Nucleic Acid Compositions Comprising Amphiphilic Oligoethylene Glycol (OEG) Conjugate Compounds and Methods of Using Such Compounds and Compositions

Abstract

The present invention relates generally to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligoethylene glycol (OEG) conjugate compounds (as an alternative to PEG lipids), and in particular to the use of such compositions for delivering nucleic acids to cells of a subject or in therapy, such amphiphilic OEG conjugate compounds, and conjugates of such amphiphilic OEG conjugate compounds.

Description

[Technical Field]

[0001] Technical Field The present invention relates generally to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligoethylene glycol (OEG) conjugate compounds (as an alternative to PEG lipids), and in particular to the use of such compositions for delivering nucleic acids to cells of a subject or in therapy, such amphiphilic OEG conjugate compounds, and conjugates of such amphiphilic OEG conjugate compounds. [Background technology]

[0002] background The use of recombinant nucleic acids (e.g., DNA or RNA) to deliver foreign genetic information to target cells is well known. Recombinant nucleic acids can be administered to a subject in need thereof in naked form; however, recombinant nucleic acids are usually administered using compositions. For example, nucleic acids such as RNA can be delivered to a subject using a variety of delivery vehicles, most of which are based on cationic polymers or lipids that form nanoparticles with the nucleic acid. Nanoparticles are intended to protect nucleic acids such as RNA from degradation, enable delivery of nucleic acids such as RNA to target sites, and promote cellular uptake and processing by target cells. The efficiency of nucleic acid delivery can depend, in part, on the molecular composition of the nanoparticles, which can depend on numerous parameters, including particle size, formulation, and charge, or grafting with molecular moieties such as polyethylene glycol (PEG) or other ligands.

[0003] Grafting with PEG is thought to reduce serum interactions, increase serum stability, and extend circulation time, which may be beneficial for certain targeting approaches. Ligands that bind to receptors at target sites may be beneficial for improving targeting efficacy. Furthermore, PEGylation can be used in particle engineering. For example, when lipid nanoparticles (LNPs) are produced by mixing an aqueous phase of nucleic acid, such as RNA, with an organic phase of lipids, a certain fraction of the lipid mixture must be PEG-conjugated (such PEG-conjugated lipids contain at least 30 consecutive ethylene glycol repeat units); otherwise, particles will aggregate during or after the mixing process. It has been shown that particle size can be tuned by varying the molar fraction of PEG-lipids containing various molar masses of PEG. Similarly, particle diameter can be tuned by varying the molar mass of the PEG moiety of PEGylated lipids. Typical accessible sizes range from 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles thus formed have the added advantage of a long circulating half-life and minimal interaction with serum components, which is desirable for many drug delivery approaches, due to the PEG fraction. Without PEG-lipids, particles of distinct sizes cannot be formed; they form large aggregates and precipitate. Therefore, one of the key roles of PEG-lipids is to promote particle self-assembly by providing steric hindrance to the surface of nascent particles formed when nucleic acids, such as RNA, are rapidly mixed with a lipid-containing ethanol solution and bind to them. The PEG steric hindrance prevents interparticle fusion and promotes the formation of a homogeneous population of LNPs, which can achieve diameters of <100 nm.

[0004] Despite these advantages, PEGylation of nanoparticles can also have several adverse effects that are detrimental to the intended drug delivery application. PEGylation of liposomes and LNPs is known to promote cellular uptake and endosomal escape, ultimately reducing overall transfection efficiency. Indeed, the PEG shell provides steric hindrance to efficient particle binding to cells and prevents endosomal release by blocking membrane fusion between liposomes and endosomal membranes. This is why the type and amount of PEG-lipid used must always be carefully adjusted. On the one hand, it must provide sufficient stealth effect for in vivo and stabilization aspects, while on the other hand, it must not interfere with transfection. This phenomenon is known as the "PEG dilemma."

[0005] In addition to reduced transfection efficiency, PEGylation is also associated with the accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and / or complement activation and storage diseases (Bendele A et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; SM Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes in rats can significantly alter the pharmacokinetic behavior of a second dose when this second dose is administered several days later (Laverman P et al., 2001, J. Pharmacol. Exp. Ther. 298(2), 607-12; Ishida et al., 2006, J. Control Release 115(3), 251-8). The phenomenon of "accelerated blood clearance" (ABC) is thought to be proportional to the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma induces rapid clearance of particles by the monophagocyte system (MPS), ultimately reducing the efficacy of the drug.

[0006] Because PEG is widely used as an ingredient in foods, cosmetics, hygiene products, and medicines, a certain percentage of the general population has "pre-existing" anti-PEG antibodies, which may be associated with reduced efficacy of PEGylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.

[0007] Because PEG can induce immune responses, multiple injections are required, which must be avoided in some applications.An example is nucleic acid (e.g., RNA, especially mRNA) for protein replacement therapy.Here, the risk is particularly high due to the potential for intrinsic immunogenicity of nucleic acid (e.g., RNA).Another example is protein knockdown therapy using inhibitory RNA (e.g., siRNA), antisense oligonucleotides, or DNA-based therapy. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need in the art for efficient compositions and methods for introducing nucleic acids, such as RNA, into cells that avoid the drawbacks associated with the use of PEG. The present invention addresses this and other needs.

[0009] The present inventors have surprisingly discovered that the compositions, methods, polymer conjugate compounds (also referred to as amphiphilic OEG conjugate compounds of the present invention) and conjugates described herein fulfill the above-mentioned needs. In particular, the polymer conjugate compounds described herein have been shown to be not bound by antibodies raised against the PEG structure and to be stable under physiological conditions. The polymer conjugate compounds and the polymer components of the polymer conjugate compounds and their conjugates can be synthesized by well-known procedures, such as solid-phase peptide synthesis (SPPS). The polymer conjugate compounds and conjugates can be end-group functionalized with various moieties for charge modulation or introduction of specific molecular moieties, such as ligands. [Means for solving the problem]

[0010] overview The invention is defined by the appended claims.

[0011] In a first aspect, the present invention provides a composition comprising: (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) (a) a polymer comprising the following general formula (I): and (b) a polymer conjugate compound comprising one or more hydrophobic chains: [ka] [During the ceremony, X 2 and X 1 together are an optionally substituted amide, an optionally substituted thioamide, an ester or a thioester, preferably an optionally substituted amide, an optionally substituted thioamide or an ester; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and and n is 1 to 100.] This composition is also referred to as the nucleic acid composition of the present invention.

[0012] As shown in the present application, anti-PEG antibodies (polyclonal and IgG and IgM anti-PEG antibodies) raised against and binding to PEG (i.e., having at least 30 consecutive ethylene glycol repeating units) do not bind to polymers comprising the structure of formula (I). Furthermore, the present invention demonstrates that polymers comprising the structure of formula (I) are stable under physiological conditions.

[0013] In certain embodiments of this first aspect (particularly with respect to formula (I)), X 2 and X 1 and together are an optionally substituted amide. Thus, in some embodiments, X 1 is -C(O)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X is alkyl. 1 Ha-NR 1 - and X 2 is -C(O)-, where R1 is hydrogen or C 1-8 It is alkyl.

[0014] In certain embodiments of this first aspect (particularly with respect to formula (I)), X 2 and X 1 together are an optionally substituted thioamide. Thus, in some embodiments, X 1 is -C(S)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X is alkyl. 1 Ha-NR 1 - and X 2 is -C(S)-, where R 1 is hydrogen or C 1-8 It is alkyl.

[0015] In certain embodiments of this first aspect (particularly with respect to formula (I)), X 2 and X 1 together form an ester. Thus, in some embodiments, X 1 is -C(O)- and X 2 is —O. In some embodiments, X 1 If is -O-, then X 2 is -C(O).

[0016] In certain embodiments of this first aspect (particularly with respect to formula (I)), X 2 and X 1 together form a thioester. Thus, in some embodiments, X 1 is -C(S)- and X 2 is —O—. In some embodiments, X 1 If is -O-, then X 2 is -C(S)-. In some embodiments, X 1 If is -C(O)-, then X 2 is -S-. In some embodiments, X 1 If is -S-, then X 2is -C(O)-.

[0017] In certain embodiments of this first aspect (particularly with respect to formula (I)), X 1 is -C(O)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8 alkyl. For example, R 1 can be hydrogen or methyl. In some embodiments, R 1 is hydrogen.

[0018] In certain embodiments of this first aspect (particularly with respect to Formula (I)), Y is -CH2- or -(CH2)2-. In certain embodiments, Y is -CH2-.

[0019] In certain embodiments of the first aspect, the polymer has the following general formula (II): [ka] [In the formula, R 1 is hydrogen or C 1-8 It is alkyl. In certain embodiments of Formula (II), R 1 is hydrogen or methyl. For example, R 1 can be hydrogen. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 can be H or methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is R in other repeating units 1 (e.g., R 1 is a particular alkyl (e.g., H), and for at least one different repeat unit, R 1 is a specific alkyl (e.g., methyl).

[0020] In certain embodiments of this first aspect (particularly with respect to either of Formulas (I) and (II)), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In certain embodiments, z is 2 to 7. In certain embodiments, z is 2 to 5. In certain embodiments, z is 2 or 3. In certain embodiments, z is 2.

[0021] In certain embodiments of the first aspect, the polymer has the following general formula (III): [ka] [In the formula, R 1 is hydrogen or C 1-8 It is alkyl. In certain embodiments of Formula (III), R 1 is hydrogen or methyl. For example, R 1 can be hydrogen. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 can be H or methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is R in other repeating units 1 (e.g., R 1 is a particular alkyl (e.g., H), and for at least one different repeat unit, R 1 is a specific alkyl (e.g., methyl).

[0022] In certain embodiments of the first aspect, the polymer has the following general formula (IV): [ka] Includes.

[0023] In certain embodiments of the first aspect, the polymer has the following general formula (IVa): [ka] Includes.

[0024] In certain embodiments of this first aspect (particularly with respect to any of Formulas (I), (II), (III), (IV), and (IVa)), n is 5 to 50, for example, 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In certain embodiments, n is 5 to 25, for example, 6 to 20 or 6 to 15. In certain embodiments, n is 7 to 16, for example, 7 to 14, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 14. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, n is 12. In certain embodiments, n is 16.

[0025] In certain embodiments of this first aspect (particularly with respect to any of formulas (I), (II), (III), (IV) and (IVa)), one or more hydrophobic chains are present in the X 1 Terminal or X 2 Located at the end.

[0026] In some embodiments of this first aspect (particularly with respect to any of Formulas (I), (II), (III), (IV), and (IVa)), one or more hydrophobic chains are independently selected from acyclic, preferably linear, hydrocarbyl groups, such as the hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the hydrocarbyl group has at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl group may be saturated or unsaturated. If the polymer conjugate compound contains two or more hydrophobic chains, these chains may be the same or different. For example, if the polymer conjugate compound contains two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In some other embodiments, the two hydrophobic chains are different, for example, one may be saturated and the other (mono)unsaturated.

[0027] In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (V) or (V'): [ka] [During the ceremony, X 2 and X 1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester; Y is -CH2-, -(CH2)2-, or -(CH2)3-; R 2 is a moiety comprising one or more hydrophobic chains; R 3 is H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 , -SR 20 , halogen, -CN, -N3, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C 1-6 The alkyl group may optionally be -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 20 is H, C 1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-3 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23, substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl), substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; z is 2 to 24; and n is 1 to 100. Includes.

[0028] In certain embodiments of Formula (V) or (V'), X 2 and X 1 and together are an optionally substituted amide. Thus, in some embodiments, X 1 is -C(O)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8In some embodiments, X is alkyl. 1 Ha-NR 1 - and X 2 is -C(O)-, where R 1 is hydrogen or C 1-8 It is alkyl.

[0029] In certain embodiments of Formula (V) or (V'), X 2 and X 1 together are an optionally substituted thioamide. Thus, in some embodiments, X 1 is -C(S)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X is alkyl. 1 Ha-NR 1 - and X 2 is -C(S)-, where R 1 is hydrogen or C 1-8 It is alkyl.

[0030] In certain embodiments of formula (V) or (V'), X 2 and X 1 together form an ester. Thus, in some embodiments, X 1 is -C(O)- and X 2 is —O. In some embodiments, X 1 If is -O-, then X 2 is -C(O).

[0031] In certain embodiments of Formula (V) or (V'), X 2 and X 1 together form a thioester. Thus, in some embodiments, X 1 is -C(S)- and X 2 is —O—. In some embodiments, X 1 If is -O-, then X 2 is —C(S). In some embodiments, X 1 If is -C(O)-, then X 2is -S-. In some embodiments, X 1 If is -S-, then X 2 is -C(O)-.

[0032] In certain embodiments of Formula (V) or (V'), X 1 is -C(O)- and X 2 Ha-NR 1 -, where R 1 is hydrogen or C 1-8 alkyl. For example, R 1 can be hydrogen or methyl. In some embodiments, R 1 is hydrogen.

[0033] In some embodiments of formula (V) or (V'), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0034] In some embodiments of Formula (V) or (V'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0035] In certain embodiments of Formula (V) or (V'), R 1 is hydrogen or methyl. For example, R 1 can be hydrogen. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 can be H or methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is R in other repeating units 1 (e.g., R 1 is a particular alkyl (e.g., H), and for at least one different repeat unit, R 1 is a specific alkyl (e.g., methyl).

[0036] In some embodiments of Formula (V) or (V'), n is 5 to 50, for example, 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, for example, 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, for example, 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

[0037] In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VI) or (VI'): [ka] [In the formula, z, n, R 2 and R 3 is as defined for formulas (V) and (V'); and R 1 is hydrogen or C 1-8 It is alkyl. Includes.

[0038] In certain embodiments of Formula (VI) or (VI'), R 1 is hydrogen or methyl. For example, R 1 can be hydrogen. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 can be H or methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is R in other repeating units 1 (e.g., R 1 is a particular alkyl (e.g., H), and for at least one different repeat unit, R 1 is a specific alkyl (e.g., methyl).

[0039] In some embodiments of Formula (VI) or (VI'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0040] In some embodiments of Formula (VI) or (VI'), n is 5 to 50, for example, 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, for example, 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, for example, 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

[0041] In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VII) or (VII'): [ka] [In the formula, n, R 2 and R 3 is as defined for formulas (V) and (V'); and R 1 is hydrogen or C 1-8 It is alkyl. Includes.

[0042] In certain embodiments of Formula (VII) or (VII'), R 1 is hydrogen or methyl. For example, R 1 can be hydrogen. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 can be H or methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is R in other repeating units 1(e.g., R 1 is a particular alkyl (e.g., H), and for at least one different repeat unit, R 1 is a specific alkyl (e.g., methyl).

[0043] In some embodiments of Formula (VII) or (VII'), n is 5 to 50, for example, 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, for example, 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, for example, 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

[0044] In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VIII), (VIIIa), (VIII'), or (VIIIa'): [ka] [In the formula, n, R 2 and R 3 is as defined for formulas (V) and (V'). Includes.

[0045] In some embodiments of Formula (VIII), (VIIIa), (VIII'), or (VIIIa'), n is 5 to 50, for example, 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, for example, 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, for example, 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

[0046] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 is R 4 or -L 1 (R 4 ) p where each 4 are independently hydrophobic chains such as hydrocarbyl groups; L 1 is a linker; and p is 1 or 2.

[0047] In one embodiment, L 1 contains at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety, and / or the alkylene group is R 4and wherein preferably each monovalent functional moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocalcium, thiocarbamate ... Bonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemi and / or each divalent functionalized moiety is independently selected from cetal, ketal, hemiketal, imide, and amide moieties; and / or each divalent functionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate , thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

[0048] In one embodiment, L 1 is [*-C(O)O] p (C 1-6-alkylene)-, [*-OC(O)] p (C 1-6 -alkylene)-, [*-NHC(O)] p (C 1-6 -alkylene)-, [*-C(O)NH] p (C 1-6 -Alkylene)-, [*-S] p (C 1-6 -Alkylene)-, [*-SS] p (C 1-6 -alkylene)-, [*-S(O)2] p (C 1-6 -alkylene)-, [(*-O) r C(OR 25 ) 3-r ]-(C 1-6 -alkylene)-, [*-C(OR 25 )2O] p (C 1-6 -alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-6 -alkylene)-, [*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-6 -alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-6 -alkylene)-, [*-N(R 26 )N(R 26 )] p (C 1-6 -alkylene)-, [*=C(=N(OH))] p (C 1-6 -alkylene)-, [*-OC(R 25 )(R 26 )O] p (C 1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) p N(R 26 ) 2-p and [*-C(O)NH](C 1-6-alkyltriyl)-, where * is R 4 represents the point of attachment to C; p is 1 or 2; C 1-6 - alkylene is divalent (when p is 1) or trivalent (when p is 2); R 25 is C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); R 26 is H, C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 substituted with one or more substituents selected from the group consisting of alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is further substituted with other hydrophobic chains R 4 directly binds to

[0049] In some embodiments, L 1 further comprises at least one further bifunctionalized moiety through which R 2 is X in formula (V) 1 (or a carbonyl group of any of formulae (VI), (VII), (VIII) and (VIIIa)) or X of formula (V') 2(or to the N atom of any of formulas (VI'), (VII'), (VIII'), and (VIIIa'). In certain embodiments, the at least one additional bifunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidinium, thiomethyl ... dino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably selected from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein L 1 further comprises at least two additional bifunctionalized moieties, these at least two additional bifunctionalized moieties are optionally C 1-6 - separated by alkylene groups.

[0050] In one embodiment, L 1 is [*-C(O)O] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-C(O)O] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)-NR 26 -, [*-C(O)O] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6-alkylene)C(O)-, [*-OC(O)] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-OC(O)] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -Alkylene)NR 26 -, [*-OC(O)] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)C(O)-, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -Alkylene)NR 26 -, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)C(O)-, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-NR 26 -, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )-O(C 1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] p (C 1-6 -alkylene)O-, [*-OC(O)] p (C1-6 -Alkylene)O-, (*-) p N(R 26 ) 2-p and [*-C(O)NH](C 1-6 -alkyltriyl)O—, where * is R 4 represents the point of attachment to [*-C(O)O]; p is 1 or 2; p (C 1-6 -alkylene), [*-OC(O)] p (C 1-6 -alkylene), [*-NHC(O)] p (C 1-6 -alkylene) and [*-C(O)NH] p (C 1-6 -alkylene)C 1-6 - alkylene is divalent (when p is 1) or trivalent (when p is 2); R 26 is H, C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); R 27 is H, C 1-6 Alkyl, aryl, aryl (C 1-6 alkyl) and a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; an ammonium (NH + or an organic cation, such as a quaternary ammonium or amine cation; 3,4-dihydro-2H-chromen-6-yl optionally contains halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 substituted with one or more substituents selected from the group consisting of alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is further substituted with other hydrophobic chains R 4 directly binds to

[0051] In one embodiment, L 1 is [*-C(O)O] p(C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-C(O)O] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)NH-, [*-C(O)O] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)C(O)-, [*-OC(O)] p (C 1-6 -alkylene)-OP(O)(OR 27 )-O(C 1-6 -alkylene)-, [*-OC(O)] p (C 1-6 -alkylene)-OP(O)(OR 27 )O(C 1-6 -alkylene)NH-, [*-OC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)C(O)-, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)NH-, [*-NHC(O)] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)C(O)-, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6 -alkylene)-, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )O(C 1-6-alkylene)NH-, [*-C(O)NH] p (C 1-6 -alkylene)OP(O)(OR 27 )-O(C 1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] p (C 1-6 -alkylene)O-, [*-OC(O)] p (C 1-6 -alkylene)O-, (*-)2N- and [*-C(O)NH](C 1-6 -alkyltriyl)O- or L 1 is(*-)(R 26 )N-, where * is R 4 represents the point of attachment to [*-C(O)O]; p is 1 or 2; p (C 1-6 -alkylene), [*-OC(O)] p (C 1-6 -alkylene), [*-NHC(O)] p (C 1-6 -alkylene) and [*-C(O)NH] p (C 1-6 -alkylene)C 1-6 - alkylene is divalent (when p is 1) or trivalent (when p is 2); R 26 is H and C 1-6 alkyl; R 27 is selected from the group consisting of H and a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH + or an organic cation, such as a quaternary ammonium or amine cation; 3,4-dihydro-2H-chromen-6-yl optionally contains halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 substituted with one or more substituents selected from the group consisting of alkyl; and C 1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is further substituted with other hydrophobic chains R 4 directly binds to

[0052] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 is [R 4 C(O)O] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)-, [R 4 C(O)O] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)NH-, [R 4 C(O)O] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)C(O)-, [R 4 OC(O)] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)-, [R 4 OC(O)] p (C 2-3 -alkylene)-OP(O)(OR 27 )-O(C 1-3 -alkylene)NH-, [R 4 OC(O)] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)C(O)-, [R 4 NHC(O)] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)-, [R 4 NHC(O)] p (C2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)NH-, [R 4 NHC(O)] p (C 2-3 -alkylene)OP(O)(OR 27 )O(C 1-3 -alkylene)C(O)-, [R 4 C(O)NH] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)-, [R 4 C(O)NH] p (C 2-3 -alkylene)-OP(O)(OR 27 )O(C 1-3 -alkylene)NH-, [R 4 C(O)NH] p (C 2-3 -alkylene)OP(O)(OR 27 )-O(C 1-3 -alkylene)C(O)-, (2-R 4 -3,4-dihydro-2H-chromen-6-yl)O-, [R 4 C(O)O] p (C 2-3 -alkylene)O-, [*-OC(O)] p (C 2-3 -alkylene)O-, (R 4 )2N- and [R 4 C(O)NH](C 2-3 -alkyltriyl)O— or R 2 (R 4 )(R 26 )N-, where p is 1 or 2; C 2-3 - alkylene is divalent (when p is 1) or trivalent (when p is 2); R 26 is H and C 1-6 alkyl; R 27is selected from the group consisting of H and a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH + or an organic cation, such as a quaternary ammonium or amine cation; 3,4-dihydro-2H-chromen-6-yl optionally contains halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 substituted with one or more substituents selected from the group consisting of alkyl; and C 2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is further substituted with other hydrophobic chains R 4 directly binds to

[0053] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety; or R 2 is a monoalkylamine moiety.

