Nucleic acid compositions comprising amphiphilic oligopolyethylene glycol (OEG) conjugated compounds and methods of using such compounds and compositions

Abstract

The present disclosure relates generally to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligomeric ethylene glycol (OEG) conjugated compounds (as substitutes for PEG lipids); uses of such compositions, in particular for delivering nucleic acids to cells of a subject or for therapy; to an amphiphilic OEG conjugated compound of this type; and conjugates of such amphiphilic OEG conjugated compounds.

Description

Technical Field

[0001] The present disclosure generally relates to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligoethylene glycol (OEG)-conjugated compounds (as an alternative to PEG lipids); uses of such compositions, particularly for delivering nucleic acids to cells of a subject or for therapeutic use; such amphiphilic OEG-conjugated compounds; and conjugates of such amphiphilic OEG-conjugated compounds. Background Art

[0002] It is well known to use recombinant nucleic acids (e.g., DNA or RNA) for delivering foreign genetic information to target cells. Recombinant nucleic acids can be applied to subjects in need thereof in naked form; however, compositions are typically used to administer recombinant nucleic acids. For example, nucleic acids, such as RNA, can be delivered to subjects using different delivery vehicles, primarily based on cationic polymers or lipids that form nanoparticles with the nucleic acid. Nanoparticles are intended to protect nucleic acids (e.g., RNA) from degradation, enable nucleic acids (e.g., RNA) to be delivered to target sites, and promote cellular uptake and processing of target cells. The efficiency of nucleic acid delivery depends in part on the molecular composition of the nanoparticles and can be affected by many parameters, including particle size, formulation, and charge or grafting with molecular moieties (e.g., polyethylene glycol (PEG) or other ligands).

[0003] Consider grafting with PEG to reduce serum interactions, to improve serum stability and extend circulation time, which may help certain targeting methods. Ligands that bind to receptors at the target site can help improve targeting efficacy. In addition, PEGylation can be used for particle modification. For example, if lipid nanoparticles (LNPs) are prepared by mixing an aqueous phase of nucleic acid (e.g., RNA) with an organic phase of lipids, a certain fraction of PEG-conjugated lipids (such PEG-conjugated lipids have at least 30 consecutive ethylene glycol repeating units) is required in the lipid mixture, otherwise the particles will aggregate during or after the mixing step. It has been shown that by changing the molar fraction of PEG lipids containing different molar masses of PEG, the size of the particles can be adjusted. Similarly, the particle size can be adjusted by changing the molar mass of the PEG portion of the PEGylated lipid. The typical size that can be obtained is 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles thus formed have the additional advantage that, due to the PEG moiety, they interact less with serum components and have a longer circulation half-life, which is desirable in many drug delivery methods. Without PEG lipids, particles of discrete sizes cannot be formed; the particles form large aggregates and precipitate. Thus, one of the main roles of PEG lipids is to facilitate particle self-assembly by providing a steric barrier on the surface of nascent particles that form when nucleic acids are rapidly mixed in an ethanolic solution containing lipids to bind nucleic acids, such as RNA. PEG steric hindrance prevents interparticle fusion and promotes the formation of a uniform LNP population, wherein diameters <100 nm can be achieved.

[0004] In spite of these advantages, the PEGylation of nanoparticles may also lead to several effects that are unfavorable for the intended use of drug delivery. The PEGylation of known liposomes and LNPs has reduced cellular uptake and endosome escape, thereby ultimately reducing overall transfection efficiency. In fact, the PEG shell provides a spatial barrier for the effective combination of particles and cells, and also hinders endosome release by stopping the membrane fusion between liposomes and the endosomal membrane. This is why the type of PEG lipid and the amount of used PEG lipid must always be carefully adjusted. It should provide enough stealth effects in vivo and in terms of stability on the one hand, while not hindering transfection on the other hand. This phenomenon is referred to as the "PEG dilemma".

[0005] In addition to reducing transfection efficiency, PEGylation has been associated with the phenomenon of accelerated blood clearance (ABC) induced by anti-PEG antibodies and / or complement activation, as well as 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 significantly altered the pharmacokinetic behavior of a second dose when the second dose was 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 "accelerated blood clearance" (ABC) phenomenon was shown to be associated with the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma induced a higher clearance of particles by the mononuclear phagocyte system (MPS), which ultimately reduced the efficacy of the drug.

[0006] Due to the widespread use of PEG as an ingredient in food, cosmetics, hygiene products, and pharmaceuticals, a certain proportion of the general population has "pre-existing" anti-PEG antibodies. Anti-PEG antibodies are associated with reduced efficacy of PEGylated drugs and allergic reactions that can lead to severe allergic symptoms.

[0007] Because PEG can induce an immune response, it needs to be avoided for certain applications that require multiple injections. Some examples are treatments using nucleic acids (e.g., RNA, particularly mRNA), such as for protein replacement therapy. Here, the risk may be particularly high due to the potential inherent immunogenicity of nucleic acids (particularly RNA). Other examples are protein knockdown treatments using inhibitory RNA (e.g., siRNA), antisense oligonucleotides, or DNA-based therapies.

[0008] Therefore, there remains a need in the art for effective compositions and methods for introducing nucleic acids (eg, RNA) into cells that avoid the disadvantages associated with the use of PEG. The present disclosure addresses these and other needs.

[0009] The present inventors have unexpectedly discovered that the compositions, methods, polymer-conjugated compounds (also referred to herein as amphiphilic OEG-conjugated compounds) and conjugates described herein meet the above requirements. In particular, it was shown that the polymer-conjugated compounds described herein are not bound by antibodies raised against PEG structures and are stable under physiological conditions. The polymer components of the polymer-conjugated compounds, as well as the polymer-conjugated compounds and their conjugates, can be synthesized by well-known methods (e.g., solid-phase peptide synthesis (SPPS)). The polymer-conjugated compounds and conjugates can be end-functionalized with different moieties to adjust the charge or introduce specific molecular moieties such as ligands. Summary of the Invention

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

[0011] In a first aspect, the present disclosure provides a composition comprising (i) a nucleic acid; (ii) a cationic lipid or a lipid that can ionize into a cation; and (iii) a polymer-conjugated compound comprising (a) a polymer comprising the following general formula (I); and (b) one or more hydrophobic chains:

[0012]

[0013] in

[0014] X 2 and X 1 taken together are optionally substituted amides, optionally substituted thioamides, esters or thioesters, preferably optionally substituted amides, optionally substituted thioamides or esters;

[0015] Y is -CH2-, -(CH2)2- or -(CH2)3-;

[0016] z is 2 to 24; and

[0017] n is 1 to 100. The composition is also referred to herein as a nucleic acid composition.

[0018] As shown in this application, anti-PEG antibodies (polyclonal antibodies 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, this application shows that polymers comprising the structure of formula (I) are stable under physiological conditions.

[0019] In some embodiments of the first aspect (particularly with respect to Formula (I)), X 2 and X 1Taken together, is an optionally substituted amide. Thus, in some embodiments, X 1 is -C(O)- and X 2 Yes-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X 1 Yes-NR 1 - and X 2 is -C(O)-, where R 1 is hydrogen or C 1-8 alkyl.

[0020] In some embodiments of the first aspect (particularly with respect to Formula (I)), X 2 and X 1 Taken together, is an optionally substituted thioamide. Thus, in some embodiments, X 1 is -C(S)- and X 2 Yes-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X 1 Yes-NR 1 - and X 2 is -C(S)-, where R 1 is hydrogen or C 1-8 alkyl.

[0021] In some embodiments of the first aspect (particularly with respect to Formula (I)), X 2 and X 1 Thus, in some embodiments, X 1 is -C(O)- and X 2 In some embodiments, X 1 If it is -O-, then X 2 It is -C(O).

[0022] In some embodiments of the first aspect (particularly with respect to Formula (I)), X 2 and X 1 Thus, in some embodiments, X 1 is -C(S)- and X 2 In some embodiments, X 1 If it is -O-, then X 2 is -C(S)-. In some embodiments, X 1 If it is -C(O)-, then X 2 In some embodiments, X 1 If it is -S-, then X 2It is -C(O)-.

[0023] In some embodiments of the first aspect (particularly with respect to Formula (I)), X 1 is -C(O)- and X 2 Yes-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 It's hydrogen.

[0024] In some embodiments of the first aspect (particularly with respect to Formula (I)), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0025] In some embodiments of the first aspect, the polymer comprises the following formula (II):

[0026]

[0027] where R 1 is hydrogen or C 1-8 In some 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 at each occurrence (ie, in each repeating unit) (e.g., in each repeating unit, R 1 can be H or methyl). In some embodiments, R in at least one repeating unit is 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., H), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0028] In some embodiments of the first aspect (particularly with respect to any one of formulas (I) and (II)), 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.

