w of exogenous antioxidants.

[0500] In some embodiments, the CLP contains natural lipids, and the natural lipids contain cardiolipin (CL) from about 0 wt / wt% to 20 wt / wt%.

[0501] Natural messenger package (NMP)

[0502] Multiple NMPs in the modified NMP formulation can be loaded with exogenous peptides, polypeptides or proteins 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 the multiple NMPs encapsulate exogenous peptides, polypeptides or proteins. In some embodiments, the NMP is derived from arthropods, fungi, archaea or bacteria.

[0503] NMP can include arthropod, plant, fungal, archaeal or bacterial EVs or segments, portions or extracts thereof, wherein the EVs have a diameter of about 5 - 2000 nm. For example, NMP can include EVs or segments, portions or extracts thereof having an average 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 - 1000 nm, about 1000 - 1250 nm, about 1250 - 1500 nm, about 1500 - 1750 nm or about 1750 - 2000 nm. In some cases, NMP includes arthropod, plant, fungal, archaeal or bacterial EVs or segments, portions or extracts thereof having an average 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 cases, the average diameter of the arthropod, plant, fungal, archaeal or bacterial EVs or segments, portions or extracts thereof is about 50 - 200 nm. In certain cases, the average diameter of the EVs or segments, portions or extracts thereof is about 50 - 300 nm. In certain cases, the average diameter of the EVs or segments, portions or extracts thereof is about 200 - 500 nm. In certain cases, the average diameter of the EVs or segments, portions or extracts thereof is about 30 - 150 nm.

[0504] In some cases, the NMP can include arthropod, plant, fungal, archaeal, or bacterial EVs or their segments, parts, or extracts having an average 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 cases, the NMP includes arthropod, fungal, archaeal, or bacterial EVs or their segments, parts, or extracts having an average diameter of: 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. Various standard methods in the art (e.g., dynamic light scattering method) can be used to measure the particle diameter of the EVs or their segments, parts, or extracts.

[0505] In some cases, the NMP can include arthropod, plant, fungal, archaeal, or bacterial EVs or their segments, parts, or extracts having an average surface area of 77 nm 2 to 3.2 x 10 6 nm 2 (e.g., 77 - 100 nm 2 , 100 - 1000 nm 2 , 1000 - 1 x 10 4 nm 2 , 1 x 10 4 - 1 x 10 5 nm 2 , 1 x 10 5 - 1 x 10 6 nm 2 or 1 x 10 6 - 3.2 x 10 6 nm 2 ). In some cases, the NMP can include arthropod, plant, fungal, archaeal, or bacterial EVs or their segments, parts, or extracts having an average volume of 65 nm 3 to 5.3 x 10 8 nm 3 (e.g., 65 - 100 nm 3 , 100 - 1000 nm 3 , 1000 - 1 x 104 nm 3 、 1x10 4 -1x10 5 nm 3 、 1x10 5 -1x10 6 nm 3 、 1x10 6 -1x10 7 nm 3 、 1x10 7 -1x10 8 nm 3 、 1x10 8 -5.3x10 8 nm 3 ) of arthropods, fungi, archaea or bacteria EVs or their segments, parts or extracts. In some cases, the NMP may include an average surface area of at least 77 nm 2 (e.g., at least 77 nm 2 、 at least 100 nm 2 、 at least 1000 nm 2 、 at least 1x10 4 nm 2 、 at least 1x10 5 nm 2 、 at least 1x10 6 nm 2 or at least 2x10 6 nm 2 ) of arthropods, plants, fungi, archaea or bacteria EVs or their segments, parts or extracts. In some cases, the NMP may include an average volume of at least 65 nm 3 (e.g., at least 65 nm 3 、 at least 100 nm 3 、 at least 1000 nm 3 、 at least 1x10 4 nm 3 、 at least 1x10 5 nm 3 、 at least 1x10 6 nm 3 、 at least 1x10 7 nm 3 、 at least 1x10 8 nm 3 、 at least 2x10 8 nm 3 、 at least 3x10 8 nm 3 、 at least 4x10 8 nm 3 or at least 5x10 8 nm3 Arthropod, fungal, archaeal, or bacterial EVs or segments, portions, or extracts thereof

[0506] In some cases, NMPs can have the same size as arthropod, plant, fungal, archaeal, or bacterial EVs or segments, extracts, or portions thereof. Alternatively, the size of NMPs can be different from the size of the initial EVs from which they are generated. For example, the diameter of NMPs can be about 5 - 2000 nm. For example, the average diameter of NMPs can be 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 - 1000 nm, about 1000 - 1200 nm, about 1200 - 1400 nm, about 1400 - 1600 nm, about 1600–1800 nm, or about 1800–2000 nm. In some cases, the average diameter of NMPs can be 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 standard methods in the art (e.g., dynamic light scattering methods) can be used to measure the particle diameter of NMPs. In some cases, the size of NMPs is determined after loading a heterologous functional agent or after other modifications to the NMPs.

[0507] In some cases, the average surface area of NMPs can be 77 nm 2 to 1.3x10 7 nm 2 (e.g., 77 - 100 nm 2 、100 - 1000 nm 2 、1000 - 1x10 4 nm 2 、1x10 4 -1x10 5 nm2 、 1x10 5 -1x10 6 nm 2 or 1x10 6 -1.3x10 7 nm 2 ). In some cases, the average volume of NMP can be 65 nm 3 to 4.2x10 9 nm 3 (e.g., 65 - 100 nm 3 、 100 - 1000 nm 3 、 1000 - 1x10 4 nm 3 、 1x10 4 -1x10 5 nm 3 、 1x10 5 -1x10 6 nm 3 、 1x10 6 -1x10 7 nm 3 、 1x10 7 -1x10 8 nm 3 、 1x10 8 -1x10 9 nm 3 or 1x10 9 -4.2x10 9 nm 3 ). In some cases, the average surface area of NMP is at least 77 nm 2 (e.g., at least 77 nm 2 、 at least 100 nm 2 、 at least 1000 nm 2 、 at least 1x10 4 nm 2 、 at least 1x10 5 nm 2 、 at least 1x10 6 nm 2 or at least 1x10 7 nm 2 )。In some cases, the average volume of NMP is at least 65 nm 3 (e.g., at least 65 nm 3 、 at least 100 nm 3 、 at least 1000 nm 3 、 at least 1x10 4 nm 3 、 at least 1x10 5 nm 3, at least 1x10 6 nm 3 , at least 1x10 7 nm 3 , at least 1x10 8 nm 3 , at least 1x10 9 nm 3 , at least 2x10 9 nm 3 , at least 3x10 9 nm 3 or at least 4x10 9 nm 3 ).

[0508] In some cases, NMP can include whole arthropods, plants, fungi, archaea, or bacterial EVs. In some embodiments, NMP can include non-plant natural sources such as algae or animal-derived organ EVs or segments, parts, or extracts thereof. Alternatively, NMP can include segments, parts, or extracts of the total surface area of the vesicles of EVs (e.g., including segments, parts, or extracts that are less than 100% of the total surface area of the vesicles (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%)). The segments, parts, or extracts can have any shape, such as circumferential segments, spherical segments (e.g., hemispherical), curved segments, straight segments, or flat segments. In the case where the segment is a spherical segment of a vesicle, the spherical segment can represent a spherical segment produced by splitting a spherical vesicle along a pair of parallel lines, or a spherical segment produced by splitting a spherical vesicle along a pair of non-parallel lines. Thus, multiple NMPs can include multiple whole EVs, multiple EV segments, parts, or extracts, or a mixture of whole EVs and their segments. Those skilled in the art will appreciate that the ratio of whole to fragmented EVs will depend on the specific separation method used. For example, compared to non-destructive extraction methods such as vacuum infiltration, grinding or blending arthropods, fungi, plants, archaea, or bacteria or parts thereof can produce NMPs containing a higher percentage of EV segments, parts, or extracts.

[0509] In the case where NMP includes segments, parts, or extracts of arthropod, fungal, archaeal, or bacterial EVs, the EV segments, parts, or extracts can have an average surface area that is less than the average surface area of the whole vesicles (e.g., the average surface area is less than 77 nm 2 , 100 nm 2 , 1000 nm 2 , 1x10 4 nm 2 , 1x10 5 nm2 、 1 x 10 6 nm 2 or 3.2 x 10 6 nm 2 ). In some cases, the surface area of the EV section, portion or extract is less than 70 nm 2 、 60 nm 2 、 50 nm 2 、 40 nm 2 、 30 nm 2 、 20 nm 2 or 10 nm 2 . In some cases, NMP can include arthropod, fungal, archaeal or bacterial EVs or fragments, portions or extracts thereof with an average volume less than the average volume of intact vesicles (e.g., an average volume less than 65 nm 3 、 100 nm 3 、 1000 nm 3 、 1 x 10 4 nm 3 、 1 x 10 5 nm 3 、 1 x 10 6 nm 3 、 1 x 10 7 nm 3 、 1 x 10 8 nm 3 or 5.3 x 10 8 nm 3 ).

[0510] In cases where the NMP includes an extract of arthropod, plant, fungal, archaeal or bacterial EVs, such as in cases where the NMP includes lipids extracted from EVs (e.g., with chloroform or ethanol), the NMP can include at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more than 99% of the lipids extracted from arthropod, fungal, archaeal or bacterial EVs (e.g., with chloroform or with ethanol). The NMPs among the plurality can include arthropod, plant, fungal, archaeal or bacterial EV sections and / or EV-extracted lipids or mixtures thereof.

[0511] Plant messenger package (PMP)

[0512] A PMP is a lipid (e.g., a lipid bilayer, monolayer, or multi-layer structure) structure that includes a plant EV or a segment, portion, or extract thereof (e.g., a lipid extract). A plant EV is a closed lipid bilayer structure that naturally occurs in plants and has a diameter of about 5 - 2000 nm. Plant EVs can be derived from multiple plant biogenesis pathways. In nature, plant EVs can be present in intracellular and extracellular compartments of plants, such as the plant apoplast, which is located outside the plasma membrane and is formed by the cell wall continuum and the extracellular space. Alternatively, a PMP can be a plant EV-enriched present in cell culture media after secretion from plant cells. Plant EVs can be isolated from plants by multiple methods further described herein, thereby providing PMPs. Additionally, a PMP can optionally include a therapeutic agent, which can be introduced in vivo or in vitro.

[0513] The PMP may comprise plant EVs or segments, parts or extracts thereof. Optionally, in addition to the lipids derived from plant EVs, the PMP may further comprise exogenous lipids (e.g., sterols (e.g., cholesterol or sitosterol), ionizable lipids, and / or PEGylated lipids). In some embodiments, the diameter of the plant EVs is about 5 - 1000 nm. For example, the PMP may comprise plant EVs or segments, parts or extracts thereof having an average 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 - 1000 nm, about 1000 - 1250 nm, about 1250 - 1500 nm, about 1500 - 1750 nm or about 1750 - 2000 nm. In some cases, the PMP comprises plant EVs or segments, parts or extracts thereof having an average diameter of: about 5 - 950 nm, about 5 - 900 nm, about 5 - 850 nm, about 5 - 800 nm, about 5 - 750 nm, about 5 - 700 nm, about 5 - 650 nm, about 5 - 600 nm, about 5 - 550 nm, about 5 - 500 nm, about 5 - 450 nm, about 5 - 400 nm, about 5 - 350 nm, about 5 - 300 nm, about 5 - 250 nm, about 5 - 200 nm, about 5 - 150 nm, about 5 - 100 nm, about 5 - 50 nm or about 5 - 25 nm. In certain cases, the average diameter of the plant EVs or segments, parts or extracts thereof is about 50 - 200 nm. In certain cases, the average diameter of the plant EVs or segments, parts or extracts thereof is about 50 - 300 nm. In certain cases, the average diameter of the plant EVs or segments, parts or extracts thereof is about 200 - 500 nm. In certain cases, the average diameter of the plant EVs or segments, parts or extracts thereof is about 30 - 150 nm.

[0514] In some cases, the PMP can include plant EVs or their segments, parts, or extracts having an average 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, or at least 1000 nm. In some cases, the PMP includes plant EVs or their segments, parts, or extracts having an average diameter of: 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. Various standard methods in the art (e.g., dynamic light scattering method) can be used to measure the particle diameter of plant EVs or their segments, parts, or extracts.

[0515] In some cases, the PMP can include plant EVs or their segments, parts, or extracts having an average surface area of 77 nm 2 to 3.2 x 10 6 nm 2 (e.g., 77 - 100 nm 2 , 100 - 1000 nm 2 , 1000 - 1 x 10 4 nm 2 , 1 x 10 4 - 1 x 10 5 nm 2 , 1 x 10 5 - 1 x 10 6 nm 2 or 1 x 10 6 - 3.2 x 10 6 nm 2 ) of plant EVs or their segments, parts, or extracts. In some cases, the PMP can include plant EVs or their segments, parts, or extracts having an average volume of 65 nm 3 to 5.3 x 10 8 nm 3 (e.g., 65 - 100 nm 3 , 100 - 1000 nm 3 , 1000 - 1 x 10 4 nm 3 , 1 x 10 4 - 1 x 10 5nm 3 、 1x10 5 -1x10 6 nm 3 、 1x10 6 -1x10 7 nm 3 、 1x10 7 -1x10 8 nm 3 、 1x10 8 -5.3x10 8 nm 3 ) of plant EVs or their segments, parts or extracts. In some cases, the PMP may include plant EVs or their segments, parts or extracts with an average surface area of at least 77 nm 2 (e.g., at least 77 nm 2 、 at least 100 nm 2 、 at least 1000 nm 2 、 at least 1x10 4 nm 2 、 at least 1x10 5 nm 2 、 at least 1x10 6 nm 2 or at least 2x10 6 nm 2 ) of plant EVs or their segments, parts or extracts. In some cases, the PMP may include plant EVs or their segments, parts or extracts with an average volume of at least 65 nm 3 (e.g., at least 65 nm 3 、 at least 100 nm 3 、 at least 1000 nm 3 、 at least 1x10 4 nm 3 、 at least 1x10 5 nm 3 、 at least 1x10 6 nm 3 、 at least 1x10 7 nm 3 、 at least 1x10 8 nm 3 、 at least 2x10 8 nm 3 、 at least 3x10 8 nm 3 、 at least 4x10 8 nm 3 or at least 5x10 8 nm 3 ) of plant EVs or their segments, parts or extracts.

[0516] In some cases, the size of the PMP can be the same as the size of the plant EV or its segments, extracts, or parts. Alternatively, the size of the PMP can be different from the size of the initial plant EV from which it is produced. For example, the diameter of the PMP can be about 5 - 2000 nm. For example, the average diameter of the PMP can be 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 - 1000 nm, about 1000 - 1200 nm, about 1200 - 1400 nm, about 1400 - 1600 nm, about 1600–1800 nm, or about 1800–2000 nm. In some cases, the average diameter of the PMP can be 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. Various standard methods in the art (e.g., dynamic light scattering method) can be used to measure the particle diameter of the PMP. In some cases, the size of the PMP is determined after loading a therapeutic agent or after other modifications to the PMP.

[0517] In some cases, the average surface area of the PMP can be 77 nm 2 to 1.3x10 7 nm 2 (e.g., 77 - 100 nm 2 、100 - 1000 nm 2 、1000 - 1x10 4 nm 2 、1x10 4 -1x10 5 nm 2 、1x10 5 -1x10 6 nm 2 or 1x10 6-1.3x10 7 nm 2 )。 In some cases, the average volume of the PMP can be 65 nm 3 to 4.2x10 9 nm 3 (e.g., 65 - 100 nm 3 , 100 - 1000 nm 3 , 1000 - 1x10 4 nm 3 , 1x10 4 -1x10 5 nm 3 , 1x10 5 -1x10 6 nm 3 , 1x10 6 -1x10 7 nm 3 , 1x10 7 -1x10 8 nm 3 , 1x10 8 -1x10 9 nm 3 or 1x10 9 -4.2x10 9 nm 3 )。 In some cases, the average surface area of the PMP is at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1x10 4 nm 2 , at least 1x10 5 nm 2 , at least 1x10 6 nm 2 or at least 1x10 7 nm 2 )。 In some cases, the average volume of the PMP is at least 65 nm 3 (e.g., at least 65 nm 3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1x10 4 nm 3 , at least 1x10 5 nm 3 , at least 1x10 6 nm 3 , at least 1x10 7 nm 3 , at least 1x108 nm 3 , at least 1x10 9 nm 3 , at least 2x10 9 nm 3 , at least 3x10 9 nm 3 or at least 4x10 9 nm 3 ).

[0518] In some cases, the PMP can include whole plant EVs. Alternatively, the PMP can include a segment, portion or extract of the total surface area of the vesicles of the plant EV (e.g., including a segment, portion or extract that is less than 100% of the total surface area of the vesicles (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%)). The segment, portion or extract can have any shape, such as a circumferential segment, a spherical segment (e.g., hemispherical), a curved segment, a straight segment or a flat segment. In the case where the segment is a spherical segment of the vesicle, the spherical segment can represent a spherical segment produced by splitting the spherical vesicle along a pair of parallel lines, or a spherical segment produced by splitting the spherical vesicle along a pair of non-parallel lines. Thus, multiple PMPs can include multiple whole plant EVs, multiple plant EV segments, portions or extracts, or a mixture of whole plant EVs and their segments. Those skilled in the art will appreciate that the ratio of whole to fragmented plant EVs will depend on the particular separation method used. For example, compared to non-destructive extraction methods, such as vacuum infiltration, grinding or blending the plant or its parts can produce PMPs containing a higher percentage of plant EV segments, portions or extracts.

[0519] In the case where the PMP includes a segment, portion or extract of the plant EV, the EV segment, portion or extract can have an average surface area that is less than the average surface area of the whole vesicle (e.g., an average surface area less than 77 nm 2 , 100 nm 2 , 1000 nm 2 , 1x10 4 nm 2 , 1x10 5 nm 2 , 1x10 6 nm 2 or 3.2x10 6 nm 2 ). In some cases, the surface area of the EV segment, portion or extract is less than 70 nm 2 , 60 nm 2 , 50 nm 2, 40 nm 2 , 30 nm 2 , 20 nm 2 or 10 nm 2 . In some cases, the PMP may include plant EVs or fragments, parts or extracts thereof with an average volume less than the average volume of intact vesicles (e.g., with an average volume less than 65 nm 3 , 100 nm 3 , 1000 nm 3 , 1x10 4 nm 3 , 1x10 5 nm 3 , 1x10 6 nm 3 , 1x10 7 nm 3 , 1x10 8 nm 3 or 5.3x10 8 nm 3 ).