[0054] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), each of the one or more hydrophobic chains (i.e., R 4Each of the groups (a) and (b) is independently an acyclic, preferably linear, hydrocarbyl group, e.g., a hydrophobic (e.g., lipophilic) chain of a natural lipid. In certain embodiments, one or more hydrocarbyl groups independently have at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. One or more hydrocarbyl groups may be saturated or unsaturated. If the polymer conjugate compound contains two or more hydrophobic chains, these chains may be the same or different. For example, if the polymer conjugate compound contains two hydrophobic chains, in certain embodiments, the two hydrophobic chains are the same. In certain other embodiments, the two hydrophobic chains are different, e.g., one may be saturated and the other (mono)unsaturated, and / or the two hydrophobic chains have different lengths. Examples of the one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, e.g., hydrocarbyl chains of naturally occurring fatty acids, having at least 8 carbon atoms. In specific examples, the one or more hydrophobic chains include hydrocarbyl chains of caprylic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol, stearic alcohol, arachidyl alcohol, behenic alcohol, lignoceryl alcohol, cerotinoyl alcohol, oleyl alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

[0055] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties or R 2 is the monomyristylamine moiety.

[0056] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 is H, C 1-6 Alkyl, C 2-6 Alkynyl, -C(O)R 21 , -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C 1-6 The alkyl group may optionally be -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl), sugars, amino acids, peptides, and members of a targeting pair.

[0057] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 is H, C 1-3 Alkyl, C 2-6 Alkynyl, -C(O)R 21 , -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and one or more substituents independently selected from the group consisting of members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 and one or more substituents independently selected from the group consisting of alkyl)2 and members of a targeting pair.

[0058] In certain embodiments of this first aspect (particularly with respect to any of formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 is H, -C(O)(C 1-3 alkyl), -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2 and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, -SH, halogen, -CN, -N, C 2-6Substituted with one or more substituents independently selected from the group consisting of alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2 and members of a targeting pair.

[0059] In certain embodiments of this first aspect (particularly with respect to any of Formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), the targeting pair is selected from the following pairs: maleimide-thiol; thiol-alkyl halide (particularly brominated); azide-alkyne (particularly in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (particularly in Diels-Alder reactions); antigen-antibody specific for the antigen (including fragments or derivatives thereof); biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α v β3 integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

[0060] In certain embodiments of the first aspect, the polymer conjugate compound has the formula: [ka] [ka] [ka] [wherein n is 5 to 25; R 3 is H, -C(O)(C 1-3 alkyl) and a member of a targeting pair, wherein C 1-3The alkyl group may optionally be -OH, -SH, halogen, -CN, -N, C 2-6 substituted with one or more substituents independently selected from the group consisting of alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2 and members of a targeting pair; R 27 is H or a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH + or an organic cation, such as a quaternary ammonium or amine cation; and in each case -C(O)C 17 H 35 is the moiety -C(O)(CH2) 16 CH3 (stearoyl), and in either case -C(O)C 15 H 31 is the moiety -C(O)(CH2) 14 CH3 (palmitoyl), and in either case -C(O)C 13 H 27 is the moiety -C(O)(CH2) 12 CH3 (myristoyl), and in either case -C 14 H 29 is the moiety -(CH2) 13 CH3 (myristyl), in either case -C 13 H 27 is the moiety -(CH2) 12 CH3 and in both cases -C(O)C 17 H 33 refers to the moiety -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). In some embodiments, n is 7 to 16, e.g., 7 to 14 (preferably 8, 10, 12, 14, or 16); and / or R 3 is H or -C(O)(C 1-3 alkyl), where C 1-3The alkyl group optionally is 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), -SH, -Br, -N, C 2-6 It is substituted with one substituent selected from the group consisting of alkynyl, antigen and antibody.

[0061] Further preferred embodiments of the polymer conjugate compound (particularly with respect to any of Formulas (I), (II), (III), (IV), (IVa), (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII'), and (VIIIa')) are set forth under the heading "A polymer conjugate compound comprising: (a) a polymer comprising a structure of Formula (I); and (b) one or more hydrophobic chains." In certain preferred embodiments of the first aspect, the polymer conjugate compound has any of Formulas (V-1), (V-5), (V-17), and (V-25).

[0062] In certain embodiments of the first aspect, the composition is substantially free of lipids or lipid-like substances comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeat units.

[0063] In certain embodiments of the first aspect, the composition is also substantially free of other polymer-conjugated lipids, in certain embodiments, the other polymer-conjugated lipids are polysarcosine-conjugated lipids and / or conjugates comprising a hydrophobic chain and a polyoxazoline (POX) and / or polyoxazine (POZ) polymer.

[0064] In certain embodiments of the first aspect, water is the major component of the composition and / or the total amount of solvents other than water in the composition is less than about 1.0% (v / v), e.g., less than about 0.5% (v / v). For example, the amount of water in the composition can be at least 50% (w / w), e.g., at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). In particular, when the composition includes a cryoprotectant, the amount of water contained in the composition can be at least 50% (w / w), e.g., at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition can be at least 95% (w / w). Additionally or alternatively, the total amount of non-water solvents in the composition may be less than about 0.5% (v / v), e.g., less than about 0.4% (v / v), less than about 0.3% (v / v), less than about 0.2% (v / v), less than about 0.1% (v / v), less than about 0.05% (v / v), less than about 0.01% (v / v), or less than about 0.005% (v / v). In this regard, a cryoprotectant that is liquid under normal conditions is considered a cryoprotectant, not a non-water solvent. In other words, the optional limitation above that the total amount of non-water solvents in the composition may be less than about 0.5% (v / v), e.g., less than about 0.4% (v / v), does not apply to a cryoprotectant that is liquid under normal conditions.

[0065] In one embodiment of the first aspect, the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 500 mg / L. In one embodiment, the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 100 mg / L. In one embodiment, the concentration of nucleic acid (particularly RNA) in the composition is about 5 mg / L to about 500 mg / L, for example, about 10 mg / L to about 400 mg / L, about 10 mg / L to about 300 mg / L, about 10 mg / L to about 200 mg / L, about 10 mg / L to about 150 mg / L, or about 10 mg / L to about 100 mg / L, preferably about 10 mg / L to about 140 mg / L, more preferably about 20 mg / L to about 130 mg / L, and more preferably about 30 mg / L to about 120 mg / L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is about 5 mg / L to about 150 mg / L, e.g., about 10 mg / L to about 140 mg / L, about 20 mg / L to about 130 mg / L, about 25 mg / L to about 125 mg / L, about 30 mg / L to about 120 mg / L, about 35 mg / L to about 115 mg / L, about 40 mg / L to about 110 mg / L, about 45 mg / L to about 105 mg / L, or about 50 mg / L to about 100 mg / L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is 1 mg / L to about 50 mg / L or about 10 mg / L to about 100 mg / L.

[0066] In certain embodiments of the first aspect, the composition comprises a cryoprotectant. In certain embodiments of the first aspect, the composition is substantially free of cryoprotectants.

[0067] In certain embodiments of the first aspect, the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

[0068] In certain embodiments of the first aspect, the cationically ionizable lipid has the formula (X): [ka] [In the formula, L 10 , L 20 , G 1 , G 2 , G 3 , R35 , R 36 and R 37 is as defined herein.] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. In some embodiments, the cationically ionizable lipid is selected from the following: Structures X-1 through X-36 (shown herein); or Structures A through G (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having Structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., a lipid having Structure G).

[0069] In certain embodiments of the first aspect, the cationically ionizable lipid has formula (XI): [ka] wherein R1, R2R3, R4, L2, G2, and m are as defined herein. In some embodiments, the cationically ionizable lipid is selected from structures (XIV-1), (XIV-2), and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-1. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-2. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-3.

[0070] In certain embodiments of the first aspect, the cationic or cationically ionizable lipid is 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ... )-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or mixtures thereof.

[0071] In certain embodiments of the first aspect, the cationically ionizable lipid is replaced in whole or in part with a cationic lipid, hi certain embodiments, the cationic lipid is selected from structures XV-1 through XV-6 (shown herein).

[0072] In certain embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol.

[0073] In certain embodiments of the first aspect, the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids and combinations thereof, more preferably a combination of a phospholipid and a steroid.

[0074] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE).

[0075] In some embodiments, it constitutes about 5 mol % to about 30 mol % of the total lipids present in the phospholipid composition.

[0076] In some embodiments, the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol. contains or is cholesterol.

[0077] In some embodiments, the steroid comprises about 10 mol % to about 60 mol % of the total lipids present in the composition.

[0078] In certain embodiments of the first aspect, the cationic or cationically ionizable lipid comprises about 20 mol% to about 70 mol% of the total lipid present in the composition; the polymer conjugate compound (amphipathic OEG conjugate compound) comprises about 0.5 mol% to about 15 mol% (e.g., about 2 mol% to about 6 mol% or about 2 mol% to about 5 mol%) of the total lipid present in the composition; the phospholipid comprises about 5 mol% to about 25 mol% of the total lipid present in the composition; and the steroid comprises about 20 mol% to about 55 mol% of the total lipid present in the composition.

[0079] In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids can include cationic lipids. In these embodiments, when cationic lipids are present, the sum of the amount of (1) the cationically ionizable lipid and the amount of (2) the cationic lipid is used in the calculation. For example, if the amount of cationically ionizable lipid in the composition should be about 20 mol% to about 70 mol%, and the composition also contains cationic lipids, the sum of the amount of (1) the cationically ionizable lipid and the amount of (2) the cationic lipid is about 20 mol% to about 70 mol%.

[0080] In certain embodiments of the first aspect, the lipids included in the composition are only polymer conjugate compounds (i.e., amphiphilic OEG conjugate compounds) comprising a cationic or cationically ionizable lipid, a steroid, a neutral lipid, and a polymer of formula (I) as defined herein, particularly polymer conjugate compounds comprising a cationic or cationically ionizable lipid, a steroid, a phospholipid, and a polymer of formula (I) as defined herein.

[0081] In certain embodiments of the first aspect, the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationic or cationically ionizable lipid, and at least a portion of a polymer conjugate compound comprising a polymer of Formula (I) as defined herein. In certain embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In certain embodiments, the particles comprise or are LNPs. In certain embodiments, the particles comprise or are liposomes. In certain embodiments, the particles comprise or are LPXs. In certain embodiments, the particles comprise or are a mixture of LNPs and liposomes. In certain embodiments, the particles comprise or are a mixture of LNPs and LPXs. In certain embodiments, the particles comprise or are a mixture of liposomes and LPXs. In certain embodiments, the particles comprise or are a mixture of LNPs, liposomes, and LPXs.

[0082] In certain embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles comprise all of the lipids present in the composition (particularly the cationic or cationically ionizable lipids, if present, and all of the polymer conjugate compounds comprising one or more additional lipids and a polymer of Formula (I) as defined herein).

[0083] In some embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles comprise at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acid (especially RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85%, of the nucleic acid (especially RNA) present in the composition.

[0084] In certain embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of nucleic acids.

[0085] In certain embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the nucleic acid (eg, RNA) is encapsulated within or associated with the particles.

[0086] In some embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles have a size of about 30 nm to about 500 nm, hi some embodiments, the particles have a size of about 50 nm to about 150 nm.

[0087] In certain embodiments of the second aspect, the nucleic acid is DNA.

[0088] In certain embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA (eg, siRNA).

[0089] In certain embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and (i) contains modified nucleosides in place of uridine; (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with an increased G / C content compared to the wild-type coding sequence. In certain embodiments, the modified nucleosides are selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0090] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and comprises at least one or more of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In some embodiments, the RNA (e.g., mRNA) comprises all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In some embodiments, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably a punctuated sequence of A nucleotides. In some embodiments, the 5' cap is a Cap 1 or Cap 2 structure.

[0091] In certain embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and encodes one or more polypeptides. In certain embodiments, the one or more polypeptides are pharmaceutically active polypeptides and / or comprise an epitope for inducing an immune response against an antigen in a subject.

[0092] In certain embodiments of the first aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a protein, immunogenic variant of the protein, or immunogenic fragment of the protein or immunogenic variant thereof of a pathogen. In certain embodiments, the pathogen is a pathogen that causes an infectious disease.

[0093] In some embodiments of the first aspect, nucleic acid is inhibitory RNA (for example, siRNA), and selectively hybridizes / or is specific to target mRNA.In some embodiments, target mRNA comprises the ORF that encodes pharmaceutically active peptide or polypeptide, particularly the pharmaceutically active peptide or polypeptide whose expression (particularly, for example, compared with the expression in healthy subjects, increased expression) is associated with disease.In some embodiments, target mRNA comprises the ORF that encodes pharmaceutically active peptide or polypeptide whose expression (particularly, for example, compared with the expression in healthy subjects, increased expression) is associated with cancer.

[0094] In a second aspect, the present invention relates to a method of delivering a nucleic acid to a cell in a subject, comprising administering to the subject a nucleic acid composition of the first aspect, it being understood that any embodiment described in the context of the first aspect also applies to any embodiment of the second aspect.

[0095] In a third aspect, the present invention relates to a method of delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It will be understood that any embodiment described in the context of the first or second aspects also applies to any embodiment of the third aspect.

[0096] In a fourth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to the nucleic acid composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. It will be understood that any embodiment described in the context of the first, second or third aspects also applies to any embodiment of the fourth aspect.

[0097] In a fifth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to the nucleic acid composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. It will be understood that any embodiment described in the context of the first, second, third or fourth aspect also applies to any embodiment of the fifth aspect.

[0098] In certain embodiments of the second through fifth aspects, the subject is a mammal, such as a human.

[0099] In a sixth aspect, the present invention provides a polymer conjugate compound (also referred to herein as an amphiphilic OEG conjugate compound) comprising: (a) a polymer comprising a structure of Formula (I); and (b) one or more hydrophobic chains. Preferred embodiments of the polymer conjugate compound of the sixth aspect are identified in the first aspect and are set forth herein under the heading "Polymer conjugate compound comprising: (a) a polymer comprising a structure of Formula (I); and (b) one or more hydrophobic chains."

[0100] In a seventh aspect, the present invention provides a conjugate of (a) the polymer conjugate compound of the sixth aspect, which comprises a member of a targeting pair; and (b) a compound comprising another member of the targeting pair. In some embodiments, the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (e.g., an antigen or epitope), or an antibody. In some embodiments of the seventh aspect, the conjugate has the following formula: [ka] [wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in any case, —C(O)C 17 H 35 is the moiety -C(O)(CH2) 16 CH3 (stearoyl); m1 and m2 are each independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for the antigen. or a salt thereof. In certain embodiments of the seventh aspect, the conjugate has one of the following formulas: [ka] [wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in any case, —C(O)C 17 H 35 is the moiety -C(O)(CH2) 16 CH3 (stearoyl); and Pept is an antigen or an antibody specific for the antigen. or a salt thereof. In certain embodiments of the seventh aspect, the polymer conjugate compound comprising a member of a targeting pair has the following formula: [ka] [wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in any case, —C(O)C 17 H 35 is the moiety -C(O)(CH2) 16 CH3 (stearoyl); and Pept is an antigen or an antibody specific for the antigen. the compound comprising the other member of the targeting pair is (i) an antibody specific for the antigen if Pept is the antigen; or (ii) a compound comprising the antigen if Pept is an antibody specific for the antigen; and the polymer conjugate compound comprising a member of the targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) the antibody specific for the antigen and (2) the antigen.

[0101] It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth or sixth aspect also applies to any embodiment of the seventh aspect.

[0102] In an eighth aspect, the present invention provides a composition comprising: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the seventh aspect.

[0103] It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth or seventh aspect also applies to any embodiment of the eighth aspect.

[0104] In a ninth aspect, the present invention relates to a method of delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject a composition of the eighth aspect, It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect also applies to any embodiment of the ninth aspect.

[0105] In a tenth aspect, the present invention relates to a method of delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspects also applies to any embodiment of the tenth aspect.

[0106] In an eleventh aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein delivery of a nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to a composition of the eighth aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of a nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspects also applies to any embodiment of the eleventh aspect.

[0107] In a twelfth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to a composition of the eighth aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh aspects also applies to any embodiment of the twelfth aspect.

[0108] In a thirteenth aspect, the present invention provides a method of transfecting cells, comprising adding a composition of the first or eighth aspect to cells and incubating the mixture of the composition and the cells for a sufficient period of time. In some embodiments, particularly where the nucleic acid is DNA or RNA (e.g., mRNA) and encodes a pharmaceutically active protein, the mixture of the composition and the cells is incubated for a sufficient period of time to allow expression of the pharmaceutically active protein. In some embodiments, particularly where the nucleic acid is an inhibitory RNA (e.g., siRNA) against a target mRNA, the mixture of the composition and the cells is incubated for a sufficient period of time to allow inhibition of transcription and / or translation of the target mRNA. In some embodiments, the sufficient period of time is at least 1 hour (e.g., at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or up to about 48 hours (e.g., up to about 36 hours or up to about 24 hours).

[0109] In certain embodiments of the thirteenth aspect, the method is performed in vivo (i.e., the cells form part of an organ, tissue, and / or organism of a subject). In certain embodiments of the thirteenth aspect, the method is performed in vitro (i.e., the cells do not form part of an organ, tissue, and / or organism of a subject, e.g., the cells are in ex vivo cell culture).

[0110] It will be understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspects also applies to any embodiment of the thirteenth aspect.

[0111] In a fourteenth aspect, the present invention provides a pharmaceutical composition comprising the conjugate of the seventh aspect or the composition of the first or eighth aspect. In certain embodiments, the pharmaceutical composition further comprises one or more of a pharmaceutically acceptable carrier, diluent, and excipient. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspects also applies to any embodiment of the fourteenth aspect.

[0112] In a further aspect, the present invention provides a kit comprising a composition of the first or eighth aspect, a polymer conjugate compound (amphiphilic OEG conjugate compound) of the sixth aspect, a conjugate of the seventh aspect, or a pharmaceutical composition described herein (e.g., a pharmaceutical composition of the fourteenth aspect). In some embodiments, the kit is for use in therapy, such as for inducing an immune response. In some embodiments, the kit is for use in inducing an immune response against a pathogen, such as for treating or preventing an infectious disease.

[0113] In a further aspect, the present invention provides a method of making an amphiphilic OEG conjugate compound, comprising the steps of: (a) providing an intermediate compound having formula (VII) or (VII′) as disclosed herein, wherein for formula (VII), R 2 is OH and R 3 is H, acetyl or Fmoc; and for formula (VII'), R 2 is H, acetyl or Fmoc, and R 3is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, particularly a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety, thereby obtaining an amphiphilic OEG conjugate compound. Preferred embodiments of this aspect are shown under the heading "(a) a polymer comprising a structure of formula (I); and (b) a polymer conjugate compound comprising one or more hydrophobic chains." It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspects also applies to any embodiment of this aspect of the method for making an amphiphilic OEG conjugate compound. [Brief explanation of the drawings]

[0114] [Figure 1] Scheme of the anti-PEG ELISA assay used to determine the binding of anti-PEG polyclonal antibodies to various antigens (PEG, AEEA, and pSAR). Biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin-NH2-AEEA14, biotin-capping-pSAR, or biotin-NH2-pSAR) were synthesized and captured on neutravidin-coated plates in wash buffer (1x PBS, 0.1% CHAPS) for 2 h at RT with slow shaking. After washing with wash buffer (4x), the plates were incubated with various amounts of rabbit anti-PEG polyclonal serum (7 ng / ml, 3 ng / ml, 167 ng / ml, or 830 ng / ml) in assay buffer (1x PBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT with slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (1:5000 dilution) and the plate was incubated for 1 h at RT with gentle shaking. After washing with wash buffer (5x), HRP substrate was added and the fluorescent signal was measured.

[0115] [Figure 2]Figure 1 Anti-PEG ELISA assay results for various biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin-NH2-AEEA14, biotin-capping-pSAR, or biotin-NH2-pSAR).

[0116] [Figure 3] Results of further anti-PEG ELISA assays using monoclonal anti-PEG IgG (A) or monoclonal anti-PEG IgM (B) and various antigens (PEG36, capping-AEEA14, NH2-AEEA14). PEG36 had a molecular weight of approximately 1.6 kDa. Capping-AEEA14 was Ac-AEEA14.

[0117] [Figure 4] Stability of pAEEA under physiological conditions. Ac-AEEA14 (A) and NH2-AEEA14 (B) were incubated in mouse or human plasma for 0 to 72 hours.

[0118] [Figure 5] Particle size and PDI of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Also included are particle size and PDI of the BM LNP formulation.

[0119] [Figure 6] Zeta potential of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Zeta potential of the BM formulation is also included.

[0120] [Figure 7] Accessible RNA of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid, as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0121] [Figure 8]Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0122] [Figure 9] Hemolysis analysis after incubation (neutral pH conditions) of human whole blood and DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid.

[0123] [Figure 10] Viability of all tested formulations after transfection with DSPE-AEEA14-AC, including LNPs using HY501 as the cationically ionizable lipid. Viability of BM_2 and other PEG-containing formulations is also included. Plotted are mean values (n=3) ± StDev.

[0124] [Figure 11] Particle size and PDI of VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Also included are particle size and PDI of the BM LNP formulation.

[0125] [Figure 12] Zeta potential of VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Zeta potential of the BM formulation is also included.

[0126] [Figure 13] Accessible RNA of VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid, as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0127] [Figure 14]Quantification of S1 protein expression after transfection with VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. (A) Variation in MFI (mean fluorescence intensity) versus VE-AEEA14-AC composition of the overall cell population. Data were fitted using a second-order polynomial function. (B) Viability of all tested formulations. MFI and viability of BM_2 and other PEG-containing formulations are included. Plotted are mean values (n=3) ± StDev.

[0128] [Figure 15] Yield and Purity of Peptide Intermediates. The peptide intermediates Ac-(AEEA)8-OH (A–D) and Ac-(AEEA)14-OH (E–H) were synthesized, and the yield and purity were determined using UPLC (A, B, E, F) and mass spectrometry (C, D, G, H). Samples for UPLC and mass spectrometry were taken immediately after cleavage from the resin, i.e., before the QC method (crude purity; Figure 15A, C, E, G) or after the QC method (final purity; Figure 15B, D, F, H).

[0129] [Figure 16] Monitoring the synthesis of Ac-(AEEA)8-α-tocopherol. Reaction samples were taken and analyzed using the methods described herein. UPLC chromatograms are shown for samples taken after (A) 5 minutes and (B) the end of the reaction time (4 hours).