[0029] In some embodiments of the first aspect, the polymer comprises the following formula (III):

[0030]

[0031] where R1 is hydrogen or C 1-8 In some 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 at each occurrence (ie, in each repeating unit) (e.g., in each repeating unit, R 1 can be H or methyl). In some embodiments, R in at least one repeating unit is 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., H), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0032] In some embodiments of the first aspect, the polymer comprises the following formula (IV):

[0033]

[0034] In some embodiments of the first aspect, the polymer comprises the following general formula (IVa):

[0035]

[0036] In some embodiments of the first aspect (particularly with respect to any one of Formulas (I), (II), (III), (IV), and (IVa)), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 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 some embodiments of the first aspect (particularly with respect to any one of Formulas (I), (II), (III), (IV) and (IVa)), one or more hydrophobic chains are located at X 1 End or X 2 end.

[0038] In some embodiments of the first aspect (particularly with respect to any one of formula (I), (II), (III), (IV) and (IVa)), one or more hydrophobic chains are independently selected from acyclic, preferably linear hydrocarbon groups, such as hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the hydrocarbon group has at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbon group can be saturated or unsaturated. If the polymer-conjugated compound comprises two or more hydrophobic chains, these chains can be the same or different. For example, if the polymer-conjugated compound comprises two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In some alternative embodiments, the two hydrophobic chains are different, for example, one can be saturated and the other can be (mono)unsaturated.

[0039] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (V) or (V'):

[0040]

[0041] in

[0042] X 2 and X 1 taken together is an optionally substituted amide, optionally substituted thioamide, ester or thioester;

[0043] Y is -CH2-, -(CH2)2-, or -(CH2)3-;

[0044] R 2 is a moiety containing one or more hydrophobic chains;

[0045] R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein C 1-6 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 20 Selected from H, C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-3 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 can be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and substituent substitutions of members of targeting pairs;

[0046] z is 2 to 24; and

[0047] n is 1 to 100.

[0048] In some embodiments of Formula (V) or (V'), X 2 and X 1 Taken together, is an optionally substituted amide. Thus, in some embodiments, X 1 is -C(O)- and X 2 Yes-NR 1 -, where R 1 is hydrogen or C1-8 In some embodiments, X 1 Yes-NR 1 - and X 2 is -C(O)-, where R 1 is hydrogen or C 1-8 alkyl.

[0049] In some embodiments of Formula (V) or (V'), X 2 and X 1 Taken together, is an optionally substituted thioamide. Thus, in some embodiments, X 1 is -C(S)- and X 2 Yes-NR 1 -, where R 1 is hydrogen or C 1-8 In some embodiments, X 1 Yes-NR 1 - and X 2 is -C(S)-, where R 1 is hydrogen or C 1-8 alkyl.

[0050] In some embodiments of Formula (V) or (V'), X 2 and X 1 Thus, in some embodiments, X 1 is -C(O)- and X 2 In some embodiments, X 1 If it is -O-, then X 2 It is -C(O).

[0051] In some embodiments of Formula (V) or (V'), X 2 and X 1 Thus, in some embodiments, X 1 is -C(S)- and X 2 In some embodiments, X 1 If it is -O-, then X 2 In some embodiments, X 1 If it is -C(O)-, then X 2 In some embodiments, X 1 If it is -S-, then X 2 It is -C(O)-.

[0052] In some embodiments of Formula (V) or (V'), X 1 is -C(O)- and X 2 Yes-NR 1 -, where R 1is hydrogen or C 1-8 Alkyl. For example, R 1 Can be hydrogen or methyl. In some embodiments, R 1 It's hydrogen.

[0053] In some embodiments of Formula (V) or (V'), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0054] 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.

[0055] In some 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 at each occurrence (ie, in each repeating unit) (e.g., in each repeating unit, R 1 can be H or methyl). In some embodiments, R in at least one repeating unit is 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., H), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0056] In some embodiments of Formula (V) or (V'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 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.

[0057] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VI) or (VI'):

[0058]

[0059] Among them, z, n, R 2 and R 3 As defined in formulas (V) and (V'); and R 1is hydrogen or C 1-8 alkyl.

[0060] In some 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 at each occurrence (ie, in each repeating unit) (e.g., in each repeating unit, R 1 can be H or methyl). In some embodiments, R in at least one repeating unit is 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., H), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0061] 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.

[0062] In some embodiments of Formula (VI) or (VI'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 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.

[0063] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VII) or (VII'):

[0064]

[0065] Where n, R 2 and R 3 As defined in formulas (V) and (V'); and R 1 is hydrogen or C 1-8 alkyl.

[0066] In some embodiments of Formula (VII) or (VII'), R 1 is hydrogen or methyl. For example, R 1 Can be hydrogen. In some embodiments, R1 is the same at each occurrence (ie, in each repeating unit) (e.g., in each repeating unit, R 1 can be H or methyl). In some embodiments, R in at least one repeating unit is 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., H), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0067] In some embodiments of Formula (VII) or (VII'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 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.

[0068] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VIII), (VIIIa), (VIII'), or (VIIIa'):

[0069]

[0070] Where n, R 2 and R 3 As defined in formulae (V) and (V').

[0071] In some embodiments of Formula (VIII), (VIIIa), (VIII') or (VIIIa'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 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.

[0072] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 It is R4 or -L 1 (R 4 ) p , where each R 4 L is independently a hydrophobic chain, such as a hydrocarbon group; 1 is a linker; and p is 1 or 2.

[0073] In some embodiments, L 1 Contains at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or an alkylene moiety substituted with R 4 An alkylene moiety attached to a divalent functional moiety at the terminal end of the linkage, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azide, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, sulfamate / ester, diselenide, sulfonamide, sulfuramide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate / ester, dithioate / ester, imidate / ester, imino , thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioa te), thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties; and / or each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfonamide, sulfuramide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioate

[0014] The present invention also includes but is not limited to, but not limited to, alkyl, alkyl, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino, alkylamino,

[0074] In some embodiments, L 1 Contains a functional moiety selected from:

[0075] [*-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-pand [*-C(O)NH](C 1-6 alkyltriyl)-,

[0076] * indicates the same as R 4 The connection point; p is 1 or 2; C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); R 26 Selected from 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 substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN, and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl group is optionally substituted with one or more -OH substituents and is combined with another hydrophobic chain R 4 Direct connection.

[0077] In some embodiments, L 1 Also comprising at least one additional bifunctional moiety, R 2 By the additional bifunctional moiety and X in formula (V) 1 (or with the carbonyl group of any one of formulas (VI), (VII), (VIII) and (VIIIa)) or with X in formula (V') 2 (or to the N atom of any one of Formulas (VI'), (VII'), (VIII') and (VIIIa'). In some embodiments, at least one additional bifunctional moiety is selected from the group consisting of ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfonamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate , guanidino, imidamido, carbamoyl, carboimidate, carbamate, dithiocarbamate, dithiocarbamate, thiocarbamate, thiocarbamate, thiocarbamate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties are preferably selected from phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioacid salt, dithioacid salt, carbonyl and thiocarbonyl, wherein if L1 further comprising at least two additional bifunctional moieties, the at least two additional bifunctional moieties being optionally C 1-6 The alkylene groups are separated from each other.

[0078] In some embodiments, L 1 Selected from

[0079] [*-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 (C1-6 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-,

[0080] * indicates the same as R 4 The attachment point of [*-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 C in alkylene) 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); R 27 Selected from H, C 1-6 Alkyl, aryl, aryl (C 1-6 alkyl) and a counter cation (e.g., the counter cation can be 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 (NH4 + ); Or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN, and -OC 1-3The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl group is optionally substituted with one or more -OH substituents and is combined with another hydrophobic chain R 4 Direct connection.

[0081] In some embodiments, L 1 Selected from

[0082] [*-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)(OR27 )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-

[0083] or L 1 Yes(*-)(R 26 )N-, where * indicates the same as R 4 The attachment point of [*-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 C in alkylene) 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 selected from H and a counter cation (e.g., the counter cation can be 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 (NH4 + ); Or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN, and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl group is optionally substituted with one or more -OH substituents and is combined with another hydrophobic chain R 4 Direct connection.

[0084] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 Selected from

[0085] [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 (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)(OR27 )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 Yes (R 4 )(R 26 )N-,

[0086] Where p is 1 or 2; C 2-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 selected from H and a counter cation (e.g., the counter cation can be 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 (NH4 + ); Or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN, and -OC 1-3 The alkyl group is substituted with a substituent; and C 2-3 The alkyltriyl group is optionally substituted with one or more -OH substituents and is combined with another hydrophobic chain R 4 Direct connection.

[0087] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 is selected from a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglycerol moiety, a dialkylamino moiety, and a ceramide moiety, or R 2 It is a monoalkylamine moiety.