[0520] In the case where the PMP includes an extract of plant EVs, e.g., in the case where the PMP includes lipids extracted from plant EVs (e.g., with chloroform or ethanol), the PMP may include at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater than 99% of the lipids extracted from plant EVs (e.g., with chloroform or ethanol). The PMPs among the plurality may include plant EV segments and / or lipids extracted from plant EVs or mixtures thereof.

[0521] Generation of NMP

[0522] NMP can be produced from arthropod, fungal, archaeal or bacterial EVs or segments, parts or extracts (e.g., lipid extracts) thereof that occur naturally in arthropods, fungi, archaea or bacteria or parts thereof (including tissues or cells). In some embodiments, the NMP can include non-plant natural sources such as algal or animal-derived organ EVs or segments, parts or extracts thereof. Exemplary methods for producing NMP include (a) providing an initial sample from a source or part thereof, wherein the source or part thereof contains EVs; and (b) isolating a crude NMP fraction from the initial sample, wherein the level of at least one contaminant or undesired component from the source or part thereof in the crude NMP fraction is reduced 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 reduced level of at least one contaminant or undesired component from arthropods, fungi, archaea or bacteria or parts thereof relative to the level in the crude EV fraction. Each production step is discussed in further detail below. Exemplary methods for the isolation and purification of NMP can be found in, for example, 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 incorporated herein by reference.

[0523] For example, multiple NMPs can be isolated from arthropods, fungi, archaea, or bacteria by a method comprising the following steps: (a) providing an initial sample from a source or a part thereof, wherein the source or the part thereof contains EVs; (b) isolating a crude NMP fraction from the initial sample, wherein the level of at least one contaminant or undesired component from arthropods, fungi, archaea, or bacteria or a part thereof in the crude NMP fraction is reduced relative to the level in the initial sample (e.g., the level is reduced 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 multiple pure NMPs, wherein the level of at least one contaminant or undesired component from the source or a part thereof in the multiple pure NMPs is reduced relative to the level in the crude EV fraction (e.g., the level is reduced 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% of the level).

[0524] The NMP provided herein may include arthropod, fungal, archaeal or bacterial EVs or segments, portions or extracts isolated from a variety of sources. NMP can be isolated from any genus of arthropods, fungi, archaea or bacteria, including but not limited to crabs, crayfish, shrimp, spiders, scorpions, crickets, grasshoppers, beetles, millipedes, ticks, mites, centipedes, ants, wasps, dragonflies, flies, mosquitoes, other insects and crustaceans, yeasts, mushrooms, puffballs, stinkhorns, boletes, smuts, bunts, polypores, jelly fungi, agarics, molds, rusts, earthstars, chanterelles, ergot, fireleaf fungi, bitter-loving fungi, methanogens, crenarchaeota, nanoarchaeota, ignicoccus, caldiarchaeum, halophiles, Escherichia, Acinetobacter, Agrobacterium, Anabaena, Epulopiscium, Aquifex, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus Liberibacter, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfovibrio, Erwinia, Francisella, Clostridium, Myxococcus, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Candida, Photobacterium, Photobaculum, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rhodobacter, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Flavobacterium, Xylella, Yersinia pestis, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Microbacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium or Thermoanaerobacter.

[0525] NMP can be produced by whole arthropods, fungi, archaea, or bacteria (e.g., whole insects, spiders, crustaceans, fungi, or single cells of archaea or bacteria) or alternatively from one or more source parts (e.g., segments, organs, eggs, spores, mycelia, tissues, membranes, or cell walls). For example, NMP can be produced from organ / structure / tissue / cell cultures (e.g., body segments, appendages, organs, eggs, exoskeletons, embryos, spores, mycelia, hyphae, thalli, suspension cultures, cell walls, inner membranes or outer membranes, gametophytes, sporophytes, polymerases, glycerol-ether lipids, metabolites, flagella, pili, ribosomes, or organelles) or their progeny. The source can be at any developmental stage. In some embodiments, NMP is produced by insects or fungi (e.g., crickets, yeast, or mushrooms). In some embodiments, NMP is produced by bacteria or archaea (e.g., E. coli). In some embodiments, NMP is produced by algae (e.g., seaweed or chlorella). In some embodiments, NMP is produced by animal organs (e.g., brain or blood).

[0526] NMP can be produced from arthropods, fungi, archaea, or bacteria or parts thereof by a variety of methods. Any method that allows the release of an EV-containing fraction from the source or other extracellular fractions containing NMPs that contain secreted EVs (e.g., cell culture medium) is suitable for the methods of the present invention. EVs can be isolated from the source or source part by destructive methods (e.g., grinding or blending) or non-destructive methods (washing or vacuum infiltration). For example, arthropods, fungi, archaea, or bacteria or parts thereof can be vacuum infiltrated, ground, blended, or a combination thereof to isolate EVs from the source or source part, thereby producing NMP. For example, the isolation step can involve (b) isolating a crude NMP fraction from an initial sample (e.g., an arthropod, fungus, archaea, or bacteria or part, or a sample derived from an arthropod, fungus, archaea, or bacteria or part) wherein the level of at least one contaminant or undesired component from the source or its part in the crude NMP fraction is reduced relative to its level in the initial sample; wherein the isolation step involves vacuum infiltrating an arthropod, fungus, archaea, or bacteria (e.g., using a vesicle isolation buffer) to release and collect the desired fraction. Alternatively, the isolation step can involve (b) grinding or blending the source to release EVs, thereby producing NMP.

[0527] When isolating arthropod, fungal, archaeal, or bacterial EVs to generate NMPs, the NMPs can be isolated or collected as a crude NMP fraction. For example, the isolation step can involve separating multiple 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 organelles. Thus, the crude NMP fraction will have a reduced number of large contaminants, including, for example, tissue fragments, cells, or organelles (e.g., nuclei, mitochondria, etc.) compared to the initial sample from the source or source part.

[0528] The crude NMP fraction can be further purified by additional purification methods to generate multiple pure NMPs. For example, the crude NMP fraction can be separated from other source components by ultracentrifugation, such as using a density gradient (iodixanol or sucrose), size exclusion, and / or other methods that remove aggregated components (e.g., precipitation or size exclusion chromatography). The 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, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof) in the resulting pure NMP can be reduced relative to one or more fractions generated during earlier isolation steps or relative to a pre-established threshold level, such as a commercial release specification. For example, the level of source organelles or cell wall components in the pure NMP can be reduced relative to the level in the initial sample (e.g., reduced by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%; or reduced by about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x, or greater than 100x). In some cases, the pure NMP is substantially free (e.g., has undetectable levels) of one or more non-NMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof. Additional examples of release and isolation steps can be found in WO2021 / 041301. The concentration of NMP can be, for example, 1x10 9 、5x10 9 、1x10 10 、5x10 10 、5x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11, 7 x 10 11 , 8 x 10 11 , 9 x 10 11 , 1 x 10 12 , 2 x 10 12 , 3 x 10 12 , 4 x 10 12 , 5 x 10 12 , 6 x 10 12 , 7 x 10 12 , 8 x 10 12 , 9 x 10 12 , 1 x 10 13 or greater than 1 x 10 13 NMP / mL.

[0529] For example, protein aggregates can be removed from the isolated NMP. For example, the isolated NMP solution can be taken out at a range of pH (e.g., as measured using a pH probe) to precipitate the protein aggregates in the solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9 or pH 11 by adding, for example, 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 by adding a charged polymer such as Polymin-P or Praestol 2640. Briefly, Polymin-P or Praestol 2640 is added to the solution and mixed with an impeller. The solution can then be filtered to remove particulates. Alternatively, the aggregates can be dissolved by increasing the salt concentration. For example, NaCl can be added to the isolated NMP solution until it reaches, for example, 1 mol / L. The solution can then be filtered to separate the NMP. Alternatively, the aggregates are dissolved by raising the temperature. For example, the isolated NMP can be heated while mixing until the solution reaches a uniform temperature of, for example, 50 °C for 5 minutes. The NMP mixture can then be filtered to separate the NMP. Alternatively, according to standard procedures, soluble contaminants can be separated from the NMP solution by a size exclusion chromatography column, where the NMP elutes in the first fraction, while the proteins and ribonucleoproteins and some lipoproteins elute subsequently. The efficiency of protein aggregate removal can be determined by measuring the protein concentration before and after removing the protein aggregates using BCA / Bradford protein quantification. In some embodiments, the protein aggregates are removed before the exogenous peptide, polypeptide or protein is encapsulated by the NMP. In other embodiments, the protein aggregates are removed after the exogenous peptide, polypeptide or protein is encapsulated by the NMP.

[0530] In some embodiments, the preparation of NMP from natural sources is carried out by an ethanol extraction method. In some aspects, a 3:2 ethyl acetate:ethanol solvent facilitates the extraction. In some embodiments, the preparation of NMP from natural sources is carried out by a modified Matyash extraction method. In some aspects, a 1:2 MeOH:MTBE solvent facilitates the extraction.

[0531] Any of the production methods described herein for producing NMP can be supplemented with any quantitative or qualitative methods known in the art to characterize or identify NMP at any step of the production process. NMP can be characterized by a variety of analytical methods to estimate NMP yield, NMP concentration, NMP purity, NMP composition, or NMP size. NMP can be evaluated by a variety of methods known in the art capable of visualizing, quantifying, or qualitatively characterizing NMP (e.g., identification of composition), such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopic analysis (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis). In certain cases, methods (e.g., mass spectrometry) can be used to identify EV markers present on NMP. To facilitate the analysis and characterization of NMP fractions, NMP can additionally be labeled or stained. For example, NMP can be stained with iodide 3,3'-dihexyloxacarbocyanine (DIOC6) (fluorescent lipophilic dye), PKH67 (Sigma Aldrich), Alexa 488 (Thermo Fisher Scientific) or DyLight TM 800 (Thermo Fisher). In the absence of sophisticated forms of nanoparticle tracking, this relatively simple method quantifies total membrane content and can be used to indirectly measure the concentration of NMP (Rutter and Innes, Plant Physiology 173(1):728-741, 2017; Rutter et al., Bio-protocol 7(17):e2533, 2017). For more precise measurements and to evaluate the size distribution of NMP, nanoparticle tracking, nanoflow cytometry, or tunable resistive pulse sensing can be used.

[0532] During the production process, NMP can optionally be prepared such that the concentration of NMP is increased relative to the EV level in a control or initial sample (e.g., increased by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100%; or increased by about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x or more than 100x). The isolated NMP can be about 0.1% to about 100% of the NMP composition, such as any 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 cases, the compositions described herein include at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher NMP, e.g., as measured by weight / volume, percent NMP protein composition and / or percent lipid composition (e.g., by measuring fluorescently labeled lipids). In some cases, a concentrate is used as a commercial product, e.g., a diluent with a significantly lower concentration of the active ingredient can be used by the end user. In some embodiments, the compositions described herein are formulated as NMP concentrated formulations, e.g., ultra-low volume concentrated formulations. In some embodiments, the concentration of NMP in the composition can effectively increase the fitness of an organism, such as a plant, animal, insect, bacterium or fungus. In other aspects, the concentration of NMP in the composition can effectively decrease the fitness of an organism, such as a plant, animal, insect, bacterium or fungus.

[0533] NMP can be produced by a variety of arthropods, fungi, archaea or bacteria or one or more parts thereof (e.g., segments, organs, eggs, spores, mycelia, tissues, membranes or cell walls). For example, NMP can be produced by an organ / structure / tissue / cell culture (e.g., body segment, appendage, organ, egg, exoskeleton, embryo, spore, mycelium, hypha, thallus, suspension culture, cell wall, inner membrane or outer membrane, gametophyte, sporophyte, polymerase, glycerol-ether lipid, metabolite, flagellum, pilus, ribosome or organelle) or its progeny. The source can be at any developmental stage. In some embodiments, NMP is produced by an insect or a fungus (e.g., cricket, yeast or mushroom). In some embodiments, NMP is produced by a bacterium or an archaeon (e.g., E. coli). In some embodiments, NMP is produced by an alga (e.g., seaweed or Chlorella). In some embodiments, NMP is produced by an animal organ (e.g., brain or blood).

[0534] NMP can be generated and purified by a variety of methods, e.g., by using density gradients (iodixanol or sucrose) in combination with ultracentrifugation and / or methods for removing aggregated contaminants such as precipitation or size exclusion chromatography.

[0535] In some cases, the NMP of the compositions and methods of the present invention can be isolated from arthropods, fungi, archaea, or bacteria or parts thereof and used without further modification of the NMP. In other cases, the NMP can be modified prior to use as further outlined herein.

[0536] Generation of PMP

[0537] PMP can be generated from plant EVs or segments, parts, or extracts thereof (such as lipid extracts) that are naturally present in plants or parts thereof (including plant tissues or plant cells). Exemplary methods for generating PMP include (a) providing an initial sample from a plant or part thereof, wherein the plant or part thereof contains EVs; and (b) isolating a crude PMP fraction from the initial sample, wherein the level of at least one contaminant or undesired component from the plant or part thereof in the crude PMP fraction is reduced relative to the level in the initial sample. The method can further include an additional step (c) that comprises purifying the crude PMP fraction, thereby generating a plurality of pure PMPs, wherein the level of at least one contaminant or undesired component from the plant or part thereof in the plurality of pure PMPs is reduced relative to the level in the crude EV fraction. Each generation step is discussed in further detail below. Exemplary methods for the isolation and purification of PMP can be found in, e.g., Rutter and Innes, Plant Physiology 173(1):728-741, 2017; Rutter et al., Bio-protocol 7(17):e2533, 2017; Regente et al., Journal of Experimental Biology 68(20):5485-5496, 2017; Mu et al., Molecular Nutrition & Food Research, 58, 1561–1573, 2014 and Regente et al., FEBS Letters. 583:3363-3366, 2009, each of which is incorporated herein by reference.

[0538] In some cases, multiple PMPs can be isolated from plants by a method including the following steps: (a) providing an initial sample from a plant or a part thereof, wherein the plant or the part thereof contains EVs; (b) isolating a crude PMP fraction from the initial sample, wherein the level of at least one contaminant or undesired component from the plant or the part thereof in the crude PMP fraction is reduced relative to the level in the initial sample (e.g., the level is reduced 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 PMP fraction, thereby producing multiple pure PMPs, wherein the level of at least one contaminant or undesired component from the plant or the part thereof in the multiple pure PMPs is reduced relative to the level in the crude EV fraction (e.g., the level is reduced 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% of the level).

[0539] PMP can include plant EVs or their segments, parts or extracts produced by a variety of plants. PMP can be produced by plants of any genus (vascular or non-vascular plants), including but not limited to angiosperms (monocots and dicots), gymnosperms, ferns, Selaginella plants, Equisetum plants, Psilophytes, Lycophytes, algae (e.g., unicellular or multicellular algae, e.g., Archaeplastida), or bryophytes. In some cases, PMP can be produced using vascular plants, such as monocots or dicots or gymnosperms. For example, PMP can be produced using: alfalfa, apple, Arabidopsis, banana, barley, 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, liliaceae plants, flaxseed, millet, melon, mustard, oats, oil palm, rapeseed, papaya, peanut, pineapple, ornamental plants, Phaseolus vulgaris, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugar cane, sunflower, strawberry, tobacco, tomato, turf grass, wheat or vegetable crops, such as lettuce, celery, broccoli, cauliflower, gourd; fruit trees and nut trees, such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazelnut; vines, such as grape, kiwi, hops; fruit shrubs and brambles, such as raspberry, blackberry, currant; forest trees, such as ash, pine, fir, maple, oak, chestnut, poplar; with alfalfa, canola, castor bean, corn, cotton, Crambe, flax, flaxseed, mustard, oil palm, rapeseed, peanut, potato, rice, safflower, sesame, soybean, sugar beet, sunflower, tobacco, tomato or wheat.

[0540] PMP can be produced using the whole plant (e.g., whole flower clusters or seedlings), or alternatively from one or more plant parts (e.g., leaves, seeds, roots, fruits, vegetables, pollen, phloem sap or xylem sap). For example, PMP can be produced using the following: vegetative organs / structures of buds (e.g., leaves, stems or tubers), roots, flowers and floral organs / structures (e.g., pollen, bracts, sepals, petals, stamens, carpels, anthers or ovules), seeds (including embryos, endosperm or seed coats), fruits (ripened ovaries), saps (e.g., phloem or xylem sap), plant tissues (e.g., vascular tissue, root tissue, tumor tissue, etc.) and cells (e.g., single cells, protoplasts, embryos, callus, guard cells, egg cells, etc.) or their progeny. For example, the isolation step can involve (a) providing a plant or a part thereof. In some instances, the plant part is a leaf of a plant of the genus Arabidopsis. The plant can be at any developmental stage. For example, PMP can be produced using seedlings, e.g., seedlings that are 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks old (e.g., seedlings of a plant of the genus Arabidopsis). Other exemplary PMPs can include PMPs produced using roots (e.g., ginger roots), fruit juices (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., phloem sap of a plant of the genus Arabidopsis) or xylem sap (e.g., xylem sap of a tomato plant).

[0541] In some embodiments, PMP is produced from algae or lemons.

[0542] PMP can be produced from a plant or a part thereof by a variety of methods. Any method that allows the release of the EV-containing apoplast fraction of the plant or other extracellular fractions containing PMPs that contain secreted EVs (e.g., cell culture medium) is suitable for the methods of the present invention. EVs can be isolated from a plant or a plant part by a destructive method (e.g., grinding or blending the plant or any plant part) or a non-destructive method (washing or vacuum infiltrating the plant or any plant part). For example, a plant or a part thereof can be vacuum infiltrated, ground, blended or a combination thereof to isolate EVs from the plant or the plant part, thereby producing PMP. For example, the isolation step can involve infiltrating the plant (e.g., with a vesicle isolation buffer) to release and collect the apoplast fraction. Alternatively, the isolation step can involve grinding or blending the plant to release EVs, thereby producing PMP.

[0543] When isolating plant EVs and thereby generating PMPs, the PMPs can be isolated or collected into a crude PMP fraction (e.g., the apoplast fraction). For example, the isolation step can involve using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate multiple PMPs into a crude PMP fraction to separate the fraction containing plant PMPs from large contaminants including plant tissue fragments or plant cells. Thus, the amount of large contaminants including plant tissue fragments or plant cells in the crude PMP fraction will be reduced compared to the initial sample from the plant or plant part. Depending on the method used, the crude PMP fraction can additionally contain reduced levels of plant organelles (e.g., nuclei, mitochondria or chloroplasts) compared to the initial sample from the plant or plant part.