[0130] [Figure 17] Purity of amphiphilic compounds Ac-(AEEA)8-α-tocopherol (A, B), Ac-(AEEA)14-α-tocopherol (C, D), Ac-(AEEA)14-DMA (E, F), Ac-(AEEA)8-DMG (G, H), and Ac-(AEEA)14-DSPE (I, J) shown by exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), respectively.

[0131] [Figure 18]Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid). Also included are particle size and PDI of BM LNP formulations.

[0132] [Figure 19] Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid). Zeta potential of BM formulations is also included.

[0133] [Figure 20] Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as the cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0134] [Figure 21] Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0135] [Figure 22] Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid)

[0136] [Figure 23] Quantification of S1 protein expression after transfection with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid). (A) Overall cell population composition vs. MFI (mean fluorescence intensity) variation. Data were fitted using a second-order polynomial function. (B) Viability of all test formulations. MFI and viability of BM_1 and BM_2 and other control formulations are included. Plotted are mean values (n=3) ± StDev.

[0137] [Figure 24] Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

[0138] [Figure 25] Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

[0139] [Figure 26] Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as the cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0140] [Figure 27] In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 27A, B), mouse macrophage cell line (Raw) (Figure 27C, D), and liver cancer cell line (HepG2) (Figure 27E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng / well, 25 ng / well, and 50 ng / well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

[0141] [Figure 28]T cell targeting using LNPs with various stealth lipids and Alf lipids. LNPs were formulated with various lipid compositions (cargo: hy1.1 RNA / Luc RNA / Np proxy Venus 1:1:2 w / w; N / P ratio: 6; lipid mixture: HY501 / cholesterol / DSPC / stealth lipid / Alf lipid). The lipid ratios were selected as [47.5 / 40.5 / 10 / 1.8 / 0.2] for the following combinations of stealth lipids and Alf lipids: C16 PEG2k ceramide / DSPE PEG2k Alfa, DSPE PEG2k / DSPE PEG2k. Alfa, DSPE-AEEA14 / DSPE-AEEA14-Alfa, or VE-AEEA8 / DSPE-AEEA14-Alfa. The lipid ratios were selected as follows: 47.5 / 38.5 / 10 / 3.8 / 0.2 for the following combinations of stealth lipid and Alf lipid: VE-PEG1k / DSPE-PEG2k-Alfa or VE-AEEA8 / DSPE-AEEA14-Alfa. LNPs were post-functionalized with aCD3 VHH × NbAlf ligands (w / w = ligand-to-cargo ratio 0.48; RNA concentration: 0.1 μg / μl). The diameters of all LNPs were 100–170 nm, as measured by DLS, with a PDI of less than 0.4.

[0142] For transfection studies, 10 μl (1000 ng dose) of each formulation was pre-diluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 6Thawed human PBMCs were diluted with 50 μl of 100% coagulation PHS and added to the nanoparticle dilutions. After 30 minutes of incubation (37°C, 5% CO2), 30 μl of each transfection reaction was transferred to a second ultra-low attachment 96-well plate, and 170 μl of X-Vivo 15 medium + 100 U / ml IL2 was added per well. Cell dilutions were cultured for an additional 18 hours (37°C, 5% CO2). Specific transfection (Thy1.1) was analyzed by flow cytometry in the following cell types: CD2-negative cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells. Percentages of transfected cells (CD2-negative cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells) within total transfected PBMCs (transfection, y-axis) are listed per formulation condition tested.

[0143] [Figure 29] Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

[0144] [Figure 30] Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

[0145] [Figure 31] Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as the cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0146] [Figure 32] Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0147] [Figure 33]Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid)

[0148] [Figure 34] In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 34A, B), mouse macrophage cell line (Raw) (Figure 34C, D), and liver cancer cell line (HepG2) (Figure 34E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng / well, 25 ng / well, and 50 ng / well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

[0149] [Figure 35] Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid).

[0150] [Figure 36] Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as the cationically ionizable lipid).

[0151] [Figure 37] Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0152] [Figure 38] Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0153] [Figure 39] Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid)

[0154] [Figure 40] In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds in skeletal muscle cell line (C2C12) (Figure 40A, B), mouse macrophage cell line (Raw) (Figure 40C, D), and liver cancer cell line (HepG2) (Figure 40E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng / well, 25 ng / well, and 50 ng / well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

[0155] [Figure 41] Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

[0156] [Figure 42] Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as the cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0157] [Figure 43] Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0158] [Figure 44]Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid)

[0159] [Figure 45] In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 45A, B), mouse macrophage cell line (Raw) (Figure 45C, D), and liver cancer cell line (HepG2) (Figure 45E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng / well, 25 ng / well, and 50 ng / well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

[0160] [Figure 46] Particle size and PDI of LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

[0161] [Figure 47] Zeta potential of LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

[0162] [Figure 48] Accessible RNA of LNPs containing amphiphilic OEG-conjugated compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0163] [Figure 49]Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0164] [Figure 50] Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP)

[0165] [Figure 51] In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing amphiphilic OEG-conjugated compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) in a skeletal muscle cell line (C2C12) (Figure 51A, B), a mouse macrophage cell line (Raw) (Figure 51C, D), and a liver cancer cell line (HepG2) (Figure 51E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng / well, 25 ng / well, and 50 ng / well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

[0166] [Figure 52] Functionalized LNPs prepared using five different OEG-conjugated compounds as stealth lipid particle sizes and PDIs.

[0167] [Figure 53] Agarose gel electrophoresis of control, untreated functionalized LNPs (top row) and functionalized LNPs treated with release solution (bottom row).

[0168] [Figure 54]Particle size and PDI of functionalized LNPs prepared with each OEG conjugate compound ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA-pAEEA14-Ac, and (E) VitE-pAEEA8-Ac) and subjected to three freeze-thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0169] [Figure 55] In vitro transfection with LNP. A) Percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) within total transfected PBMCs (transfection, y-axis) per test formulation. B) Total cell counts acquired by flow cytometry within 20 seconds. Counts are corrected for counting beads. BD FACS Lyric was used for sample acquisition. FlowJo was used for data analysis.

[0170] [Figure 56] Ligand-mediated transfection of T cells in vivo. B6-hCD3EDG transgenic mice were intramuscularly injected with naked or LNP-formulated luciferase- and Thy1.1-encoding RNA mixtures (1:1 weight-to-weight mixture) (1 μg RNA dose / injected side; 2 μg total RNA dose / mouse). Analysis was performed 18 h after injection. (A) Drainage was analyzed via ex vivo bioluminescence imaging of popliteal, inguinal, axillary, and brachial lymph nodes and the spleen. (B) Cell-type-specific transfection was analyzed by flow cytometry via detection of delivered Thy1.1 RNA expression in immune cell subtypes within popliteal and inguinal lymph nodes and the spleen. (C) T cell activation status was analyzed by the mean fluorescence intensity of CD69 surface expression within the indicated organs (after staining with anti-CD69-APC antibody). LN, lymph node; LNP, lipid nanoparticle; NK, natural killer; PMN, polymorphonuclear cell.

[0171] Sequence description The following table provides a list of certain sequences cited herein. [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0172] Detailed Description Although the present invention is described in further detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0173] Below, the elements of the present invention are described in more detail. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The description is understood to support and encompass embodiments combining any number of the disclosed and / or preferred elements with the explicitly described embodiments. Furthermore, any permutation and combination of all elements described herein is construed to be disclosed by the description of this application, unless the context indicates otherwise.

[0174] Preferably, the terms used herein are those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0175] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, as described in the literature of the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. (See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0176] All methods described herein can be performed in any suitable order unless otherwise indicated herein or contrary to context. Any and all examples or exemplary language (e.g., "etc.") provided herein are intended merely to better illustrate the invention and are not intended to impose limitations on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0177] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring to each separate value falling within the range, and unless otherwise stated herein, each separate value is included herein to the same extent as if it were individually written herein.

[0178] Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific articles, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0179] definition The following provides definitions that apply to all aspects of the present invention. The following terms have the following meanings unless otherwise specified: Any undefined term has its art-recognized meaning.

[0180] Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprise" and variations thereof, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" means the exclusion of any other member, integer, or step in any essential sense. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each instance herein, the term "comprising" can be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each instance herein, the term "consisting essentially of" can be replaced with the term "consisting of."

[0181] As used in the context of describing the invention, singular and similar expressions (particularly in the context of the claims) should be construed to encompass both the singular and the plural unless otherwise stated herein or clearly contradicted by the context.

[0182] As used herein, "and / or" shall be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" shall be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, to the same extent as if each were individually set forth herein.

[0183] In the present invention, the term "about" refers to an accuracy interval that is understood by those skilled in the art to still innovate the technical effect of the characteristic in question. This term typically refers to a deviation of ±5% of the numerical value indicated, for example, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example, ±0.01%. As those skilled in the art will recognize, the specific deviation of the numerical value for a certain technical effect depends on the nature of the technical effect. For example, natural or biological technical effects generally have a larger deviation than artificial or engineered technical effects.

[0184] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar terms includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0185] As used herein, "enhancement" and "increase" refer to the ability to cause an overall increase or enhancement of levels, for example, by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more. In some embodiments, these terms refer to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0186] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0187] As used herein, "physiological conditions" refers to the conditions (especially pH and temperature) in a living subject, particularly a human. Preferably, physiological conditions refer to physiological pH and / or a temperature of about 37°C.

[0188] As used herein, "%(w / v)" (or "% w / v") refers to weight / volume percent, which is a unit of concentration of the amount of solute measured in grams (g) expressed as a percentage of the total volume of the solution in milliliters (ml).

[0189] As used herein, "volume percent" or "%(v / v)" (or "%v / v") refers to volume percent, which is a unit of concentration of a liquid substance measured in milliliters (ml), expressed as a percentage of the total volume of a solution in milliliters (ml).

[0190] As used herein, "weight percent" or "%(w / w)" (or "% w / w") refers to weight percent, which is a unit of concentration of an amount of a substance measured in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

[0191] As used herein, "mol %" is defined as the ratio of the number of moles of a component to the total number of moles of all components multiplied by 100.

[0192] As used herein, "mol % of total lipid" is defined as the ratio of the number of moles of a lipid component to the total number of moles of total lipid multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

[0193] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species and their respective charges in a particular solution. Thus, ionic strength, I, is defined by the formula:

number

[0194] According to the present invention, the term "ionic strength" relates in certain embodiments to the presence of monovalent ions.

[0195] In some embodiments, the concentration or effective concentration (the presence of free ions) of divalent inorganic ions, particularly divalent inorganic cations, due to the presence of chelating agent is low enough to prevent RNA degradation.In some embodiments, the concentration or effective concentration of divalent inorganic ions is below the catalytic level of the hydrolysis of the phosphodiester bond between RNA nucleotides.In some embodiments, the concentration of free divalent inorganic ions is 20 μM or less.In some embodiments, there is no or essentially no free divalent inorganic ions.

[0196] A "monovalent" compound refers to a compound that has only one functional group of interest. For example, a monovalent anion refers to a compound that has only one negatively charged group, preferably under physiological conditions.

[0197] A "divalent" or "dibasic" compound refers to a compound that has two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is Ca. 2+ is.

[0198] A "polyhydric" or "polybasic" compound refers to a compound having three or more functional groups of interest. For example, a polybasic organic acid has three or more acid groups.

[0199] A "monovalent moiety" refers to a monoradical, i.e., a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, and the like.

[0200] A "divalent moiety" or "divalent moiety" refers to a diradical, i.e., a moiety having a valence of two. Exemplary divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, and the like. A further example of a divalent moiety is the group [*-S] when p is 1. p (C 1-6 -alkylene)- in C 1-6 -Alkylene moiety (group *-S(C 1-6 -alkylene)-, for example *-S-(CH2)6- or *-S-CH2- (where * is R 4 represents the point of attachment to

[0201] A "polyvalent moiety" refers to a polyradical, i.e., a moiety having a valence of at least 3. For example, a "trivalent moiety" refers to a triradical, i.e., a moiety having a valence of 3. For example, by further removing an H atom from an alkylene group, the resulting alkylene is trivalent. A further example of a trivalent moiety is the group [*-S] if p is 2. p (C 1-6 -alkylene)- in C 1-6 -Alkylene moiety (group [*-S]2(C 1-6 -alkylene)-, for example *-S-CH(S-*)(CH2)5- or *-S-CH(S-*)(CH2)- (where * is R 4 Another example of a trivalent moiety is the group [*-C(O)NH](C 1-6 -alkyltriyl)- in C 1-6 -alkyltriyl moiety (where the [*-C(O)NH] moiety and the further hydrophobic chain are C 1-6 -alkyltriyl moiety, which in turn is linked to X 1 (for formula (V)) or X 2 (for formula (V')) directly or via at least one further bifunctional moiety). 1An example of [*-C(O)NH](C 1-6 -alkyltriyl) moiety (wherein C 1-6 -Alkyltriyl is another hydrophobic chain R 4 (directly attached to) may include at least the following structure: [ka] [During the ceremony, [ka] One of them is C 1-6 - represents a bond in which alkyltriyl is bonded to [*-C(O)NH]; [ka] The other is C 1-6 represents a bond that connects -alkyltriyl to the remainder of the compound (e.g., a compound of formula (V)) (directly or via a further bifunctionalized moiety). 1-6 When the -alkyltriyl is also substituted with one -OH moiety, at least the following structures are included: [ka]

[0202] As used herein, "molar ratio" refers to the ratio of molar amounts between any two substances. For example, if a first substance is present in a composition in an amount of 1 millimole (mmol) and a second substance is present in a composition in an amount of 2 millimoles (mmol), the molar ratio of the first substance to the second substance is 1:2, or 0.5.

[0203] "Osmolality" refers to the concentration of a particular solute expressed as osmoles per kilogram of solute in a solvent.

[0204] The term "lyophilize" or "lyophilization" refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or less than 1 Pa) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilization" and "freeze-drying" are used interchangeably herein.

[0205] The term "spray drying" refers to the spray drying of a material by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer) where the solvent from the formed droplets evaporates, resulting in a dry powder.

[0206] The term "reconstitution" relates to the addition of a solvent, such as water, to a dry product to return it to a liquid state, such as its original liquid state.

[0207] The term "freezing" refers to the solidification of a liquid, usually by the removal of heat. In some embodiments, freezing is the opposite of thawing.

[0208] The term "thawing" refers to the liquefaction of a solid, usually by the application of heat. In some embodiments, thawing is the reverse of freezing.

[0209] The term "aqueous phase," as used herein with respect to compositions / formulations comprising particles, particularly LNPs, liposomes, and / or lipoplexes, refers to a mobile or liquid phase, i.e., a continuous aqueous phase, that includes all components dissolved therein but (morphologically) excludes particles. Thus, if particles such as LNPs are dispersed in an aqueous phase, and the aqueous phase is substantially free of compound X, the aqueous phase is virtually and practically free of X in a manner that is feasible, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is possible that particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

[0210] The term "recombinant" in the context of the present invention means "produced through genetic engineering." In certain embodiments, a "recombinant entity" in the context of the present invention is not naturally occurring.

[0211] As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), is isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "naturally occurring" means "occurring in nature," and includes known entities as well as entities that have not been discovered and / or isolated in nature, but may be discovered and / or isolated from nature in the future.

[0212] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and include temperatures of at least about 15°C, preferably from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0213] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon monoradical. Preferably, an alkyl group has 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, C 1-12 abbreviated as alkyl (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, C 1-10alkyl), more preferably containing 1 to 8 carbon atoms, for example 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, iso-propyl (also known as 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" refers to an alkylene group in which one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein. Examples of substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0214] The term "alkylene" refers to a diradical of a saturated straight-chain or branched hydrocarbon. Preferably, the alkylene contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Examples of alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-isopentylene, , 1,1-sec-pentyl, 1,1-neo-pentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-hexylene, isomers include 1,1-butylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1-isoheptylene), octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene), etc. A straight-chain alkylene moiety having at least three carbon atoms and a free valence at each end may also be named by the number of methylenes (e.g., 1,4-butylene may also be called tetramethylene).Generally, instead of using the terminal "ylene" for the alkylene moieties specified above, the terminal "diyl" can also be used (e.g., 1,2-butylene can also be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0215] The term "alkenyl" refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2; if the alkenyl group has an odd number of carbon atoms, the result is rounded down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenyl group contains 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group contains 2 to 12 (C 2-12Alkenyl groups (e.g., 2 to 10) contain carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the cis (Z) or trans (E) configuration. Examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, and 5-nonenyl. , 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If the alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom."Substituted alkenyl" means that one or more (e.g., 1 up to the maximum number of hydrogen atoms attached to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkenyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0216] The term "alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. An alkynyl group may optionally have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group is equal to the number of carbon atoms in the alkynyl group divided by two, and if the number of carbon atoms in the alkynyl group is odd, the result is rounded down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2, carbon-carbon triple bonds. "Substituted alkynyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkynyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0217] The term "alkenylene" refers to an unsaturated straight-chain or branched hydrocarbon diradical having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenylene group by 2; if the number of carbon atoms in the alkenylene group is odd, the result of the division is rounded down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group contains 2 to 12 (e.g. 2 to 10) carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, e.g. 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, the alkenylene group contains 2 to 12 (e.g., 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the cis (Z) or trans (E) configuration. Examples of alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. If the alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom."Substituted alkenylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkenylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0218] The term "cycloalkyl" refers to cyclic, non-aromatic versions of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. A cycloalkyl group can consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). "Substituted cycloalkyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the cycloalkyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0219] The term "cycloalkylene" refers to a cyclic, non-aromatic version of "alkylene," and is a geminal, vicinal, or isolated diradical. In certain embodiments, the cycloalkylene is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and / or (ii) 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 3- to 12-, or 3-10-membered). In certain embodiments, the cycloalkylene is a mono-, bi-, or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 3- to 12-, or 3-10-membered) cycloalkylene. Generally, instead of using the terminal "ylene" for the cycloalkylene moieties identified above, the terminal "diyl" may be used (e.g., 1,2-cyclopropylene may also be referred to as cyclopropane-1,2-diyl). Exemplary cycloalkylene groups are cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3.1.1]diyl). 3,7 ]decane-2,2-diyl). "Substituted cycloalkylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0220] The term "cycloalkenylene" refers to a cyclic, non-aromatic version of "alkenylene," and may be a geminal, vicinal, or isolated diradical. Generally, the maximum number of carbon-carbon double bonds in a cycloalkenylene group may be equal to the integer obtained by dividing the number of carbon atoms in the cycloalkenylene group by 2; if the number of carbon atoms in the cycloalkenylene group is odd, the result of the division is rounded down to the next integer. For example, for a cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. In certain embodiments, the cycloalkenylene is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and / or (ii) 3- to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3-12 or 3-10 members). In certain embodiments, the cycloalkenylene is a mono-, bi-, or tricyclic 3- to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3-12 or 3-10 members) cycloalkenylene. Exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. "Substituted cycloalkenylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of a cycloalkenylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0221] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, e.g., 5, 6, or 10) carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Examples of aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerene. "Substituted aryl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an aryl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents specified herein. Examples of substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl).

[0222] The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group, as defined above, in which one or more carbon atoms of the aryl group are replaced with an O, S, or N heteroatom. Preferably, heteroaryl is a 5- or 6-membered aromatic monocyclic ring in which one, two, or three carbon atoms are replaced with an O, N, or S heteroatom. Alternatively, heteroaryl refers to an aromatic bicyclic or tricyclic ring system in which one, two, three, four, or five carbon atoms are replaced with the same or different O, N, or S heteroatoms. Preferably, in each ring of a heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, and the like. Includes zolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolidinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl."Substituted heteroaryl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of a heteroaryl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0223] The term "heterocyclyl" or "heterocyclic ring" refers to a cycloalkyl group, as defined above, in which one, two, three, or four carbon atoms of the cycloalkyl group are replaced by a heteroatom of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. A heterocyclyl group preferably has one or two rings containing 3 to 10, e.g., 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of a heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also intended to include partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the above heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also known as piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactone, lactam, cyclic imide, and cyclic anhydride. "Substituted heterocyclyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the heterocyclyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0224] The term "heterocycloalkylene," as used herein, refers to a heterocyclyl group, as defined above, containing at least one ring heteroatom (e.g., selected from the group consisting of O, S, N, B, Si, and P), wherein one hydrogen atom has been removed to result in a geminal, vicinal, or isolated diradical. In certain embodiments, the heteroatom of a heterocycloalkylene group is selected from the group consisting of O, S, and N. For example, a heterocycloalkylene can be an O / S-heterocycloalkylene, e.g., an O-heterocycloalkylene. In certain embodiments, in each ring of a heterocycloalkylene group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. A heterocycloalkylene can be monocyclic or polycyclic (e.g., bi- or tricyclic). In certain embodiments, heterocycloalkylene is a mono-, bi-, or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 4- to 12- or 4- to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also intended to encompass partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the above heteroaryl groups in which one hydrogen atom is removed from the same carbon atom to provide a geminal diradical (preferably, partially or fully hydrogenated forms of the above mono-, bi-, or tricyclic heteroaryl groups). Thus, in certain embodiments, heterocycloalkylene is saturated or unsaturated (i.e., contains one or more double bonds of the heterocycloalkylene group within the ring), but cannot be aromatic. "Substituted heterocycloalkylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

[0225] As used herein, the phrase "partially hydrogenated form" of an unsaturated compound or group means that some unsaturation has been removed by formally adding hydrogen to an originally unsaturated compound or group without removing all of the unsaturation. The term "fully hydrogenated form" of an unsaturated compound or group is used interchangeably herein with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to an originally unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl group (containing two double bonds in the ring, such as furan) includes a dihydro form of the 5-membered heteroaryl group (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while a tetrahydro form of the 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a fully hydrogenated (or perhydro) form of the 5-membered heteroaryl group. Similarly, for a 6-membered heteroaryl group having three double bonds in the ring (e.g., pyridyl), the partially hydrogenated forms include the di- and tetrahydro forms (e.g., di- and tetrahydropyridyl), while the hexahydro forms (e.g., piperidinyl in the case of heteroarylpyridyl) are fully hydrogenated (or perhydro) derivatives of the 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl may only be considered as a partially hydrogenated form according to the present invention if the aryl or heteroaryl contains at least four unsaturated moieties consisting of double and triple bonds between ring atoms.