[0088] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), each of the one or more hydrophobic chains (i.e., R 4 Each of) is independently acyclic, preferably linear, hydrophobic chains, such as hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, one or more hydrocarbyl groups independently have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. One or more hydrocarbyl groups can be saturated or unsaturated. If the polymer-conjugated compound comprises two or more hydrophobic chains, these chains can be identical or different. For example, if the polymer-conjugated compound comprises two hydrophobic chains, in some embodiments, the two hydrophobic chains are identical. In some alternative embodiments, the two hydrophobic chains are different, for example, one can be saturated and the other can be (single) unsaturated, and / or the two hydrophobic chains are of different lengths. Some examples of one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, such as naturally occurring fatty acids and hydrocarbyl chains with at least 8 carbon atoms. Specific examples of the one or more hydrophobic chains include the following hydrocarbyl chains: caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleicalcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric alcohol, cerotic alcohol, oleicalcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

[0089] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 2 a moiety selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopherol, DMG (1,2-dimyristoylglycerol), DMA (dimyristamide) and palmitoylceramide, or R 2 It is the monomyristamide moiety.

[0090] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein C 1-6 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; and R 22 and R 23Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 can be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and substituents of members of targeting pairs.

[0091] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 Selected from 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 members of a targeting pair, where C 1-3 The alkyl group is optionally substituted by one or more groups independently selected from OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 and a substituent that is substituted with a member of a targeting pair; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a substituent that is substituted with a member of a targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R23 can be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and substituted with a substituent targeting a member of the pair.

[0092] In some embodiments of the first aspect (particularly with respect to any one of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R 3 Selected from 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 is optionally substituted with one or more substituents independently selected from the group consisting of: -OH, -SH, halogen, -CN, -N3, C 2-6 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.

[0093] In some embodiments of the first aspect (particularly with respect to any one of formula (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (particularly brominated) alkyl; azide-alkyne (particularly in a copper(I)-catalyzed reaction); conjugated diene-substituted olefin (dienophile) (particularly in a Diels-Alder reaction); antigen-antibody (including fragments or derivatives thereof) 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-α v β3 integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

[0094] In some embodiments of the first aspect, the polymer-conjugated compound has one of the following formulae:

[0095]

[0096]

[0097]

[0098] wherein n is 5 to 25; R 3 Selected from H, -C(O)(C 1-3 alkyl) and a member of a targeting pair, wherein C 1-3 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and substituents of members of the targeted pair; R 27 is H or a counter cation (e.g., the counter cation can be 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 (NH4 + ); or an organic cation, such as a quaternary ammonium or amine cation); and in each case -C(O)C 17 H 35 Refers to -C(O)(CH2) 16 CH3(stearoyl) moieties, in each case -C(O)C 15 H 31 Refers to -C(O)(CH2) 14 CH3(palmitoyl) moieties, in each case -C(O)C 13 H 27 Refers to -C(O)(CH2) 12 CH3(myristoyl) moieties, in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moieties, in each case -C 13 H 27 refers to -(CH2) 12 CH3 moieties, and in each case -C(O)C 17 H 33 refers to a -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl) moiety. In some embodiments, n is 7 to 16, such as 7 to 14 (preferably 8, 10, 12, 14 or 16); and / or R 3 is H or -C(O)(C 1-3 alkyl), wherein C1-3 The alkyl group is optionally substituted with a substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimido), -SH, -Br, -N3, C 2-6 Alkynyl groups, antigens and antibodies.

[0099] Additional preferred embodiments of polymer-conjugated compounds (particularly with respect to any one of Formula (I), (II), (III), (IV), (IVa), (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII'), and (VIIIa')) are given under the heading "Polymer-conjugated compounds comprising (a) a polymer comprising a structure of Formula (I); and (b) one or more hydrophobic chains". In some preferred embodiments of the first aspect, the polymer-conjugated compound has any one of Formula (V-1), (V-5), (V-17), and (V-25).

[0100] In some embodiments of the first aspect, the composition is substantially free of a lipid or lipid-like substance comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive repeating units of ethylene glycol.

[0101] In some embodiments of the first aspect, the composition is also substantially free of another polymer-conjugated lipid. In some embodiments, the another polymer-conjugated lipid is a polysarcosine-conjugated lipid and / or a lipid comprising a hydrophobic chain and a poly Oxazoline (POX) and / or poly Conjugates of oxazine (POZ) polymers.

[0102] In some embodiments of the first aspect, water is the major component in the composition, and / or the total amount of solvent other than water included in the composition is less than about 1.0% (v / v), for example, less than about 0.5% (v / v). For example, the amount of water included in the composition can be at least 50% (w / w), for example, 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, if the composition comprises a cryoprotectant, the amount of water included in the composition can be at least 50% (w / w), for example, 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 included in the composition can be at least 95% (w / w). Additionally or alternatively, the total amount of solvent other than water included in the composition can be less than about 0.5% (v / v), for example, 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, cryoprotectants that are liquid under normal conditions will not be considered solvents other than water, but rather will be considered cryoprotectants. In other words, the above optional limitation, i.e., the total amount of solvent other than water included in the composition may be less than about 0.5% (v / v), such as less than about 0.4% (v / v), does not apply to cryoprotectants that are liquid under normal conditions.

[0103] In some embodiments of the first aspect, the concentration of the nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 500 mg / l. In some embodiments, the concentration of the nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 100 mg / l. In some embodiments, the concentration of the nucleic acid (particularly RNA) in the composition is from about 5 mg / l to about 500 mg / l, for example, from about 10 mg / l to about 400 mg / l, from about 10 mg / l to about 300 mg / l, from about 10 mg / l to about 200 mg / l, from about 10 mg / l to about 150 mg / l, or from about 10 mg / l to about 100 mg / l, preferably from about 10 mg / l to about 140 mg / l, more preferably from about 20 mg / l to about 130 mg / l, more preferably from about 30 mg / l to about 120 mg / l. In some embodiments, the concentration of nucleic acids (particularly RNA) in the composition is from about 5 mg / l to about 150 mg / l, for example, from about 10 mg / l to about 140 mg / l, from about 20 mg / l to about 130 mg / l, from about 25 mg / l to about 125 mg / l, from about 30 mg / l to about 120 mg / l, from about 35 mg / l to about 115 mg / l, from about 40 mg / l to about 110 mg / l, from about 45 mg / l to about 105 mg / l, or from about 50 mg / l to about 100 mg / l. In some embodiments, the concentration of nucleic acids (particularly RNA) in the composition is from 1 mg / l to about 50 mg / l or from about 10 mg / l to about 100 mg / l.

[0104] In some embodiments of the first aspect, the composition comprises a cryoprotectant. In some embodiments of the first aspect, the composition is substantially free of a cryoprotectant.

[0105] In some embodiments of the first aspect, the lipid ionizable to release a cation comprises a head group comprising at least one tertiary amine moiety.

[0106] In some embodiments of the first aspect, the lipid that can ionize into a cation has a structure of formula (X):

[0107]

[0108] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 10 、L 20 , G 1 , G 2 , G 3 、R 35 、R 36 and R 37As defined herein. In some embodiments, the lipid that can ionize to release a cation is selected from the following: Structures X-1 to X-36 (shown herein); or Structures A to G (shown herein). In some embodiments, the lipid that can ionize to release a cation is a lipid having structure X-3. In some embodiments, the lipid that can ionize to release a cation is DPL-14 (i.e., a lipid having structure G).

[0109] In some embodiments of the first aspect, the lipid that can ionize into a cation has the structure of Formula (XI):

[0110]

[0111] Wherein R1, R2 R3, R4, L2, G2 and m are as defined herein. In some embodiments, the lipid that can ionize to give a cation is selected from structures (XIV-1), (XIV-2) and (XIV-3) (shown herein). In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-1. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-2. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-3.

[0112] In some embodiments of the first aspect, the cationic lipid or the lipid that can ionize to release cations comprises 2,3-dioleoyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleoyl-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-dioleyloxy)propyl)-N,N , N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyloxy-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.

[0113] In some embodiments of the first aspect, the lipid that ionizes into a cation is completely or partially replaced by a cationic lipid. In some embodiments, the cationic lipid is selected from Structures XV-1 to XV-6 (shown herein).

[0114] In some embodiments of the first aspect, the cationic lipid or lipid that can ionize into a cation comprises about 20 mol% to about 80 mol% of the total lipids present in the composition.

[0115] In some 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 phospholipids and steroids.

[0116] In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), dioctano ... The following are the phosphatidylcholine compounds: dioleoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), ditetracosanoylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestyrylsuccinyl-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).

[0117] In some embodiments, the phospholipids comprise from about 5 mol% to about 30 mol% of the total lipids present in the composition.

[0118] In some embodiments, the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol.

[0119] In some embodiments, the steroid comprises from about 10 mol% to about 60 mol% of the total lipids present in the composition.

[0120] In some embodiments of the first aspect, the cationic lipids or lipids that can ionize into cations comprise about 20 mol% to about 70 mol% of the total lipids present in the composition; the polymer-conjugated compound (amphiphilic OEG-conjugated 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 lipids present in the composition; the phospholipids comprise about 5 mol% to about 25 mol% of the total lipids present in the composition; and the steroids comprise about 20 mol% to about 55 mol% of the total lipids present in the composition.