[0544] In some cases, the isolation step can involve using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate multiple PMPs into a crude PMP fraction to separate the fraction containing PMPs from plant cells or cell fragments. In such cases, the amount of plant cells or cell fragments in the crude PMP fraction will be reduced compared to the initial sample from the source plant or plant part.

[0545] The crude PMP fraction can be further purified by additional purification methods to generate multiple pure PMPs. For example, the crude PMP fraction can be separated from other plant components by ultracentrifugation, e.g., using a density gradient (iodixanol or sucrose) and / or using other methods to remove aggregated components (e.g., precipitation or size exclusion chromatography). The level of contaminants or other unwanted components from the source plant (e.g., one or more non-PMP components such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles or combinations thereof) in the resulting pure PMPs can be reduced relative to one or more fractions generated during earlier isolation steps or relative to a pre-established threshold level, e.g., a commercial release specification. For example, the level of plant organelles or cell wall components in the pure PMPs can be reduced (e.g., reduced by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100%; or reduced by about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x or greater than 100x) relative to the level in the initial sample. In some cases, the pure PMPs are substantially free (e.g., have undetectable levels) of one or more non-PMP components such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles or combinations thereof. The concentration of the PMPs can be, for example, 1x10 9 、5x10 9, 1 x 10 10 , 5 x 10 10 , 5 x 10 10 , 1 x 10 11 , 2 x 10 11 , 3 x 10 11 , 4 x 10 11 , 5 x 10 11 , 6 x 10 11 , 7 x 10 11 , 8 x 10 11 , 9 x 10 11 , 1 x 10 12 , 2 x 10 12 , 3 x 10 12 , 4 x 10 12 , 5 x 10 12 , 6 x 10 12 , 7 x 10 12 , 8 x 10 12 , 9 x 10 12 , 1 x 10 13 or greater than 1 x 10 13 PMP / mL.

[0546] For example, protein aggregates can be removed from PMP. For example, PMP can be taken out at a range of pH (e.g., as measured using a pH probe) to precipitate protein aggregates in the solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9, or pH 11 by adding, for example, sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it can be filtered to remove particulates. Alternatively, PMP can be flocculated using an added charged polymer such as Polymin-P or Praestol 2640. Briefly, Polymin-P or Praestol 2640 is added to the solution and mixed with an impeller. The solution can then be filtered to remove particulates. Alternatively, the aggregates can be dissolved by increasing the salt concentration. For example, NaCl can be added to PMP until it reaches, for example, 1 mol / L. The solution can then be filtered to separate the PMP. Alternatively, the aggregates can be dissolved by raising the temperature. For example, PMP can be heated while mixing until the solution reaches a uniform temperature of, for example, 50 °C for 5 minutes. The PMP mixture can then be filtered to separate the PMP. Alternatively, according to standard procedures, soluble contaminants can be separated from the PMP solution by size exclusion chromatography columns, where PMP elutes in the first fraction, while proteins and ribonucleoproteins and some lipoproteins elute subsequently. The efficiency of protein aggregate removal can be determined by measuring protein concentration before and after removing protein aggregates using BCA / Bradford protein quantification and comparing them.

[0547] Any generation method in the generation methods described herein can be supplemented with any quantitative or qualitative method known in the art to characterize or identify PMPs at any step of the generation process. PMPs can be characterized by a variety of analytical methods to estimate PMP yield, PMP concentration, PMP purity, PMP composition, or PMP size. PMPs can be evaluated by a variety of methods known in the art capable of achieving visualization, quantitative, or qualitative characterization of PMPs (e.g., identification of composition), such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopic analysis (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis). In some cases, methods (e.g., mass spectrometry) can be used to identify plant EV markers present on PMPs, such as the markers disclosed in the appendix. To facilitate the analysis and characterization of PMP fractions, PMPs can additionally be labeled or stained. For example, PMPs can be stained with iodide 3,3'-dihexyloxacarbocyanine (DIOC6) (fluorescent lipophilic dye), PKH67 (Sigma-Aldrich), Alexa 488 (Thermo Fisher Scientific), or DyLight TM 800 (Thermo Fisher). In the absence of sophisticated forms of nanoparticle tracking, this relatively simple method quantifies total membrane content and can be used to indirectly measure PMP concentration (Rutter and Innes, Plant Physiology 173(1):728-741, 2017; Rutter et al., BioProtocol 7(17):e2533, 2017). For more precise measurements and to evaluate the size distribution of PMPs, nanoparticle tracking can be used.

[0548] During production, PMP can optionally be prepared such that the concentration of PMP is increased relative to the EV level in a control or initial sample (e.g., increased by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100%; or increased by about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x or more than 100x). PMP can be about 0.1% to about 100% of the PMP composition, such as any 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% or about 75% to about 100%. In some cases, the compositions described herein include at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher PMP, e.g., as measured by weight / volume, percentage of PMP protein composition and / or percentage of lipid composition (e.g., by measuring fluorescently labeled lipids). In some cases, a concentrate is used as a commercial product, e.g., the end user can use a diluent with a significantly lower concentration of the active ingredient. In some embodiments, the compositions described herein are formulated as agricultural concentrate formulations, e.g., ultra-low volume concentrate formulations.

[0549] Lipid-reconstituted native messenger packaging (LNMP)

[0550] Lipid-reconstituted NMP (LNMP) is used herein. LNMP refers to NMP that has been derived from a lipid structure (e.g., lipid bilayer, monolayer, multi-layer structure; e.g., vesicle lipid structure) from a natural source (e.g., enriched, isolated or purified therefrom), wherein the lipid structure is disrupted (e.g., by lipid extraction) and reassembled or reconstituted in a liquid phase (e.g., a liquid phase containing cargo) using standard methods, e.g., reconstituted by methods including lipid membrane hydration and / or solvent injection to produce LNMP as described herein. If desired, the method can further include sonication, freeze / thaw treatment and / or lipid extrusion, e.g., to reduce the size of the reconstituted LNMP. Alternatively, a microfluidic device (such as IGNITE TM a microfluidic instrument (Precision NanoSystems Inc.)) can be used to produce LNMP.

[0551] In some embodiments, LNMP is produced by a process comprising the steps of: (a) providing a plurality of purified NMPs (e.g., NMP purified as described in Section IA herein); (b) treating the plurality of NMPs to produce a lipid membrane; (c) reconstituting the lipid membrane in an organic solvent or solvent combination, thereby producing a lipid solution; and (d) treating the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, thereby producing LNMP.

[0552] In some cases, treating the plurality of NMPs to produce a lipid membrane includes extracting lipids from the plurality of NMPs, e.g., using the Bligh-Dyer method (Bligh and Dyer, J Biolchem Physiol, 37:911-917, 1959). The extracted lipids can be provided as a stock solution, e.g., a chloroform:methanol solution. Producing the lipid membrane can include, for example, evaporating the solvent with a stream of inert gas (e.g., nitrogen).

[0553] Lipid-reconstituted plant messenger package (LPMP)

[0554] Lipid-reconstituted PMP (LPMP) is used herein. LPMP refers to a PMP that has been derived from a lipid structure (e.g., lipid bilayer, monolayer, multi-layer structure; e.g., vesicle lipid structure) from a plant source (e.g., enriched, isolated, or purified therefrom), wherein the lipid structure is disrupted (e.g., by lipid extraction) and reassembled or reconstituted in a liquid phase (e.g., a liquid phase containing cargo) using standard methods, e.g., by methods comprising lipid membrane hydration and / or solvent injection, to produce LPMP as described herein. If desired, the method can further comprise sonication, freeze / thaw treatment, and / or lipid extrusion, e.g., to reduce the size of the reconstituted LPMP. Alternatively, a microfluidic device (such as IGNITE TM a microfluidic instrument (Precision NanoSystems)) can be used to produce LPMP.

[0555] In some embodiments, LPMP is produced by a process comprising the steps of: (a) providing a plurality of purified PMPs (e.g., PMP purified as described in Section IA herein); (b) treating the plurality of PMPs to produce a lipid membrane; (c) reconstituting the lipid membrane in an organic solvent or solvent combination, thereby producing a lipid solution; and (d) treating the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, thereby producing LPMP.

[0556] In some cases, processing multiple PMPs to produce a lipid membrane includes extracting lipids from the multiple PMPs, for example, using the Bligh-Dyer method (Bligh and Dyer, Journal of Biological Chemistry and Physiology, 37:911-917, 1959). The extracted lipids can be provided as a stock solution, for example, a chloroform:methanol solution. Producing the lipid membrane can include, for example, evaporating the solvent with a stream of an inert gas (e.g., nitrogen).

[0557] Natural lipid

[0558] The LNMP can contain from 10% to 100% of the lipids derived from lipid structures from natural sources (e.g., lemon or algae), for example, can 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% of the lipids derived from lipid structures from natural sources. The LNMP can contain all or a portion of the lipid species present in the lipid structures from a source (e.g., lemon or algae), for example, the LNMP can 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 structures from a source. The LNMP can not contain, contain a portion or all of the protein species present in the lipid structures from a source (e.g., lemon or algae), for example, can 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 structures from natural sources (e.g., lemon or algae). In some cases, the lipid bilayer of the LNMP does not contain proteins. In some cases, relative to the lipid structures of natural sources, the lipid structures of the LNMP contain a reduced amount of proteins.

[0559] In some embodiments, the natural lipids of the LNMP are extracted from plant sources such as lemon or algae. In some embodiments, the natural lipids are replaced with synthetic structural lipids to form an LNP formulation.

[0560] Exogenous lipid

[0561] LNMP can be modified to contain a heterologous agent (e.g., a cell-penetrating agent) that is capable of increasing cellular 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 unmodified LNMP. For example, the modified LNMP can comprise a plant cell-penetrating agent such as an ionizable lipid (e.g., loaded with the plant cell-penetrating agent, e.g., encapsulating the plant cell-penetrating agent or conjugating with the plant cell-penetrating agent) or formulated together with the plant cell-penetrating agent (e.g., suspended or resuspended in a solution containing the cell-penetrating agent). Each modified LNMP in the modified LNMP can comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0562] LNMP can comprise one or more exogenous lipids, e.g., lipids that are exogenous to a plant (e.g., from a source that is not the plant or plant part from which the LNMP is produced). The lipid composition of LNMP can comprise 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 lipids. In some instances, the amount of exogenous lipid (e.g., ionizable lipid) added is 25% or 40% (w / w) of the total lipids in the formulation. In some instances, the exogenous lipid is added to the formulation prior to step (b), e.g., mixed with the extracted NMP lipids prior to step (b).

[0563] Exemplary exogenous lipids include ionizable lipids. The ionizable lipids in the LNMP compositions herein include one or more of the compounds from groups i)-iv) as described herein.

[0564] The exogenous lipid can further comprise a cationic lipid.

[0565] In some cases, the exogenous lipid may further comprise an ionizable lipid or a cationic lipid selected from the following: 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), cationic sulfonamide amino lipid, 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.

[0566] In some embodiments, the exogenous lipid may further comprise an ionizable lipid or a cationic lipid selected from the following: C12-200, MC3, DODAP, DC-cholesterol, DOTAP, ethyl PC, GL67, KC2, MD1, OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5 (Moderna), cationic sulfonamide amino lipid, and amphiphilic zwitterionic amino lipid, or a combination thereof. In some embodiments, the ionizable lipid is selected from C12-200, MC3, DODAP, and DC-cholesterol, or a combination thereof. In some cases, 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 methyl (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-oate, DLin-MC3-DMA (MC3). In some cases, the exogenous lipid is a cationic lipid. In some embodiments, the cationic lipid is DC-cholesterol or dioleoyl-3-trimethylammonium propane (DOTAP).

[0567] In some cases, the LNMP comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the ionizable lipid.

[0568] In some cases, the LNMP comprises ionizable lipids in 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 greater than 90%, for example, ionizable lipids in a molar ratio of 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90%, for example, ionizable lipids in a molar ratio of about 30%-75% (e.g., ionizable lipids in a molar ratio of about 30%-75%). In some embodiments, the LNMP comprises 25% ionizable lipids. In some embodiments, the LNMP comprises ionizable lipids in a molar ratio of 35%. In some embodiments, the LNMP comprises ionizable lipids in a molar ratio of 50%. In some embodiments, the LNMP comprises 40% MC3. In some embodiments, the LNMP comprises ionizable lipids in a molar ratio of 50%. In some embodiments, the LNMP comprises 20% or 40% DC-cholesterol. In some embodiments, the LNMP comprises 25% or 40% DOTAP.

[0569] The agent can increase the uptake of the overall LNMP or can increase the uptake of a portion or component of the LNMP carried by the LNMP (e.g., an mRNA therapeutic agent). The degree of increased cellular uptake can vary depending on the plant or plant part to which the compositions described herein are delivered, the LNMP formulation, and other modifications made to the LNMP. For example, relative to an unmodified LNMP, the cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of a modified LNMP can be increased by at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some cases, the increased cellular uptake is an increase in cellular uptake that is at least 2x, 4x, 5x, 10x, 100x or 1000x that of an unmodified LNMP.

[0570] In some embodiments, an LNMP modified with ionizable lipids encapsulates negatively charged polynucleotides more efficiently than an LNMP that has not been modified with ionizable lipids. In some aspects, the biodistribution of an LNMP modified with ionizable lipids is altered relative to an LNMP that has not been modified with ionizable lipids. In some aspects, the fusion of an LNMP modified with ionizable lipids with the endosomal membrane of target cells has been altered (e.g., increased) relative to an LNMP that has not been modified with ionizable lipids.

[0571] Ionizable lipid

[0572] In some embodiments, the ionizable lipid has at least one (e.g., one, two, three, four, or all five) of the following listed properties:

[0573] (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, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ionizable amines);

[0574] (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, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 lipid tails), wherein the length of each lipid tail in the lipid tails is independently at least 6 carbon atoms (e.g., the length is 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, such as the length is 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);

[0575] (iii) An acid dissociation constant (pKa) of about 4.5 to about 7.5 (e.g., the pKa is 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., the pKa is about 6.5 to about 7.5 (e.g., the pKa is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5)));

[0576] (iv) Ionizable amines and heteroorganic groups; and

[0577] (v) An N:P (amine of ionizable lipid:phosphate of mRNA) ratio of at least 3 (or at least 4);

[0578] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group. In some embodiments, the heteroorganic group is a hydroxyl group. 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, an optionally substituted C1-C6 alkoxy group, or fluorine.

[0579] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

[0580] The ionizable lipid in the LNMP composition comprises one of the compounds from groups i) to iv) as discussed below.

[0581] Ionizable lipid compound i)

[0582] In some embodiments, the ionizable lipid is represented by the following formula I:

[0583]

[0584] Its pharmaceutically acceptable salt or a stereoisomer of any of the foregoing,

[0585] where

[0586] Each A is independently a C1-C 16 branched or unbranched alkyl group or a C1-C 16 branched or unbranched alkenyl group, said alkyl or alkenyl group being optionally substituted with a heteroatom or substituted with OH, SH, or a halogen;

[0587] Each B is independently a C1-C 16 branched or unbranched alkyl group or a C1-C 16 branched or unbranched alkenyl group, said alkyl or alkenyl group being optionally substituted with a heteroatom or substituted with OH, SH, or a halogen;

[0588] Each X is independently a biodegradable moiety; and

[0589] W is where

[0590] R5 is OH, SH, NR 10 R 11 ;

[0591] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0592] Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, NR 10 R 11 , where each R 10 and R 11 are independently H, a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle;

[0593] Each s is independently 1, 2, 3, 4, or 5;

[0594] Each u is independently 1, 2, 3, 4, or 5;

[0595] t is 1, 2, 3, 4, or 5;

[0596] Each Z independently does not exist, is O, S, or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl group, or a C2-C7 branched or unbranched alkenyl group, provided that when Z does not not exist, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH; and

[0597] Q is O, S, or NR 13 , where each R 13 is H, a C1-C5 alkyl group.

[0598] In some embodiments, B is a C3-C 20 alkyl group.

[0599] In some embodiments, W in formula (I) can alternatively be where:

[0600] V is a branched or unbranched C2-C 10 alkylene group, a C2-C 10 alkenylene group, a C2-C 10 alkynylene group, or a C2-C 10 heteroalkylene group, and the alkylene group, alkenylene group, alkynylene group, or heteroalkylene group is optionally substituted with one or more OH, SH, and / or halogen groups;

[0601] Each R6 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or a cycloalkyl group;

[0602] Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, (CH2) v R 17 or NR10 R 11 , wherein each R 10 and R 11 is independently H, C1-C3 alkyl, or R 10 and R 11 together form a heterocycle;

[0603] Each v is independently 0, 1, 2, 3, 4 or 5;

[0604] R 17 is OH, SH or N(CH3)2; and

[0605] Each u is independently 1, 2, 3, 4 or 5.

[0606] In some embodiments, W in formula (I) may alternatively be wherein:

[0607] V is C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene;

[0608] Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl or cycloalkyl; and

[0609] Each u is independently 1, 2, 3, 4 or 5.

[0610] In some embodiments, W in formula (I) may alternatively be wherein:

[0611] R 14 is a heterocycle;

[0612] Each v is independently 0, 1, 2, 3, 4 or 5; and

[0613] Each u is independently 1, 2, 3, 4 or 5.

[0614] In some embodiments, W in formula (I) may alternatively be wherein:

[0615] Z is O, S, -C((CH2) v N(R 15 )2)- or N(R 15 ), wherein R 15 is H, C1-C4 branched or unbranched alkyl, and v is 0, 1, 2, 3, 4 or 5;

[0616] Each R 10 is independently H or C1-C3 alkyl; and

[0617] Each u is independently 0, 1, 2, 3, 4 or 5.

[0618] In some embodiments, W in formula (I) may alternatively be wherein:

[0619] Each Y is a divalent heterocycle;

[0620] Q is O, S or NH; and

[0621] Each u is independently 1, 2, 3, 4 or 5.

[0622] In some embodiments, W in formula (I) may alternatively be wherein:

[0623] R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 or C(S)NR 10 R 11 where each R 10 and R 11 are independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, said alkyl, cycloalkyl, cycloalkenyl optionally substituted by one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle;

[0624] R 16 is H, ═O, ═S or CN;

[0625] Each v is independently 0, 1, 2, 3, 4 or 5; and

[0626] Each u is independently 1, 2, 3, 4 or 5.