[0226] The term "aromatic," as used in the context of hydrocarbons, requires that the entire molecule be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this invention. Similarly, if a bicyclic or polycyclic aryl (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this invention (even though one ring, as in 1,2-dihydronaphthyl, is still aromatic). A similar distinction is made herein between heteroaryl and heterocyclyl. For example, indolinyl, i.e., the dihydro variant of indolyl, is classified as a heterocyclyl for purposes of this invention because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0227] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removal of one H atom from a hydrocarbon molecule. In certain embodiments, the hydrocarbyl group is acyclic, e.g., linear (straight chain) or branched. Representative examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Specific examples of hydrocarbyl groups include C 1-30 Alkyl (e.g., C 6-30 Alkyl, C 8-24 Alkyl or C 10-20 alkyl), C with 1, 2 or 3 double bonds 2-30 Alkenyl (e.g., C 6-30 Alkenyl, C 8-24 Alkenyl or C 10-20 alkenyl), aryl and aryl (C 1-6In certain embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more first-level substituents, one or more second-level substituents, or one or more third-level substituents as defined herein), so long as the overall polarity of the hydrocarbon remains relatively non-polar. In certain embodiments, the hydrocarbyl group is a hydrocarbyl chain of a naturally occurring fatty acid and can have at least 8 carbon atoms, for example, the hydrocarbyl group can be a (preferably straight-chain) C 8-20 Alkyl chains, e.g., (preferably linear) C 10-18 alkyl chain).

[0228] The term "optionally substituted" refers to a group in which one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4 or 1-3 or 1 or 2) hydrogen atoms are substituted with a group (i.e., first level substituents) other than hydrogen, such as alkyl (preferably C 1-6 alkyl), alkenyl (preferably C 2-6 alkenyl), alkynyl (preferably C 2-6 alkynyl), aryl (preferably 6- to 14-membered aryl), heteroaryl (preferably 3- to 14-membered heteroaryl), cycloalkyl (preferably 3- to 14-membered cycloalkyl), heterocyclyl (preferably 3- to 14-membered heterocyclyl), halogen, -CN, azide, -NO2, -OR 71 , -N(R 72 )(R 73 ), -S(O) 0-2 R 71 , -S(O) 1-2 OR 71 , -OS(O) 1-2 R 71 , -OS(O) 1-2 OR 71 , -S(O) 1-2 N(R 72 )(R 73 ), -OS(O) 1-2 N(R 72 )(R 73 ), -N(R 71 )S(O)1-2 R 71 , -NR 71 S(O) 1-2 OR 71 , -NR 71 S(O) 1-2 N(R 72 )(R 73 ), -OP(O)(OR 71 )2, -C(=X1)R 71 , -C(=X1)X1R 71 , -X1C(=X1)R 71 and -X1C(=X1)X 1 R 71 and / or any two first-level substituents attached to the same carbon atom of a cycloalkyl or heterocyclyl group can together form =X1, where each of the first-level substituent alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups can itself be replaced by C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl, halogen, -CF3, -CN, azide, -NO2, -OR 81 , -N(R 82 )(R 83 ), -S(O) 0-2 R 81 , -S(O) 1-2 OR 81 , -OS(O) 1-2 R 81 , -OS(O) 1-2 OR 81 , -S(O) 1-2 N(R 82 )(R 83 ), -OS(O) 1-2 N(R 82 )(R 83 ), -N(R 81 )S(O) 1-2 R 81 , -NR 81 S(O) 1-2 OR 81 , -NR 81 S(O) 1-2 N(R 82 )(R83 ), -OP(O)(OR 81 )2, -C(=X2)R 81 , -C(=X2)X2R 81 , -X2C(=X2)R 81 and -X2C(=X2)X2R 81 and / or any two second-level substituents attached to the same carbon atom of a cycloalkyl or heterocyclyl group of the first-level substituents can together form =X2, where C of the second-level substituents 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, and 3- to 14-membered heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3and / or any two third-level substituents attached to the same carbon atom of a 3- to 14-membered cycloalkyl or heterocyclyl group of the second-level substituents are together ═O, ═S, ═NH, or ═N(C 1-3 alkyl); where: R 71 , R 72 and R 73 Each of the groups independently represents H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 selected from the group consisting of alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 3- to 7-membered heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, =O, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl; R 81 , R 82 and R 83 Each of these is H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 independently selected from the group consisting of alkynyl, 3- to 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl, wherein C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Each of the alkynyl, 3- to 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 3- to 6-membered heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, =O, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl; and Each of X1 and X2 independently represents O, S, and N(R 84 ), where R 84 is H or C 1-3 It is alkyl.

[0229] Typical first level substituents are preferably C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 6- to 14-membered (e.g., 6- to 10-membered) aryl, 3- to 14-membered (e.g., 5- or 6-membered) heteroaryl, 3- to 14-membered (e.g., 3- to 7-membered) cycloalkyl, 3- to 14-membered (e.g., 3- to 7-membered) heterocyclyl, halogen, -CN, azide, -NO2, -OR 71 , -N(R 72 )(R 73 ), -S(O) 0-2 R 71 , -S(O) 1-2 OR 71 , -OS(O) 1-2 R 71 , -OS(O) 1-2 OR 71 , -S(O) 1-2 N(R 72 )(R 73 ), -OS(O) 1-2 N(R 72 )(R 73 ), -N(R 71 )S(O) 1-2 R 71 , -NR 71 S(O) 1-2 OR 71 , -C(=X1)R 71 , -C(=X1)X1R 71 , -X1C(=X1)R 71 and -X1C(=X1)X1R 71 , e.g. C 1-4 Alkyl, C 2-4 Alkenyl, C2-4 Alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7-membered cycloalkyl, 3- to 7-membered (e.g., 5- or 6-membered) heterocyclyl, halogen, —CF3, —CN, azide, —NO2, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 alkyl is independently selected from the group consisting of methyl, ethyl, propyl, or isopropyl; where X is independently selected from O, S, NH, and N(CH); and R 71 , R 72 and R 73 are as defined above or, preferably, independently H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 and selected from the group consisting of alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 In some embodiments, the first level substituent is C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH and -C(=O)OCH3 (where z is 0, 1 or 2, C 1-3 wherein alkyl is methyl, ethyl, propyl, or isopropyl. In some embodiments, the first level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF, such as halogen (e.g., F, Cl, or Br) and —CF.

[0230] Typical second level substituents are preferably C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4Alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF3, —CN, azido, —NO2, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 and alkyl is independently methyl, ethyl, propyl, or isopropyl. Particular examples of second level substituents are C 1-3 Alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF3, —CN, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2(C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z (wherein each z is independently 0, 1, or 2; and each C 1-3 and alkyl is independently selected from the group consisting of methyl, ethyl, propyl, or isopropyl. Particularly preferred second-level substituents are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, ═O, and ═S.

[0231] Typical third level substituents are preferably C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH and -C(=O)OCH3 (where z is 0, 1 or 2, C 1-3 and alkyl is selected from the group consisting of methyl, ethyl, propyl, or isopropyl. Particularly preferred third level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF, such as halogen (e.g., F, Cl, or Br) and —CF.

[0232] As used herein, the term "tertiary amine moiety" refers to a moiety that includes a nitrogen atom substituted with three organic substituents, where the substituents may be the same or different from one another. In certain embodiments, the organic substituents are optionally substituted (e.g., with one or more first-level substituents, one or more second-level substituents, or one or more third-level substituents as defined herein) with a hydrocarbyl group (e.g., an alkyl group, particularly C 1-6 alkyl groups).

[0233] As used herein, the term "filtration" refers to any process involving the removal or separation of at least one component (e.g., permeable molecules such as salts, small proteins, solvents, etc.) of a liquid composition based on the molecular size of the component contained in the composition. This separation may use small molecule-permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., dialysis). Thus, examples of filtration include dialysis, tangential flow filtration, and diafiltration.

[0234] As used herein, the phrase "substantially free of X" means that a mixture (e.g., a composition described herein or an aqueous phase thereof) is, in a manner that is virtually and practically feasible, free of X. For example, if a mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the mixture.

[0235] For example, as used herein, "substantially free of lipids containing polyethylene glycol (PEG), wherein PEG has at least 30 consecutive ethylene glycol repeat units" means that the mixture (e.g., a composition described herein or its aqueous phase) is virtually and practically free of lipids containing at least 30 consecutive ethylene glycol repeat units. For example, if the mixture is substantially free of lipids containing at least 30 consecutive ethylene glycol repeat units, the amount of lipids containing at least 30 consecutive ethylene glycol repeat units in the mixture can be less than 1 wt.% (e.g., less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, less than 0.1 wt.%, less than 0.09 wt.%, less than 0.08 wt.%, less than 0.07 wt.%, less than 0.06 wt.%, less than 0.05 wt.%, less than 0.04 wt.%, less than 0.03 wt.%, less than 0.02 wt.%, less than 0.01 wt.%, less than 0.005 wt.%, or less than 0.001 wt.%) based on the total weight of the mixture. Similar considerations apply to phrases that include the term "substantially free" (e.g., "substantially free of sarcosylated lipids," "substantially free of (POX)-conjugated and / or polyoxazine (POZ)-conjugated lipids," and "substantially free of any polymer-conjugated lipids other than amphiphilic OEG-conjugated compounds").

[0236] The phrase "nucleic acid integrity" refers to the percentage of full-length (i.e., unfragmented) nucleic acids relative to the total amount of nucleic acids (i.e., unfragmented + fragmented nucleic acids) contained in a sample. Nucleic acid integrity can be determined by chromatographically separating the nucleic acids (e.g., using capillary electrophoresis), determining the peak area of the main nucleic acid peak (i.e., the peak area of the full-length (i.e., unfragmented) nucleic acid), determining the peak area of the total nucleic acid, and dividing the peak area of the main nucleic acid peak by the peak area of the total nucleic acid. Similarly, the phrase "RNA integrity" refers to the percentage of full-length (i.e., unfragmented) RNA relative to the total amount of RNA (i.e., unfragmented + fragmented RNA) contained in a sample. RNA integrity can be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of the major RNA peak (i.e., the peak area of full-length (i.e., unfragmented) RNA), determining the peak area of total RNA, and dividing the peak area of the major RNA peak by the peak area of total RNA.

[0237] The term "cryoprotectant" relates to a substance added to a formulation (eg, a formulation or composition) to protect the active ingredients of the formulation during the freezing step.

[0238] The term "lyoprotectant" refers to a substance added to a formulation to protect the active ingredient during the drying step.

[0239] According to the present invention, the term "peptide" includes oligo- and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids joined together via peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids, and the terms "peptide," "polypeptide," and "protein" are generally used synonymously herein.

[0240] A "therapeutic peptide or protein," when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic peptide or protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "therapeutic peptide or protein" includes whole peptides or proteins, as well as therapeutically active fragments thereof. It can also include therapeutically active variants of peptides or proteins. Examples of therapeutically active peptides or proteins include, but are not limited to, vaccination antigens and immune stimulants such as cytokines. The terms "therapeutic peptide or protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

[0241] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may designate a contiguous or discontinuous fraction of the structure.

[0242] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a continuous element of the structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0243] A "fragment" of an amino acid sequence (peptide, polypeptide, or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing the amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon for initiating translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, a sequence of at most 8, in particular at most 10, at most 12, at most 15, at most 20, at most 30 or at most 55 consecutive amino acids of the amino acid sequence.

[0244] According to the present invention, a portion or fragment of a peptide, polypeptide, or protein preferably possesses at least one functional property of the peptide, polypeptide, or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides, polypeptides, or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide, polypeptide, or protein possesses at least one pharmacological activity of the peptide, polypeptide, or protein from which it is derived. A portion or fragment of a peptide, polypeptide, or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of at most 8, particularly at most 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

[0245] As used herein, a "variant" with respect to an amino acid sequence (peptide, polypeptide, or protein) refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid or a modification of the same amino acid). The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence or a modified version of the wild-type amino acid sequence. In certain embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences, and preferably 1 to about 10 or 1 to about 5 amino acid differences compared to the parent.

[0246] "Wild-type" or "WT" or "native" with respect to an amino acid sequence refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, polypeptide, or protein has an amino acid sequence that has not been intentionally modified. Similarly, "wild-type" or "WT" or "native" with respect to a nucleic acid sequence refers to a nucleic acid sequence that has been found in nature, including allelic variations. For example, a wild-type coding sequence refers to a coding sequence that is found in nature and has not been intentionally modified.

[0247] As used herein, "coding sequence" refers to a portion of a nucleic acid (e.g., the DNA or RNA of a gene) that encodes a protein.

[0248] The phrase "guanosine / cytosine (G / C) content" or "G / C content" refers to the percentage of bases in a DNA or RNA molecule that are guanine (G) or cytosine (C). The G / C content can be given for a specific portion of DNA or RNA or for the entire genome. When G / C content refers to a portion, it can refer to the G / C content of an individual gene or portion of a gene (domain), a group of genes or gene clusters, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0249] For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes total mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0250] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-terminus of the protein are also referred to herein as N- and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. It is preferred to modify and / or replace amino acids with others having similar properties at positions in the amino acid sequence that are not conserved between homologous proteins or peptides. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of amino acids with similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of a family of amino acids with related side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0251] In some embodiments, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably shown for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0252] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0253] The terms "% identical" and "% identity" or similar terms refer specifically to the percentage of nucleotides or amino acids that are identical in optimal alignment between the compared sequences. The percentage is purely statistical; the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithms available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In one embodiment, the algorithm parameters used in the BLASTN algorithm on the NCBI website include: (i) a prediction threshold setting of 10; (ii) a word length setting of 28; (iii) a query range maximum match setting of 0; (iv) a match / mismatch score setting of 1, -2; (v) a gap cost setting of linear; and (vi) use of a filter for low complexity regions. In one embodiment, the algorithm parameters used in the BLASTP algorithm on the NCBI website include: (i) a prediction threshold setting of 10; (ii) a word length setting of 3; (iii) a query range maximum match setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap costs settings of presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0254] Percentage identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence), and multiplying the result by 100.

[0255] In some embodiments, the degree of similarity or identity is shown for at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is shown for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, the degree of similarity or identity is shown for the entire length of the reference sequence.

[0256] Homologous amino acid sequences according to the present invention exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0257] The amino acid sequence variants described herein can be readily produced by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0258] In some embodiments, a fragment or variant of an amino acid sequence (peptide, polypeptide, or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigenic sequence, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered, particularly compared to the amino acid sequence of the parent molecule or sequence, and still retains one or more functions of the parent molecule or sequence, such as inducing an immune response (immunogenic fragment). In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0259] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, those skilled in the art will understand that antigens suitable for use herein can be altered from the naturally occurring or native sequence from which they are derived while maintaining the desired activity of the native sequence.

[0260] In some embodiments, "isolated" refers to modification or removal (e.g., purification) from a natural state or an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell. In some embodiments, the RNA (e.g., mRNA) used in the present invention is in a substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of RNA (e.g., mRNA) in a substantially purified form comprises a first buffer system.

[0261] The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid.

[0262] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" may also include the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids described herein can be in vitro (e.g., cell culture) or in vivo; for example, the cells may form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. For some transfection applications, it is sufficient if the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during transfection are usually not integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection should be performed.Such stable transfection can be achieved using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into cells.RNA can be transfected into cells to transiently express its encoded protein.

[0263] The present invention includes analogs of peptides, polypeptides, or proteins. According to the present invention, a peptide, polypeptide, or protein analog is a modified form of the peptide, polypeptide, or protein derived from the peptide, polypeptide, or protein and retains at least one functional property. For example, a pharmacologically active peptide, polypeptide, or protein analog retains at least one pharmacological activity of the peptide, polypeptide, or protein from which the analog is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the protein, polypeptide, or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In certain embodiments, a "protein, polypeptide, or peptide" "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to antibodies or other cellular ligands. The term "analog" also extends to fully functional chemical equivalents of the proteins, polypeptides, and peptides.

[0264] As used herein, the terms "linked," "fused," or "fused" are used interchangeably and refer to the joining of two or more elements, components, or domains.

[0265] According to various embodiments of the present invention, a nucleic acid, such as an RNA (e.g., mRNA), encoding a peptide, polypeptide, or protein is taken up or introduced, i.e., transfected or transduced, into a cell, which may be in vitro or present in a subject, resulting in expression of the peptide, polypeptide, or protein. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or nucleus) and may secrete and / or express the encoded peptide, polypeptide, or protein on its surface.

[0266] According to the present invention, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide, polypeptide or protein, the nucleic acid can be expressed to produce the peptide, polypeptide or protein when present in the appropriate environment, preferably within a cell.

[0267] As used herein, "activation" or "stimulation" refers to the state of a cell (e.g., an immune effector cell such as a T cell) that has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell undergoing cell division.

[0268] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0269] The term "clonal expansion" or "expansion" refers to the process of amplifying a specific entity. In certain embodiments, the term is preferably used in the context of an immunological response, in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize the antigen are expanded. In certain embodiments, expansion leads to differentiation of immune effector cells.

[0270] According to the present invention, "antigen" refers to any substance that induces an immune response and / or any substance to which an immune response or immune mechanism, such as a cellular response and / or a humoral response, is directed. This also includes situations in which an immune response or immune mechanism is directed against one or more antigenic peptides, if the antigen is processed into antigenic peptides and presented, particularly in the context of MHC molecules. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" can include any molecule that contains at least one epitope, such as a T cell epitope. In certain embodiments, an antigen in the context of the present invention is a molecule that, after optional processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In certain embodiments, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0271] According to the present invention, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a humoral or cellular immune response, or both. In the context of certain embodiments of the present invention, the antigen is presented by a cell, preferably an antigen-presenting cell, in the context of an MHC molecule, resulting in an immune response against the antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present invention, the antigen can correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

[0272] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens are molecules that contain epitopes that stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, such as tumor antigens, typically tumors.

[0273] In some embodiments, the antigen is a tumor antigen, i.e., a part of tumor cells, particularly, a part that mainly occurs as an intracellular or surface antigen of tumor cells.In other embodiments, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as a virus, a bacterium, a single-cell organism, or a parasite, for example, a viral antigen such as a viral ribonucleoprotein or coat protein.In particular, the antigen should be presented by MHC molecules, which leads to the regulation, particularly activation, of immune system cells, preferably CD4+ and CD8+ lymphocytes, particularly through the regulation of the activity of T cell receptors.

[0274] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to an antigen produced as an intracellular or surface antigen in a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-ferroprotein and γ-fetoprotein, as well as various viral tumor antigens. According to one embodiment of the present invention, tumor antigens include any antigen that is characteristic of a tumor or cancer and that is characteristic of tumor or cancer cells with respect to type and / or expression level.

[0275] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0276] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0277] The term "epitope" refers to an antigenic determinant on a molecule such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can comprise a continuous or discontinuous portion of the protein and can be, for example, about 5 to about 100, about 5 to about 50, about 8 to about 0, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, an epitope in the context of the present invention is a T cell epitope.

[0278] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and refer to, for example, an incomplete presentation of an antigen, which, for example, elicits an immune response against the antigen or enables a cell to express, contain, and present the antigen. In certain embodiments, the term refers to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically binds) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0279] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized when presented in the context of a T cell's MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. In the case of Class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, and particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may be effective.

[0280] Peptide and protein antigens can be, for example, 2 to 100 amino acids in length, including 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0281] A peptide or protein antigen can be any peptide or protein for which the immune system is capable of generating antibodies and / or augmenting a T cell response against the peptide or protein.

[0282] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, if a vaccine antigen is recognized by immune effector cells, it can, in the presence of an appropriate costimulatory signal, induce the stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of this embodiment of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell.

[0283] In certain embodiments, the antigen is expressed on a diseased cell (e.g., a tumor cell or an infected cell).

[0284] In some embodiments, the antigen is presented by diseased cells (e.g., tumor cells or infected cells). In some embodiments, the antigen receptor is a TCR that binds to the epitope of the antigen presented in the context of MHC. In some embodiments, when expressed by T cells and / or presented to T cells, the binding of TCR to the antigen presented by cells such as antigen-presenting cells leads to the stimulation, priming and / or expansion of the T cells. In some embodiments, when expressed by T cells and / or presented to T cells, the binding of TCR to the antigen presented by diseased cells leads to the cytolysis and / or apoptosis of diseased cells, wherein the T cells preferably release cytotoxic factors, such as perforin and granzyme.

[0285] In some embodiments, the antigen is expressed on the surface of diseased cells (e.g., tumor cells or infected cells). In some embodiments, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In some embodiments, the CAR binds to the epitope of a natural antigen present on the surface of living cells. In some embodiments, when expressed by T cells and / or presented to T cells, the CAR binds to the antigen presented on cells such as antigen-presenting cells, resulting in the stimulation, priming and / or expansion of the T cells. In some embodiments, when expressed by T cells and / or presented to T cells, the CAR binds to the antigen presented on diseased cells, resulting in the cytolysis and / or apoptosis of diseased cells, where the T cells preferably release cytotoxic factors, such as perforin and granzyme.

[0286] In certain embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope on the antigen. In certain embodiments, the antibody or B cell receptor binds to an epitope on a native antigen.

[0287] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, e.g., by an antibody located outside the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interactions with any other proteins, lipids, saccharides, or other structures that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule that associates with the cell surface can be a transmembrane protein having an extracellular portion, or a protein that associates with the cell surface through interactions with another protein that is a transmembrane protein.

[0288] "Cell surface" or "surface of a cell" is used according to its usual meaning in the art, and thus includes the outside of a cell that is accessible for binding by proteins and other molecules.An antigen is expressed on the surface of a cell if it is located on the surface of the cell, for example, accessible for binding by an antigen-specific antibody added to the cell.In some embodiments, the antigen expressed on the cell surface is an integral membrane protein that has an extracellular portion that can be recognized by CAR.

[0289] The term "extracellular portion" or "extracellular domain" in the context of the present invention refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, e.g., by a binding molecule, such as an antibody, that is located on the outside of the cell. In certain embodiments, the term refers to one or more of the extracellular loops or domains or fragments thereof.

[0290] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes its target antigen, particularly when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, in the context of an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if the cell kills a target cell that expresses the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the present invention, the RNA (particularly mRNA) encodes at least one epitope.

[0291] The term "target" refers to a factor, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present antigens or antigen epitopes, i.e., peptide fragments derived from antigens. In some embodiments, target cells are cells that express antigens, preferably presenting the antigens with class I MHC.

[0292] "Antigen processing" refers to the breakdown of an antigen into processing products that are fragments of the antigen (e.g., breakdown of a protein into peptides) and the association (e.g., via binding) of these fragments with MHC molecules for presentation to specific T cells by one or more cells, preferably antigen-presenting cells. Antigen-presenting cells can be divided into professional and non-professional antigen-presenting cells.