[0121] In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids may comprise a cationic lipid. In these embodiments in which a cationic lipid is present, the amount of (1) the lipid that can ionize to produce a cation and the amount of (2) the cationic lipid are used for calculation. For example, if the amount of the lipid that can ionize to produce a cation in the composition should be from about 20 mol % to about 70 mol %, and the composition should also contain a cationic lipid, then the amount of (1) the lipid that can ionize to produce a cation and the amount of (2) the cationic lipid should be from about 20 mol % to about 70 mol %.

[0122] In some embodiments of the first aspect, the only lipids contained in the composition are cationic lipids or lipids that can be ionized to release cations, steroids, neutral lipids and polymer-conjugated compounds comprising polymers of formula (I) as defined herein (i.e., amphiphilic OEG-conjugated compounds), in particular cationic lipids or lipids that can be ionized to release cations, steroids, phospholipids and polymer-conjugated compounds comprising polymers of formula (I) as defined herein.

[0123] In some 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 lipid or a lipid that can ionize cations, and at least a portion of a polymer-conjugated compound comprising a polymer of formula (I) as defined herein. In some embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipid complexes (LPXs), and mixtures thereof. In some embodiments, the particles comprise LNPs or are LNPs. In some embodiments, the particles comprise liposomes or are liposomes. In some embodiments, the particles comprise LPXs or are LPXs. In some embodiments, the particles comprise a mixture of LNPs and liposomes or are a mixture of LNPs and liposomes. In some embodiments, the particles comprise a mixture of LNPs and LPXs or are a mixture of LNPs and LPXs. In some embodiments, the particles comprise a mixture of liposomes and LPXs or are a mixture of liposomes and LPXs. In some embodiments, the particles comprise a mixture of LNPs, liposomes, and LPXs or are a mixture of LNPs, liposomes, and LPXs.

[0124] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the particles comprise substantially all of the lipids present in the composition (particularly all of the cationic lipids or lipids ionizable to release cations, one or more additional lipids (if present) and the polymer-conjugated compound comprising a polymer of formula (I) as defined herein).

[0125] In some embodiments of the first aspect, wherein 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 acids (particularly RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85% of the nucleic acids (particularly RNA) present in the composition.

[0126] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of nucleic acids.

[0127] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the nucleic acid (eg, RNA) is encapsulated within or associated with the particles.

[0128] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the particles have a size of about 30 nm to about 500 nm. In some embodiments, the particles have a size of about 50 nm to about 150 nm.

[0129] In some embodiments of the second aspect, the nucleic acid is DNA.

[0130] In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA (eg, siRNA).

[0131] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and (i) comprises a modified nucleoside that replaces 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 some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0132] 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 poly-A 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 poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably interrupted by A nucleotides. In some embodiments, the 5' cap is a cap 1 or cap 2 structure.

[0133] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and encodes one or more polypeptides. In some embodiments, the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response to an antigen in a subject.

[0134] In some embodiments of the first aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of said protein, or an immunogenic fragment of said protein or its immunogenic variant. In some embodiments, the pathogen is a pathogen that causes an infectious disease.

[0135] In some embodiments of the first aspect, the nucleic acid is an inhibitory RNA (e.g., siRNA) and selectively hybridizes to and / or is specific for a target mRNA. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, particularly an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (particularly, for example, increased expression compared to expression in healthy subjects) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (particularly, for example, increased expression compared to expression in healthy subjects) is associated with cancer.

[0136] In a second aspect, the present disclosure relates to a method for delivering a nucleic acid to a cell of a subject, the method comprising administering to the subject the nucleic acid composition of the first aspect. It should be understood that any embodiment described herein in the context of the first aspect may also be applied to any embodiment of the second aspect.

[0137] In a third aspect, the disclosure relates to a method for 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 a therapeutic peptide or protein. It should be understood that any embodiment described herein in the context of the first or second aspect may also be applied to any embodiment of the third aspect.

[0138] In a fourth aspect, the disclosure relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering the nucleic acid composition of the first aspect to the subject, wherein delivery of the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or condition in a subject, wherein delivery of the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or condition. It should be understood that any embodiment described herein in the context of the first, second, or third aspects may also be applied to any embodiment of the fourth aspect.

[0139] In a fifth aspect, the present disclosure relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject the nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the present disclosure relates to a nucleic acid composition of the first aspect for use in a method for treating or preventing a disease or condition in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition. It should be understood that any embodiment described herein in the context of the first, second, third, or fourth aspects may also be applied to any embodiment of the fifth aspect.

[0140] In some embodiments of the second to fifth aspects, the subject is a mammal, such as a human.

[0141] In a sixth aspect, the present disclosure provides a polymer-conjugated compound (also referred to herein as an amphiphilic OEG-conjugated compound) comprising (a) a polymer comprising a structure of formula (I); and (b) one or more hydrophobic chains. Preferred embodiments of the polymer-conjugated compound of the sixth aspect are described in detail in the first aspect and are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer comprising a structure of formula (I); and (b) one or more hydrophobic chains".

[0142] In a seventh aspect, the present disclosure provides a conjugate of (a) a polymer-conjugated compound comprising a member of a targeting pair of the sixth aspect; and (b) a compound comprising the other 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 one of the following formulae:

[0143]

[0144] or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35 Refers to -C(O)(CH2) 16 CH3 (stearoyl) moiety; each of m1 and m2 is independently 1, 2, 3, 4 or 5; and Pept is an antigen or an antibody specific for the antigen. In some embodiments of the seventh aspect, the conjugate has one of the following formulae:

[0145]

[0146] or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35 Refers to -C(O)(CH2) 16 CH3 (stearoyl) moiety; and Pept is an antigen or an antibody specific for the antigen. In some embodiments of the seventh aspect, the polymer-conjugated compound containing a member of the targeting pair has the following formula:

[0147]

[0148] wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35Refers to -C(O)(CH2) 16 a CH3(stearoyl) moiety; and Pept is an antigen or an antibody specific for said antigen; the compound comprising the other member of the targeting pair is a compound comprising: (i) an antibody specific for said antigen if Pept is said antigen; or (ii) an antigen if Pept is an antibody specific for said antigen; and the polymer-conjugated compound comprising one member of the targeting pair is conjugated to the compound comprising the other member of the targeting pair via an interaction between (1) said antibody specific for said antigen and (2) said antigen.

[0149] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspects may also be applied to any embodiment of the seventh aspect.

[0150] In an eighth aspect, the present disclosure provides a composition comprising (i) a nucleic acid (eg, DNA or RNA); (ii) a cationic lipid or a lipid that can ionize into a cation; and (iii) the conjugate of the seventh aspect.

[0151] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth or seventh aspects may also be applied to any embodiment of the eighth aspect.

[0152] In a ninth aspect, the present disclosure relates to a method for delivering a nucleic acid to a cell of a subject, the method comprising administering to the subject the composition of the eighth aspect. It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspects may also be applied to any embodiment of the ninth aspect.

[0153] In a tenth aspect, the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject the composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect may also be applied to any embodiment of the tenth aspect.

[0154] In an eleventh aspect, the present disclosure relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering the composition of the eighth aspect to the subject, wherein delivery of the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or condition in a subject, wherein delivery of the nucleic acid to the cells of the subject is beneficial for treating or preventing the disease or condition. It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspect may also be applied to any embodiment of the eleventh aspect.

[0155] In a twelfth aspect, the present disclosure relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject the composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the present disclosure relates to a composition of the eighth aspect for use in a method for treating or preventing a disease or condition in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition. It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect may also be applied to any embodiment of the twelfth aspect.

[0156] In the 13th aspect, present disclosure provides the method for transfecting cells, it includes adding the composition of the first or eighth aspect to the cell; and the mixture of the composition and the cell is incubated for a sufficient amount of time. In some embodiments, particularly in those embodiments where nucleic acid is DNA or RNA (such as mRNA) and encodes pharmaceutically active protein, the mixture of the composition and the cell is incubated for a sufficient amount of time so that the pharmaceutically active protein is expressed. In some embodiments, particularly in those embodiments where nucleic acid is an inhibitory RNA (such as siRNA) for target mRNA, the mixture of the composition and the cell is incubated for a sufficient amount of time so that the target mRNA is transcribed and / or translated. In some embodiments, the sufficient amount of time is at least one 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).

[0157] In some 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 some 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 an ex vivo cell culture).

[0158] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect may also be applied to any embodiment of the thirteenth aspect.

[0159] In a fourteenth aspect, the present disclosure provides a pharmaceutical composition comprising the conjugate of the seventh aspect or the composition of the first or eighth aspect. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and excipients. It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect may also be applied to any embodiment of the fourteenth aspect.