[0627] In some embodiments, W in formula (I) may alternatively be wherein:

[0628] T is –NHC(O)O–, –OC(O)NH– or a divalent heterocycle optionally substituted by one or more -(CH2) v OH, -(CH2) v SH and / or -(CH2) v -halogen groups;

[0629] Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R17 or NR 10 R 11 , wherein each R 10 and R 11 is independently H, C1-C3 alkyl, or R 10 and R 11 together form a heterocycle;

[0630] R 17 is OH, SH or N(CH3)2;

[0631] each v is independently 0, 1, 2, 3, 4 or 5; and

[0632] each u is independently 1, 2, 3, 4 or 5.

[0633] In some embodiments, W in formula (I) can alternatively be wherein:

[0634] T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle; and

[0635] each u is independently 1, 2, 3, 4 or 5.

[0636] In some embodiments, when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH.

[0637] In some embodiments, the heterocycle is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bipiperazine, aromatic or heteroaromatic.

[0638] In some embodiments, the ionizable lipid is represented by formula (IX):

[0639] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein

[0640] each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R 11 , or

[0641] each R1 and each R2 independently together with the carbon atom to which it is attached form a ring;

[0642] each R 10 and R 11 is independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle;

[0643] Each R3 and each R4 are independently H, C2-C 14 branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl) or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0644] Each X is independently a biodegradable moiety;

[0645] Each q is independently 2, 3, 4 or 5;

[0646] V is branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, and the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted with one or more OH, SH and / or halogen groups;

[0647] Each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl or cycloalkyl;

[0648] Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 , where each v is independently 0, 1, 2, 3, 4 or 5, and R 17 is OH, SH or N(CH3)2; and

[0649] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0650] In some embodiments, V is branched or unbranched C2-C3 alkylene. In some embodiments, V is C2-C3 alkylene substituted with OH. In some embodiments, V is branched or unbranched C2-C3 alkenylene. In some embodiments, each R6 is independently H or methyl.

[0651] In some embodiments, the ionizable lipid is represented by one of the following formulas:

[0652]

[0653] where the definitions of the variables are the same as those in formula (X).

[0654] In some embodiments, the present disclosure relates to an ionizable lipid of formula (XI): its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein

[0655] each R1 and each R2 are independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH or NR 10 R 11 ; or

[0656] each R1 and each R2 independently together with the carbon atom to which it is attached form a ring;

[0657] each R 10 and R 11 are independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle;

[0658] each R3 and each R4 are independently H, C2-C 14 branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl) or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H;

[0659] each X is independently a biodegradable moiety;

[0660] each s is independently 1, 2, 3, 4 or 5;

[0661] T is –NHC(O)O–, –OC(O)NH– or a divalent heterocycle optionally substituted by one or more -(CH2) v OH, -(CH2) v SH, -(CH2) v -halo groups;

[0662] each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 wherein R 17 is OH, SH or N(CH3)2;

[0663] each v is independently 0, 1, 2, 3, 4 or 5; and

[0664] each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0665] In some embodiments, T is a divalent heterocycle optionally substituted by -(CH2) v OH (e.g., divalent piperazine or divalent dioxopiperazine), where v is independently 0, 1, or 2.

[0666] In some embodiments, in each of the above formulas, X is –OC(O)-, -C(O)O-, -SS-, -N(R 18 )C(O)-, -C(O)N(R 18 )-, -C(O-R 13 )-O-, -C(O)O(CH2) a -, -OC(O)(CH2) a -, -C(O)N(R 18 )(CH2) a -, -N(R 18 )C(O)(CH2) a -, -C(O-R 13 )-O-(CH2) a -, where each R 18 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R 13 is independently C3-C 10 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-.

[0667] In the group of ionizable lipid compounds i), more embodiments of the ionizable lipids of formula (I) can 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. Specifically, all ionizable lipids of formulas (I)-(XII) of PCT Application No. PCT / US22 / 50725 are suitable for use as ionizable lipids in the present disclosure and are incorporated herein by reference in their entirety.

[0668] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table I below.

[0669] Table I: Exemplary ionizable lipid compounds.

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702] Ionizable lipid compound ii)

[0703] In some embodiments, the ionizable lipid is represented by Formula II below:

[0704]

[0705] Its pharmaceutically acceptable salt or stereoisomer of any of the foregoing,

[0706] wherein:

[0707] is a cyclic or heterocyclic moiety;

[0708] Y is alkyl, hydroxy, hydroxyalkyl or

[0709] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle;

[0710] Each of X and Z is independently absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-;

[0711] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl or aminoalkyl;

[0712] Each M is independently a biodegradable moiety;

[0713] R 30 、R 40 、R 50 、R 60 、R 70 、R 80 、R 90 、R100 , R 110 and R 120 Each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by OH, SH, or a halogen, or a cycloalkyl or a substituted cycloalkyl;

[0714] Each of l and m is an integer from 1 to 10;

[0715] t1 is an integer from 0 to 10; and

[0716] W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic group or heteroaryl group.

[0717] In some embodiments, Y is a hydroxyl group or

[0718] In some embodiments, selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine, and dioxane. In some embodiments, selected from the group consisting of:

[0719] In some embodiments, the ionizable lipid is represented by the following formula:

[0720] All variables in this formula have been defined and exemplified as those described in the above embodiments.

[0721] In some embodiments, the ionizable lipid is represented by the following formula:

[0722]

[0723] Wherein:

[0724] Each m1 is independently an integer from 3 to 6,

[0725] Each l1 is independently an integer from 4 to 8,

[0726] m2 and l2 are each independently an integer from 0 to 3,

[0727] R 80 and R 90 are each independently an unsubstituted C5-C8 alkyl group; or R 80 is H or an unsubstituted C1-C4 alkyl group, and R 90is an unsubstituted C5-C 11 alkyl group; and

[0728] R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl group; or R 110 is H or an unsubstituted C1-C4 alkyl group, and R 120 is an unsubstituted C5-C 11 alkyl group. All other variables in these formulas have been defined and exemplified as those described in the above embodiments. In some embodiments, in these formulas, R 80 is H or an unsubstituted C1-C2 alkyl group, and R 90 is an unsubstituted C6-C 10 alkyl group; and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl group. In some embodiments, R 80 、R 90 、R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl group.

[0729] In some embodiments, in the above formulas, A is absent, is -O-, -N(R 7 )-, N(R 7 )C(O)-, -OC(O)- or -C(O)O-, where R 6 is independently H, an alkyl group, a hydroxyl group, a hydroxyalkyl group, an amino group, an aminoalkyl group, a thiol, a thioalkyl group or N + (R 7 )3-alkylene-Q-; and R 7 is H or a C1-C3 alkyl group.

[0730] In some embodiments, in the above formulas, t1 is 0, 1, 2, 3 or 4; and t is 0, 1 or 2.

[0731] In some embodiments, in the above formulas, W is a hydroxyl group, a hydroxyalkyl group or one of the following: wherein:

[0732] each Q is independently absent, is -O-, -C(O)-, -C(S)-, -C(O)O-, -C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 )-;

[0733] Each R 6 is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, mercapto, thioalkyl or N + (R 7 )3–alkylene-Q-;

[0734] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, mercapto or thioalkyl, or two Rs 8 together with the nitrogen atom can form a ring;

[0735] Each q is independently 0, 1, 2, 3, 4 or 5; and

[0736] Each p is independently 0, 1, 2, 3, 4 or 5.

[0737] In some embodiments, in the above formula,

[0738] X is absent, is -O- or –C(O)-;

[0739] Z is –O-, –C(O)O- or –OC(O)-;

[0740] M is -OC(O)- or -C(O)O-;

[0741] Y or is:

[0742] OH,

[0743] Each R c is independently H or C1-C3 alkyl;

[0744] Each t1 is independently 1, 2, 3 or 4;

[0745] R 30 , R 40 , R 50 and R 60 each of which is H or C1-C4 branched or unbranched alkyl;

[0746] R 70 is H; and R 80 and R 90 each of which is independently H or C1-C 12 branched or unbranched alkyl;

[0747] R 100 is H; and R 110 and R 120 each of which is independently H or C1-C 12A branched or unbranched alkyl group, provided that R 80 and R 90 at least one of which is not H, and R 110 and R 120 at least one of which is not H;

[0748] l is from 3 to 7; and

[0749] m is from 1 to 5.

[0750] In some embodiments, in the above formula, Y or is: OH,

[0751] In the group of ionizable lipid compounds ii), further embodiments of the ionizable lipids of formula (II) can be found in PCT Application No. PCT / US23 / 16300 filed on March 24, 2023, the content of which PCT application is incorporated herein by reference in its entirety. Specifically, all 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 ionizable lipids in the present disclosure and are incorporated herein by reference in their entirety.

[0752] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table II below.

[0753] Table II: Exemplary Ionizable Lipid Compounds.

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] Ionizable lipid compound iii)

[0777] In some embodiments, the ionizable lipid is represented by formula (III), its pharmaceutically acceptable salts, and stereoisomers of any of the foregoing, wherein:

[0778] R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl, or

[0779] R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, which ring is optionally substituted with R a ;

[0780] R a is H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, a halogen, OH, or SH;

[0781] Each R1 and each R2 are independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, OH, a halogen, SH, or NR 10 R11 or

[0782] R1 and R2 together form a ring;

[0783] Each R 10 and R 11 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, or

[0784] R 10 and R 11 together form a heterocycle;

[0785] n is 0, 1, 2, 3 or 4;

[0786] Y is O or S;

[0787] Z is absent, is O, S or N(R 12 )(R 12 ), where each R 12 is independently H, a C1-C7 branched or unbranched alkyl group or a C2-C7 branched or unbranched alkenyl group, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH;

[0788] Each A is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen;

[0789] Each B is independently a C1-C 16 branched or unbranched alkyl group or a C2-C 16 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen;

[0790] Each X is independently a biodegradable moiety.

[0791] In some embodiments, R 20 and R 30 are each independently H or a C1-C3 branched or unbranched alkyl group. In some embodiments, R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, and the ring is optionally substituted by R a . In some embodiments, R a is H, a C1-C3 branched or unbranched alkyl group or OH. In one embodiment, R a is H or OH.

[0792] In some embodiments, Z is absent and is S, O, or NH. In some embodiments, n is 0, 1, or 2.

[0793] In some embodiments, the ionizable lipid is represented by formula (V):

[0794] its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein

[0795] R1 is H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, OH, halogen, SH, or NR 10 R 11， and

[0796] R2 is H, OH, halogen, SH, or NR 10 R 11， or

[0797] R1 and R2 together form a ring;

[0798] R 10 and R 11 are each independently H or a C1-C3 alkyl, or R 10 and R 11 together form a heterocycle;

[0799] Q is OH or -(OCH2CH2) u NR 20 R 30 ,

[0800] R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl, or a C2-C5 branched or unbranched alkenyl, or

[0801] R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, which ring is optionally substituted with R a ;

[0802] R a is H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, halogen, OH, or SH;

[0803] u is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0804] v is 0, 1, 2, 3, or 4;

[0805] y is 0, 1, 2, 3, or 4;

[0806] Each A is independently a C1-C 16A branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen;

[0807] Each B is independently a C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen; and

[0808] Each X is independently a biodegradable moiety.

[0809] In some embodiments, the present disclosure relates to an ionizable lipid of one of the following formulas:

[0810]

[0811] 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 the above formulas (III) and (V).

[0812] In some embodiments, in the above formula, X is –OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) s -, -OC(O)(CH2) s -, -C(O)N(R 7 )(CH2) s -, -N(R 7 )C(O)(CH2) s -, -C(O-R 13 )-O-(CH2) s -, wherein each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl, each R 13 is independently a C3-C 10 alkyl, and each s is independently 0-16. In some embodiments, X is –OC(O)-, –C(O)O-, -C(O)O(CH2) s - or -OC(O)(CH2) s -. In some embodiments, s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0813] Among the ionizable lipid compounds of group (iii), further embodiments of the ionizable lipids of formula (III) or (V) can 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. Specifically, all ionizable lipids of formulas (IO)-(VIIO) and (I)-(VIID) of PCT Application No. PCT / US22 / 50111 are suitable for use as ionizable lipids in the present disclosure and are incorporated herein by reference in their entirety.

[0814] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table III below.

[0815] Table III: Exemplary Ionizable Lipid Compounds.

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834] Ionizable lipid compound iv)

[0835] 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'):

[0836] Its pharmaceutically acceptable salt or stereoisomer of any of the foregoing,

[0837] Wherein:

[0838] Each E is independently a biodegradable group;

[0839] R a Each is independently a C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0840] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

[0841] R t Each is independently H, C1-C 16 Branched or unbranched alkyl or C1-C 16 Branched or unbranched alkenyl, said alkyl or alkenyl optionally interrupted by a heteroatom or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

[0842] Represents the bond connecting the tail group to the head group; and

[0843] Wherein the pKa of the lipid is from about 4 to about 8.

[0844] In some embodiments, each E is independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, wherein each R 7 Is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 Is a branched or unbranched C3-C 10 Alkyl; and r is 1, 2, 3, 4 or 5. In some embodiments, each E is independently -OC(O)-, -C(O)O-, -N(R7 )C(O)- or -C(O)N(R 7 )-, where R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl.

[0845] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII):

[0846] where u3 and u4 are each independently 0, 1, 2, 3 or 4. The definitions of the other variables in (TII) are the same as those defined above in (TI).

[0847] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII):

[0848] (e.g.,

[0849] where u3 is 0, 1, 2, 3, 4, 5, 6 or 7; and R b is independently H or C1-C4 alkyl in each case. The definitions of the other variables in (TIII) are the same as those defined above in (TI).

[0850] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV):

[0851] where u3 and u4 are each independently 0, 1, 2, 3 or 4. The definitions of the other variables in (TIV) are the same as those defined above in (TI).

[0852] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TV), (e.g., where u3 is 0, 1, 2, 3, 4, 5, 6 or 7; R 7 is independently H or methyl; and R b is independently H or C1-C4 alkyl in each case. The definitions of the other variables in (TV) are the same as those defined above in (TI).

[0853] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII'), (e.g., where u3 is 0, 1, 2, 3, 4, 5, 6 or 7; and R bIndependently in each case is H or a C1-C4 alkyl group. The definitions of the other variables in (TII') are the same as those defined above in (TI').

[0854] In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII'), (e.g., ), where u3 is 0, 1, 2, 3, 4, 5, 6 or 7; R 7 are each independently H or methyl; and R b Independently in each case is H or a C1-C4 alkyl group. The definitions of the other variables in (TIII') are the same as those defined above in (TI').

[0855] In some embodiments, the lipid comprises at least one tail group of formula (TII), (TIII), (TIV), (TV), (TII') and (TIII'), where

[0856] R 7 are each independently H or methyl;

[0857] R b Independently in each case is H or a C1-C4 alkyl group;

[0858] u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; and

[0859] where the pKa of the lipid is from about 4 to about 8.

[0860] In some embodiments, the lipid comprises two, three, four or more tail groups having formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII') and / or (TIII'), and each tail group can be the same or different.

[0861] In some embodiments, in any one of the above formulas (T), (TI), (TII) and (TIII), (TIV), (TV), (TI'), (TII') and / or (TIII'), R a are each independently a C1-C5 branched or unbranched alkyl group, a C2-C5 branched or unbranched alkenyl group or a C2-C5 branched or unbranched alkynyl group. In some embodiments, R a are each independently a C1-C3 branched or unbranched alkyl group. In one embodiment, each R a is methyl.

[0862] In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u1 is 3, 4, or 5. In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u2 is 0, 1, 2, or 3. In some embodiments, in any one of the above formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'), u3 and u4 are each independently 1-7; for example, u3 and u4 are each independently 1, 2, 3, or 4.

[0863] In some embodiments, the lipid comprises at least one tail of formula (TIII), wherein each R a is methyl, and R b is independently H, ethyl, or butyl in each case, 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 two tails of formula (TIII), wherein the two tails of formula (TIII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIII), wherein each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TIII), wherein each tail can be the same or different. In some embodiments, in each tail of formula (TIII), each R a is methyl, and u1 is 3, u2 is 2, and u3 is 4.

[0864] In some embodiments, the lipid comprises at least one tail of formula (TII), wherein each R a is methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4. In some embodiments, the lipid has at least two tails of formula (TII), wherein the two tails of formula (TII) are the same. In some embodiments, the lipid has at least two tails of formula (TII), wherein the two tails of formula (TII) are different. In some embodiments, the lipid comprises at least three tails of formula (TII), wherein each tail can be the same or different. In some embodiments, the lipid has four tails of formula (TII), wherein each tail can be the same or different.

[0865] In some embodiments, the lipid comprises at least one tail of formula (TIV), wherein each R ais 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 can be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), wherein each tail can be the same or different.

[0866] In some embodiments, the lipid comprises at least two tails of formula (TV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TV), wherein each tail can be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TV), wherein each tail can be the same or different.

[0867] In some embodiments, the lipid has at least two tails of formula (TII'), wherein each tail can be the same or different. In some embodiments, the lipid has at least three tails of formula (TII'), wherein each tail can be the same or different. In some embodiments, the lipid has at least four tails of formula (TII'), wherein each tail can be the same or different.

[0868] In some embodiments, the lipid has at least two tails of formula (TIII'), wherein each tail can be the same or different. In some embodiments, the lipid has at least three tails of formula (TIII'), wherein each tail can be the same or different. In some embodiments, the lipid has at least four tails of formula (TIII'), wherein each tail can be the same or different.

[0869] In some embodiments, the lipid has at least one tail of formula (TII) and / or at least one tail of formula (TIII); the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail, and the at least one tail does not contain a geminal di - functional group bonded to the same carbon adjacent to E (e.g., -C(O)O-).

[0870] In some embodiments, the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail, and the at least one tail does not contain a geminal di - functional group bonded to the same carbon adjacent to E.

[0871] In some embodiments, the lipid further comprises at least one tail that does not have the formula (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). That is, the lipid further comprises at least one tail that does not contain a geminal di-functional group bonded to the same carbon next to E.

[0872] In some embodiments, the lipid further comprises at least one tail of formula (TNG-I):

[0873] wherein

[0874] E is each independently a biodegradable group as described herein (e.g., -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -S-S-, or -C(O)N(R 7 ));

[0875] u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and

[0876] R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

[0877] In some embodiments, the at least one tail of formula (TNG-I) can be represented by

[0878] wherein

[0879] u3 and u4 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; and

[0880] R b is in each case independently H or a C1-C4 alkyl.

[0881] As described above, all embodiments of the definitions of E, R b , R t , u1, u2, u3, and u4 that relate to tail groups containing a geminal di-functional group of formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII'), or (TIII') bonded to the same carbon next to E also apply to tail groups that do not contain a geminal di-functional group of formula (TNG-I), (TNG-II), or (TNG-III) bonded to the same carbon next to E.