[0293] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0294] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon-gamma. Examples of non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0295] The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with a presentable antigen, they are activated into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on the cell surface using MHC molecules. Simultaneously, they upregulate cell surface receptors that act as coreceptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate via the bloodstream to the spleen or via the lymphatic system to lymph nodes. Here, by presenting antigens, dendritic cells act as antigen-presenting cells, and together with non-antigen-specific costimulatory signals, activate helper T cells, killer T cells, and B cells. Therefore, dendritic cells can actively induce T cell or B cell-related immune responses. In some embodiments, dendritic cells are splenic dendritic cells.

[0296] The term "macrophage" refers to a subgroup of phagocytes produced by the differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products nonspecifically phagocytose and kill foreign pathogens within the macrophage through hydrolytic and obstetric attacks, causing the pathogens to degrade. Peptides from degraded proteins are presented on the macrophage cell surface, which can be recognized by T cells and can directly interact with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0297] "Antigen-responsive CTL" refers to a CD8 CTL that responds to an antigen presented on the surface of an antigen-presenting cell together with class I MHC or a peptide derived from the antigen.+ It means T cells.

[0298] According to the present invention, CTL responsiveness can induce sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic cell death of tumor antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0299] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the body's integrated response to an antigen, and may refer to a cellular immune response, a humoral immune response, or both. According to the present invention, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, directed against the agent. An immune response includes the development of antibodies against one or more antigens and the production of CD4 + and CD8 + T lymphocytes, e.g., CD8 + The reaction may include one or more responses selected from the group consisting of expansion of antigen-specific T lymphocytes, such as T lymphocytes, which may be detected in vitro by various proliferation or cytokine production tests.

[0300] In the context of the present invention, the terms "induction of an immune response" and "elicitation of an immune response" and similar terms refer to the induction of an immune response, for example, the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / preventive / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B, or C), CMV, or RSV)). "Induction" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was a certain level of immune response against a particular antigen or pathogen before induction, and that the immune response is enhanced after induction. Thus, "induction of an immune response" in this context includes "enhancing an immune response." In some embodiments, after induction of an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease state is alleviated by the induction of an immune response.

[0301] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are intended to include a cellular response directed against cells characterized by expression of antigens and / or presentation of antigens with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (CD4 + T cells (also called killer cells) play a central role in regulating immune responses and are classified as cytotoxic T cells, cytolytic T cells, and CD8 + T cells or CTLs (also called T cells or CTLs) kill cells, such as diseased cells.

[0302] The term "humoral immune response" refers to the process in living organisms by which antibodies are generated in response to factors and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to a single specific antigen. After binding to the appropriate antigen and receiving various other activation signals, B lymphocytes divide and produce memory B cells as well as antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain quiescent until subsequently activated by a specific antigen. These lymphocytes provide the cellular basis of memory and result in an increased antibody response when re-exposed to the specific antigen.

[0303] As used herein, the term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, capable of specifically binding to an epitope of an antigen under typical physiological conditions, preferably with a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days or more, etc., or any other relevant, functionally defined period of time (e.g., a period of time sufficient for the antibody to induce, promote, enhance, and / or modulate an antigen-associated physiological response to the antigen and / or a period of time sufficient for the antibody to recruit effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3, and the CDRs of VL are referred to as LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3).The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or may be immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0304] The variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to the region that interacts with an antigen and includes both the VH and VL regions. As used herein, antibodies include not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions.

[0305] As noted above, the term antibody herein includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to an antigen, unless otherwise specified or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains or the monovalent antibodies disclosed in WO2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single-arm antibody; and (v) dAb fragments consisting essentially of the VH domain (Ward et al., Nature 2009, 144:145-150). 341, 544-546 (1989)) and also known as domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24); and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be produced using recombinant methods as a single protein chain that allows the VL and VH domains to pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, e.g., Bird et al., Science 2000). 242 , 423-426 (1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988). Such single chain antibodies are included within the scope of the term antibody unless otherwise specified or clearly contradicted by the context. Although such fragments are generally included within the meaning of antibody, collectively and each independently are unique features of the present invention and exhibit various biological properties and utilities. These and other useful antibody fragments in the context of the present invention, as well as bispecific forms of such fragments, are further described herein. The term antibody refers to a fragment of an antibody, unless otherwise specified. It should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to the antigen, which may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0306] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, e.g., antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins and are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are mainly V L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domains and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H The immunoglobulin chains consist of immunoglobulin domains such as α, δ, ε, γ, and μ, which constitute the different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at the carboxy terminus. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the various immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0307] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons, by administering to the individual one or more of the immunogens or antigens described herein or derivatives thereof, particularly in the form of RNA (especially mRNA) encoding same, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by presentation of said one or more immunogens or antigens.

[0308] "Cells characterized by antigen presentation" or "cell-presenting antigen" or "MHC molecule that causes antigen to be presented on the surface of an antigen-presenting cell" or similar expressions means that cells or antigen-presenting cells, such as diseased cells, particularly tumor cells or infected cells, present the antigen or antigenic peptide, either directly or after processing, in the context of an MHC molecule, preferably an MHC class I and / or MHC class II molecule, most preferably an MHC class I molecule.

[0309] In the present invention, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA (especially mRNA), which can then be translated into peptides, polypeptides or proteins.

[0310] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0311] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0312] In the present invention, the term "RNA encodes" means that the RNA, when present in an appropriate environment, such as within the cells of a target tissue, is capable of directing the assembly of amino acids to produce the peptide or protein that it encodes during the process of translation.

[0313] As used herein, the term "serum" refers to a fluid resulting from the removal of cells and clotting factors from whole blood, such as whole blood obtained from a human or mouse. In certain embodiments, the serum is human serum or mouse serum.

[0314] The pharmaceutical preparations, particularly kits, described herein may include instructional materials or directions. As used herein, "instruction materials" or "directions" includes publications, records, diagrams, or any other means of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional materials of the kits of the present invention may, for example, be attached to a container containing a composition of the present invention or shipped together with a container containing the composition. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the instructional materials and the composition be used cooperatively by the recipient.

[0315] As used herein, the term "optionally" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and the description includes instances where the event, circumstance, or condition occurs and instances where it does not occur.

[0316] A prodrug of a particular compound described herein is a compound that undergoes chemical conversion under physiological conditions to provide the particular compound after administration to an individual. Furthermore, a prodrug can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with, for example, a suitable enzyme or chemical reagent. Exemplary prodrugs are in vivo hydrolyzable esters (using an alcohol or carboxy group contained in the particular compound) or amides (using an amino or carboxy group contained in the particular compound). Specifically, certain amino groups contained in the particular compound that bear at least one hydrogen atom can be converted to a prodrug form. Exemplary N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0317] In this specification, the structural formula of a compound may represent a certain isomer of the compound. However, it should be understood that the present invention includes all isomers and isomer mixtures, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and other structurally occurring isomers, and is not limited to the description of the formula. Furthermore, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it should be understood that the present invention includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0318] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of equal amounts of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert into each other by the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is a keto-enol-tautomeric isomer. "Conformers" are stereoisomers that can only be interconverted morphologically by rotation about a single bond, leading to—in particular—different three-dimensional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0319] As used herein, the term "solvate" refers to an addition complex of a dissolved substance in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), wherein the addition complex exists in crystalline or mixed crystalline form. The amount of solvent present in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate where the solvent is water.

[0320] In an isotopically labeled compound, one or more atoms are replaced with corresponding atoms having the same number of protons but different neutrons. For example, a hydrogen atom may be replaced with a deuterium or tritium atom. Exemplary isotopes that can be used in the present invention are deuterium, tritium, 11 C. 13 C. 14 C. 15 N, 18 F, 32 P, 32 S, 35 S, 36 Cl and 125 Includes I.

[0321] The term "average diameter" refers to the diameter measured by dynamic light scattering (DLS), resulting in the so-called Z 平均 The "average diameter", "diameter" or "size" of a particle refers to the average hydrodynamic diameter of the particle after data analysis using the so-called cumulant algorithm, which provides the dimensions of length and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle refers to the particle size, Z 平均 Used synonymously with this value.

[0322] In one embodiment, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis described in the definition of "average diameter." Under certain conditions, it can be interpreted as a measure of the size distribution of the nanoparticles as a whole.

[0323] The "cross-sectional radius of gyration" of the particle with respect to the axis of rotation (here R g ) is the radial distance from the point of rotation at which the moment of inertia about an axis is assumed to be the same as the actual mass distribution if the total mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle component from the center of mass or some axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at iFor a macromolecule consisting of n mass elements (i=1, 2, 3, . . . , n), R g is the s over all mass elements i 2 is the square root of the mass average of and can be calculated as follows:

number

[0324] The cross-sectional radius of gyration can be empirically determined or calculated, for example, using light scattering. In particular, the small scattering vector

number

number

[0325] "D10 value" refers to the diameter at which 10% of particles have a diameter smaller than this value, particularly in relation to the quantitative size distribution of particles. The D10 value is meant to refer to the proportion of the smallest particles within a particle population (e.g., within a particle peak obtained by a flow field separation method).

[0326] "D50 value" refers to the diameter of the particle population (e.g., within a particle peak obtained by a flow field separation method) at which 50% of the particles have a smaller diameter, particularly in relation to the quantitative size distribution of the particles.

[0327] The "D90 value" is the diameter below which 90% of the particles have a diameter, particularly in relation to the quantitative size distribution of particles. The "D95", "D99" and "D100" values have corresponding meanings. The D90, D95, D99 and D100 values are meant to refer to the proportion of large particles within a particle population (e.g., within a particle peak obtained by a flow field separation method).

[0328] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a small, highly hydrated charged particle may have a larger hydrodynamic radius than a large, weakly hydrated charged particle. This is because the small particle will entrain a greater number of water molecules as it moves through the solution. Because the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is determined using the Stokes-Einstein equation:

number

[0329] The term "aggregate" as used herein refers to a mass of particles where the particles are identical or very similar and are adhered to each other in a non-covalent manner (e.g., ionic interactions, H-bridge interactions, dipole interactions and / or van der Waals interactions).

[0330] As used herein, the expression "light scattering" refers to the physical process by which light is deviated from its linear trajectory by one or more paths due to localized non-uniformities in the medium through which the light passes.

[0331] The term "UV" means ultraviolet light and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, ie, shorter than visible light but longer than X-rays.

[0332] As used herein, the expression "multi-angle light scattering" or "MALS" refers to a technique for measuring by scattering light from a sample at multiple angles. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles measured, for example, by a single detector moving over a range that includes a selected specific angle, or by a series of detectors fixed at a specific angular position that includes the specific angle. In a preferred embodiment, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the cross-sectional radius of gyration (R) can be calculated using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ), thus making it possible to determine the size of the particles. Preferably, the Zimm plot is calculated using the following formula (or its reciprocal):

number

number

number

[0333] As used herein, the phrase "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is projected onto a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution are struck by the light, causing it to diffract in all directions. The light diffracted from the particles interferes constructively (bright areas) or destructively (dark areas). This process is repeated at short intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity of each spot over time.

[0334] As used herein, the phrase "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's cross-sectional radius of gyration and / or particle molar mass. High-intensity monochromatic light, usually a laser, is directed into a solution containing the particles. One or multiple detectors are used to measure the scattered intensity at one or multiple angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and the size of the radius of the entire macromolecule. Thus, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), is generally considered the standard practice of static light scattering.

[0335] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is the component of the immune system that is relatively nonspecific and immediate. Along with the adaptive immune system, it is one of the two major components of the vertebrate immune system.

[0336] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0337] As used herein, the term "exogenous" refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

[0338] The term "repeating unit" refers to a basic unit that repeats itself periodically along the polymer chain of a polymer and is derived from one monomer. The structures of a repeating unit and its corresponding monomer often coincide, but can differ from each other.

[0339] As used herein, the term "functionalized moiety" refers to a group of atoms in a molecule having a distinctive chemical property, where the atoms of the functionalized moiety are covalently bonded to each other and to the rest of the molecule. Preferably, the atoms of the functionalized moiety include at least one atom selected from the group consisting of O, N, and S. The functionalizing moiety can be monovalent (e.g., hydroxy, cyano, nitro, or amido (e.g., -C(O)NHCH3)) or divalent (e.g., amide (e.g., -C(O)NH-), carbonyl (-C(O)-), or ester (e.g., -OC(O)-). In certain embodiments, the functionalizing moiety provides hydrophilicity and / or at least one charge (positive or negative) to the group to which the functionalizing moiety is attached, for example, by providing at least one hydrogen bond acceptor / donor. In certain embodiments, the functionalizing moiety comprises a hydrogen bond acceptor (e.g., a carbonyl moiety), a hydrogen bond donor (e.g., a hydroxyl moiety, -NH- (e.g., in an amide moiety) or a thiol moiety), or both (e.g., in an amide moiety) and / or a charge (e.g., a phosphate, amino, or ammonium moiety). Examples of monovalent functionalizing moieties include hydroxy, ether, halogen, cyano, azido, nitro, amino, , ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionyl a These include imido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.Examples of divalent functionalized moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino(imine), imidothioate, thionylamide, carbonate, Carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

[0340] The term "hydroxyl" or "hydroxy" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group --OH.

[0341] The term "halogen" as used herein, particularly with respect to a functionalized moiety as a component of a linker, means fluoro, chloro, bromo, or iodo.

[0342] The term "cyano" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group --CN.

[0343] As used herein, particularly with respect to functionalized moieties as components of linkers, the term "azido" refers to the group N3.

[0344] The term "nitro" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group -NO2.

[0345] The term "amino," as used herein with respect to functionalized moieties, particularly as components of linkers, includes unsubstituted amino (i.e., the group -NH) and substituted amino (i.e., mono- or di-substituted amino in which one or two of the hydrogen atoms have been replaced with a group other than hydrogen). Amino groups can be monovalent (e.g., -NRR, where each R is an R group as defined below). 72 or R 73 and the like are independently H or an organic group) or divalent (e.g., —NR—, where R is R as defined below). 72 In certain embodiments, the term "amino" refers to the group -N(R 72 )(R 73 ), where R 72 and R 73 is independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 72 and R 73 together with the nitrogen atom to which they are attached form the group -N=CR 75 R 76 wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups may optionally be selected from independently selected R 70 is substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); R 75 and R 76 are independently -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NH y R 80 2-y or R 75 and R 76may form a ring together with the atoms to which they are attached, which may optionally be independently selected from one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) R 70 wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with an independently selected R 70 is substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); y is an integer from 0 to 2; R 80 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups optionally contains an independently selected R 70 (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); and R 70 is other than H, preferably a first level substituent, second level substituent, or third level substituent disclosed herein. In certain embodiments, R 72 and R 73 each independently represents H or H, C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl), wherein each of the hydrocarbyl groups (e.g., C 1-6 Alkyl, aryl and aryl(C 1-6Each of the alkyl groups may optionally contain one or more (e.g., 1 to 10) hydrocarbyl groups (e.g., C 1-6 Alkyl, aryl or aryl(C 1-6 up to the maximum number of hydrogen atoms bonded to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2, independently selected R 70 is replaced by .

[0346] As used herein, particularly with respect to functionalized moieties as components of linkers, the term "ammonium" refers to the group -N + (R 72 )2(R 73 ) (where R 72 and R 73 is as defined for the term "amino".

[0347] The term "thiol" or "sulfanyl" as used herein with respect to a functionalized moiety, particularly as a component of a linker, refers to the group --SH.

[0348] The term "disulfanyl" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group -SSH.

[0349] The term "carboxyl" or "carboxy" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group --COOH.

[0350] The term "amide" or "amido" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a structure comprising the structure -C(O)NH- (including its isomeric arrangement, the structure -NHC(O)-, unless specified to the contrary). Preferably, each end of the amide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker) (if both ends are bonded to the same organic group, the amide moiety also describes a lactam). The amide group is monovalent (e.g., -C(O)NRR or -NRC(O)R, where each R is H or an R as defined above in the definition of the term "amino." 72 or a divalent (e.g., —C(O)NR— or —NRC(O)—, where R is H or R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0351] The term "ester," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to a group comprising the structure -C(O)O- (including its isomeric arrangements of the structure -OC(O)-, unless specified to the contrary). Preferably, each end of the ester structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). (If both ends are bonded to the same organic group, the ester moiety may also be referred to as a lactone.) The ester group is monovalent (e.g., -C(O)OR or -OC(O)R, where R is independently selected from the R groups listed above in the definition of the term "amino." 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (e.g., —C(O)O— or —OC(O)—).

[0352] The term "ether," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -O-, where each end of the ether structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). An ether group is monovalent (e.g., -OR, where R is an R group as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (eg, —O—).

[0353] The term "sulfide" or "thioether," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -S-, where each end of the sulfide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker). The sulfide group is monovalent (e.g., -SR, where R is an R group as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (eg, -S-).

[0354] The term "disulfide," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to a group comprising the structure -SS-, where each end of the disulfide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). A disulfide group is monovalent (e.g., -SSR, where R is an R group as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (eg, -SS-).

[0355] The term "diselenide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SeSe-, where each end of the diselenide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). A diselenide group is monovalent (e.g., -SeSeR, where R is an R group as defined above in the definition of the term "amino"). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (e.g., -SeSe-).

[0356] The term "sulfoxide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group that includes the sulfinyl structure -S(O)-, where each end of the sulfoxide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfoxide group is monovalent (e.g., -S(O)R, where R is an R group as set forth above in the definition of the term "amino." 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (as, for example, -S(O)-).

[0357] The term "sulfone," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group that includes the sulfonyl structure -S(O)-, where each end of the sulfone structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfone group is monovalent (e.g., -S(O)R, where R is an R group as set forth above in the definition of the term "amino." 72 is an organic group such as one of the organic groups specified in the definition of (I)) or divalent (e.g., as in -S(O)2-).

[0358] The term "sulfite," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OS(O)O-, where one of the two ends of the sulfite structure is covalently bonded to an atom of an organic group and the other end is covalently bonded to H or a C atom of the same or another organic group (e.g., an alkylene group as a further component of the linker). The sulfite group is monovalent (e.g., -OS(O)OR, where R is H or an R atom as set forth above in the definition of the term "amino." 72 (e.g., —OS(O)O—), or divalent (e.g., —OS(O)O—).

[0359] The term "sulfate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OS(O)2O-, where one of the two ends of the sulfate structure is covalently bonded to an atom of an organic group and the other end is covalently bonded to H or a C atom of the same or another organic group (e.g., an alkylene group as a further component of the linker). Sulfate groups are monovalent (e.g., -OS(O)2OR, where R is H or an R atom as set forth above in the definition of the term "amino." 72 (e.g., —OS(O)2O—), or divalent (e.g., —OS(O)2O—).

[0360] The term "phosphate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OP(O)(OR)O-, where one of the two ends of the phosphate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where R is H or R as set forth above in the definition of the term "amino"). 72 The phosphate group is a monovalent (e.g., —OP(O)(OR)2, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or a divalent (e.g., —OP(O)(OR)O—, where the term R is H or an R group as set forth above in the definition of the term “amino.”72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0361] The term "sulfinamide" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -S(O)N(R)-, where the S terminus of the sulfinamide is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the N terminus is covalently bonded to H or a C atom of the same or another organic group (R is H or R as indicated above in the definition of the term "amino"). 72 The sulfinamide group is a monovalent (e.g., —S(O)N(R)2, where each R is H or an R group as shown above in the definition of the term “amino.” 72 (e.g., —S(O)N(R)—, where R is H or R as shown above in the definition of the term “amino”) or divalent (e.g., —S(O)N(R)—, where R is H or R as shown above in the definition of the term “amino”). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0362] The term "sulfonamide" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -S(O)N(R)-, where the S terminus of the sulfonamide is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the N terminus is covalently bonded to H or a C atom of the same or another organic group (R is H or R as set out above in the definition of the term "amino"). 72 The sulfonamide group is a monovalent (e.g., —S(O)N(R), where each R is H or an R group as shown above in the definition of the term “amino.” 72 (e.g., —S(O)N(R)—, where R is H or an R group as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0363] The term "sulfamate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OS(O)N(R)- (including its isomeric arrangements, -N(R)S(O)O-, unless specified to the contrary), in which one end of the sulfamate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (R is H or R as defined above in the definition of the term "amino"). 72 The sulfamate group is a monovalent (e.g., —OS(O)N(R) or —N(R)S(O)OR, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or a divalent (e.g., —OS(O)N(R)— or —N(R)S(O)O—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0364] The term "sulfite diamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)S(O)N(R)-, where one end of the sulfite diamide structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as defined above in the definition of the term "amino"). 72 The sulfite diamide group is a monovalent (e.g., —N(R)S(O)N(R)2, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)S(O)N(R)—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0365] The term "acidous diamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)S(O)N(R)-, where one end of the acidous diamide structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as defined above in the definition of the term "amino"). 72 The acid diamide group is a monovalent (e.g., —N(R)S(O)N(R) where each R is H or an R group as shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)S(O)N(R)—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0366] The term "urea," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(O)N(R)-, where one end of the urea structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as set forth above in the definition of the term "amino"). 72 The urea group is a monovalent (e.g., —N(R)C(O)N(R)2, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or a divalent (e.g., —N(R)C(O)N(R)—, where each R is H or an R as shown above in the definition of the term “amino” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0367] The term "thiourea," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(S)N(R)-, where one end of the thiourea structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as defined above in the definition of the term "amino"). 72 The thiourea group is a monovalent (e.g., —N(R)C(S)N(R)2, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or a divalent (e.g., —N(R)C(S)N(R)—, where each R is H or an R as defined above in the definition of the term “amino” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0368] The term "carbonyl," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)-, where one end of the carbonyl structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (if both ends are bonded to C atoms of organic groups, the carbonyl moiety is also referred to as a "keto" moiety). A carbonyl group is monovalent (e.g., -C(O)R, where R is H or R as defined above in the definition of the term "amino"). 72 (e.g., —C(O)—), or divalent (e.g., —C(O)—).

[0369] The term "thiocarbonyl," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(S)-, where one end of the thiocarbonyl structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group. A thiocarbonyl group is monovalent (e.g., -C(S)R, where R is H or R as defined above in the definition of the term "amino."72 (e.g., —C(S)—) or divalent (e.g., —C(S)—).