[0160] In another aspect, the present disclosure provides a kit comprising the composition of the first or eighth aspect, the polymer-conjugated compound of the sixth aspect (amphiphilic OEG-conjugated compound), the conjugate of the seventh aspect, or a pharmaceutical composition as described herein (e.g., the pharmaceutical composition of the fourteenth aspect). In some embodiments, the kit is used for treatment, e.g., for inducing an immune response. In some embodiments, the kit is used to induce an immune response against a pathogen, e.g., for treating or preventing an infectious disease.

[0161] In another aspect, the present disclosure provides a method for preparing an amphiphilic OEG-conjugated 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 diacylglycerol moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety) to obtain an amphiphilic OEG-conjugated compound. Preferred embodiments of this aspect are given under the heading "Polymer-conjugated compound comprising (a) a polymer comprising a structure of formula (I); and (b) one or more hydrophobic chains". It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect may also be applied to any embodiment of this aspect of the method for preparing an amphiphilic OEG-conjugated compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 : Protocol for anti-PEG ELISA assay for determining the binding of anti-PEG polyclonal antibodies to different antigens (PEG, AEEA, pSAR). Biotin-labeled antigens (biotin-PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NH2-pSAR) were synthesized and captured on neutravidin-coated plates in wash buffer (1× PBS, 0.1% CHAPS) for 2 hours at room temperature with slow shaking. After washing with wash buffer (4×), the plates were incubated with different amounts of rabbit anti-PEG polyclonal serum (7 ng / ml, 3 ng / ml, 167 ng / ml, or 830 ng / ml) in assay buffer (1× PBS, 0.1% CHAPS, 0.2% BSA) for 2 hours at room temperature with slow shaking. After washing with wash buffer (5×), anti-rabbit-HRP secondary antibody (diluted 1:5000) was added and the plate was incubated for 1 hour at room temperature with slow shaking. After washing with wash buffer (5×), HRP substrate was added and the fluorescence signal was measured.

[0163] Figure 2 : Different biotin-labeled antigens (biotin-PEG36K, biotin-blocked-AEEA14, biotin-NH2-AEEA14, biotin-blocked-pSAR or biotin-NH2-pSAR) Figure 1 The results of anti-PEG ELISA assay.

[0164] Figure 3: Results of additional anti-PEG ELISA assays using monoclonal anti-PEG IgG (A) or monoclonal anti-PEG IgM (B) and different antigens (PEG36, blocked-AEEA14, NH2-AEEA14). PEG36 has a molecular weight of approximately 1.6 kDa. Blocked AEEA14 is Ac-AEEA14.

[0165] 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.

[0166] Figure 5 : Particle size and PDI of LNPs containing DSPE-AEEA14-AC using HY501 as the ionizable lipid. Also included are the particle size and PDI of BM LNP formulations.

[0167] Figure 6 : Zeta potential of LNPs containing DSPE-AEEA14-AC using HY501 as the ionizable lipid. Also included is the zeta potential of the BM formulation.

[0168] Figure 7 : Accessible RNA of DSPE-AEEA14-AC-containing LNPs using HY501 as the ionizable lipid, measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0169] Figure 8 : Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0170] Figure 9 : Hemolytic analysis after incubation (under neutral pH conditions) of human whole blood with LNPs containing DSPE-AEEA14-AC using HY501 as the lipid that can ionize into cations.

[0171] Figure 10 : Viability of all tested formulations after transfection with LNPs containing DSPE-AEEA14-AC using HY501 as the ionizable lipid. Also included are the viabilities of BM_2 and other PEG-containing formulations. Mean values ​​(n=3) + / - StDev are plotted.

[0172] Figure 11: Particle size and PDI of LNPs containing VE-AEEA14-AC using HY501 as the ionizable lipid. Also included are the particle size and PDI of BM LNP formulations.

[0173] Figure 12 : Zeta potential of LNPs containing VE-AEEA14-AC using HY501 as the ionizable lipid. Also included is the zeta potential of the BM preparation.

[0174] Figure 13 : Accessible RNA of VE-AEEA14-AC-containing LNPs using HY501 as the lipid ionizable to release cations as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0175] Figure 14 Quantification of S1 protein expression following transfection with VE-AEEA14-AC-containing LNPs using HY501 as the ionizable lipid. (A) Change in VE-AEEA14-14-AC composition of the total cell population vs. MFI (mean fluorescence intensity). Data were fitted using a quadratic polynomial function. (B) Viability of all tested formulations. MFI and viability of BM-2 and other PEG-containing formulations are also included. Mean values ​​(n=3) + / - StDev are plotted.

[0176] Figure 15 : Yield and purity of peptide intermediates. The peptide intermediates Ac-(AEEA)8-OH (A to D) and Ac-(AEEA) 14 -OH (E to H), and their 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 directly after cleavage from the resin (i.e., before QC procedures) (crude purity; Figure 15 A, C, E, G) or obtained after QC method (final purity; Figure 15 B, D, F, H).

[0177] Figure 16 : Monitoring of the synthesis of Ac-(AEEA)8-α-tocopherol. Reaction samples were obtained and analyzed using the methods described herein. UPLC chromatograms of samples obtained (A) after 5 minutes and (B) at the end of the reaction time (4 hours) are shown.

[0178] Figure 17 :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) purity as shown in exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), respectively.

[0179] Figure 18 : Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations). Also included are the particle size and PDI of BM LNP formulations.

[0180] Figure 19 : Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations). Also included is the zeta potential of BM formulations.

[0181] Figure 20 : Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid ionizable to release cations) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0182] Figure 21 : Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0183] Figure 22 : Hemolytic analysis after incubation (at neutral pH) of human whole blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 as a lipid that ionizes into cations.

[0184] Figure 23 Quantification of S1 protein expression following transfection with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (a lipid that ionizes cations). (A) Changes in overall cell population composition vs. mean fluorescence intensity (MFI). Data were fitted using a quadratic polynomial function. (B) Viability of all tested formulations. MFI and viability of BM_1 and BM_2, as well as other control formulations, are also included. Mean values ​​(n=3) + / - StDev are plotted.

[0185] Figure 24 : Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations).

[0186] Figure 25: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations).

[0187] Figure 26 : Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid ionizable to release cations) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0188] Figure 27 : LNPs containing various amphiphilic OEG-conjugated compounds were expressed in skeletal muscle cell lines (C2C12) ( Figure 27 A, B), mouse macrophage cell line (Raw) ( Figure 27 C, D) and liver cancer cell line (HepG2) ( Figure 27 In vitro expression (A, C, E) and viability (B, D, F) in Figure 5. Firefly luciferase expression 24 hours after incubation with 12.5, 25, and 50 ng of mRNA-loaded LNPs per well. Mean values ​​(n=3) + / - StDev are plotted.

[0189] Figure 28 : T cell targeting using LNPs with different stealth lipids and α-lipids. LNPs were formulated with different lipid compositions (carrier: Thy1.1 RNA / Luc RNA / Np proxy Venus1:1:2 w / w; N / P ratio: 6; lipid mixture: HY501 / cholesterol / DSPC / stealth lipid / α-lipid. For the following stealth lipid and α-lipid combinations, the lipid ratios were selected as [47.5 / 40.5 / 10 / 1.8 / 0.2]: C16 PEG2k ceramide / DSPE PEG2kα, DSPE PEG2k / DSPEPEG2kα, DSPE-AEEA14 / DSPE-AEEA14-α, or VE-AEEA8 / DSPE-AEEA14-α. For the following stealth lipid and α-lipid combinations, lipid ratios were selected as [47.5 / 38.5 / 10 / 3.8 / 0.2]: VE-PEG1k / DSPE-PEG2k-α or VE-AEEA8 / DSPE-AEEA14-α. LNPs were post-functionalized with aCD3 VHH XNbα ligand [w / w* = ligand to cargo ratio 0.48]; RNA concentration: 0.1 μg / μl]. All LNPs had diameters ranging from 100 to 170 nm, with a PDI below 0.4 as determined by DLS.

[0190] For transfection studies, 10 μl (1000 ng dose) of the corresponding preparation was pre-diluted in 50 μl X-Vivo 15 in ultra-low adhesion 96-well plates.6 Individual thawed human PBMCs were diluted in 50 μl 100% condensed PHS and added to nanoparticle diluents. After incubation (37°C, 5% CO2) for 30 minutes, 30 μl of each transfection reaction was transferred to a second ultra-low adhesion 96-well plate, and 170 μl X-Vivo 15 culture medium + 100 U / ml IL2 was added to each well. The cell dilutions were cultured for another 18 hours (37°C, 5% CO2). In the following, cell type specific transfection (Thy1.1) was analyzed by flow cytometry. The percentage of transfected cells (CD2 negative cells, CD19+B cells, CD4+T cells and CD8+T cells) in all transfected PBMCs (transfection, y axis) under each test formulation condition is shown.

[0191] Figure 29 : Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations).

[0192] Figure 30 : Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations).

[0193] Figure 31 : Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid ionizable to release cations) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0194] Figure 32 : Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0195] Figure 33 : Hemolytic analysis after incubation (at neutral pH) of human whole blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 as a lipid that ionizes into cations.