[0882] In some embodiments, the lipid further comprises at least two tails that do not have the formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). In some embodiments, the lipid comprises two tail groups of formula (TNG-II) or (TNG-III), and each of the tail groups may be the same or different,

[0883] In some embodiments, the lipid further comprises at least three tails that do not have the formulas (T), (TI), (TII), (TIII), (TIV), (TV), (TI'), (TII'), and / or (TIII'). In some embodiments, the lipid comprises three tail groups of formula (TNG-II) or (TNG-III), and each of the tail groups may be the same or different,

[0884] In some embodiments, the head group of the lipid has a structure of formula (HA-I):

[0885]

[0886] Wherein:

[0887] R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl group, or a C2-C5 branched or unbranched alkenyl group, and the alkyl or alkenyl group is optionally interrupted by one or more heteroatoms or substituted with OH, SH, halogen, or cycloalkyl; or

[0888] R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, and the heterocyclic or heteroaromatic ring is optionally substituted with one or more OH, SH, halogen, alkyl, or cycloalkyl;

[0889] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, halogen, SH, or NR 10 R 11 ; or R1 and R2 together form a ring;

[0890] R 10 and R 11 are each independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle;

[0891] n is 0, 1, 2, 3, or 4;

[0892] Z is absent, or is O, S or NR 12 , where R 12 is H or a C1-C7 branched or unbranched alkyl group; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH; and

[0893] represents the bond connecting the head group to the tail group.

[0894] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-I): 20 and R 30 together with the adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl groups.

[0895] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-I):

[0896]

[0897] wherein:

[0898] Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring;

[0899] R 10 and R 11 are each independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocyclic ring;

[0900] m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0901] n is 0, 1, 2, 3 or 4;

[0902] Z is absent, or is O, S or NR 12 , where R 12 is H or a C1-C7 branched or unbranched alkyl group; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH; and

[0903] represents the bond connecting the head group to the tail group.

[0904] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-III): where Z is absent, is O, S or NR 12 ; and R 12 is H or a C1-C7 branched or unbranched alkyl group. The definitions of the other variables in (HA-III) are the same as those defined in (HA-IA) above.

[0905] In some embodiments, wherein the head group has the following structure: wherein:

[0906] Rc is H or an alkyl group, optionally substituted with OH; and

[0907] m1 is 1, 2 or 3.

[0908] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-V):

[0909]

[0910] wherein:

[0911] R1 is H, a C1-C3 alkyl group, OH, halogen, SH or NR 10 R 11 ;

[0912] R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring;

[0913] R 10 and R 11 are each independently H or a C1-C3 alkyl group; or R 10 and R 11 can combine together to form a heterocycle;

[0914] R 20 and R 30 are each independently H, a C1-C5 branched or unbranched alkyl group, a C2-C5 branched or unbranched alkenyl group; or R 20 and R 30 can combine together to form a ring; and

[0915] each of v and y is independently 1, 2, 3 or 4.

[0916] In some embodiments, the head group of the ionizable lipid has the structure of formula (HA-VI): The definitions of all variables in (HA-VI) are the same as those defined above in (HA-V).

[0917] In some embodiments, in any one of the above formulas (HA-V) or (HA-VI), each R 20 and R 30 is independently a C1-C3 alkyl. In one embodiment, each R 20 and R 30 is independently methyl.

[0918] In some embodiments, the head group of the ionizable lipid has the structure of formula (HB-I):

[0919]

[0920] where W is

[0921] where

[0922] R5 is OH, SH, (CH2) s OH or NR 10 R 11 ;

[0923] each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl or cycloalkyl;

[0924] each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring;

[0925] each R 20 is independently H or a C1-C3 branched or unbranched alkyl;

[0926] R 14 is a heterocycle, NR 10 R 11 , C(O)NR 10 R 11 , NR 10 C(O)NR10 R 11 or NR 10 C(S)NR 10 R 11， where each R 10 and R 11 is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, said alkyl, cycloalkyl, cycloalkenyl being optionally substituted by one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle;

[0927] R 16 is H, =O, =S or CN;

[0928] each of s, u and t is independently 1, 2, 3, 4 or 5;

[0929] each v is independently 0, 1, 2, 3, 4 or 5;

[0930] each Y is a divalent heterocycle;

[0931] each Z independently does not exist, is O, S or NR 12 , where R 12 is H, C1-C7 branched or unbranched alkyl or C2-C7 branched or unbranched alkenyl;

[0932] Q is O, S, CH2 or NR 13 , where each R 13 is H, C1-C5 alkyl;

[0933] V is branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, said alkylene, alkenylene, alkynylene or heteroalkylene being optionally substituted by one or more OH, SH and / or halogen groups; and

[0934] T is –NHC(O)O-, –OC(O)NH- or a divalent heterocycle.

[0935] In some embodiments, in formula (HB-I), W is where:

[0936] each of R6, R7 and R8 is independently H or methyl; and

[0937] each of u and t is independently 1, 2 or 3.

[0938] In some embodiments, in formula (HB-I), W is Wherein:

[0939] R 16 is H or =O;

[0940] R 14 is a 5- or 6-membered nitrogen-containing heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H or a C1-C3 alkyl; and

[0941] each of u and v is independently 1, 2 or 3.

[0942] In some embodiments, in formula (HB-I), W is Wherein:

[0943] each R6 is independently H or methyl;

[0944] each u is independently 1, 2 or 3; and

[0945] V is a C2-C6 alkylene or a C2-C6 alkenylene.

[0946] In some embodiments, in formula (HB-I), W is Wherein:

[0947] each R6 is independently H or methyl;

[0948] each R7 is independently H;

[0949] each R8 is methyl;

[0950] each u is independently 1, 2 or 3; and

[0951] V is a C2-C6 alkylene or a C2-C6 alkenylene.

[0952] In some embodiments, in formula (HB-I), W is Wherein:

[0953] each u is independently 1, 2 or 3; and

[0954] T is a divalent 5- or 6-membered nitrogen-containing heterocycle.

[0955] In some embodiments, in formula (HB-I), W is

[0956] Wherein:

[0957] Each u is independently 1, 2, or 3;

[0958] Q is O;

[0959] Each Z is independently NR 12 ; and

[0960] R 12 is H or C1-C3 alkyl.

[0961] In some embodiments, the head group has the following structure:

[0962]

[0963] Where each of u and t is independently 1 or 2.

[0964] In some embodiments, the head group of the ionizable lipid has the structure of formula (HC-I):

[0965] Where

[0966] is a cyclic or heterocyclic moiety;

[0967] Y is alkyl, hydroxy, hydroxyalkyl,

[0968] A is absent, is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-,-S-, or -S-S-;

[0969] Each of X and Z is independently absent, is -O-, -C(O)-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, or -S-;

[0970] Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl;

[0971] t is 0, 1, 2 or 3;

[0972] t1 is an integer from 0 to 10; and

[0973] W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic group or heteroaryl group.

[0974] In some embodiments, the head group has a structure of the following formula:

[0975]

[0976] In some embodiments, in the above formula,

[0977] A is absent, is -O-, -N(R 7 )-, -OC(O)- or -C(O)O-;

[0978] X is absent, is O- or –C(O)-; and

[0979] Z is –O-, –C(O)O- or –OC(O)-.

[0980] In some embodiments, the head group has a structure of the following formula:

[0981] wherein t1 is 0, 1, 2 or 3.

[0982] In some embodiments, W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, or one of the following moieties:

[0983]

[0984] wherein

[0985] each Q independently is absent, is -O-, -C(O)-, -C(S)-, -C(O)O-, -(CH2) q C(R 7 )2-, -C(O)N(R 7 )-, -C(S)N(R 7 )- or -N(R 7 );

[0986] R 6 is independently H, alkyl, hydroxyl, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N(R 7 )2, amino, alkylamino, aminoalkyl, thiol, thiolalkyl or N + (R 7 )3–alkylene-Q;

[0987] Each R 8 is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol or thioalkyl, heterocyclic group, heteroaryl, or two Rs 8 together with the nitrogen atom can form a ring, which is optionally substituted by one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl;

[0988] q is 0, 1, 2, 3, 4 or 5; and

[0989] p is 0, 1, 2, 3, 4 or 5.

[0990] In some embodiments, W is one of the following:

[0991] OH,

[0992] In some embodiments, the ionizable lipid is represented by the formula its pharmaceutically acceptable salts and stereoisomers of any of the foregoing, wherein

[0993] each R1 is independently H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R1 and R2 can combine together to form a ring; R 10 and R 11 are each independently H, C1-C3 alkyl, and R 10 and R 11 can combine together to form a heterocycle;

[0994] each R2 is independently H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R1 and R2 can combine together to form a ring; R 10 and R 11 are each independently H, C1-C3 alkyl, and R 10 and R 11 can combine together to form a heterocycle;

[0995] m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0996] n is 0, 1, 2, 3 or 4;

[0997] each r is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0998] each R3 is independently H or C3-C 10 alkyl;

[0999] Each R4 is independently H or C3-C 10 alkyl; provided that at least one of R3 and R4 is not H;

[1000] Z is absent, is O, S or NR 12 ; wherein R 12 is C1-C7 alkyl;

[1001] Each X is independently X', provided that at least one X in the formula is and

[1002] X' is a biodegradable moiety.

[1003] In some embodiments, each X is In some embodiments, X' is -OCO-, -COO-, -NR 7 CO-, -CONR 7 -, -C(O-R 13 )-O-(acetal), -COO(CH2) s -, -CONH(CH2) s -, or -C(O-R 13 )-O-(CH2) s -; wherein R 7 is H or C1-C3 alkyl; and R 13 is C3-C 10 alkyl.

[1004] In some embodiments, at least one X in the formula is wherein R 7 is H or methyl. In one embodiment, each X is In one embodiment, each X is wherein R 7 is H or methyl.

[1005] In some embodiments, m = 3. In some embodiments, n = 0 or 1. In some embodiments, each of R, R1 and R2 is H. In some embodiments, Z is absent.

[1006] In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z is NH.

[1007] In some embodiments, r is 3. In some embodiments, r is 4.

[1008] Among the ionizable lipid compounds of group iv), more embodiments of the above ionizable lipids comprising at least one head group (e.g., the head groups of formulas (HA-I) to (HA-VII), (HB-I), and (HC-I) to (HC-IIIE)) and at least one tail group of formula (TI) or (T1') (e.g., the tail groups of formulas (TII), (TIII), (TIV), (TV), (TII'), or (TIII')) can be found in PCT Application No. PCT / US23 / 31669 filed on August 31, 2023, which is incorporated herein by reference in its entirety. Additionally, all ionizable lipids of formulas (LA-I)-(LA-VII), (LB-1)-(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 ionizable lipids in the present disclosure and are incorporated herein by reference in its entirety.

[1009] Certain exemplary ionizable lipid compounds disclosed herein are set forth in Table IV below.

[1010] Table IV: Exemplary Ionizable Lipid Compounds.

[1011]

[1012]

[1013]

[1014]

[1015]

[1016]

[1017]

[1018]

[1019]

[1020]

[1021]

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

[1029]

[1030]

[1031]

[1032]

[1033]

[1034]

[1035]

[1036]

[1037]

[1038]

[1039]

[1040]

[1041]

[1042]

[1043]

[1044]

[1045]

[1046]

[1047]

[1048]

[1049]

[1050]

[1051]

[1052]

[1053]

[1054]

[1055]

[1056]

[1057]

[1058]

[1059]

[1060]

[1061]

[1062]

[1063]

[1064]

[1065]

[1066]

[1067]

[1068] In some embodiments, the lipid membrane of the LNMP (or LNP) comprises at least 35% of the lipid compounds from group i), such as at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% of the lipid compounds from group i), such as 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90% of the lipid compounds from group i).

[1069] In some embodiments, the lipid membrane of LNMP (or LNP) comprises at least 35% of the lipid compounds from group (ii), such as at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 90% of the lipid compounds from group (ii), such as 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90% of the lipid compounds from group (ii).

[1070] In some embodiments, the lipid membrane of LNMP (or LNP) comprises at least 35% of the lipid compounds from group (iii), such as at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 90% of the lipid compounds from group (iii), such as 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90% of the lipid compounds from group (iii).

[1071] In some embodiments, the lipid membrane of LNMP (or LNP) comprises at least 35% of the lipid compounds from group (iii), such as at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 90% of the lipid compounds from group (iii), such as 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90% of the lipid compounds from group (iv).

[1072] In some cases, LNMP (or LNP) comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% of ionizable lipid.

[1073] In some cases, LNMP (or LNP) comprises an ionizable lipid in 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 greater than 90%, such as, an ionizable lipid in a molar ratio of 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80% or 80%-90%, such as an ionizable lipid in a molar ratio of about 25%-75% (e.g., an ionizable lipid in a molar ratio of about 25%-75%).

[1074] Other ionizable lipids

[1075] In an LNMP or LNP formulation, more than one ionizable lipid can be used for the ionizable lipid component: one or more ionizable lipids from the ionizable lipids of the formula compounds in groups i)-iv) can be used alone or in combination with different ionizable lipids from the formula compounds in groups i)-iv).

[1076] In some embodiments, the ionizable lipid is not selected from 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5 (Moderna), and 98N12-5.

[1077] 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.

[1078] In some embodiments, the additional ionizable lipid is represented by the following formula III:

[1079] wherein R is C8-C 14 alkyl.

[1080] The ionizable lipids described herein may include an amine core substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) lipid tails as described herein. In some embodiments, the ionizable lipids described herein include at least 3 lipid tails. The lipid tails may be C8-C 18 hydrocarbons (e.g., C6-C 18 alkyl or C6-C 18 alkanoyl). The amine core may be substituted with one or more lipid tails at the nitrogen atom (e.g., one hydrogen atom attached to the nitrogen atom may be replaced by a lipid tail).

[1081] In some embodiments, the amine core has the following structure:

[1082]

[1083] In some embodiments, the amine core has the following structure:

[1084]

[1085] In some embodiments, the amine core has the following structure:

[1086]

[1087] In some embodiments, the amine core has the following structure:

[1088]

[1089] In some embodiments, the amine core has the following structure:

[1090]

[1091] In some embodiments, the amine core has the following structure:

[1092] In some embodiments, the amine core has the following structure:

[1093] In some embodiments, the amine core has the following structure:

[1094] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2016 / 118725, which is incorporated herein by reference in its entirety.

[1095] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1096] And their pharmaceutically acceptable salts.

[1097] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2016 / 118724, which is incorporated herein by reference in its entirety.

[1098] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1099] And their pharmaceutically acceptable salts.

[1100] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids having the formula 14,25 - bis(tridecyl)-15,18,21,24 - tetraaza - octatriacontane and their pharmaceutically acceptable salts.

[1101] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publications WO2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference in its entirety.

[1102] In some embodiments, the RNA compositions and methods for their preparation and use include lipids of the following formula:

[1103] or a pharmaceutically acceptable salt thereof, wherein each instance of R L is independently an optionally substituted C6-C40 alkenyl.

[1104] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structure:

[1105] and pharmaceutically acceptable salts thereof.

[1106] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structure:

[1107] and pharmaceutically acceptable salts thereof.

[1108] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structure:

[1109] and pharmaceutically acceptable salts thereof.

[1110] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structure:

[1111] and pharmaceutically acceptable salts thereof.

[1112] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2015 / 184256, which is incorporated herein by reference in its entirety. In some embodiments, the RNA compositions and methods for their preparation and use include lipids of the following formula:

[1113] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R AIndependently is hydrogen, an optionally substituted C1-50 alkyl group, an optionally substituted C2-50 alkenyl group, an optionally substituted C2-50 alkynyl group, an optionally substituted C3-10 carbocyclic group, an optionally substituted 3-14 membered heterocyclic group, an optionally substituted C6-14 aryl group, an optionally substituted 5-14 membered heteroaryl group or a halogen; and each RB is independently hydrogen, an optionally substituted C1-50 alkyl group, an optionally substituted C2-50 alkenyl group, an optionally substituted C2-50 alkynyl group, an optionally substituted C3-10 carbocyclic group, an optionally substituted 3-14 membered heterocyclic group, an optionally substituted C6-14 aryl group, an optionally substituted 5-14 membered heteroaryl group or a halogen.

[1114] In certain embodiments, the RNA compositions and methods for their preparation and use include a lipid "Target 23" having the following compound structure:

[1115] (Target 23), and pharmaceutically acceptable salts thereof.

[1116] Other suitable lipids for RNA compositions and methods for their preparation and use include the lipids described in International Patent Publication WO2016 / 004202, which International Patent Publication is incorporated herein by reference in its entirety.

[1117] In some embodiments, the RNA compositions and methods for their preparation and use include a lipid having the following compound structure:

[1118] or a pharmaceutically acceptable salt thereof.

[1119] In some embodiments, the RNA compositions and methods for their preparation and use include a lipid having the following compound structure:

[1120] or a pharmaceutically acceptable salt thereof.

[1121] In some embodiments, the RNA compositions and methods for their preparation and use include a lipid having the following compound structure:

[1122] or a pharmaceutically acceptable salt thereof.

[1123] Other suitable lipids for RNA compositions and methods for their preparation and use include the lipids described in U.S. Provisional Patent Application Serial No. 62 / 758,179, which U.S. Provisional Patent Application is incorporated herein by reference in its entirety.

[1124] In some embodiments, the RNA compositions and methods for their preparation and use include a lipid of the following formula:

[1125] or a pharmaceutically acceptable salt thereof, wherein each R1 and R 2 independently is 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 L 1 independently is an ester, thioester, disulfide or anhydride group; each L 2 independently is C2-C10 aliphatic; each X 1 independently is H or OH; and each R 3 independently is C6-C20 aliphatic.

[1126] In some embodiments, the RNA compositions and methods for their preparation and use include lipids of the following formula:

[1127]

[1128] or a pharmaceutically acceptable salt thereof.

[1129] In some embodiments, the RNA compositions and methods for their preparation and use include lipids of the following formula:

[1130] or a pharmaceutically acceptable salt thereof.

[1131] In some embodiments, the RNA compositions and methods for their preparation and use include lipids of the following formula:

[1132] or a pharmaceutically acceptable salt thereof.

[1133] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in the following documents: J. McClellan, M. C. King, *Cell* 2010, 141, 210-217 and Whitehead et al., *Nature Communications* (2014) 5:4277, which are incorporated herein by reference in their entirety.

[1134] In certain embodiments, the lipids of the RNA compositions and methods for their preparation and use include lipids having the following compound structure:

[1135] and pharmaceutically acceptable salts thereof.

[1136] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2015 / 199952, which is incorporated herein by reference in its entirety.