[0370] The term "orthoester," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to an ester of an ester containing three alkoxy groups (i.e., -OR, where R is an alkoxy group as defined above in the definition of the term "amino"). 72 An exemplary formula for an orthoester is the structure (-O) r C(OR) 3-r -, where each R is an R as defined above in the definition of the term "amino". 72 wherein each of the orthoesters has the structure (-O) independently selected from the organic groups specified in the definition of (-O) and ... r C(OR 25 ) 3-r where each R 25 is independently C 1-6 Alkyl, aryl and aryl(C 1-6 and r is 1 or 2; and each end of the orthoester structure is covalently bonded to a C atom of an additional organic group or two further apart organic groups. The orthoester group is monovalent (e.g., —C(OR)3 or —OC(OR)2R, where each R is an R group as set forth above in the definition of the term “amino”). 72 (R) or -OC(OR)-, where each R is an R as defined above in the definition of the term "amino." 72 (e.g., an organic group independently selected from the organic groups specified in the definition of

[0371] The term "thioate" or "thioester," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(O)- (including its isomeric arrangements -C(O)S-, -OC(S) and -C(S)O-, unless specified to the contrary), where each end of the thioate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The thioate group is monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, where each R is H or an R as set forth above in the definition of the term "amino." 72 (e.g., —S—C(O)— or —C(O)S— or —O—C(S)— or —C(S)O—).

[0372] The term "dithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)- (including its isomeric arrangement, -C(S)S-, unless specified to the contrary), where each end of the dithioate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). Dithioate groups are monovalent (e.g., -SC(S)R or -C(S)SR, where each R is H or an R as set forth above in the definition of the term "amino." 72 (S)-- or (C(S)S--).

[0373] The term "imidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)- (including its isomeric arrangements of the structure -C(=NR)O-, unless specified to the contrary), where each end of the imidate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker) (each R is independently H or R as set forth above in the definition of the term "amino"). 72 The imidate group is a monovalent organic group (e.g., —OC(═NR)R′ or —C(═NR)OR′, where each R is independently H or an R group as defined above in the definition of the term “amino.” 72 and each R' is independently an organic group such as one of the organic groups specified above in the definition of the term "amino." 72 or divalent (e.g., —OC(═NR)— or —C(═NR)O—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0374] The term "imino" or "imine," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group that includes the structure -C(=NR)-, where one end of the imino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as defined above in the definition of the term "amino"). 72 The moiety -C(=NR)H is also known as an "aldimine" and the moiety -C(=NR)R' (where R' is an organic group such as one of the organic groups specified above in the definition of the term "amino"). 72 An imino group is an organic group such as one of the organic groups specified in the definition of the term "amino." Another name for this group is "ketimine." An imino group is a monovalent (e.g., -C(=NR)R, where each R is independently H or an R group as defined above in the definition of the term "amino." 72and each R' is independently an organic group such as one of the organic groups specified above in the definition of the term "amino." 72 or divalent (e.g., —C(═NR)—, where R is H or R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0375] The term "imidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(=NR)S- (including its isomeric arrangements, -SC(=NR)-, unless specified to the contrary), wherein one end of the imidothioate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as set forth above in the definition of the term "amino"). 72 The imidothioate group is a monovalent (e.g., —C(═NR)SR or —SC(═NR)R, where each R is H or an R group as shown above in the definition of the term “amino.” 72 or divalent (e.g., —C(═NR)S— or —SC(═NR)—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0376] The term "thionylamino," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(S)NR- (including its isomeric arrangements of the structure -N(R)C(S)-, unless specified to the contrary), wherein one end of the thionylamino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an R as defined above in the definition of the term "amino"). 72A thionylamino group is a monovalent (e.g., —C(S)NRR or —N(R)C(S)R, where each R is H or an R as defined above in the definition of the term “amino.” 72 or divalent (e.g., —C(S)NR— or —N(R)C(S)—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0377] The term "carbonate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(O)O-, where each end of the carbonate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonate group is monovalent (e.g., -OC(O)OR', where R' is an R group as set forth above in the definition of the term "amino." 72 (e.g., —O—C(O)O—), or divalent (e.g., —O—C(O)O—).

[0378] The term "carbonothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -OC(S)O- or -OC(O)S- (including its isomeric arrangements, -SC(O)O-, unless specified to the contrary), where each end of the carbonothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonothioate group is monovalent (e.g., -OC(S)OR' or -OC(O)SR' or -SC(O)OR', where each R' is independently an R as set forth above in the definition of the term "amino." 72 (e.g., —OC(S)O— or —OC(O)S— or —SC(O)O—) or divalent (e.g., —OC(S)O— or —OC(O)S— or —SC(O)O—).

[0379] The term "carbonodithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -SC(O)S- or -OC(S)S- (including its isomeric arrangements of the structure -SC(S)O-, unless specified to the contrary), where each end of the carbonodithioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonodithioate group is monovalent (e.g., -SC(O)SR'-OC(S)SR' or -SC(S)OR', where each R' is independently an R as defined above in the definition of the term "amino." 72 (e.g., —SC(O)S— or —OC(S)S— or —SC(S)O—) or divalent (e.g., —SC(O)S— or —OC(S)S— or —SC(S)O—).

[0380] The term "carbonotrithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)S-, where each end of the carbonotrithioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonotrithioate group is monovalent (e.g., -SC(S)SR, where R is an R group as set forth above in the definition of the term "amino." 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl) or divalent (e.g., -SC(S)S-).

[0381] The term "guanidino" or "imidamide" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group having the structure -N(R)C(=NR)NR-, where each R is independently H or any of the R groups set forth above in the definition of the term "amino." 72 In the context of a group containing a guanidino structure, one of the ends of the guanidino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of a linker) and the other end is covalently bonded to H or a C atom of the same or another organic group. The guanidino group is monovalent (e.g., —N(R)C(═NR)NR—R, where each R is H or an R as defined above in the definition of the term “amino.” 72or divalent (e.g., —N(R)C(═NR)NR—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0382] The term "carbamidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)NR- (including its isomeric arrangements, -N(R)C(=NR)O-, unless specified to the contrary), in which the O terminus of the carbamidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other (N) terminus is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or R as set forth above in the definition of the term "amino"). 72 The carbamimidate group is a monovalent (e.g., —OC(═NR)NRR or —N(R)C(═NR)OR′, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(═NR)NR— or —N(R)C(═NR)O—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0383] The term "carbonimidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)O-, where each of the termini of the carbonimidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an R as set forth above in the definition of the term "amino"). 72The carbonimidate group is a monovalent (e.g., —OC(═NR)OR′, where R is one of the R groups identified above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or a divalent (e.g., —OC(═NR)O—, where R is H or R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0384] The term "carbamate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(O)NR- (including its isomeric arrangements, -N(R)C(O)O-, unless specified to the contrary), in which the O-terminus of the carbamate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or R as set forth above in the definition of the term "amino"). 72 The carbamate group is a monovalent (e.g., —OC(O)NRR or —N(R)C(O)OR′, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(O)NR— or —N(R)C(O)O—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0385] The term "carbamodithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)NR- (including its isomeric arrangements -N(R)C(S)S-, unless specified to the contrary), in which the S-terminus of the carbamodithioate is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or R as set forth above in the definition of the term "amino"). 72 The carbamodithioate group is a monovalent (e.g., —SC(S)NRR or —N(R)C(S)SR′, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —SC(S)NR— or —N(R)C(S)S—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0386] The term "carbonodithioimidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(=NR)S-, where each of the termini of the carbonodithioimidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an R as set forth above in the definition of the term "amino"). 72 The carbonodithioimidate group is a monovalent (e.g., —SC(═NR)SR′, where R is one of the R groups identified above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72or divalent (e.g., —SC(═NR)S—, where R is H or R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0387] The term "carbamimidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(=NR)NR- (including its isomeric arrangements of the structure -N(R)C(=NR)S-, unless specified to the contrary), in which the S-terminus of the carbamimidothioate is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or R as set forth above in the definition of the term "amino"). 72 The carbamimidothioate group is a monovalent (e.g., —SC(═NR)NRR or —N(R)C(═NR)SR′, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or a divalent (e.g., —SC(═NR)NR— or —N(R)C(═NR)S—, where each R is independently H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0388] The term "carbamothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including the isomeric arrangements thereof -SC(O)NR- or -OC(S)NR-, unless specified to the contrary), where the O / S terminus of the carbamothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to a C atom of the same or another organic group (where each R is independently H or an R as defined above in the definition of the term "amino"). 72 A carbamothioate group is a monovalent (e.g., —N(R)C(O)SR′ or —N(R)C(S)OR′ or —SC(O)NRR or —OC(S)NRR, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and each R' is independently an organic group such as one of the organic groups specified above in the definition of the term "amino." 72 or divalent (e.g., —N(R)C(O)S— or —N(R)C(S)O— or —SC(O)NR— or —OC(S)NR—, where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0389] The term "carbonimidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)S- (including its isomeric arrangements, -SC(=NR)O-, unless specified to the contrary), in which the O / S terminus of the carbonimidothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or R as set forth above in the definition of the term "amino"). 72The carbonimidothioate group is a monovalent (e.g., —OC(═NR)SR′ or —SC(═NR)OR′, where each R is independently H or an R group as set forth above in the definition of the term “amino.” 72 and each R' is independently an organic group such as one of the organic groups specified above in the definition of the term "amino." 72 or divalent (e.g., —OC(═NR)S— or —SC(═NR)O—, where each R is H or an R as defined above in the definition of the term “amino.” 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0390] The term "acylhydrazone," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -C(R')(=NN(R)C(O)-) (including its isomeric arrangements (-C(O)(N(R)-N=)C(R')-) unless specified to the contrary) and / or =C(=NN(R)C(O)R'), where each R' is independently an R as set forth above in the definition of the term "amino." 72 an organic group such as one of the organic groups specified in the definition; each R is H or an R as shown above in the definition of the term "amino" 72 and each end of the acylhydrazone structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In some embodiments, the acylhydrazone has the structure -C(R 25 )(=NN(R 26 )C(O)-) (Unless otherwise specified, the isomerically arranged structure (-C(O)(N(R 26 )-N=)C(R 25 )-) and / or =C(=NN(R 26 )C(O)R 25 ), where each R 25 is independently C 1-6 Alkyl, aryl and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more first-level, second-level, or third-level substituents as defined herein); 26 are independently H or C 1-6 Alkyl, aryl and aryl(C 1-6 The acylhydrazone is a hydrocarbyl group such as alkyl, which is optionally substituted (e.g., with one or more first-level, second-level, or third-level substituents as defined herein); and each of the ends of the acylhydrazone structure is covalently bonded to a C atom of an additional organic group or two further organic groups. An exemplary chemical structure of an acylhydrazone is shown below: [ka] [During the ceremony, [ka] represents the bond covalently linking the acylhydrazone to a further organic group (e.g., an alkylene group as a further component of the linker). An acylhydrazone group is monovalent (e.g., —C(R′)(═NN(R)C(O)R′) or —C(O)(N(R)—N═)C(R′)2, where each R is independently H or an R as defined above in the definition of the term “amino.” 72 and each R' is independently an organic group such as one of the organic groups specified above in the definition of the term "amino." 72 is an organic group such as one of the organic groups specified in the definition of 26 )C(O)R 25 where each R' is independently selected from the R groups listed above in the definition of the term "amino." 72 and each R is H or an R as set forth above in the definition of the term "amino." 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0391] The term "hydrazine," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -N(R)N(R)-, where each R is independently H or an R as set forth above in the definition of the term "amino." 72 and each end of the hydrazine structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In some embodiments, the hydrazine has the structure -N(R 26 )N(R 26 )-, where each R 26 are independently H or C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the ends of the hydrazine structure is covalently bonded to a C atom of an additional organic group or two further apart organic groups. The hydrazine group is monovalent (e.g., —N(R)N(R)2, where each R is H or an R as defined above in the definition of the term “amino.” 72 or a divalent (e.g., —N(R)N(R)—, where each R′ is independently an R group as defined above in the definition of the term “amino”); 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0392] The term "oxime," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure =C(=N(OH)), where each end of the oxime structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). An exemplary chemical formula of an oxime is shown below: [ka] [During the ceremony, [ka] represents the bond covalently linking the oxime to an additional organic group. An oxime group is a monovalent (e.g., —C(═N(OH))(R), where each R is H or an R as shown above in the definition of the term “amino.” 72 (wherein ═C(═N(OH))) or divalent (e.g., ═C(═N(OH))).

[0393] The term "acetal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -OCH(R')O-, where R' is one of the R groups set forth above in the definition of the term "amino." 72 and each of the two O atoms of the acetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In some embodiments, the acetal has the structure -OCH(R 25 )O—, where R 25 is C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the O atoms of the acetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups. The acetal group is monovalent (e.g., —OCH(R′)OR′, where each R′ is independently an R group as defined above in the definition of the term “amino”). 72 (e.g., —OCH(R′)O—, where R′ is an R group as set forth above in the definition of the term “amino”) or divalent (e.g., —OCH(R′)O—, where R′ is an R group as set forth above in the definition of the term “amino”). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0394] The term "hemiacetal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OCH(OH)-, where each end of the hemiacetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of the linker). A hemiacetal group is monovalent (e.g., -OCH(OH)OR', where R' is an R group as set forth above in the definition of the term "amino." 72 (e.g., —OCH(OH)—) or divalent (e.g., —OCH(OH)—).

[0395] The term "ketal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -OC(R')(R')O-, where each R' is an R group as set forth above in the definition of the term "amino." 72 and each of the O atoms of the ketal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In some embodiments, the ketal has the structure -OC(R 25 )(R 25 )O—, where each R 25 is independently C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the O atoms of the ketal structure is covalently bonded to a C atom of an additional organic group or two further apart organic groups. The ketal group is monovalent (e.g., —OC(R′)(R′)OR′, where each R′ is independently an R group as set forth above in the definition of the term “amino.” 72 or a divalent (e.g., —OC(R′)(R′)O—, where each R′ is independently selected from the R groups identified above in the definition of the term “amino”); 72(wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0396] The term "hemiketal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -OCR'(OH)-, where R' is any of the R groups set forth above in the definition of the term "amino." 72 and each of the ends of the hemiketal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In some embodiments, the hemiketal has the structure -OCR 25 (OH)—, where R 25 is C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the ends of the hemiketal structure is covalently bonded to a C atom of an additional organic group or two further apart organic groups. The hemiketal group is monovalent (e.g., —OC(R′)2(OH), where each R′ is independently an R group as set forth above in the definition of the term “amino.” 72 and R' is an organic group, such as an alkyl group, independently selected from the organic groups specified in the definition of the term "amino" or a divalent (e.g., -OCR'(OH)-, where R' is an alkyl group selected from the organic groups specified above in the definition of the term "amino"). 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0397] The term "imido," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -C(O)N(R)C(O)-, where R is any of the R groups set forth above in the definition of the term "amino." 72and each end of the imide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker). The imide group is monovalent (e.g., —C(O)N(R)C(O)R′, where R is independently H or R as defined above in the definition of the term “amino.” 72 and R' is an organic group such as one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —C(O)N(R)C(O)—, where R is H or R as shown above in the definition of the term “amino”); 72 (wherein the aryl group is an organic group such as one of the organic groups specified in the definition of aryl).

[0398] The term "acyclic," as used herein in the context of organic groups, refers to an open-chain organic group that does not contain a ring. An "open-chain" or "acyclic" organic group can be straight-chained (i.e., containing only one unbranched chain without any side chains) or branched (i.e., the main chain "contains" one or more side chains).

[0399] An organic group that is "substituted with one or more substituents" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the organic group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the one or more substituents can be selected from the first-level substituents, second-level substituents, or third-level substituents described herein.

[0400] As used herein, the term "hydrogen bond" or "H bond" refers to a non-covalent bond (in some embodiments, primarily electrostatic attraction) between (i) a hydrogen atom covalently bonded to a more electronegative atom or group and (ii) a lone pair of another electronegative atom. In some embodiments, the more electronegative atom or group includes a nitrogen atom and an oxygen atom; thus, examples of groups in which a hydrogen atom is covalently bonded to a more electronegative atom or group include an amino group, an -NH- group of an amide group, a hydroxyl group (e.g., as in an alcohol) or as part of another functional group (e.g., as part of a carboxyl (-COOH) group)), and a sulfanyl group (e.g., as in a thiol) or as part of another functional group (e.g., as part of a disulfanyl (-SSH) or thioester (-C(OSH)) group)) that bears at least one covalently bonded hydrogen atom. In some embodiments, the lone pair of another electronegative atom is the lone pair of the oxygen atom present in a carbonyl group or the lone pair of the nitrogen atom present in a primary, secondary, or tertiary amino group.

[0401] As used herein, the phrase "hydrogen bond donor" refers to an atomic, ionic, or molecular component of a hydrogen bond that provides a bridging (covalent) hydrogen atom. In some embodiments, hydrogen bond donors include amino groups, -NH- groups of amide groups, hydroxyl groups (e.g., as in alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (e.g., as in thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH)) groups)) that bear at least one covalently bonded hydrogen atom.

[0402] As used herein, the phrase "hydrogen bond acceptor" refers to an atomic, ionic, or molecular component of a hydrogen bond that does not contribute a bridging (covalent) hydrogen atom. In some embodiments, the hydrogen bond acceptor contains at least one lone pair of electrons. Examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary, and tertiary amino groups.

[0403] The term "stealth" as used herein refers to the ability of the particles described herein to not be detected, then sequestered and / or degraded, or to be barely detected, then sequestered and / or degraded, and / or to be subsequently detected, then sequestered and / or degraded, by the immune system of the host to which they are administered.

[0404] The term "phosphatidylethanolamine" refers to a compound having the following formula: [ka] or a salt thereof, wherein in each case, acyl refers to an acyl moiety (e.g., a -C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably linear). In certain embodiments, each acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moieties may be saturated or unsaturated (e.g., monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In certain embodiments, one acyl is saturated and the other is unsaturated (e.g., monounsaturated). An example of an acyl moiety is -C(O)(CH2). 16 CH3 (Stearoyl), -C(O)(CH2) 14 CH3 (palmitoyl), -C(O)(CH2) 12 CH3 (myristoyl) and -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The term "phosphatidylethanolamine moiety" refers to a monovalent radical of phosphatidylethanolamine, preferably one in which the hydrogen atom of the amino group has been removed.

[0405] The term "DSPE" refers to a compound of the formula: [ka] or a salt thereof, wherein in each case -C(O)C 17 H 35 is the moiety -C(O)(CH2) 16CH3 (stearoyl). The term "distearoylphosphatidylethanolamine moiety" refers to a monovalent radical of DSPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0406] The term "DPPE" refers to a compound of the formula: [ka] or a salt thereof, wherein in each case —C(O)C 15 H 31 is the moiety -C(O)(CH2) 14 CH3 (palmitoyl). The term "dipalmitoylphosphatidylethanolamine moiety" refers to a monovalent radical of DPPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0407] The term "DOPE" refers to a compound of the formula: [ka] or a salt thereof, wherein in each case —C(O)C 17 H 33 refers to the moiety -cis-C(O)(CH)-CH=CH-(CH)CH (oleoyl). The term "dioleoylphosphatidylethanolamine moiety" refers to the monovalent radical of DOPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0408] The term "POPE" refers to a group having the formula: [ka] or a salt thereof, wherein —C(O)C 15 H 31 is the moiety -C(O)(CH2) 14 CH3 (palmitoyl); and -C(O)C 17 H 33refers to the moiety -cis-C(O)(CH)-CH=CH-(CH)CH (oleoyl). The term "palmitoyloleoylphosphatidylethanolamine moiety" refers to the monovalent radical of POPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0409] The term "tocopherol" refers to a compound of the formula: [ka] (i.e., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol), where R t1 and R t2 Each of R is independently H or methyl. t1 and R t2 are both methyl; in β-tocopherol, R t1 is methyl and R t2 is H; in γ-tocopherol, R t1 is H and R t2 is methyl; and in δ-tocopherol, R t1 and R t2 are both H. The term "tocopherol moiety" or "tocopheryl moiety" means a monovalent radical of tocopherol, preferably one in which the hydrogen atom of the hydroxy group has been removed.

[0410] The term "DAG" refers to a DAG with the following formula: [ka] or a salt thereof, wherein in each case acyl refers to an acyl moiety (e.g., a -C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably linear). In certain embodiments, each acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moieties may be saturated or unsaturated (e.g., monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In certain embodiments, one acyl is saturated and the other is unsaturated (e.g., monounsaturated). An example of an acyl moiety is -C(O)(CH2). 16 CH3 (Stearoyl), -C(O)(CH2) 14 CH3 (palmitoyl), -C(O)(CH2) 12 For example, DMG is 1,2-dimyristoylglycerol, i.e., both acyl groups are -C(O)(CH). 12 The term "diacylglyceride moiety" refers to a diacylglyceride of the above formula, which is CH3 (myristoyl). The term "diacylglyceride moiety" refers to a monovalent radical of a diacylglyceride, preferably one in which the hydrogen atom of a hydroxy group has been removed.

[0411] The term "DAA" refers to a dialkylamine or salt thereof having the formula HN(alkyl), wherein each alkyl moiety is preferably linear. In some embodiments, each alkyl moiety has at least 8 carbon atoms. Preferably, each alkyl moiety is an alkyl moiety of a fatty acid alcohol, more preferably, each alkyl moiety is an alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. An example of an alkyl moiety is -(CH). 17 CH3 (Stearyl), -(CH2) 15 CH3 (palmityl) and -(CH2) 13 For example, DMA is 1,2-dimyristylamine, i.e., both alkyl groups are -(CH). 13means a dialkylamine of the above formula, CH3 (myristyl). The term "dialkylamine moiety" means a monovalent radical of a dialkylamine, preferably one in which the hydrogen atom of the amino group has been removed.

[0412] The term "ceramide" refers to a compound having the formula: [ka] or a salt thereof, wherein -C 13 H 27 is the moiety -(CH2) 12 and acyl refers to an acyl moiety (e.g., a -C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably linear). In some embodiments, the acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety can be saturated or unsaturated (e.g., monounsaturated). An example of an acyl moiety is -C(O)(CH) 16 CH3 (Stearoyl), -C(O)(CH2) 14 CH3 (palmitoyl), -C(O)(CH2) 12 C(O)(CH)-CH=CH-(CH)CH (oleoyl). For example, palmitoyl ceramide has an acyl group of -C(O)(CH) 14 The term "ceramide moiety" refers to a monovalent radical of ceramide, preferably one in which the hydrogen atom of a hydroxy group (preferably the hydrogen of the terminal (primary) hydroxy group) has been removed.