[0196] Figure 34 :In skeletal muscle cell line (C2C12) ( Figure 34 A, B), mouse macrophage cell line (Raw) ( Figure 34 C, D) and liver cancer cell line (HepG2) ( Figure 34E, F), in vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds. Firefly luciferase expression after 24 hours of incubation with 12.5, 25, and 50 ng of mRNA-loaded LNPs per well. Mean values ​​(n=3) + / - StDev are plotted.

[0197] Figure 35 : Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugated compounds and HY501 (as a lipid that can ionize cations).

[0198] Figure 36 : Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugated compounds and HY501 (as a lipid that can ionize cations).

[0199] Figure 37 : Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugated compounds and HY501 (as a lipid ionizable to release cations) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0200] Figure 38 : Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0201] Figure 39 : Hemolytic analysis after incubation (at neutral pH) of human whole blood with LNPs containing various amphiphilic VE-(AEEA)n-AC conjugated compounds and HY501 as a lipid that can ionize into cations.

[0202] Figure 40 :In skeletal muscle cell line (C2C12) ( Figure 40 A, B), mouse macrophage cell line (Raw) ( Figure 40 C, D) and liver cancer cell line (HepG2) ( Figure 40 In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugated compounds (E, F). Firefly luciferase expression after 24 hours of incubation with 12.5, 25, and 50 ng of mRNA-loaded LNPs per well. Mean values ​​(n=3) + / - StDev are plotted.

[0203] Figure 41 : Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid that can ionize cations).

[0204] Figure 42 : Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as a lipid ionizable to release cations) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0205] Figure 43 : Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0206] Figure 44 : Hemolytic analysis after incubation (at neutral pH) of human whole blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 as a lipid that ionizes into cations.

[0207] Figure 45 :In skeletal muscle cell line (C2C12) ( Figure 45 A, B), mouse macrophage cell line (Raw) ( Figure 45 C, D) and liver cancer cell line (HepG2) ( Figure 45 E, F), in vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds. Firefly luciferase expression after 24 hours of incubation with 12.5, 25, and 50 ng of mRNA-loaded LNPs per well. Mean values ​​(n=3) + / - StDev are plotted.

[0208] Figure 46 : Particle size and PDI of LNPs containing amphiphilic OEG-conjugated compounds and various cationic lipids or lipids that can ionize cations (DODMA, DODAB, DOTMA, DOTAP).

[0209] Figure 47 : Zeta potential of LNPs containing amphiphilic OEG-conjugated compounds and various cationic lipids or lipids that can ionize cations (DODMA, DODAB, DOTMA, DOTAP).

[0210] Figure 48 : Accessible RNA of LNPs containing amphiphilic OEG-conjugated compounds and various cationic lipids or lipids that can ionize out cations (DODMA, DODAB, DOTMA, DOTAP) measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

[0211] Figure 49: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0212] Figure 50 : Hemolysis analysis after incubation (at neutral pH) of human whole blood with LNPs containing amphiphilic OEG-conjugated compounds and various cationic lipids or lipids that can ionize cations (DODMA, DODAB, DOTMA, DOTAP).

[0213] Figure 51 :In skeletal muscle cell line (C2C12) ( Figure 51 A, B), mouse macrophage cell line (Raw) ( Figure 51 C, D) and liver cancer cell line (HepG2) ( Figure 51 In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing amphiphilic OEG-conjugated compounds and various cationic lipids or lipids that can ionize cations (DODMA, DODAB, DOTMA, DOTAP) in E, F). Firefly luciferase expression after 24 hours of incubation with 12.5, 25, and 50 ng of mRNA-loaded LNPs per well. Mean values ​​(n=3) + / - StDev are plotted.

[0214] Figure 52 : Particle size and PDI of functionalized LNPs prepared with five different OEG-conjugated compounds as stealth lipids.

[0215] Figure 53 : Agarose gel electrophoresis of control, untreated functionalized LNPs (upper row), and functionalized LNPs treated with release solution (lower row).

[0216] Figure 54 : Particle size and PDI of functionalized LNPs prepared with OEG-conjugated compounds ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA-pAEEA14-Ac, and (E) VitE-pAEEA8-Ac) subjected to three freeze-thaw cycles from -20°C to room temperature and from -80°C to room temperature, respectively.

[0217] Figure 55Figure 2: In vitro transfection with LNPs. A) Percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) among all transfected PBMCs (transfection, y-axis) in each test formulation. B) Total cell counts obtained by flow cytometry within 20 seconds. Numbers were corrected for counting beads. Samples were collected using a BD FACS Lyric. Data were analyzed using FlowJo.

[0218] Figure 56 : In vivo ligand-mediated T cell transfection. B6-hCD3EDG transgenic mice were injected intramuscularly with a mixture of RNA encoding luciferase and Thy1.1 (1:1 weight ratio mixture), either naked or formulated as LNPs (1 μg RNA dose per flank; 2 μg total RNA dose per mouse). Analysis was performed 18 hours after injection. (A) Drainage analyzed by ex vivo bioluminescence imaging of popliteal, inguinal, axillary, and brachial lymph nodes, as well as the spleen. (B) Cell type-specific transfection analyzed by flow cytometry by detecting expression of delivered Thy1.1 RNA in immune cell subsets in the popliteal and inguinal lymph nodes and spleen. (C) T cell activation status analyzed by mean fluorescence intensity of CD69 surface expression in the organs (after staining with anti-CD69-APC antibody). LN, lymph node; LNP, lipid nanoparticle; NK, natural killer; PMN, polymorphonuclear cell.

[0219] DETAILED DESCRIPTION

[0220] Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing certain specific embodiments only and are not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0221] Hereinafter, the key elements of the present disclosure will be described in more detail. These key elements are listed together with some specific embodiments, however, it should be understood that they can be combined in any way and in any quantity to produce other embodiments. The examples and preferred embodiments of the multiple descriptions should not be construed as limiting the present disclosure to only some embodiments clearly described. This specification sheet should be understood as supporting and encompassing the embodiments that will clearly describe the embodiment and any number of disclosed and / or preferred key element combinations. In addition, unless the context indicates otherwise, any arrangement and combination of all described key elements in this application should be considered to be disclosed by the specification sheet of the application.

[0222] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0223] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the art (see, e.g.,

[0224] Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuchder Organischen Chemie", S.Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme VerlagStuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J.Sambrook et al.eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989.

[0225] Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure that is otherwise claimed. The language in this specification should not be interpreted as indicating any unclaimed element necessary for practicing the present disclosure.

[0226] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0227] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, guides, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0228] definition

[0229] The following definitions are provided for all aspects of this disclosure. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0230] Throughout this specification and the appended claims, unless the context requires otherwise, the words "comprises / comprising" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated members, integers or steps or groups of members, integers or steps, but not the exclusion of any other members, integers or steps or groups of members, integers or steps. The term "consisting essentially of is meant to exclude any other members, integers or steps in any substantial sense. The term "comprises / comprising" encompasses the term "consisting essentially of", which in turn encompasses the term "consisting of". Therefore, the term "comprises / comprising" may be replaced with the term "consisting essentially of" or "consisting of" at each occurrence in this application. Similarly, the term "consisting essentially of" may be replaced with the term "consisting of" at each occurrence in this application.

[0231] Unless otherwise indicated herein or clearly contradicted by context, unmodified nouns and similar referents used in the context of describing the disclosure (especially in the context of the claims) are understood to cover both the singular and the plural.

[0232] As used herein, "and / or" is considered specific disclosure that each of the two specified features or components has or does not have the other. For example, "X and / or Y" is considered specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually listed herein.

[0233] In the context of the present disclosure, the term "about" represents an accuracy interval that will be understood by those of ordinary skill in the art, to still ensure the technical effect of the feature in question. The term generally represents a deviation from the indicated numerical value of ±5%, such as ±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 such as ±0.01%. As will be understood by those of ordinary skill in the art, the specific such deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can generally have greater such deviations than artificial or engineered technical effects.

[0234] As used herein, terms such as "reduce / reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, such as a decrease of 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 phrases include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

[0235] As used herein, terms such as "enhance" or "increase" refer to the ability to cause an overall increase in levels or enhance, for example, 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 relate 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%.

[0236] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is between 7.3 and 7.5. In some embodiments, the physiological pH is between 7.35 and 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0237] As used herein, "physiological conditions" refer to conditions (particularly pH and temperature) in a living subject (particularly a human). Preferably, physiological conditions mean physiological pH and / or a temperature of about 37°C.

[0238] As used herein, "% (w / v)" (or "% w / v") refers to weight to volume percentage, which is a unit of concentration measuring the amount of solute in grams (g) as a percentage of the total volume of a solution in milliliters (ml).

[0239] As used herein, "volume %" or "% (v / v)" (or "% v / v") refers to volume percent, which is a unit of concentration measuring the amount of a liquid substance (in milliliters (ml)) expressed as a percentage of the total volume of a solution (in milliliters (ml)).