[1137] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1138] and pharmaceutically acceptable salts thereof.

[1139] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1140] and pharmaceutically acceptable salts thereof.

[1141] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1142] and pharmaceutically acceptable salts thereof.

[1143] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1144] and pharmaceutically acceptable salts thereof.

[1145] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1146] and pharmaceutically acceptable salts thereof.

[1147] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1148] and pharmaceutically acceptable salts thereof.

[1149] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1150] and pharmaceutically acceptable salts thereof.

[1151] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1152]

[1153] and pharmaceutically acceptable salts thereof.

[1154] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1155] and their pharmaceutically acceptable salts.

[1156] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1157] and their pharmaceutically acceptable salts.

[1158] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1159] and their pharmaceutically acceptable salts.

[1160] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1161] and their pharmaceutically acceptable salts.

[1162] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1163] and their pharmaceutically acceptable salts.

[1164] Other suitable lipids for RNA compositions and methods for their preparation and use include the lipids described in International Patent Publication WO2017 / 004143, which is incorporated herein by reference in its entirety.

[1165] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1166] and their pharmaceutically acceptable salts.

[1167] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1168] and their pharmaceutically acceptable salts.

[1169] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1170] and their pharmaceutically acceptable salts.

[1171] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1172] and their pharmaceutically acceptable salts.

[1173] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1174] and their pharmaceutically acceptable salts.

[1175] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1176] and their pharmaceutically acceptable salts.

[1177] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1178] and their pharmaceutically acceptable salts.

[1179] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1180] and their pharmaceutically acceptable salts.

[1181] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1182] and their pharmaceutically acceptable salts.

[1183] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1184] and their pharmaceutically acceptable salts.

[1185] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1186] and their pharmaceutically acceptable salts.

[1187] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1188] and its pharmaceutically acceptable salts.

[1189] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1190] and its pharmaceutically acceptable salts.

[1191] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1192] and its pharmaceutically acceptable salts.

[1193] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1194] and its pharmaceutically acceptable salts.

[1195] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1196] and its pharmaceutically acceptable salts.

[1197] In some embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1198] and its pharmaceutically acceptable salts.

[1199] Other suitable lipids for the RNA compositions and methods of making and using the same include the lipids described in International Patent Publication WO2017 / 075531, which is incorporated herein by reference in its entirety.

[1200] In some embodiments, the RNA compositions and methods of making and using the same include lipids of the following formula:

[1201] or its pharmaceutically acceptable salts, wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -S-S-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a-or -NR a C(=O)O-; and L 1 or L 2 in which the other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or -NR a C(=O)O- or a direct bond; G 1 and G 2 each independently is unsubstituted C1-C 12 alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; x is 0, 1 or 2.

[1202] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2017 / 117528, which is incorporated herein by reference in its entirety. In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1203] and their pharmaceutically acceptable salts.

[1204] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1205] and its pharmaceutically acceptable salts.

[1206] In some embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1207] and its pharmaceutically acceptable salts.

[1208] Other suitable lipids for RNA compositions and methods for their preparation and use include lipids described in International Patent Publication WO2017 / 049245, which is incorporated herein by reference in its entirety.

[1209] In some embodiments, the lipids of the RNA compositions and methods for their preparation and use include compounds of one of the following formulas:

[1210]

[1211] and

[1212] and its pharmaceutically acceptable salts. For any one of the four formulas, R4 is independently selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -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 heterocycles; and n is 1, 2, or 3.

[1213] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1214] and its pharmaceutically acceptable salts.

[1215] In certain embodiments, the RNA compositions and methods for their preparation and use include lipids having the following compound structures:

[1216] and its pharmaceutically acceptable salts.

[1217] In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1218] and their pharmaceutically acceptable salts.

[1219] In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1220] and their pharmaceutically acceptable salts.

[1221] Other suitable lipids for RNA compositions and methods of making and using the same include lipids described in International Patent Publications WO2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference in its entirety. In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1222] and their pharmaceutically acceptable salts.

[1223] In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1224] and their pharmaceutically acceptable salts.

[1225] In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1226] and their pharmaceutically acceptable salts.

[1227] In certain embodiments, the RNA compositions and methods of making and using the same include lipids having the following compound structures:

[1228] and their pharmaceutically acceptable salts.

[1229] In some embodiments, the LNMPs described herein may include ionizable lipids as described in the following, may be formulated as described in the following, or may comprise the following or comprise a composition as described in the following: WO2016118724, WO2016118725, WO2016187531, WO2017176974, WO2018078053, WO2019027999, WO2019036030, WO2019089828, WO2019099501, WO2020072605, WO2020081938, WO2020118041, WO2020146805 or WO2020219876, each of which is incorporated herein by reference in its entirety.

[1230] The ionizable lipids disclosed herein can be used to form LNMP compositions together with one or more natural lipids disclosed herein. In some embodiments, the LNMP compositions are formulated to further comprise one or more therapeutic agents. In some embodiments, the LNMP compositions are lipid nanoparticles encapsulating or associated with the one or more therapeutic agents. In some embodiments, the therapeutic agent is one or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems.

[1231] In some embodiments, the N / P ratio of the RNA compositions disclosed herein is at least 3, e.g., the N / P ratio is from 3 to 100, from 3 to 50, from 3 to 30, from 3 to 20, from 3 to 15, from 3 to 12, from about 3 to about 10, from 6 to 30, from 6 to 20, from 6 to 15 or from 6 to 12. For example, the N / P ratio can be about 6 ± 1, or the N / P ratio can be about 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 N / P ratio of the RNA compositions is from about 12 to about 17, e.g., 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).

[1232] In some embodiments, the present disclosure relates to a composition comprising (i) one or more compounds selected from ionizable lipids of formula (I)-(III), 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.

[1233] In some embodiments, the present disclosure relates to a composition comprising (i) one or more lipid nanoparticles and (ii) one or more lipid components.

[1234] In some embodiments, the one or more lipid components comprise one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component comprises one or more helper lipids, one or more PEG lipids and one or more neutral lipids.

[1235] Non-limiting examples of neutral lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 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, dilinoleoyl phosphatidylcholine and mixtures thereof. Other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids may be acyl groups derived from fatty acids having C 10 -C 24 carbon chains, for example, lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

[1236] In some embodiments, the RNA composition comprises phytosterols or a combination of phytosterols and cholesterol. In some embodiments, the phytosterols are selected from the group consisting of: β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterols are selected from the group consisting of: β-sitosterol, β-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 phytosterols are 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 some embodiments, the phytosterols are a combination of compound S-141, compound S-140, compound S-143, and compound S-148. In some embodiments, the phytosterols comprise sitosterol or a salt or ester thereof. In some embodiments, the phytosterols comprise stigmasterol or a salt or ester thereof.

[1237] Other Lipids and Other Agents

[1238] The exogenous lipid can be a cell permeant, can be capable of increasing the delivery of the polypeptide to cells via LNMP, and / or can be capable of increasing the loading of the polypeptide (e.g., loading efficiency or loading capacity). Additional exemplary exogenous lipids include sterols and PEGylated lipids.

[1239] The LNMP 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 properties of the LNMP. For example, the LNMP can be further modified with stabilizing molecules that increase the stability of the LNMP (e.g., remaining stable for at least one day at room temperature and / or stable for at least one week at 4°C).

[1240] In some embodiments, the LNMP is modified with a sterol, such as sitosterol, dihydro-sitosterol, β-sitosterol, 7α-hydroxy cholesterol, pregnenolone, cholesterol (e.g., sheep cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or an analogue of any sterol (e.g., glycoside, ester, or peptide). In some instances, the exogenous sterol is added to the formulation before step (b), e.g., mixed with the extracted NMP lipids before step (b). The amount of exogenous sterol added can be, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% (w / w) of the total lipids and sterols in the formulation.

[1241] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the LNMP contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or greater than 60% sterol (e.g., cholesterol or sitosterol) on a molar basis, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60% sterol. In some embodiments, the LNMP contains sterol (e.g., cholesterol or sitosterol) at a molar ratio of about 35%-50%, e.g., sterol at a molar ratio of about 36%, 38.5%, 42.5%, or 46.5%. In some embodiments, the LNMP contains sterol at a molar ratio of about 20%-40%.

[1242] In some embodiments, the stability of the sterol-modified LNMP is altered (e.g., increased) relative to the LNMP that has not been modified with a sterol. In some aspects, the fusion ratio of the sterol-modified LNMP with the membrane of the target cells is greater relative to the LNMP that has not been modified with a sterol.

[1243] In some cases, the LNMP contains exogenous lipids and exogenous sterols.

[1244] In some embodiments, the LNMPs are modified with PEGylated lipids. The polyethylene glycol (PEG) length can vary between 1 kDa and 10 kDa; in some aspects, PEG with a length of 2 kDa is used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some cases, the LNMPs contain 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 greater than 50% of the PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., a molar ratio of 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% of the PEGylated lipid. In some embodiments, the LNMPs contain a molar ratio of about 0.1%-10% of the PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., a molar ratio of about 1%-3% of the PEGylated lipid, e.g., a molar ratio of about 1.5% or about 2.5% of the PEGylated lipid. In some embodiments, the stability of the LNMPs modified with PEGylated lipids is altered (e.g., increased) relative to the LNMPs that have not been modified with PEGylated lipids. In some embodiments, the particle size of the LNMPs modified with PEGylated lipids is altered relative to the LNMPs that have not been modified with PEGylated lipids. In some embodiments, the LNMPs modified with PEGylated lipids are less likely to be phagocytosed relative to the LNMPs that have not been modified with PEGylated lipids. The addition of the PEGylated lipid can also affect stability in the GI tract and enhance particle migration through mucus. The PEG can be used as a means to attach targeting moieties.

[1245] In some embodiments, the LNMPs are modified with one or both of an ionizable lipid and a sterol (e.g., cholesterol or sitosterol) and a PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k).

[1246] In some embodiments, the LNMP comprises natural lipids in a molar ratio of about 5%-50% (e.g., natural lipids in a molar ratio of about 10%-20%, e.g., natural lipids in a molar ratio of about 10%, 12.5%, 16%, or 20%); ionizable lipids in a molar ratio of about 30%-75% (e.g., ionizable lipids in a molar ratio of about 35% or about 50%); sterols in a molar ratio of about 35%-50% (e.g., sterols in a molar ratio of about 36%, 38.5%, 42.5%, or 46.5%); and PEGylated lipids in a molar ratio of about 0.1%-10% (e.g., PEGylated lipids in a molar ratio of about 1%-3%, e.g., PEGylated lipids in a molar ratio of about 1.5% or about 2.5%).

[1247] In some embodiments, the modified LNMP comprises natural lipids in a molar ratio of about 5%-60% (e.g., natural lipids in a molar ratio of about 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60%, e.g., natural lipids in a molar ratio of about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60%); ionizable lipids in a molar ratio of about 25%-75% (e.g., ionizable lipids in a molar ratio of about 35% or about 50%); sterols in a molar ratio of about 10%-50% (e.g., sterols in a molar ratio of about 10%, 12.5%, 14%, 16%, 18%, 20%, 36%, 38.5%, 42.5%, or 46.5%); and PEGylated lipids in a molar ratio of about 0.1%-10% (e.g., PEGylated lipids in a molar ratio of about 0.5%-5%, e.g., PEGylated lipids in a molar ratio of about 1%-3% or PEGylated lipids in a molar ratio of about 1.5% or about 2.5%).

[1248] In some embodiments, the ionizable lipids, natural lipids, sterols, and PEGylated lipids respectively comprise about 25%-75%, about 20%-60%, about 10%-45%, and about 0.5%-5% of the lipids in the modified NMP.

[1249] In some embodiments, the ionizable lipids, natural lipids, sterols, and PEGylated lipids respectively comprise about 30%-75%, about 20%-50%, about 10%-45%, and about 1%-5% of the lipids in the modified NMP.

[1250] In some embodiments, the ionizable lipids, natural lipids, sterols, and PEGylated lipids respectively comprise about 35%-75%, about 20%-50%, about 10%-45%, and about 1%-5% of the lipids in the modified NMP.

[1251] In some embodiments, the ionizable lipid, natural lipid, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:12.5:2.5.

[1252] In some embodiments, the ionizable lipid, natural lipid, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:50:11.5:3.5.

[1253] In some embodiments, the ionizable lipid, natural lipid, sterol, and PEGylated lipid are formulated at a molar ratio of about 35:20:42.5:2.5.

[1254] In some embodiments, compared to LNMPs that have not been modified with cationic lipids and sterols and / or PEGylated lipids, LNMPs that have been modified with cationic lipids and sterols and / or PEGylated lipids more effectively encapsulate negatively charged cargo (e.g., nucleic acids). The encapsulation efficiency of the modified LNMPs for cargo (e.g., nucleic acids, e.g., RNA or DNA) can be at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%, e.g., the encapsulation efficiency can be 5%-30%, 30%-50%, 50%-70%, 70%-80%, 80%-90%, 90%-95%, or 95%-100%.

[1255] The cellular uptake of the modified LNMPs can be measured by a variety of methods known in the art. For example, the LNMPs or their components can be labeled with a marker (e.g., a fluorescent marker), and the marker can be detected in isolated cells to confirm uptake.

[1256] In some embodiments, the LNMP formulations provided herein comprise two or more different modified LNMPs, e.g., modified LNMPs derived from different unmodified LNMPs (e.g., unmodified LNMPs from two or more different natural sources) and / or modified LNMPs comprising different species and / or different ratios of ionizable lipids, sterols, and / or PEGylated lipids.

[1257] In some cases, the organic solvent that dissolves the lipid membrane is dimethylformamide:methanol (DMF:MeOH). Alternatively, the organic solvent or solvent combination can be, for example, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol, or methylformamide:methanol.

[1258] The aqueous phase can be any suitable solution, such as a citrate buffer (e.g., a citrate buffer with a pH of about 3.2), water, or phosphate buffered saline (PBS). The aqueous phase can further contain nucleic acids (e.g., siRNA or siRNA precursors (e.g., dsRNA), miRNA or miRNA precursors, mRNA, or plasmid (pDNA)) or small molecules.

[1259] The lipid solution and the aqueous phase can be mixed in a microfluidic device at any suitable ratio. In some examples, the aqueous phase and the lipid solution are mixed at a volume ratio of 3:1.

[1260] The LNMP can optionally include additional agents, such as cell-penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. The LNMP can carry or associate with additional agents in a variety of ways, enabling delivery of the agents to a target plant, for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure, or associating the agent with the surface of the lipid bilayer structure (e.g., by conjugation). Nucleic acid molecules can be incorporated into the LNMP in vivo (e.g., in a plant) or in vitro (e.g., in tissue culture, in cell culture, or synthetically incorporated).

[1261] ζ potential

[1262] The ζ-potential of an LNMP containing an ionizable lipid and optionally a cationic lipid (e.g., DC-cholesterol or DOTAP) can be, for example, greater than -30 mV in the absence of cargo, greater than -20 mV, greater than -5 mV, greater than 0 mV, or about 30 mV. In some examples, the LNMP has a negative ζ-potential in the absence of cargo, such as a ζ-potential less than 0 mV, less than -10 mV, less than -20 mV, less than -30 mV, less than -40 mV, or less than -50 mV. In some examples, the LNMP has a positive ζ-potential in the absence of cargo, such as a ζ-potential greater than 0 mV, greater than 10 mV, greater than 20 mV, greater than 30 mV, greater than 40 mV, or greater than 50 mV. In some examples, the ζ-potential of the LNMP is about 0.

[1263] The ζ-potential of the LNMP can be measured using any method known in the art. The ζ-potential is typically measured indirectly, for example, using a theoretical model and calculated from 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 adjustable resistive pulse sensing. Electrophoretic light scattering is based on dynamic light scattering. Generally, the ζ-potential can be obtained from dynamic light scattering (DLS) measurements, also known as photon correlation spectroscopy or quasi-elastic light scattering.

[1264] Plant EV-marker

[1265] LPMPs in RNA compositions and their preparation and use methods can have a series of markers that identify the LPMPs as being produced using plant EVs and / or comprising segments, portions, or extracts 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 a plant EV in the plant, such as a plant protein, plant nucleic acid, plant small molecule, plant lipid, or a combination thereof. Examples of plant EV-markers can be found in, for example, Rutter and Innes, Plant Physiology 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., Mol. 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.

[1266] Additional examples of suitable plant EV markers include those described and listed in International Patent Application Publication No. WO 2021 / 041301, which is incorporated herein in its entirety by reference.

[1267] Bacterial EV-marker

[1268] Bacterial components (e.g., bacterial lipids) in bacterially-derived lipid compositions and their preparation and use methods can have a series of markers that identify the bacterial components as being produced. As used herein, the term "bacterial EV-marker" refers to a component that is naturally associated with a bacterial EV and incorporated into or onto the bacterial EV, such as a bacterial protein, bacterial nucleic acid, bacterial small molecule, bacterial lipid, or a combination thereof.

[1269] EV-marker of natural origin

[1270] NMP can have a series of markers that identify the NMP as being produced by EVs from a specific source and / or include segments, portions, or extracts thereof. As used herein, the term "EV-marker" refers to a component that is naturally associated with a specific source and is incorporated into or onto the EV in vivo, such as a protein, nucleic acid, small molecule, 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, β-glucan, pekilo, fungal proteins, cerato-platanins, exotoxins, diacylglycerol, triglycerides, phosphatidylcholine, phosphatidylinositol, ornithine lipids, glycolipids, sphingolipids, hopanoids, or ergosterol.

[1271] Source EV markers can include lipids. Examples of lipid markers that can be found in NMP include lipid A, lipopolysaccharide, ergosterol, ornithine lipid (OL), sulfolipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), glycolipid (GL), diacylglycerol (DAG), hopanoids (HOP), glucosylceramide, sterol glycoside, ether-linked lipids, or a combination thereof.

[1272] Other EV markers can include lipids that accumulate in the source in response to abiotic or biotic stressors.

[1273] Alternatively, the source EV marker can include a protein. In some cases, the protein EV marker can be an antimicrobial or antiviral protein naturally produced by the source, including proteins secreted in response to abiotic or biotic stressors. Some examples of protein EV markers include, but are not limited to, bacteriocins, moricin, defensins, proline- and glycine-rich peptides, fungal immunomodulatory proteins, flagellin, encapsulin, streptavidin, invasin, pilin, halocin, or archaeocin. In some cases, the EV marker can include a protein involved in lipid metabolism. In some cases, the protein EV marker is a cellular transport protein in the source. In certain cases where the EV marker is a protein, the protein marker may lack the signal peptide typically associated with secreted proteins. Unconventional secreted proteins seem to have several common features, such as (i) lack of a leader sequence, (ii) absence of PTMs specific to the ER or Golgi apparatus, and / or (iii) secretion is not affected by brefeldin A, which blocks the classical ER / Golgi-dependent secretion pathway. Those skilled in the art can use a variety of freely available tools to evaluate the signal sequence or the lack thereof of a protein.