[0413] The term "MAA" refers to a monoalkylamine or salt thereof having the formula HN(alkyl), wherein the alkyl moiety is preferably linear. In some embodiments, the alkyl moiety has at least 8 carbon atoms. Preferably, the alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. An example of an alkyl is -(CH)17 CH3 (Stearyl), -(CH2) 15 CH3 (palmityl) and -(CH2) 13 For example, MMA is myristylamine, i.e., the alkyl group is -(CH2). 13 means a monoalkylamine of the above formula, which is CH3 (myristyl). The term "monoalkylamine moiety" means a monovalent radical of a monoalkylamine, preferably one in which one of the hydrogen atoms of the amino group has been removed.

[0414] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded molecules and as linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present invention, means that a nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) for DNA or in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0415] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0416] The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are commonly abbreviated to the single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly referred to as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides may be found in both RNA and DNA therapies. In RNA, they are represented as A, C, and G, while in DNA, they are represented as dA, dC, and dG.

[0417] The modified purine (A or G) or pyrimidine (C, T or U) base moiety preferably contains one or more alkyl groups, more preferably one or more C 1-4 More preferably, the base moiety is modified with an alkyl group, and even more preferably with one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0418] In some embodiments of all aspects of the invention, the nucleic acid is DNA.

[0419] As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA, e.g., partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the ends of DNA. It is also contemplated herein that the nucleotides of DNA may be chemically synthesized nucleotides or non-standard nucleotides, such as ribonucleotides. In the context of the present invention, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0420] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0421] RNA In some embodiments of all aspects of the invention, the nucleic acid is RNA.

[0422] According to the present invention, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In a preferred embodiment, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA. It is also contemplated herein that the nucleotides of the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the context of the present invention, these modified / modified nucleotides (or modified nucleosides) may be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA containing such modified / modified nucleotides or nucleosides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0423] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (e.g., small activating RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0424] In a preferred embodiment, RNA comprises an open reading frame (ORF) that encodes a peptide, polypeptide or protein. The RNA can express the encoded peptide, polypeptide or protein. For example, the RNA can be the RNA that encodes and expresses a pharmacologically active peptide or protein. In some embodiments, RNA can interact with cellular translation machinery and enable the translation of peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), or can secrete or produce the encoded peptide or protein on the surface. Alternatively, RNA can be non-coding RNA, such as antisense RNA, microRNA (miRNA) or siRNA.

[0425] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0426] mRNA In some embodiments of all aspects of the invention, the nucleic acid is mRNA.

[0427] According to the present invention, the term "mRNA" refers to "messenger RNA" and includes "transcripts" that can be produced using a DNA template. Generally, mRNA encodes a peptide, polypeptide, or protein. Typically, mRNA comprises a 5' UTR, a peptide / protein coding region, and a 3' UTR. In the present invention, mRNA is preferably produced by in vitro transcription (IVT) from a DNA template. As mentioned above, in vitro transcription methods are known to those skilled in the art, and a variety of in vitro transcription kits are commercially available.

[0428] Although mRNA is single stranded, it may contain self-complementary sequences that allow it to fold back on itself and pair with itself to form a double helix.

[0429] According to the present invention, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0430] In a preferred embodiment of the present invention, mRNA relates to an RNA transcript that encodes a peptide, polypeptide or protein.

[0431] In one embodiment, preferably the RNA encoding the peptide, polypeptide or protein has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0432] As is well-known in the art, RNA (e.g., mRNA) generally comprises a 5' untranslated region (5' UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3' UTR). In some embodiments, RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. In some embodiments, RNA (e.g., mRNA) is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art and are described, for example, in Molecular Cloning: A Laboratory Manual, 2000; nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Additionally, a variety of in vitro transcription kits are available, for example, from Thermo Fisher Scientific (e.g., Transcription Aid TM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (e.g., HiScribe TM T7 Kit, HiScribe TM T7 ARCA mRNA kit), Promega (e.g., RiboMAX TM , HeLaScribe®, Riboprobe® systems), Jena Bioscience (e.g., SP6 or T7 transcription kits) and Epicentre (e.g., AmpliScribe TM ) For reference to modified RNA (e.g., mRNA), correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription) or modifications can be made and / or added to the mRNA post-transcriptionally.

[0433] In some embodiments, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for transcription control can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and then introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0434] In certain embodiments of the present invention, the RNA (e.g., mRNA) is a "replicon RNA" (e.g., a "replicon mRNA") or simply a "replicon," particularly a "self-replicating RNA" (e.g., a "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (e.g., a self-replicating mRNA) is derived from or contains elements from a ssRN virus, particularly a positive-strand sRN virus such as an alphavirus. Alphaviruses are a typical example of a positive-strand RN virus. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (virion formation). There are typically two open reading frames (ORFs) in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded by a first ORF that together initiates near the 5' end of the genome, while the alphavirus structural proteins are encoded by a second ORF that together is found downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, roughly in a 2:1 ratio. In cells infected with alphaviruses, only the nucleic acid sequences encoding nonstructural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules that mimic eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124).Following infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts like messenger RNA to directly translate an open reading frame encoding the nonstructural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced with an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems utilize alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the name "trans-replication system"). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated in trans by the replicase must contain certain alphavirus sequence elements to enable recognition by the alphavirus replicase and RNA synthesis.

[0435] In certain embodiments of the present invention, the RNA (e.g., mRNA) described herein (e.g., included in the compositions and / or used in the methods of the present invention) contains one or more modifications, e.g., to increase stability and / or translation efficiency and / or reduce cytotoxicity. For example, to increase expression of an RNA (e.g., mRNA), modifications can be made within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without changing the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO2007 / 036366 and PCT / EP2019 / 056502, and include: 5' cap structures; extension or shortening of naturally occurring poly(A) tails; alteration of the 5' and / or 3' untranslated regions (UTRs), such as the introduction of UTRs unrelated to the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the G / C content of the RNA). According to the present invention, the term "modified" in the context of modified mRNA preferably relates to any modification of the mRNA that does not naturally occur in said RNA (e.g. mRNA).

[0436] In some embodiments, the RNA (e.g., mRNA) described herein comprises a 5' cap structure. In some embodiments, the mRNA does not have an uncapped 5'-triphosphate. In some embodiments, the RNA (e.g., mRNA) described herein comprises a conventional 5' cap and / or a 5' cap analog. The term "conventional 5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp) attached at the triphosphate moiety to the 5' end of the next nucleotide in the mRNA (i.e., the guanosine is attached to the remainder of the mRNA via a 5'-5' triphosphate linkage). Guanosine is an N 7 can be methylated at the cap structure m 7 The term "5' cap analog" is based on the conventional 5' cap, but to avoid incorporation of the 5' cap analog in the reverse orientation, 7This refers to a 5' cap modified at the 2' or 3' position of the guanosine structure (such 5' cap analogs are also referred to as anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as phosphorothioate-modified 5' cap analogs of the β-phosphate (e.g., m2 ), as described in PCT / EP2019 / 056502. 7,2’O G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Addition of a 5' cap structure as described herein to an RNA (e.g., mRNA) can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, where the 5' cap structure is co-transcriptionally incorporated into the produced RNA (e.g., mRNA) strand, or the RNA (e.g., mRNA) can be produced, for example, by in vitro transcription, and the 5' cap structure can be post-transcriptionally attached to the mRNA using a capping enzyme, e.g., vaccinia virus capping enzyme.

[0437] In some embodiments, the RNA (e.g., mRNA) is 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G and m2 7,3’-O Gppp(m1 2’-O In some embodiments, the RNA encoding a peptide, polypeptide, or protein comprising an antigen or epitope comprises a 5' cap structure selected from the group consisting of m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer).

[0438] In some embodiments, the RNA (e.g., mRNA) comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present invention, the term "cap 0" refers to the structure "m 7 GpppN" where N is any nucleoside bearing an OH moiety at the 2' position. According to the present invention, the...

Claims

1. A composition comprising: (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) (a) a polymer comprising the following general formula (I): and (b) a polymer conjugate compound comprising one or more hydrophobic chains: 【Chemical 1】 [During the ceremony, X ２ and X １ together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester; Y is -CH ２ -, -(CH ２ ) ２ - or -(CH ２ ) ３ - and; z is 2 to 24; and n is 1 to 100.

2. (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ - If so, X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ - If so, X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; (vi) X １ is -O-, then X ２ is —C(O)—; (vii) X １ If is -C(S)-, then X ２ is —O—; (viii) X １ is -O-, then X ２ is -C(S)-; (ix) X １ is -C(O)-, then X ２ is -S-; or (x)X １ If is -S-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ alkyl; preferably (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ - If so, X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ - If so, X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; or (vi) X １ is -O-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl, The composition of claim 1.

3. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ is hydrogen or C １－８ 3. The composition of claim 1 or 2, wherein the alkyl is alkyl.

4. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ The composition of any one of claims 1 to 3, wherein is hydrogen or methyl.

5. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ The composition of any one of claims 1 to 4, wherein is hydrogen.

6. Y is -CH ２ - or -(CH ２ ) ２ The composition of any one of claims 1 to 5, wherein

7. Y is -CH ２ The composition of any one of claims 1 to 6, wherein

8. The polymer has the following general formula (II): 【Chemistry 2】 [In the formula, R １ is hydrogen or C １－８ It is alkyl. The composition of any of claims 1 to 7, comprising:

9. 9. The composition of any one of claims 1 to 8, wherein z is 2 to 10, for example 2 to 7.

10. 10. The composition of claim 1, wherein z is 2 to 5.

11. 11. The composition of any one of claims 1 to 10, wherein z is 2 or 3.

12. 12. The composition of any of claims 1 to 11, wherein z is 2.

13. The polymer has the following general formula (III): 【Chemistry 3】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

13. The composition of any of claims 1 to 12, comprising:

14. R １ The composition of any of claims 8 to 13, wherein is hydrogen or methyl.

15. R １ The composition of any of claims 8 to 14, wherein is hydrogen.

16. The polymer has the following general formula (IV): 【Chemistry 4】 16. The composition of any of claims 1 to 15, comprising:

17. 17. The composition of any one of claims 1 to 16, wherein n is 5 to 50.

18. 18. The composition of any one of claims 1 to 17, wherein n is 5 to 25.

19. 19. The composition of any preceding claim, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

20. One or more hydrophobic chains are attached to the X １ Terminal or X ２ The composition of any one of claims 1 to 19, wherein the composition is terminally located.

21. 21. The composition of any preceding claim, wherein the one or more hydrophobic chains are acyclic, preferably straight-chain, hydrocarbyl groups, more preferably having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.

22. The polymer conjugate compound has the following general formula (V) or (V'): 【Chemistry 5】 [During the ceremony, X ２ and X １ together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester; Y is -CH ２ -, -(CH ２ ) ２ - or -(CH ２ ) ３ - and; R ２ is a moiety comprising one or more hydrophobic chains; R ３ H, C １－６ Alkyl, C ２－６ Alkynyl, -OR ２０ , -SR ２０ , halogen, -CN, -N ３ , -OC(O)R ２１ , -C(O)R ２１ , -NR ２２ R ２３ , -COOH, -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２０ H, C １－３ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－３ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.

22. The composition of any of claims 1 to 21, comprising:

23. (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ - If so, X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ - If so, X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; (vi) X １ is -O-, then X ２ is —C(O)—; (vii) X １ If is -C(S)-, then X ２ is —O—; (viii) X １ is -O-, then X ２ is -C(S)-; (ix) X １ is -C(O)-, then X ２ is -S-; or (x)X １ If is -S-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl preferably (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ - If so, X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ - If so, X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; or (vi) X １ is -O-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl, 23. The composition of claim 22.

24. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ is hydrogen or C １－８ 24. The composition of claim 22 or 23, wherein the alkyl is alkyl.

25. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ The composition of any of claims 22 to 24, wherein is hydrogen or methyl.

26. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ 26. The composition of any of claims 22 to 25, wherein is hydrogen.

27. Y is -CH ２ - or -(CH ２ ) ２ The composition of any one of claims 22 to 26, wherein

28. Y is -CH ２ The composition of any one of claims 22 to 27, wherein

29. The polymer conjugate compound has the following general formula (VI) or (VI'): 【Chemistry 6】 [In the formula, R １ is hydrogen or C １－８ It is alkyl. The composition of any of claims 22 to 28, comprising:

30. 30. The composition of any of claims 22 to 29, wherein z is 2 to 10, for example 2 to 7.

31. 31. The composition of any of claims 22 to 30, wherein z is 2 to 5.

32. 32. The composition of any of claims 22 to 31, wherein z is 2 or 3.

33. 33. The composition of any of claims 22 to 32, wherein z is 2.

34. The following general formula (VII) or (VII'): 【Chemistry 7】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

34. The composition of any of claims 22 to 33, comprising:

35. R １ The composition of any of claims 29 to 34, wherein is hydrogen or methyl.

36. R １ 36. The composition of any of claims 29 to 35, wherein is hydrogen.

37. The polymer conjugate compound has the following general formula (VIII) or (VIII'): 【Chemistry 8】 37. The composition of any of claims 22 to 36, comprising:

38. 38. The composition of any of claims 22 to 37, wherein n is 5 to 50.

39. 39. The composition of any of claims 22 to 38, wherein n is 5 to 25.

40. 40. The composition of any of claims 22 to 39, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

41. R ２ is R ４ or -L １ (R ４ ) ｐ where each ４ are independently hydrophobic chains such as hydrocarbyl groups; L １ The composition of any of claims 22 to 40, wherein: is a linker; and p is 1 or 2.

42. L １ contains at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety, and / or the alkylene group is R ４ and wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, ester, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, and / or each divalent functionalized moiety is independently selected from ketal, hemiketal, imide, and amide moieties; and / or each divalent functionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide , carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

43. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-, [*-NHC(O)] ｐ (C １－６ -alkylene)-, [*-C(O)NH] ｐ (C １－６ -alkylene)-, [*-S] ｐ (C １－６ -alkylene)-, [*-SS] ｐ (C １－６ -alkylene)-, [*-S(O) ２ ] ｐ (C １－６ -alkylene)-, [(*-O) ｒ C(OR ２５ ) ３－ｒ ](C １－６ -alkylene)-, [*-C(OR ２５ ) ２ O] ｐ (C １－６ -alkylene)-, [*-C(R ２５ )(=N-N(R ２６ )C(O)-)] ｐ (C １－６ -alkylene)-, [*-C(O)(N(R ２６ )-N=)C(R ２５ )-] ｐ (C １－６ -alkylene)-, [*=C(=N-N(R ２６ )C(O)(R ２５ ))] ｐ (C １－６ -alkylene)-, [*-N(R ２６ )N(R ２６ )] ｐ (C １－６ -alkylene)-, [*=C(=N(OH))] ｐ (C １－６ -alkylene)-, [*-OC(R ２５ )(R ２６ )O] ｐ (C １－６ -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) ｐ N(R ２６ ) ２－ｐ and [*-C(O)NH](C １－６ -alkyltriyl)-, where * is R ４ represents the point of attachment to １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２５ is C １－６ Alkyl, aryl and aryl(C １－６ alkyl); R ２６ H, C １－６ Alkyl, aryl and aryl(C １－６ alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ 43. The composition of claim 41 or 42, which directly binds to

44. L １ further comprises at least one additional bifunctional moiety through which R ２ is X in formula (V) １ or X in formula (V') ２ The composition of any one of claims 41 to 43, which binds to

45. At least one further difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide). , carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein L １ further comprises at least two additional bifunctional moieties, these at least two additional bifunctional moieties are optionally C １－６ The composition of claim 44, wherein the alkylene groups separate the aryl groups.

46. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )-O(C １－６ -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] ｐ (C １－６ -alkylene)O-, [*-OC(O)] ｐ (C １－６ -alkylene)O-, (*-) ｐ N(R ２６ ) ２－ｐ and [*-C(O)NH](C １－６ -alkyltriyl)O—, where * is R ４ represents the point of attachment to [*-C(O)O]; p is 1 or 2; ｐ (C １－６ -alkylene), [*-OC(O)] ｐ (C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene) and [*-C(O)NH] ｐ (C １－６ - alkylene) C １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ H, C １－６ Alkyl, aryl and aryl(C １－６ alkyl); R ２７ H, C １－６ Alkyl, aryl, aryl(C １－６ alkyl) and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ The composition of any one of claims 41 to 45, which directly binds to

47. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )-O(C １－６ -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] ｐ (C １－６ -alkylene)O-, [*-OC(O)] ｐ (C １－６ -alkylene)O-, (*-) ２ N- and [*-C(O)NH](C １－６ -alkyltriyl)O— or L １ (*-)(R ２６ )N-, where * is R ４ represents the point of attachment to [*-C(O)O]; p is 1 or 2; ｐ (C １－６ -alkylene), [*-OC(O)] ｐ (C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene) and [*-C(O)NH] ｐ (C １－６ - alkylene) C １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ is H and C １－６ alkyl; R ２７ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ The composition of any one of claims 41 to 46, which directly binds to

48. R ２ [R ４ C(O)O] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene), [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene), [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )-O(C １－３ -alkylene)C(O)-, (2-R ４ -3,4-dihydro-2H-chromen-6-yl)O-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)O-, [*-OC(O)] ｐ (C ２－３ -alkylene)O-, (R ４ ) ２ N- and [R ４ C(O)NH](C ２－３ -alkyltriyl)O- or R ２ (R ４ )(R ２６ )N-, where p is 1 or 2; C ２－３ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ is H and C １－６ alkyl; R ２７ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C ２－３ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ The composition of any one of claims 22 to 47, which is directly bound to

49. R ２ is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or R ２ The composition of any of claims 22-48, wherein is a monoalkylamine moiety.

50. Each R ４ 50. The composition of any of claims 41-49, wherein is independently an acyclic, preferably straight-chain, hydrocarbyl group.

51. Each R ４ The composition of any of claims 41 to 50, wherein are independently hydrocarbyl groups having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.

52. R ２ is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or R ２ 52. The composition of any of claims 22-51, wherein is a monomyristylamine moiety.

53. R ３ H, C １－６ Alkyl, C ２－６ Alkynyl, —C(O)R ２１ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ 53. The composition of any of claims 22 to 52, substituted with one or more substituents independently selected from the group consisting of: sugars, amino acids, peptides and members of targeting pairs.

54. R ３ H, C １－３ Alkyl, C ２－６ Alkynyl, —C(O)R ２１ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and one or more substituents independently selected from the group consisting of members of a targeting pair; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ 54. The composition of any of claims 22-53, substituted with one or more substituents independently selected from the group consisting of: and members of a targeting pair.

55. R ３ is H, -C(O)(C １－３ alkyl), -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , —C(O)NH ２ , —C(O)NHCH ３ , —C(O)NH(CH ２ ) ２ NH ２ 55. The composition of any of claims 22-54, substituted with one or more substituents independently selected from the group consisting of: and members of a targeting pair.

56. The targeting pair is selected from the following pairs: maleimide-thiol; thiol-alkyl halide (especially brominated); azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-alpha ｖ β ３ 56. The composition of any of claims 22 to 55, comprising an integrin; an asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; or a galactose-asialoglycoprotein receptor.

57. The polymer conjugate compound has the formula: 【Chemistry 9】 【Chemistry 10】 [During the ceremony, n is 5 to 25; R ３ is H, -C(O)(C １－３ alkyl) and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , —C(O)NH ２ , —C(O)NHCH ３ , —C(O)NH(CH ２ ) ２ NH ２ and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R ２７ is H or a countercation; and In either case, -C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl), and in either case -C(O)C １５ H ３１ is the moiety -C(O)(CH ２ ) １４ CH ３ (palmitoyl), and in either case -C(O)C １３ H ２７ is the moiety -C(O)(CH ２ ) １２ CH ３ (myristoyl), in either case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (myristyl), in either case -C １３ H ２７ is the part -(CH ２ ) １２ CH ３ In either case, -C(O)C １７ H ３３ The moiety -cis-C(O)(CH ２ ) ７ -CH=CH-(CH ２ ) ７ CH ３ (Oleoyl).

57. The composition of any of claims 1 to 56, comprising one of:

58. 58. The composition of claim 57, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14 or 16.

59. R ３ is H or -C(O)(C １－３ alkyl), where C １－３ The alkyl group optionally is 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N ３ , C ２－６ 60. The composition of claim 57 or 58, substituted with one substituent selected from the group consisting of alkynyl, antigen, or antibody.

60. The polymer conjugate compound has the formula: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 [wherein in each case -C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl), and in either case -C(O)C １５ H ３１ is the moiety -C(O)(CH ２ ) １４ CH ３ (palmitoyl), and in either case -C(O)C １３ H ２７ is the moiety -C(O)(CH ２ ) １２ CH ３ (myristoyl), in either case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (myristyl), in either case -C １３ H ２７ is the part -(CH ２ ) １２ CH ３ and in each case n is 8, 10, or 14.

60. The composition of any of claims 1 to 59, comprising one of:

61. The polymer conjugate compound has the formula: (i) 【Chemistry 14】 wherein n is 14 and in each case is —C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (Stearoyl) (ii) 【Chemistry 15】 wherein n is 14 and in each case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (Myristyl). (iii) 【Chemistry 16】 wherein n is 8, 12, 14, or 16.

61. The composition of any of claims 1 to 60, comprising one of:

62. 62. The composition of any of claims 1-61, wherein the composition is substantially free of lipids or lipid-like substances comprising polyethylene glycol (PEG), wherein PEG has one having at least 30 consecutive ethylene glycol repeat units.

63. 63. The composition of any of claims 1 to 62, wherein water is the major component of the composition and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

64. 64. The composition of any of claims 1 to 63, wherein the concentration of the nucleic acid in the composition is from about 1 mg / L to about 500 mg / L, for example from about 1 mg / L to about 100 mg / L, from about 5 mg / L to about 100 mg / L, or from about 10 mg / L to about 100 mg / L.

65. 65. The composition of any of claims 1-64, wherein the cationically ionizable lipid comprises a head group comprising at least one tertiary amine moiety.

66. The cationic or cationically ionizable lipid is represented by the formula (X) 【Chemistry 17】 [During the ceremony, L １０ and L ２０ One of the groups 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 １０ and L ２０ 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 are each independently unsubstituted C １ -C １２ Alkylene or C ２－１２ alkenylene; G ３ is C １－２４ Alkylene, C ２－２４ Alkenylene, C ３－８ Cycloalkylene or C ３－８ is cycloalkenylene; R ａ is H or C １－１２ is alkyl; R ３５ and R ３６ are each independently C ６－２４ Alkyl or C ６－２４ alkenyl; R ３７ H, OR ５０ , CN, -C(=O)OR ４０ , -OC(=O)R ４０ or -NR ５０ C(=O)R ４０ and R ４０ is C １－１２ is alkyl; R ５０ is H or C １－６ is alkyl; and x is 0, 1 or 2.