[0240] As used herein, "weight %" or "% (w / w)" (or "% w / w") refers to weight percent, which is a unit of concentration measuring the amount of a substance (in grams (g)) expressed as a percentage of the total weight (in grams (g)) of the total composition.

[0241] "Mol %" as used in this disclosure is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0242] "Mol % of total lipids" as used in this disclosure is defined as the ratio of the moles of one lipid component to the total moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0243] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Therefore, the ionic strength, I, is mathematically represented by the following formula:

[0244]

[0245] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is taken over all the different ions (i) in the solution.

[0246] According to the present disclosure, in some embodiments, the term "ionic strength" refers to the presence of monovalent ions.

[0247] With respect to the presence of divalent inorganic ions, particularly divalent inorganic cations, in one embodiment, the concentration or effective concentration (presence of free ions) is sufficiently low (presence of free ions) due to the presence of the chelating agent to prevent RNA degradation. In one embodiment, the concentration or effective concentration of the divalent inorganic ions is below the catalytic level for hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent inorganic ions is 20 μM or less. In one embodiment, free divalent inorganic ions are absent or substantially absent.

[0248] A "monovalent" compound refers to a compound having only one functional group of interest. For example, a monovalent anion refers to a compound having only one negatively charged group, preferably under physiological conditions.

[0249] A "divalent" or "dibasic" compound refers to a compound having two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is Ca 2+ .

[0250] A "polyvalent" 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.

[0251] "Monovalent moiety" refers to a monovalent group, ie, a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, and the like.

[0252] "Divalent moiety" or "bivalent moiety" refers to a diradical, i.e., a moiety with a valence of 2. Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, and the like. Another example of a divalent moiety is the group [*-S] p (C 1-6 Alkylene)-C 1-6 Alkylene moiety, if p is 1 (yielding group *-S(C 1-6 Alkylene)-, such as *-S-(CH2)6- or *-S-CH2-, wherein * represents 4 connection point).

[0253] A "polyvalent moiety" refers to a polyradical, i.e. a moiety with a valence of at least 3. For example, a "trivalent moiety" refers to a triradical, i.e. a moiety with a valence of 3. For example, by removing an additional H atom from an alkylene group, the resulting alkylene group is trivalent. Another example of a trivalent moiety is the group [*-S] p (C 1-6 Alkylene)-C 1-6 Alkylene moiety, if p is 2 (yielding the group [*-S]2(C 1-6 Alkylene)-, such as *-S-CH(S-*)(CH2)5- or *-S-CH(S-*)(CH2)-, wherein * represents the same 4 Another example of a trivalent moiety is the group [*-C(O)NH](C 1-6 Alkyltriyl)-C 1-6 The alkyl trialkyl moiety (here, the [*-C(O)NH] moiety and another hydrophobic chain are connected to the C 1-6 The alkyl triyl portion is combined, and then the C 1-6 The alkyltriyl moiety is directly or through at least one additional difunctional moiety and is bound to X 1 (For formula (V)) or X 2 (For formula (V')) connection). Therefore, [*-C(O)NH](C 1-6 Alkyltriyl) part as L 1 An example where C 1-6 Alkyl trialkyl and another hydrophobic chain R 4 Direct connection, the L 1 It may contain at least the following structures:

[0254]

[0255] in One of the C 1-6 The bond connecting the alkyltriyl group to [*-C(O)NH] and another Indicates C 1-6 A bond connecting an alkyltriyl group to the remainder of a compound (e.g., a compound of formula (V)) (directly or through another bifunctional moiety). 1-6 When the alkyl trialkyl group is also substituted with a -OH moiety, it at least covers the following structures:

[0256]

[0257] As used herein, "molar ratio" refers to the ratio between the molar amounts of 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 the composition in an amount of 2 millimoles (mmol), then the molar ratio of the first substance to the second substance is 1:2 or 0.5.

[0258] "Osmolarity" refers to the concentration of a specific solute, expressed as osmoles of solute per kilogram of solvent.

[0259] The term "lyophilization" and variations thereof refer to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the freezing medium in the substance to sublime directly from the solid phase to the gas phase. Thus, the terms "lyophilization" and "freeze drying" are used interchangeably herein.

[0260] The term "spray drying" refers to the spray drying of a substance by mixing a (heated) gas with an atomizing (spray-like) fluid in a vessel (spray dryer), wherein the solvent from the formed droplets evaporates to produce a dry powder.

[0261] The term "reconstitution" relates to the addition of a solvent (eg, water) to a dried product to return it to a liquid state, eg, its original liquid state.

[0262] The term "freezing" refers to the solidification of a liquid, usually with the removal of heat. In some embodiments, freezing is the opposite action of thawing.

[0263] The term "thawing" refers to the liquefaction of a solid, typically by the addition of heat. In some embodiments, thawing is the reverse of freezing.

[0264] As used herein, the term "aqueous phase" in relation to compositions / formulations comprising particles (particularly LNPs, liposomes and / or lipoplexes) refers to the mobile or liquid phase, i.e., the continuous aqueous phase that includes all components dissolved therein but (formally) does not include the particles. Thus, if the particles (e.g., LNPs) are dispersed in the aqueous phase, and the aqueous phase is substantially free of compound X, then the aqueous phase is free of X in such a manner that it is practically feasible and practicable, for example, the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is possible that the particles dispersed in the aqueous phase may contain compound X in an amount exceeding 1% by weight.

[0265] The term “recombinant” in the context of this disclosure means “prepared by genetic engineering.” In some embodiments, a “recombinant object” in the context of this disclosure does not occur in nature.

[0266] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in a laboratory is naturally occurring. The term "found in nature" means "occurring in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0267] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15° C., preferably about 15° C. to about 35° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 17° C. to about 22° C. Such temperatures would include 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., and 22° C.

[0268] The term "alkyl" refers to a monovalent group of a saturated straight or branched hydrocarbon. Preferably, the alkyl group 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, abbreviated as C 1-12 Alkyl, (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, abbreviated as C 1-10alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. "Substituted alkyl" means an alkylene group in which one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are 1st-stage substituents, 2nd-stage substituents, or 3rd-stage substituents as specified herein. Some examples of substituted alkyl groups include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0269] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group 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. Some exemplary 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-pentylene, 1,1-neopentyl), 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-heptylene, 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 3 carbon atoms and one free valence at each end may also be referred to as a multiple methylene group (e.g., 1,4-butylene may also be referred to as a tetramethylene group). Generally speaking, as an alternative to using the ending "ylene" of the above alkylene moieties, the ending "diyl" may also be used (e.g., 1,2-butylene may also be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0270] The term "alkenyl" refers to a monovalent group of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is an odd number, the division result is rounded to the next integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an 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, an alkenyl group comprises 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, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group contains 2 to 12 carbon atoms (abbreviated as C 2-12The alkylene group comprises an alkylene group (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 bonds may be in a cis (Z) or trans (E) configuration. Some exemplary alkenyl groups include ethenyl, 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, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonen ...9-nonenyl, 10-nonenyl, 11-nonenyl, 12-nonenyl, 13-nonenyl, 14-nonenyl, 15-nonenyl, 16-nonenyl, 17-nonenyl, 18-nonenyl, 19-nonenyl, 20-nonenyl, 21-nonenyl, 22-nonenyl, 23-nonenyl, 24-nonenyl, 25-nonenyl, 26-nonenyl, 27-nonenyl, 28-nonenyl, 29-nonenyl, 30-nonenyl, 31-non The double bond may be alkyl, 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, etc. If the alkenyl group is attached to a nitrogen atom, the double bond may not be alpha to the nitrogen atom. "Substituted alkenyl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkenyl 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 the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0271] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total carbon atoms can be six to thirty, typically six to twenty, typically six to eighteen. An alkynyl group may optionally have one or more carbon-carbon double bonds. Generally speaking, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is an odd number, the division result is rounded to the next integer. For example, for an alkynyl group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, an 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 to the maximum number of hydrogen atoms bound to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0272] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, and if the number of carbon atoms in the alkenylene group is an odd number, the division result is rounded to the next integer. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an 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, an alkenylene group comprises 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, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises 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, it comprises 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 bonds may be in cis (Z) or trans (E) configuration. Some exemplary 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, allylene, 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., 1 to the maximum number of hydrogen atoms bound to the alkenylene 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 the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0273] The term "cycloalkyl" refers to a cyclic non-aromatic form of an alkyl group having preferably 3 to 14 carbon atoms, such as 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 cycloalkyls include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl and adamantyl. Cycloalkyls can be composed of one ring (monocycle), two rings (bicycle) or more than two rings (polycycle). "Substituted cycloalkyl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0274] The term "cycloalkylene" refers to a cyclic, non-aromatic form of an "alkylene" group, and is a pair, ortho or separated diradical. In certain embodiments, the cycloalkylene group (i) is monocyclic or polycyclic (e.g., bicyclic or tricyclic) and / or (ii) is 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-membered or 3 to 10-membered). In one embodiment, the cycloalkylene group 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-membered or 3 to 10-membered) cycloalkylene group. Generally speaking, as an alternative to using the ending "sub-" of the above-mentioned cycloalkylene moiety, the ending "di-" can also be used (e.g., 1,2-cyclopropylene can also be referred to as cyclopropane-1,2-diyl). Some exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantylene (e.g., tricyclo[3.3.1.1 3,7] decane-2,2-diyl). "Substituted cycloalkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the cycloalkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., from 1 to 5, from 1 to 4, or from 1 to 3, or from 1 to 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0275] The term "cycloalkenylene" represents a cyclic non-aromatic form of an alkenylene group, and is a paired, ortho or separated diradical. Generally speaking, the maximum number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2, and if the number of carbon atoms in the cycloalkenylene group is an odd number, the division result is rounded to the next integer. For example, for the cycloalkenylene group with 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 group (i) is a monocycle or polycycle (e.g., a bicycle or tricycle) and / or (ii) is 3 to 14 yuan (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 yuan, such as 3 to 12 yuan or 3 to 10 yuan). In one embodiment, 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, such as 3- to 12-membered or 3- to 10-membered) cycloalkenylene. Some exemplary cycloalkenylenes include cyclohexene, cycloheptenene, cyclopropenene, cyclobutenene, cyclopentenene and cyclooctenene. "Substituted cycloalkenylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the cycloalkenylene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are 1st, 2nd or 3rd level substituents as specified herein.