[1274] In cases where the EV marker is a protein, the protein can have an amino acid sequence with 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.

[1275] In some cases, the EV marker includes nucleic acids, RNA, DNA, or PNA encoded in a source such as an arthropod, plant, fungus, archaea, or bacterium. For example, the NMP can include dsRNA, mRNA, viral RNA, microRNA (miRNA), or small interfering RNA (siRNA) encoded by the source. In some cases, the nucleic acid can be associated with a protein that promotes long-distance transport of RNA. In some cases, the nucleic acid EV marker can be a marker involved in host-induced gene silencing (HIGS), which is a process by which the source silences foreign transcripts of a pathogen. In some cases, the nucleic acid can be a microRNA.

[1276] In the case where the EV marker is a nucleic acid, the nucleic acid can have a nucleotide sequence with 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.

[1277] In some cases, the EV marker includes a compound produced by the source. For example, the compound can be a component of the cell wall (e.g., lipopolysaccharide). For example, the compound can be a defense compound produced in response to abiotic or biotic stressors such as pathogens or extreme environmental stress.

[1278] In some cases, NMP can also be identified as being produced by source EVs based on the absence of certain markers (e.g., lipids, polypeptides or polynucleotides) that are not typically produced by these sources but are typically associated with other organisms (e.g., markers of animal EVs or plant EVs). For example, in some cases, NMP lacks lipids that are typically found in animal EVs or plant EVs.

[1279] Any method known in the art for identifying small molecules (e.g., mass spectrometry, mass spectrometry analysis), lipids (e.g., mass spectrometry, mass spectrometry analysis), proteins (e.g., mass spectrometry, immunoblotting) or nucleic acids (e.g., PCR analysis) can be used to identify EV markers. In some cases, the NMP compositions described herein include a detectable amount (e.g., a predetermined threshold amount) of the EV markers described herein.

[1280] Loading of agents (e.g., nucleic acids)

[1281] LNMP is modified to include an RNA agent (e.g., a gene editing system; a nucleic acid molecule such as mRNA or gRNA) to form an RNA composition. LNMP can carry or associate with such agents by a variety of means such that the agent can be delivered to a target organism (e.g., a target animal), for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure or associating the component with the surface of the lipid bilayer structure of LNMP (e.g., by conjugation). In some cases, the agent is included in an LNMP formulation as described herein.

[1282] The agent can be incorporated into or loaded onto or into LNMP by any method known in the art that permits direct or indirect association between LNMP and the agent. The agent can be incorporated into LNMP by in vivo methods (e.g., in planta, e.g., by producing LNMP from a transgenic plant or source containing the agent) or in vitro methods (e.g., in tissue culture or in cell culture) or both in vivo and in vitro methods.

[1283] In some cases, loading of LNMP is carried out in vitro. Substances can be loaded onto or into (e.g., encapsulated by) LNMP using physical, chemical, and / or biological methods, including but not limited to, in tissue culture or in cell culture. For example, agents can be incorporated into LNMP by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction, or extrusion. In some cases, a microfluidic device is used, such as a method where natural lipids are provided in an organic phase, a heterologous functional agent is provided in an aqueous phase, and the organic and aqueous phases are combined in the microfluidic device to produce LNMP containing the heterologous functional agent, to incorporate agents into LNMP. Multiple methods, such as HPLC (e.g., for evaluating small molecules), immunoblotting (e.g., for evaluating proteins), and / or quantitative PCR (e.g., for evaluating nucleotides), can be used to evaluate the loaded LNMP to confirm the presence or level of the loaded agent. However, those skilled in the art will appreciate that loading the substance of interest into LNMP is not limited to the methods described above.

[1284] In some cases, an agent can be conjugated to LNMP, where the agent is indirectly or directly linked or joined to the LNMP. For example, one or more agents can be chemically linked to the LNMP such that the one or more agents are directly joined to the lipid bilayer of the LNMP (e.g., by a covalent or ionic bond). In some cases, conjugation of various agents to LNMP can be achieved by first mixing one or more agents in a suitable solvent with a suitable crosslinking agent (e.g., N-ethylcarbodiimide (“EDC”), which is commonly used as a carboxyl activator for amide bonding with primary amines and also reacts with phosphate groups). After an incubation period sufficient to allow the agent to link to the crosslinking agent, the crosslinker / agent mixture can then be combined with the LNMP, and after another incubation period, subjected to a sucrose gradient (e.g., an 8%, 30%, 45%, and 60% sucrose gradient) to separate free agent, free LNMP, and agent-conjugated LNMP. As part of combining the mixture with the sucrose gradient and the accompanying centrifugation step, the LNMP conjugated with the agent is then visualized as a band in the sucrose gradient, such that the conjugated LNMP can then be collected, washed, and dissolved in a suitable solution for use as described herein.

[1285] In some cases, LNMP is stably associated with an agent before and after delivery of the LNMP to, for example, a plant or an animal. In other cases, the agent is conjugated to the LNMP after delivery of the LNMP to, for example, a plant or an animal, such that the agent dissociates from the LNMP.

[1286] The LNMP can be loaded with various concentrations of a medicament, or the LNMP can be formulated with various concentrations of a medicament, depending on the particular medicament or use. For example, in some cases, the LNMP is loaded or the LNMP is formulated such that the LNMP formulation disclosed herein comprises 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 the medicament. In some cases, the LNMP is loaded or the LNMP is formulated such that the LNMP formulation comprises 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 the medicament. For example, the LNMP formulation can comprise from about 0.001 to about 0.01 wt%, from about 0.01 to about 0.1 wt%, from about 0.1 to about 1 wt%, from about 1 to about 5 wt%, or from about 5 to about 10 wt%, from about 10 to about 20 wt% of the medicament. In some cases, the LNMP can be loaded with or the LNMP can be formulated with the following medicaments: about 1, 5, 10, 50, 100, 200, or 500, 1,000, 2,000 (or any range between about 1 and 2,000) or more μg / ml of the medicament. The LNMP of the present invention can be loaded with or the LNMP can be formulated with the following medicaments: about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range between about 2,000 and 1) or less μg / ml of the medicament.

[1287] In some cases, the LNMP is loaded or the LNMP is formulated such that the LNMP formulations disclosed herein comprise at least 0.001 wt%, at least 0.01 wt%, at least 0.1 wt%, at least 1.0 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt% or at least 95 wt% of the agent. In some cases, the LNMP can be loaded with or formulated with the following agents: at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 200 μg / ml, at least 500 μg / ml, at least 1,000 μg / ml, at least 2,000 μg / ml of the agent.

[1288] In some cases, the LNMP is formulated with the agent by suspending the LNMP in a solution comprising or consisting of the agent, for example, by vigorously mixing to suspend or resuspend the LNMP. The agent (e.g., a cell-penetrating agent such as a nucleic acid, an enzyme, a detergent, an ionic liquid, a fluorinated liquid or a zwitterionic liquid or an ionizable lipid) can comprise, for example, less than 1% or at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the solution.

[1289] Drug formulation

[1290] The modified LNMP is formulated into a pharmaceutical composition (i.e., an RNA composition), for example, for administration to an animal (e.g., a human). The pharmaceutical composition can be administered to an animal (e.g., a human) with a pharmaceutically acceptable diluent, carrier and / or excipient. Depending on the mode of administration and the dose, the pharmaceutical composition of the methods described herein will be formulated into a suitable pharmaceutical composition to allow for easy delivery. The single dose can be in unit dosage form as needed.

[1291] The LNMP / RNA compositions can be formulated for, for example, oral administration to an animal, intravenous administration (e.g., injection or infusion), intramuscular or subcutaneous administration. For injectable formulations, a variety of effective pharmaceutical carriers are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd Edition, (2012) and ASHP Handbook on Injectable Drugs, 18th Edition, (2014)).

[1292] Suitable pharmaceutically acceptable carriers and excipients are non-toxic to the recipient at the dosages and concentrations employed. Acceptable carriers and excipients can include: buffers such as phosphate, citrate, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyl dimethyl benzyl 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.

[1293] The LNMP / RNA compositions can be formulated according to conventional pharmaceutical practice. The concentration of the compounds in the formulation will vary depending on a number of factors, including the dosage of the active agents (e.g., LNMP and nucleic acid) to be administered and the route of administration.

[1294] For oral administration to an animal, the LNMP / RNA composition can be prepared in the form of an oral formulation. Formulations for oral use can include tablets, cachets, capsules, syrups, or oral liquid dosage forms containing the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (such as cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (such as sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pre-gelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and anti-adherent agents (such as magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients can be coloring agents, flavoring agents, plasticizers, humectants, buffering agents, etc. Formulations for oral use can also be provided in unit dosage forms as chewable tablets, non-chewable tablets, cachets, capsules (e.g., as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium). The compositions disclosed herein can further include immediate-release, extended-release, or delayed-release formulations.

[1295] For parenteral administration to an animal, the LNMP / RNA composition can be formulated as a liquid solution or suspension and administered by a parenteral route (e.g., subcutaneous, intravenous, or intramuscular). The pharmaceutical composition can be formulated for injection or infusion. The pharmaceutical composition 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, normal saline, or cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), and F-12 medium). Formulation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd Edition) Taylor & Francis Group, CRC Press (2009).

[1296] Polynucleotide

[1297] The LNMP / RNA composition comprises one or more nucleic acid molecules, such as polynucleotides, which encode one or more wild-type or engineered proteins, peptides or polypeptides. Exemplary polynucleotides (e.g., polynucleotide constructs) include gene editing systems encoding RNA polynucleotides, such as mRNA and gRNA.

[1298] Examples of polypeptides that can be used herein can include enzymes (e.g., metabolic recombinases, helicases, integrases, ribonucleases, deoxyribonucleases or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas systems, TALENs or zinc fingers), ribonucleoproteins, protein aptamers or chaperone proteins.

[1299] The polypeptides included herein can include naturally occurring polypeptides or recombinantly produced variants. In some cases, the polypeptide can be a functional fragment or a variant thereof (e.g., an enzymatically active fragment or a variant thereof). For example, the polypeptide can be a functionally active variant of any of the polypeptides described herein, which, for example, has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of the polypeptide described herein or a naturally occurring polypeptide, in a specified region or over the entire sequence. In some cases, the polypeptide can have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or higher) identity to the protein of interest.

[1300] The LNMP / RNA composition can include any number or type (e.g., species) of polypeptides, such as at least about any one of 1 polypeptide, 2, 3, 4, 5, 10, 15, 20 or more polypeptides. The appropriate concentration of each polypeptide in the LNMP / RNA composition depends on factors such as efficacy, stability of the polypeptide, the number of different polypeptides in the formulation and the method of application of the formulation. In some cases, each polypeptide in the liquid formulation is from about 0.1 ng / mL to about 100 mg / mL. In some cases, each polypeptide in the solid formulation is from about 0.1 ng / g to about 100 mg / g.

[1301] Nucleic acid encoding a peptide

[1302] In some cases, the LNMP / RNA composition includes a heterologous nucleic acid encoding a polypeptide. The length of the nucleic acid encoding the polypeptide can be from about 10 to about 50,000 nucleotides (nt), about 25 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200 to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 to about 5000 nt, about 5000 to about 6000 nt, about 6000 to about 7000 nt, about 7000 to about 8000 nt, about 8000 to about 9000 nt, about 9000 to about 10,000 nt, about 10,000 to about 15,000 nt, about 10,000 to about 20,000 nt, about 10,000 to about 25,000 nt, about 10,000 to about 30,000 nt, about 10,000 to about 40,000 nt, about 10,000 to about 45,000 nt, about 10,000 to about 50,000 nt, or any range therebetween.

[1303] The LNMP / RNA composition can also include an active variant of the nucleic acid sequence of interest. In some cases, the variant of the nucleic acid has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of the nucleic acid of interest, for example, in a specified region or over the entire sequence. In some cases, the present invention includes an active polypeptide encoded by a nucleic acid variant as described herein. In some cases, the active polypeptide encoded by the nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of the polypeptide of interest or the sequence of a naturally-derived polypeptide, for example, in a specified region or over the entire sequence.

[1304] Certain methods for expressing nucleic acids encoding proteins can involve expression in cells under the control of a suitable promoter, said cells including insect, yeast, plant, bacterial or other cells. Expression vectors can include non-transcribed elements such as origins of replication, suitable promoters and enhancers, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' untranslated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, can be used to provide other genetic elements required for expressing heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

[1305] Genetic modification using recombinant methods is generally known in the art. Nucleic acid sequences encoding a desired gene can be obtained using recombinant methods known in the art, such as screening a library from cells expressing the gene, deriving the gene from a vector known to include the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than by cloning.

[1306] Expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The expression vector can be suitable for replication and expression in bacteria. The expression vector can also be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to express the desired nucleic acid sequence.

[1307] Additional promoter elements (e.g., enhancers) regulate the frequency of transcriptional initiation. Generally, these promoter elements are located in the region 30 - 110 base pairs (bp) upstream of the start site, but recently it has been shown that many promoters also contain functional elements located downstream of the start site. The spacing between promoter elements is usually flexible such that promoter function is retain...

Claims

1. A method for delivering a gene editing system to a subject in need thereof, the method comprising administering to the subject an RNA composition, the RNA composition comprising: One or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides being formulated within: (a) A plurality of lipid nanoparticles (LNPs) that comprise synthetic structural lipids and ionizable lipids; or (b) Lipid-reconstituted natural messenger packages (LNMPs) that comprise natural lipids and ionizable lipids, Wherein the ionizable lipid has two or more of the following listed properties: (i) At least 2 ionizable amines; (ii) At least 3 lipid tails; wherein each lipid tail has a length of at least 6 carbon atoms; (iii) A pKa of about 4.5 to about 7.5; (iv) An ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and (v) An N:P ratio of at least 3.

2. A gene editing method, which comprises: Contacting a cell with an RNA composition or administering the RNA composition to a subject, the RNA composition comprising: One or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems, the one or more polynucleotides being formulated within: (a) A plurality of lipid nanoparticles (LNPs) that comprise synthetic structural lipids and ionizable lipids; or (b) Lipid-reconstituted natural messenger packages (LNMPs) that comprise natural lipids and ionizable lipids, Wherein the ionizable lipid has two or more of the following listed properties: (i) At least 2 ionizable amines; (ii) At least 3 lipid tails; wherein each lipid tail has a length of at least 6 carbon atoms; (iii) A pKa of about 4.5 to about 7.5; (iv) An ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and (v) An N:P ratio of at least 3, Wherein the one or more components of the gene editing system or the one or more gene editing systems are delivered to the cell or the subject to modify the genome of the cell or the subject.

3. The method according to claim 1 or 2, wherein the gene editing system is a CRISPR-Cas gene editing system.

4. The method according to any one of claims 1 to 3, wherein the RNA composition further comprises at least one template nucleic acid.

5. The method according to claim 1 or 2, wherein the RNA composition is administered at least once.

6. The method according to claim 1 or 2, wherein the RNA composition is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, at least twenty times or more.

7. The method according to claim 1 or 2, wherein the RNA composition is administered 2 - 8 times.

8. The method according to claim 6 or 7, wherein the delivery of the gene editing system or the result of gene editing is improved after multiple administrations.

9. The method according to claim 1 or 2, wherein at least two RNA compositions are administered to the subject or contacted with the cells: a first RNA composition comprising mRNA; and a second RNA composition comprising a guide RNA nucleic acid.

10. The method according to claim 9, wherein the first RNA composition and the second RNA composition are administered simultaneously.

11. The method according to claim 9, wherein the first RNA composition and the second RNA composition are administered sequentially.

12. The method according to claim 1 or 2, wherein a single RNA composition is contacted with the cells or administered to the subject, and the single RNA composition comprises mRNA and a guide RNA nucleic acid.

13. The method according to claim 1 or 2, wherein the RNA composition is administered by oral, intravenous, intramuscular, intranasal or subcutaneous routes.

14. The method according to claim 1 or 2, wherein the RNA composition is administered more than once within at least one week, at least two weeks, at least three weeks or at least four weeks between administrations.

15. The method according to claim 1 or 2, wherein the RNA composition is formulated with b) LNMP.

16. The method according to claim 15, wherein the ionizable lipid is C12 - 200.

17. The method according to claim 1 or 2, wherein the RNA composition is formulated with a) LNP.

18. The method according to claim 15 or 17, wherein the ionizable lipid is selected from one of the following groups of compounds: i) A compound of formula , a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing Wherein: Each A is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally substituted with a heteroatom or substituted with OH, SH or a halogen; Each B is independently C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally substituted with a heteroatom or substituted with OH, SH or a halogen; Each X is independently a biodegradable moiety; and W is wherein R5 is OH, SH, NR 10 R 11 ; Each R6 is independently H, a C1 - C3 branched or unbranched alkyl, a C2 - C3 branched or unbranched alkenyl or cycloalkyl; Each R7 and each R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, NR 10 R 11 , where each R 10 and R 11 is independently H, a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; Each s is independently 1, 2, 3, 4 or 5; Each u is independently 1, 2, 3, 4 or 5; t is 1, 2, 3, 4 or 5; Each Z is independently absent, or is O, S or NR 12 , where R 12 is H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl, and Q is O, S or NR 13 , wherein each R 13 is H, C1-C5 alkyl; ii) a compound of formula , a pharmaceutically acceptable salt thereof or a stereoisomer of any of the foregoing, wherein: is a cyclic or heterocyclic moiety; Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group or A is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle; Each of X and Z is independently absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-; -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl or aminoalkyl; Each M is independently a biodegradable moiety; R 30 、R 40 、R 50 、R 60 、R 70 、R 80 、R 90 、R 100 、R 110 and R 120 Each of which is independently H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by 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 a hydroxyl group, a substituted or unsubstituted hydroxyalkyl, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl or a substituted or unsubstituted heterocyclic group or heteroaryl; and iii) Formula a compound, a pharmaceutically acceptable salt thereof and stereoisomers of any of the foregoing, wherein: R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl or a C2-C5 branched or unbranched alkenyl, or R 20 and R 30 together with the adjacent N atom forms a 3- to 7-membered ring, which ring is optionally substituted by R a substituted; R a is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH or SH; Each R1 and each R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, halogen, SH or NR 10 R 11 , or R1 and R2 together form a ring; Each R 10 and R 11 independently is H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle; n is 0, 1, 2, 3 or 4; Y is O or S; Z is absent, and is O, S or N(R 12 )(R 12 ), where each R 12 is independently H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH; u is 0, 1, 2, 3, 4, 5, 6, 7 or 8; v is 0, 1, 2, 3 or 4; y is 0, 1, 2, 3 or 4; Each A is independently a C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen; Each B is independently C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl, said alkyl or alkenyl being optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen; and Each X is independently a biodegradable moiety; and iv) A lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'); its pharmaceutically acceptable salts or stereoisomers of any of the foregoing, Wherein: E is independently -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -,-C(O)N(R 7 )(CH2) r -,-S-S- or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl group; r is 1, 2, 3, 4 or 5; R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6, or 7; R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl being optionally interrupted by a heteroatom or being substituted by OH, SH or a halogen or a cycloalkyl or a substituted cycloalkyl; represents a bond connecting the tail group and the head group; and wherein the pKa of the lipid is from about 4 to about 8.