66. The composition of any of claims 1 to 65, having the structure of: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

67. The cationic or cationically ionizable lipid has the formula (XI): 【Chemistry 18】 [During the ceremony, R １ and R ２ Each of the groups independently represents R ５ or -G １ -L １ -R ６ where R １ and R ２ At least one of the groups is -G １ -L １ -R ６ and R ３ and R ４ Each of these is C １－６ Alkyl, C ２－６ Alkenyl, aryl and C ３－１０ independently selected from the group consisting of cycloalkyl; R ５ and R ６ each independently is an acyclic hydrocarbyl group having at least 10 carbon atoms; G １ and G ２ each independently being an unsubstituted C １－１２ Alkylene or C ２－１２ alkenylene; L １ and L ２ Each of these is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ independently selected from the group consisting of -, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-; Ra is H or C １－１２ is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1 or 2.

66. The composition of any of claims 1 to 65, having the structure:

68. Cationic or cationic ionizable lipids include 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), and N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP).

66. The composition of any of claims 1-65, comprising dimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

69. 69. The composition of any of claims 1-68, wherein the cationic or cationically ionizable lipid comprises from about 20 mol% to about 80 mol% of the total lipid present in the composition.

70. 70. The composition of any of claims 1 to 69, further comprising one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids and combinations thereof, more preferably a combination of phospholipids and steroids.

71. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 71. The composition of claim 70, wherein the phosphatidylethanolamine is selected from the group consisting of 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE).

72. 72. The composition of claim 70 or 71, comprising from about 5 mol% to about 30 mol% of the total lipids present in the phospholipid composition.

73. 73. The composition of any of claims 70 to 72, wherein the steroid comprises a sterol such as cholesterol.

74. 74. The composition of any of claims 70 to 73, wherein the steroid comprises from about 10 mol% to about 60 mol% of the total lipids present in the composition.

75. 75. The composition of any of claims 70-74, wherein the cationic or cationically ionizable lipid comprises about 20 mol% to about 70 mol% of the total lipid present in the composition; the polymer conjugate compound comprises about 0.5 mol% to about 15 mol% of the total lipid present in the composition; the phospholipid comprises about 5 mol% to about 25 mol% of the total lipid present in the composition; and the steroid comprises about 20 mol% to about 55 mol% of the total lipid present in the composition.

76. 76. The composition of any of claims 1-75, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationic or cationically ionizable lipid, and at least a portion of a polymer-conjugated compound.

77. 77. The composition of claim 76, wherein the particle is selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.

78. 78. The composition of claim 76 or 77, wherein the particles constitute at least 50%, preferably at least 75%, more preferably at least 85% of the nucleic acids present in the composition.

79. 79. The composition of any of claims 76-78, wherein the particles have a size of about 30 nm to about 500 nm.

80. 80. The composition of any of claims 1 to 79, wherein the nucleic acid is RNA, preferably mRNA.

81. 81. The composition of claim 80, wherein the RNA (1) comprises modified nucleosides in place of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (2) has a codon-optimized coding sequence; and / or (3) has a coding sequence with an increased G / C content compared to a wild-type coding sequence.

82. 82. The composition of claim 80 or 81, wherein the RNA comprises at least one, and preferably all, of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

83. 83. The composition of claim 82, wherein the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

84. The composition of claim 82 or 83, wherein the 5' cap is a cap 1 or cap 2 structure.

85. 85. The composition of any of claims 80 to 84, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and / or comprise an epitope for inducing an immune response against an antigen in a subject.

86. 86. The composition of claim 85, wherein the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a protein, an immunogenic variant of the protein, or an immunogenic fragment of the protein or an immunogenic variant thereof of a pathogen.

87. 87. A method of delivering a nucleic acid to a cell in a subject, comprising administering to the subject a composition of any of claims 1-86.

88. 87. A method for delivering a therapeutic peptide or protein to a subject, comprising administering to the subject the composition of any of claims 1-86, wherein the nucleic acid encodes the therapeutic peptide or protein.

89. 87. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of any of claims 1-86, wherein delivery of a nucleic acid to cells of the subject is beneficial to the treatment or prevention of the disease or disorder.

90. 100. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject the composition of any of claims 1-86, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

91. 91. The method of any of claims 87 to 90, wherein the subject is a mammal.

92. 92. The method of claim 91, wherein the mammal is a human.

93. (a) a compound of the following general formula (I): 【Chemistry 19】 [During the ceremony, X ２ and X １ together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester; Y is -CH ２ -, -(CH ２ ) ２ - or -(CH ２ ) ３ - and; z is 2 to 24; and n is 1 to 100. and (b) one or more hydrophobic chains.

94. (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ -If it is, then X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ -If it is, then X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; (vi) X １ is -O-, then X ２ is —C(O)—; (vii) X １ If is -C(S)-, then X ２ is —O—; (viii) X １ is -O-, then X ２ is -C(S)-; (ix) X １ is -C(O)-, then X ２ is -S-; or (x)X １ If is -S-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl preferably (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ -If it is, then X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ - If so, X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; (vi) X １ is -O-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl, 94. The polymer conjugate compound of claim 93.

95. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ is hydrogen or C １－８ 95. The polymer conjugate compound of claim 93 or 94, wherein said alkyl is alkyl.

96. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ 96. The polymer conjugate compound of any of claims 93-95, wherein is hydrogen or methyl.

97. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ 97. The polymer conjugate compound of any of claims 93-96, wherein is hydrogen.

98. Y is -CH ２ - or -(CH ２ ) ２ 98. The polymer conjugate compound of any one of claims 93 to 97, wherein

99. Y is -CH ２ The polymer conjugate compound of any of claims 93 to 98, wherein

100. The polymer has the following general formula (II): 【Chemistry 20】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

100. The polymer conjugate compound of any of claims 93-99, comprising:

101. 101. The polymer conjugate compound of any of claims 93 to 100, wherein z is 2 to 10, such as 2 to 7.

102. 102. The polymer conjugate compound of any of claims 93-101, wherein z is 2-5.

103. 103. The polymer conjugate compound of any of claims 93-102, wherein z is 2 or 3.

104. 104. The polymer conjugate compound of any of claims 93-103, wherein z is 2.

105. The polymer has the following general formula (III): 【Chemical 21】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

105. The polymer conjugate compound of any of claims 93-104, comprising:

106. R １ 106. The polymer conjugate compound of any of claims 100-105, wherein is hydrogen or methyl.

107. R １ 107. The polymer conjugate compound of any of claims 100-106, wherein is hydrogen.

108. The polymer has the following general formula (IV): 【Chemical 22】 108. The polymer conjugate compound of any of claims 93 to 107, comprising:

109. The polymer conjugate compound of any of claims 93 to 108, wherein n is 5 to 50.

110. 110. The polymer conjugate compound of any one of claims 93 to 109, wherein n is 5 to 25.

111. 111. The polymer conjugate compound of any of claims 93 to 110, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

112. One or more hydrophobic chains are attached to the X １ Terminal or X ２ The polymer conjugate compound of any one of claims 93 to 111, wherein the polymer conjugate compound is located at a terminal end.

113. 113. The polymer conjugate compound of any of claims 93 to 112, wherein one or more hydrophobic chains are acyclic, preferably straight chain, hydrocarbyl groups, more preferably having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.

114. The following general formula (V) or (V'): 【Chemical 23】 [During the ceremony, X ２ and X １ together are optionally substituted amides, optionally substituted thioamides, esters, and thioesters; Y is -CH ２ -, -(CH ２ ) ２ - or -(CH ２ ) ３ - and; R ２ is a moiety comprising one or more hydrophobic chains; R ３ H, C １－６ Alkyl, C ２－６ Alkynyl, -OR ２０ , -SR ２０ , halogen, -CN, -N ３ , -OC(O)R ２１ , -C(O)R ２１ , -NR ２２ R ２３ , -COOH, -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２０ H, C １－３ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－３ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.

114. The polymer conjugate compound of any of claims 93 to 113, comprising:

115. (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ -If it is, then X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ -If it is, then X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; (vi) X １ is -O-, then X ２ is —C(O)—; (vii) X １ If is -C(S)-, then X ２ is —O—; (viii) X １ is -O-, then X ２ is -C(S)-; (ix) X １ is -C(O)-, then X ２ is -S-; or (x)X １ If is -S-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl preferably (i) X １ is -C(O)-, then X ２ Ga-NR １ - and; (ii) X １ Ga-NR １ -If it is, then X ２ is —C(O)—; (iii) X １ If is -C(S)-, then X ２ Ga-NR １ - and; (iv) X １ Ga-NR １ -If it is, then X ２ is -C(S)-; (v) X １ is -C(O)-, then X ２ is —O—; or (vi) X １ is -O-, then X ２ is —C(O)—; Here, R １ is hydrogen or C １－８ is alkyl, 115. The polymer conjugate compound of claim 114.

116. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ is hydrogen or C １－８ 116. The polymer conjugate compound of claim 114 or 115, wherein said alkyl is alkyl.

117. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ 117. The polymer conjugate compound of any of claims 114-116, wherein is hydrogen or methyl.

118. X １ is —C(O)—, and X ２ Ga-NR １ -, where R １ 118. The polymer conjugate compound of any of claims 114-117, wherein is hydrogen.

119. Y is -CH ２ - or -(CH ２ ) ２ The polymer conjugate compound of any of claims 114 to 118, wherein

120. Y is -CH ２ 120. The polymer conjugate compound of any one of claims 114 to 119, wherein

121. The compound represented by the following general formula (VI) or (VI'): 【Chemistry 24】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

121. The polymer conjugate compound of any of claims 114-120, comprising:

122. 122. The polymer conjugate compound of any of claims 114-121, wherein z is 2 to 10, such as 2 to 7.

123. 123. The polymer conjugate compound of any of claims 114-122, wherein z is 2-5.

124. 124. The polymer conjugate compound of any of claims 114-123, wherein z is 2 or 3.

125. 125. The polymer conjugate compound of any of claims 114-124, wherein z is 2.

126. The following general formula (VII) or (VII'): 【Chemistry 25】 [In the formula, R １ is hydrogen or C １－８ It is alkyl.

126. The polymer conjugate compound of any of claims 114-125, comprising:

127. R １ 127. The polymer conjugate compound of any of claims 121-126, wherein is hydrogen or methyl.

128. R １ 128. The polymer conjugate compound of any of claims 121-127, wherein is hydrogen.

129. The following general formula (VIII) or (VIII'): 【Chemical 26】 129. The polymer conjugate compound of any of claims 114-128, comprising:

130. 130. The polymer conjugate compound of any one of claims 114 to 129, wherein n is 5 to 50.

131. 131. The polymer conjugate compound of any of claims 114-130, wherein n is 5 to 25.

132. 132. The polymer conjugate compound of any of claims 114 to 131, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

133. R ２ is R ４ or -L １ (R ４ ) ｐ where each ４ are independently hydrophobic chains such as hydrocarbyl groups; L １ is a linker; and p is 1 or 2.

134. L １ contains at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety, and / or the alkylene group is R ４ and wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, thioisopropyl ... ester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemithioate and / or each divalent functionalized moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, 134. The polymer conjugate compound of claim 133, wherein the aryl group is independently selected from a carboxylate, a carboxyl group ...

135. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-, [*-NHC(O)] ｐ (C １－６ -alkylene)-, [*-C(O)NH] ｐ (C １－６ -alkylene)-, [*-S] ｐ (C １－６ -alkylene)-, [*-SS] ｐ (C １－６ -alkylene)-, [*-S(O) ２ ] ｐ (C １－６ -alkylene)-, [(*-O) ｒ C(OR ２５ ) ３－ｒ ](C １－６ -alkylene)-, [*-C(OR ２５ ) ２ O] ｐ (C １－６ -alkylene)-, [*-C(R ２５ )(=N-N(R ２６ )C(O)-)] ｐ (C １－６ -alkylene)-, [*-C(O)(N(R ２６ )-N=)C(R ２５ )-] ｐ (C １－６ -alkylene)-, [*=C(=N-N(R ２６ )C(O)(R ２５ ))] ｐ (C １－６ -alkylene)-, [*-N(R ２６ )N(R ２６ )] ｐ (C １－６ -alkylene)-, [*=C(=N(OH))] ｐ (C １－６ -alkylene)-, [*-OC(R ２５ )(R ２６ )O] ｐ (C １－６ -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) ｐ N(R ２６ ) ２－ｐ and [*-C(O)NH](C １－６ -alkyltriyl)-, where * is R ４ represents the point of attachment to １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２５ is C １－６ Alkyl, aryl and aryl(C １－６ alkyl); R ２６ H, C １－６ Alkyl, aryl and aryl(C １－６ alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ 135. The polymer conjugate compound of claim 133 or 134, which is directly bound to

136. L １ further comprises at least one additional bifunctional moiety through which R ２ is X in formula (V) １ or X in formula (V') ２ 136. The polymer conjugate compound of any of claims 133 to 135, which binds to:

137. At least one further difunctionalized moiety is an ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imide) amide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably selected from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein L １ further comprises at least two additional bifunctional moieties, these at least two additional bifunctional moieties are optionally C １－６ - The polymer conjugate compound of claim 136, separated by alkylene groups.

138. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ - alkylene)NR ２６ -, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )-O(C １－６ -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] ｐ (C １－６ -alkylene)O-, [*-OC(O)] ｐ (C １－６ -alkylene)O-, (*-) ｐ N(R ２６ ) ２－ｐ and [*-C(O)NH](C １－６ -alkyltriyl)O—, where * is R ４ represents the point of attachment to [*-C(O)O]; p is 1 or 2; ｐ (C １－６ -alkylene), [*-OC(O)] ｐ (C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene) and [*-C(O)NH] ｐ (C １－６ - alkylene) C １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ H, C １－６ Alkyl, aryl and aryl(C １－６ alkyl); R ２７ H, C １－６ Alkyl, aryl, aryl(C １－６ alkyl) and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ 138. The polymer conjugate compound of any of claims 133 to 137, wherein the polymer conjugate compound is directly bound to

139. L １ is [*-C(O)O] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-C(O)O] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-OC(O)] ｐ (C １－６ -alkylene)-OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-NHC(O)] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)C(O)-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene), [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )O(C １－６ -alkylene)NH-, [*-C(O)NH] ｐ (C １－６ -alkylene)OP(O)(OR ２７ )-O(C １－６ -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] ｐ (C １－６ -alkylene)O-, [*-OC(O)] ｐ (C １－６ -alkylene)O-, (*-) ２ N- and [*-C(O)NH](C １－６ -alkyltriyl)O— or L １ (*-)(R ２６ )N-, where * is R ４ represents the point of attachment to [*-C(O)O]; p is 1 or 2; ｐ (C １－６ -alkylene), [*-OC(O)] ｐ (C １－６ -alkylene), [*-NHC(O)] ｐ (C １－６ -alkylene) and [*-C(O)NH] ｐ (C １－６ - alkylene) C １－６ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ is H and C １－６ alkyl; R ２７ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C １－６ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ 139. The polymer conjugate compound of any of claims 133-138, wherein the polymer conjugate compound is directly bound to

140. R ２ [R ４ C(O)O] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ O.C. (O)] ｐ (C ２－３ -alkylene)-OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O-(C １－３ -alkylene), [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ NHC(O)] ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)C(O)-, [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene), [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )O(C １－３ -alkylene)NH-, [R ４ C(O)NH ｐ (C ２－３ -alkylene)OP(O)(OR ２７ )-O(C １－３ -alkylene)C(O)-, (2-R ４ -3,4-dihydro-2H-chromen-6-yl)O-, [R ４ C(O)O] ｐ (C ２－３ -alkylene)O-, [*-OC(O)] ｐ (C ２－３ -alkylene)O-, (R ４ ) ２ N- and [R ４ C(O)NH](C ２－３ -alkyltriyl)O- or R ２ (R ４ )(R ２６ )N-, where p is 1 or 2; C ２－３ alkylene is divalent (when p is 1) or trivalent (when p is 2); R ２６ is H and C １－６ alkyl; R ２７ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally selected from the group consisting of halogen, C １－３ Alkyl, —OH, —CN and —OC １－３ substituted with one or more substituents selected from the group consisting of alkyl; and C ２－３ -alkyltriyl is optionally substituted with one or more -OH substituents, and other hydrophobic chains R ４ 140. The polymer conjugate compound of any of claims 114 to 139, wherein the polymer conjugate compound is directly bound to

141. R ２ is selected from phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or R ２ 141. The polymer conjugate compound of any of claims 114-140, wherein is a monoalkylamine moiety.

142. Each R ４ 142. The polymer conjugate compound of any of claims 133-141, wherein are independently acyclic, preferably linear, hydrocarbyl groups.

143. Each R ４ 143. The polymer conjugate compound of any of claims 133-142, wherein are independently hydrocarbyl groups having at least 8 carbon atoms, e.g., at least 10 carbon atoms, or at least 12 carbon atoms.

144. R ２ is selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or R ２ 144. The polymer conjugate compound of any of claims 114-143, wherein is a monomyristylamine moiety.

145. R ３ H, C １－６ Alkyl, C ２－６ Alkynyl, —C(O)R ２１ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ 145. The polymer conjugate compound of any of claims 114-144, substituted with one or more substituents independently selected from the group consisting of: a sugar, an amino acid, a peptide, and a member of a targeting pair.

146. R ３ H, C １－３ Alkyl, C ２－６ Alkynyl, —C(O)R ２１ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NR ２２ R ２３ , —C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and one or more substituents independently selected from the group consisting of members of a targeting pair; R ２１ is C １－６ selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —NR ２２ R ２３ and a member of a targeting pair; and R ２２ and R ２３ each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may be taken together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally contains -OH, -SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, —COOH, —NH ２ , -NH(C １－３ alkyl), -N(C １－３ alkyl) ２ and members of a targeting pair.

147. R ３ is H, -C(O)(C １－３ alkyl), -NH(C １－３ alkyl) and -N(C １－３ alkyl) ２ and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , —C(O)NH ２ , —C(O)NHCH ３ , —C(O)NH(CH ２ ) ２ NH ２ and members of a targeting pair.

148. The targeting pair is selected from the following pairs: maleimide-thiol; thiol-alkyl halide (especially brominated); azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-alpha ｖ β ３ 148. The polymer conjugate compound of any of claims 114-147, comprising an integrin; an asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; or a galactose-asialoglycoprotein receptor.

149. The following formula: 【Chemical 27】 【Chemical Formula 28】 [During the ceremony, n is 5 to 25; R ３ is H, -C(O)(C １－３ alkyl) and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, —COOH, —COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , —C(O)NH ２ , —C(O)NHCH ３ , —C(O)NH(CH ２ ) ２ NH ２ and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R ２７ is H or a countercation; and In either case, -C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl), and in either case -C(O)C １５ H ３１ is the moiety -C(O)(CH ２ ) １４ CH ３ (palmitoyl), and in either case -C(O)C １３ H ２７ is the moiety -C(O)(CH ２ ) １２ CH ３ (myristoyl), in either case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (myristyl), in either case -C １３ H ２７ is the part -(CH ２ ) １２ CH ３ In either case, -C(O)C １７ H ３３ The moiety -cis-C(O)(CH ２ ) ７ -CH=CH-(CH ２ ) ７ CH ３ (Oleoyl).

149. The polymer conjugate compound of any of claims 93-148, having the formula:

150. 150. The polymer conjugate compound of claim 149, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

151. R ３ is H or -C(O)(C １－３ alkyl), where C １－３ The alkyl group optionally is 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N ３ and C ２－６ 151. The polymer conjugate compound of claim 149 or 150, substituted with one substituent selected from the group consisting of alkynyl.

152. The following formula: 【Chemical 29】 【Chemistry 30】 【Chemical 31】 [wherein in each case -C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl), and in either case -C(O)C １５ H ３１ is the moiety -C(O)(CH ２ ) １４ CH ３ (palmitoyl), and in either case -C(O)C １３ H ２７ is the moiety -C(O)(CH ２ ) １２ CH ３ (myristoyl), in either case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (myristyl), in either case -C １３ H ２７ is the part -(CH ２ ) １２ CH ３ and in each case n is 8, 10, 12, 14, or 16.

152. The polymer conjugate compound of any of claims 93-151, having the formula:

153. The following formula: (i) 【Chemical Formula 32】 wherein n is 14 and in each case is —C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (Stearoyl) (ii) 【Chemical Formula 33】 wherein n is 14 and in each case -C １４ H ２９ is the part -(CH ２ ) １３ CH ３ (Myristyl). (iii) 【Chemical 34】 wherein n is 8, 12, 14, or 16.

153. The polymer conjugate compound of any of claims 93-152, having the formula:

154. 154. A conjugate compound of any of claims 93-153, comprising (a) a member of a targeting pair; and (b) a conjugate compound comprising another member of the targeting pair.

155. 155. The conjugate of claim 154, wherein the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (e.g., an antigen or epitope), or an antibody.

156. The following formula: 【Chemistry 35】 wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case, —C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl); m1 and m2 are each independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific to the antigen. or a salt thereof.

157. The following expression (i) 【Chemical 36】 wherein m1 is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2; (ii) 【Chemical 37】 wherein m1 and m2 each independently represent 1, 2, or 3, and preferably m1 is 1 and m2 is 2, or m1 is 2 and m2 is 1.

157. The conjugate of claim 156, having one of:

158. The following formula: 【Chemical 38】 wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case, —C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl); and Pept is an antigen or an antibody specific to the antigen. or a salt thereof.

159. The polymer conjugate compound comprising a member of a targeting pair has the formula: 【Chemical 39】 wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case, —C(O)C １７ H ３５ is the moiety -C(O)(CH ２ ) １６ CH ３ (stearoyl); and Pept is an antigen or an antibody specific to the antigen. having the compound comprising the other member of the targeting pair is (i) an antibody specific for the antigen if Pept is the antigen; or (ii) a compound comprising the antigen if Pept is an antibody specific for the antigen; and The conjugate of claim 154, wherein the polymer conjugate compound comprising a member of a targeting pair is conjugated to a compound comprising another member of the targeting pair through the interaction of (1) the antibody specific for the antigen and (2) the antigen.

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