19. The method according to claim 18, wherein the ionizable lipid is a compound in Table I, Table II, Table III, or Table IV.

20. The method according to claim 19, wherein the ionizable lipid is 2272, 2320, 2439, 2356, 2243, 2431, 2455, 2454, 2424, 2433, 2425, 2275, 2220, or 2335.

21. An RNA composition for gene editing, the RNA composition comprising: one or more polynucleotides that encode one or more components of a gene editing system or one or more gene editing systems, wherein the one or more polynucleotides are formulated within a lipid-reconstituted native messenger package (LNMP) that comprises a native lipid and an ionizable lipid, wherein the ionizable lipid has two or more of the following listed properties: (i) at least 2 ionizable amines; (ii) at least 3 lipid tails; wherein each of the lipid tails has a length of at least 6 carbon atoms; (iii) a pKa of from about 4.5 to about 7.5; (iv) an ionizable amine and a heteroorganic group separated by a chain having at least two atoms; and (v) an N:P ratio of at least 3.

22. The RNA composition according to claim 21, 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(dodecane-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

23. The RNA composition according to claim 21, wherein the ionizable lipid is C12-200.

24. The RNA composition according to claim 21, wherein the ionizable lipid is wherein R is a C8-C14 alkyl group.

25. The RNA composition according to claim 21, wherein the ionizable lipid is selected from one of the following groups of compounds: i) A compound of formula , a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing wherein: Each A is independently a C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, where the alkyl or alkenyl is optionally substituted with a heteroatom or substituted with OH, SH or a halogen; Each B is independently C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally substituted with a heteroatom or substituted with OH, SH or a halogen; each X is independently a biodegradable moiety; and W is wherein R5 is OH, SH, NR 10 R 11 ; each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or a cycloalkyl; Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, NR 10 R 11 , where each R 10 and R 11 are independently H, a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S or NR 12 , where R 12 is H, C1-C7 branched or unbranched alkyl or C2-C7 branched or unbranched alkenyl, and Q is O, S or NR 13 , where each R 13 is H, C1-C5 alkyl; ii) A compound of formula , a pharmaceutically acceptable salt thereof or a stereoisomer of any of the foregoing, wherein: is a cyclic or heterocyclic moiety; Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group or A is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S-, -S-S- or a divalent heterocycle; Each of X and Z is independently absent, is -O-, -CO-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; Each R 7 independently is H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl or aminoalkyl; each M is independently a biodegradable moiety; R 30 、R 40 、R 50 、R 60 、R 70 、R 80 、R 90 、R 100 、R 110 and R 120 Each of which independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, and the alkyl or alkenyl is optionally interrupted by a heteroatom or substituted by 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 a hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclic or heteroaryl group; and iii) formula a compound, a pharmaceutically acceptable salt thereof and a stereoisomer of any of the foregoing, wherein: R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl or a C2-C5 branched or unbranched alkenyl, or R 20 and R 30 together with the adjacent N atom forms a 3- to 7-membered ring, which ring is optionally substituted by R a substituted; R a is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH or SH; Each R1 and each R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 , or R1 and R2 together form a ring; Each R 10 and R 11 independently is H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl, or R 10 and R 11 together form a heterocycle; n is 0, 1, 2, 3, or 4; Y is O or S; Z is absent, and is O, S or N(R 12 )(R 12 ), where each R 12 is independently H, a C1-C7 branched or unbranched alkyl or a C2-C7 branched or unbranched alkenyl, provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 or SH; u is 0, 1, 2, 3, 4, 5, 6, 7 or 8; v is 0, 1, 2, 3 or 4; y is 0, 1, 2, 3 or 4; Each A is independently a C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen; Each B is independently a C1-C 16 branched or unbranched alkyl or C2-C 16 branched or unbranched alkenyl, wherein the alkyl or alkenyl is optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen; and each X is independently a biodegradable moiety; and iv) a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'); a pharmaceutically acceptable salt or a stereoisomer of any of the foregoing, wherein: E is independently -OC(O)-, -C(O)O-, -N(R 7 )(O)C-, -C(O)N(R 7 )-, -C(O-R 13 )-O-, -C(O)O(CH2) r -, -C(O)N(R 7 )(CH2) r -, -S-S- or -C(O-R 13 )-O-(CH2) r -, where each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl; R 13 is a branched or unbranched C3-C 10 alkyl group; r is 1, 2, 3, 4 or 5; R a each independently is a C1-C5 alkyl group, a C2-C5 alkenyl group or a C2-C5 alkynyl group; u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7; R t each independently is H, C1-C 16 branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl, said alkyl or alkenyl being optionally interrupted by a heteroatom or being substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl; represents a bond connecting the tail group and the head group; and wherein the pKa of the lipid is from about 4 to about 8.

26. The RNA composition according to claim 25, wherein the ionizable lipid is a compound of group (i) represented by the formula , its pharmaceutically acceptable salts, and stereoisomers of any of the foregoing, wherein: Each R1 and each R2 is independently H, a C1-C3 branched or unbranched alkyl group, OH, halogen, SH or NR 10 R 11 , or Each R1 and each R2 independently together with the carbon atom to which it is attached form a ring; Each R 10 and R 11 independently is H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle; Each R3 and each R4 are independently H, C2-C 14 branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl) or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4 or 5; V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, where the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl or cycloalkyl; Each R7 and each R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 , where each v is independently 0, 1, 2, 3, 4 or 5, and R 17 is OH, SH or N(CH3)2; and each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

27. The RNA composition according to claim 26, wherein V is a branched or unbranched C2-C3 alkylene, and each R6 is independently H or methyl.

28. The RNA composition according to claim 25, wherein the ionizable lipid is a compound of group (i) represented by the formula , a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing, wherein: Each R1 and each R2 is independently H, a C1-C3 branched or unbranched alkyl group, OH, halogen, SH or NR 10 R 11 , or Each R1 and each R2 independently together with the carbon atom to which it is attached form a ring; Each R 10 and R 11 independently is H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 together form a heterocycle; Each R3 and each R4 is independently H, C2-C 14 branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl) or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each s is independently 1, 2, 3, 4 or 5; T is –NHC(O)O–, –OC(O)NH– or a divalent heterocycle optionally substituted by one or more –(CH2) v OH, –(CH2) v SH or –(CH2) v -halogen group Each R7 and each R8 are independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, OH, SH, (CH2) v R 17 or NR 10 R 11 , where R 17 is OH, SH or N(CH3)2; each v is independently 0, 1, 2, 3, 4 or 5; and each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

29. The RNA composition according to claim 28, wherein T is divalent piperazine or divalent dioxopiperazine.

30. The RNA composition according to any one of claims 26 to 29, wherein X is -OCO-, -COO-, -CONH- or -NHCO-.

31. The RNA composition according to claim 25, wherein the ionizable lipid is a compound of group ii) represented by one of the following formulas: wherein: 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 integers from 0 to 3, R 80 and R 90 each independently is an unsubstituted C5-C8 alkyl or alkenyl; or R 80 is H or an unsubstituted C1-C4 alkyl or alkenyl, and R 90 is an unsubstituted C5-C 11 alkyl or alkenyl; and R 110 and R 120 are each independently an unsubstituted C5-C8 alkyl or alkenyl; or R 110 is H or an unsubstituted C1-C4 alkyl or alkenyl, and R 120 is an unsubstituted C5-C 11 alkyl or alkenyl.

32. The RNA composition according to claim 31, wherein: M is -OC(O)- or -C(O)O-; is: OH, Each R c is independently H or C1-C3 alkyl; each t1 is independently 1, 2, 3 or 4; R 80 and R 90 each independently is H or a C1-C 12 branched or unbranched alkyl; and R 110 and R 120 each independently is H or a C1-C 12 branched or unbranched alkyl, provided that R 80 and R 90 at least one of which is not H, and R 110 and R 120 at least one of which is not H.

33. The RNA composition according to claim 25, wherein the ionizable lipid is a compound of group iii), wherein R1 and R2 are each H, or each R1 is H and one of the R2 variables is OH; and X is –OC(O)- or –C(O)O-.

34. The RNA composition according to claim 33, wherein the ionizable lipid is a compound of group iii) represented by formula (III), wherein: R 20 and R 30 each independently is H or a C1-C3 branched or unbranched alkyl group; or R 20 and R 30 together with the adjacent N atom form a 3- to 7-membered ring, which ring is optionally substituted by R a substituted; R a is H or OH; Z is absent, is S, O or NH; and n is 0, 1 or 2.

35. The RNA composition according to claim 33, wherein the ionizable lipid is a compound of group iii) represented by formula (V).

36. The RNA composition according to claim 25, wherein the ionizable lipid is a compound of group (iv) comprising at least one head group and at least one tail group, wherein: the tail group has a structure of formula (TI) (or TI'); and the head group has a structure of one of the following formulas: i) wherein: R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group or a C2-C5 branched or unbranched alkenyl group, said alkyl or alkenyl group being optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl; or R 20 and R 30 together with an adjacent N atom form a 3- to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, which heterocyclic or heteroaromatic ring is optionally substituted by one or more OH, SH, halogen, alkyl or cycloalkyl; Each of R1 and R2 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, OH, a halogen, SH or NR 10 R 11 ; or R1 and R2 together form a ring; R 10 and R 11 each independently is H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group; or R 10 and R 11 together form a heterocycle; n is 0, 1, 2, 3 or 4; and Z is absent, and is O, S or NR 12 , wherein R 12 is H or a C1-C7 branched or unbranched alkyl; provided that when Z is not absent, adjacent R1 and R2 cannot be OH, NR 10 R 11 , SH; ii) wherein: R1 is H, C1-C3 alkyl, OH, halogen, SH or NR 10 R 11 ; R2 is OH, halogen, SH or NR 10 R 11 ; or R1 and R2 can combine together to form a ring; R 10 and R 11 each independently is H or C1-C3 alkyl; or R 10 and R 11 are capable of combining together to form a heterocycle; R 20 and R 30 each independently is H, a C1-C5 branched or unbranched alkyl group, a C2-C5 branched or unbranched alkenyl group; or R 20 and R 30 are capable of combining together to form a ring; and each of v and y is independently 1, 2, 3 or 4; iii) where W is where R5 is OH, SH, (CH2) s OH or NR 10 R 11 ; each R6 is independently H, a C1-C3 branched or unbranched alkyl, a C2-C3 branched or unbranched alkenyl or cycloalkyl; Each R7 and R8 is independently H, a C1-C3 branched or unbranched alkyl group, a C2-C3 branched or unbranched alkenyl group, a halogen, (CH2) v OH, (CH2) v SH, (CH2) s N(CH3)2 or NR 10 R 11 , where each R 10 and R 11 is independently H or a C1-C3 alkyl group, or R 10 and R 11 together form a heterocycle; or R7 and R8 together form a ring; Each R 20 is independently H or a C1-C3 branched or unbranched alkyl group; R 14 is a heterocycle, NR 10 R 11 、C(O)NR 10 R 11 、NR 10 C(O)NR 10 R 11 or NR 10 C(S)NR 10 R 11， wherein each R 10 and R 11 is independently H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, said alkyl, cycloalkyl, cycloalkenyl being optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 together form a heterocycle; R 16 is H, =O, =S or CN; each of s, u and t is independently 1, 2, 3, 4 or 5; each v is independently 0, 1, 2, 3, 4 or 5; each Y is a divalent heterocycle; Each Z is independently absent, O, S or NR 12 , where R 12 is H, C1-C7 branched or unbranched alkyl or C2-C7 branched or unbranched alkenyl; Q is O, S, CH2 or NR 13 , wherein each R 13 is H, C1-C5 alkyl; V is a branched or unbranched C2-C 10 alkylene, C2-C 10 alkenylene, C2-C 10 alkynylene or C2-C 10 heteroalkylene, wherein the alkylene, alkenylene, alkynylene or heteroalkylene is optionally substituted by one or more OH, SH and / or halogen groups; and T is –NHC(O)O–, –OC(O)NH– or a divalent heterocycle; and iv) wherein: is a cyclic or heterocyclic moiety; Y is an alkyl group, a hydroxyl group, a hydroxyalkyl group, A is absent and is -O-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )-, -N(R 7 )C(O)N(R 7 )-, -S- or -S-S-; Each of X and Z is independently absent, is -O-, -C(O)-, -N(R 7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N(R 7 )C(O)-, -C(O)N(R 7 )- or -S-; Each R 7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl or aminoalkyl; t1 is an integer from 0 to 10; and W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl or a substituted or unsubstituted heterocyclic group or heteroaryl; and wherein the pKa of the lipid is from about 4 to about 8.

37. The RNA composition according to claim 36, wherein the ionizable lipid is a compound of group (iv), and wherein at least one tail group of the lipid has one of the following formulas: wherein: R 7 each independently is H or methyl; R b independently in each case is H or C1-C4 alkyl; 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: i) where m is 1, 2, 3, 4, 5, 6, 7 or 8; ii) iii) and iv) 38. The RNA composition according to claim 37, wherein 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, wherein u3 and u4 are each independently 1-7, and R a are each independently methyl.

39. The RNA composition according to claim 25, wherein the ionizable lipid is a compound in Table I, Table II, Table III or Table IV.

40. The RNA composition according to claim 39, wherein the ionizable lipid is 41. The RNA composition according to claim 21, wherein the natural lipid is extracted from lemon or algae.

42. The RNA composition according to claim 21, wherein the LNMP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

43. The RNA composition according to claim 42, wherein the sterol is cholesterol.

44. The RNA composition according to claim 42, wherein the PEG-lipid conjugate comprises PEG-2k.

45. The RNA composition according to claim 42, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

46. The RNA composition according to claim 42, wherein the PEG-lipid conjugate is PEG2k-DMG or PEG-2k-PE.

47. The RNA composition according to claim 21, wherein the LNMP comprises: about 20 mol% to about 50 mol% of the ionizable lipid, about 20 mol% to about 60 mol% of the natural 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.

48. The RNA composition according to claim 47, wherein the LNMP comprises an ionizable lipid: natural lipid: sterol: PEG-lipid molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:16:46.5:2.5, about 50:10:38.5:1.5 or about 50:20:28.5:1.

5.

49. The RNA composition according to claim 47, wherein the amount of the PEG-lipid conjugate is about 1.5 - 2.5 mol%.

50. The RNA composition according to claim 47, wherein the amount of the ionizable lipid is about 30 - 50 mol%.

51. The RNA composition according to claim 50, wherein the amount of the ionizable lipid is about 50 mol% or 35 mol%.

52. The RNA composition according to claim 47, wherein the N / P ratio is 6 ± 1.

53. The RNA composition according to claim 47, wherein the N / P ratio is 3 ± 1.

54. The RNA composition according to claim 47, wherein the N / P ratio is 15 ± 1.

55. The RNA composition according to any one of the preceding claims, wherein the gene editing system comprises an RNA-guided DNA binder.

56. The RNA composition according to claim 55, wherein the gene editing system is a CRISPR-Cas gene editing system.

57. The RNA composition according to claim 55, wherein the one or more polynucleotides comprise mRNA or modified mRNA.

58. The RNA composition according to claim 55, wherein the RNA-guided DNA binder is Cas nuclease mRNA.

59. The RNA composition according to claim 58, wherein the Cas nuclease mRNA is a class II Cas nuclease mRNA.

60. The RNA composition according to claim 59, wherein the class II Cas nuclease is Cas9 nuclease mRNA.

61. The RNA composition according to claim 55, wherein the one or more polynucleotides comprise gRNA or modified gRNA.

62. The RNA composition according to claim 55, wherein the one or more polynucleotides comprise gRNA and class II Cas nuclease mRNA.

63. The RNA composition according to claim 55, wherein the one or more polynucleotides comprise RNA, the RNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine content of the open reading frame is in the range from its minimum uridine content to 150% of the minimum uridine content.

64. The RNA composition according to claim 63, wherein the one or more polynucleotides comprise mRNA, the mRNA comprises an open reading frame encoding an RNA-guided DNA binder, and the uridine dinucleotide content of the open reading frame ranges from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

65. The RNA composition according to claim 61 or 62, wherein the gRNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

66. The RNA composition according to claim 61, wherein the gRNA is a modified gRNA, and the modified gRNA comprises modifications selected from the group consisting of: 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, and 2'-fluoro (2'-F) modified nucleotides.

67. The RNA composition according to claim 61, wherein the gRNA is a modified gRNA, and the modified gRNA comprises a modification at one or more nucleotides among the first five nucleotides at the 5' end or the 3' end.

68. The RNA composition according to claim 61, wherein the gRNA is a modified gRNA, and the modified gRNA comprises a PS bond between the first four nucleotides or the last four nucleotides.

69. The RNA composition according to any one of claims 66 to 68, wherein the modified gRNA further comprises a 2'-O-Me modified nucleotide at the first three nucleotides at the 5' end or the 3' end.

70. The RNA composition according to claim 62, wherein the gRNA and the type II Cas nuclease mRNA are present in a ratio in the range of about 10:1 to about 1:10 by weight.

71. The RNA composition according to claim 62, wherein the gRNA and the type II Cas nuclease mRNA are present in a ratio in the range of about 5:1 to about 1:5 by weight.

72. The RNA composition according to claim 62, wherein the gRNA and the type II Cas nuclease mRNA are present in a ratio in the range of about 2:1 to about 1:2 by weight.

73. The RNA composition according to claim 62, wherein the gRNA and the type II Cas nuclease mRNA are present in a ratio of about 2:1 or about 1:1 by weight.

74. The RNA composition according to claim 55, which further comprises at least one template nucleic acid.

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