[0276] The term "aryl" refers to a monovalent group of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9 or 10, such as 5, 6 or 10) carbon atoms, which may be arranged in one ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropane, The term "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Some preferred examples are phenyl and naphthyl. Aryl does not include fullerenes. "Substituted aryl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the aryl, 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 the aryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-order substituent, a second-order substituent, or a third-order substituent as specified herein. Some examples of substituted aryl groups include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilino, 4-hydroxyphenyl, and methoxyphenyl (ie, 2-, 3-, or 4-methoxyphenyl).

[0277] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by O, S or N heteroatoms. Preferably, heteroaryl refers to a five- or six-membered aromatic monocyclic ring in which 1, 2 or 3 carbon atoms are replaced by the same or different O, N or S heteroatoms. Alternatively, it means an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4 or 5 carbon atoms are replaced by the same or different O, N or S heteroatoms. Preferably, in each ring of the 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, Azolyl, iso Azolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzo Azolyl, indole Indoxazinyl, benzylisothiazolinyl oxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenanthroline Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophene, pyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, anthranyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophene, Azolyl, iso Azolyl, The term "substituted heteroaryl" refers to a group consisting of 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., 1 to the maximum number of hydrogen atoms bound to the heteroaryl 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 the heteroaryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-order substituent, a second-order substituent, or a third-order substituent, as specified herein.

[0278] The term "heterocyclyl" or "heterocycle" means a cycloalkyl group as defined above in which 1, 2, 3 or 4 of the carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus or sulfur, preferably O, S or N. The heterocyclyl group preferably has 1 or 2 rings containing 3 to 10, for example 3, 4, 5, 6 or 7 ring atoms. Preferably, in each ring of the 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 meant to encompass partially or fully hydrogenated forms (e.g., dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Some exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also known as piperidyl), piperazinyl, dihydrofuranyl and tetrahydrofuranyl, dihydrothiophenyl and tetrahydrothiophenyl, dihydropyranyl and tetrahydropyranyl, hexamethylenetetramine (urotropinyl), lactones, lactams, cyclic imides, and cyclic anhydrides. "Substituted heterocyclyl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the heterocyclyl, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent or a 3rd level substituent, as specified herein.

[0279] As used herein, the term "heterocycloalkylene" means a heterocyclic radical as defined above comprising at least one heteroatom (e.g., a heteroatom selected from O, S, N, B, Si, and P) and wherein a hydrogen atom has been removed to produce a paired, ortho, or separated diradical. In some embodiments, the heteroatom of heterocycloalkylene is selected from O, S, and N. For example, heterocycloalkylene can be an O / S-heterocycloalkylene, such as an O-heterocycloalkylene. In some embodiments, in each ring of heterocycloalkylene, 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. Heterocycloalkylene can be monocyclic or polycyclic (e.g., bicyclic or tricyclic). In some embodiments, heterocycloalkylene is a single, double, or tricyclic 4 to 14-membered (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, such as 4 to 12-membered or 4 to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also meant to encompass partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the above-mentioned heteroaryl groups (preferably partially or fully hydrogenated forms of the above-mentioned mono-, bi-, or tricyclic heteroaryl groups) in which one hydrogen atom has been removed from the same carbon atom to produce a paired diradical. Thus, in some embodiments, heterocycloalkylene is saturated or unsaturated (i.e., contains one or more double bonds in the ring), but may not be aromatic. "Substituted heterocycloalkylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

[0280] As used herein, the expression "partially hydrogenated form" of an unsaturated compound or group means a portion of the unsaturation is removed by formally adding hydrogen to the original unsaturated compound or group without removing all of the unsaturated portions. As used herein, the phrase "fully hydrogenated form" of an unsaturated compound or group is used interchangeably with the term "perhydro" and means the removal of all unsaturations by formally adding hydrogen to the original unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl (comprising 2 double bonds in the ring, such as furan) includes a dihydro form of the 5-membered heteroaryl (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while a tetrahydro form of the 5-membered heteroaryl (e.g., tetrahydrofuran, i.e., THF) is a fully hydrogenated (or perhydro) form of the 5-membered heteroaryl. Similarly, for a 6-membered heteroaryl having 3 double bonds in the ring (e.g., pyridyl), the partially hydrogenated forms include dihydro and tetrahydro forms (e.g., dihydropyridyl and tetrahydropyridyl), while the hexahydro forms (in the case of heteroaryl pyridyl, e.g., piperidyl) are fully hydrogenated (or perhydro) derivatives of the 6-membered heteroaryl. Thus, if the aryl or heteroaryl group contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms, the hexahydro form of the aryl or heteroaryl group can only be considered as a partially hydrogenated form according to the present disclosure.

[0281] The term "aromatic" used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if monocyclic aryl is hydrogenated (partially or completely hydrogenated), then for the purposes of this disclosure, the gained hydrogenated cyclic structure is classified as cycloalkyl. Similarly, if bicyclic or polycyclic aryl (such as naphthyl) is hydrogenated, then for the purposes of this disclosure, the gained hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as cycloalkyl (even if one ring (such as in 1,2-dihydronaphthyl) is still aromatic). In the present application, similar differences are arranged between heteroaryl and heterocyclic radical. For example, for the purposes of this disclosure, dihydroindole, i.e. the dihydro variant of indole is classified as heterocyclic radical, because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0282] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, the hydrocarbyl group is non-cyclic, such as linear (straight chain) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Some specific examples of hydrocarbyl groups are C 1-30 Alkyl (e.g. C 6-30 Alkyl, C 8-24 Alkyl or C 10-20 alkyl), C having 1, 2 or 3 double bonds 2-30 Alkenyl (e.g. C 6-30 Alkenyl, C 8-24Alkenyl or C 10-20 alkenyl)), aryl and aryl (C 1-6 In some embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more primary substituents, one or more secondary substituents, one or more tertiary substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively non-polar. In some embodiments, the hydrocarbyl group is a hydrocarbyl chain of a naturally occurring fatty acid and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) C 8-20 Alkyl chains, such as (preferably linear) C 10-18 alkyl chain).

[0283] The term "optionally substituted" indicates that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms may be replaced by a group other than hydrogen (i.e., a primary substituent), such as an alkyl group (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, azido, -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 bound to the same carbon atom of a cycloalkyl or heterocyclyl group may be linked together to form =X 1 , wherein each of the first-level substituents of alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl may itself be substituted with one or more (e.g., one, two or three) substituents (i.e., second-level substituents) selected from the following: 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, azido, -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- 2N(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 )(R 83 ), -OP(O)(OR 81 )2,-C(=X2)R 81 , -C(=X2)X2R 81 , -X2C(=X2)R 81 , and -X2C(=X2)X2R 81

[0284] and / or any two second-stage substituents bonded to the same carbon atom of the cycloalkyl or heterocyclic group as the first-stage substituent may be linked together to form =X2, wherein the C 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 is optionally substituted with one or more (e.g., one, two, or three) substituents (i.e., third-level substituents) independently selected from the group consisting of: C 1-3 Alkyl, halogen, -CF3, -CN, azido, -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 1-3 The alkyl group is independently methyl, ethyl, propyl or isopropyl, and / or any two third-level substituents bonded to the same carbon atom of the 3- to 14-membered cycloalkyl or heterocyclic group as the second-level substituent may be linked together to form =O, =S, =NH or =N(C 1-3 alkyl);

[0285] in

[0286] R 71 、R 72 and R 73 Each of which is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 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 2-6Alkenyl, 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 is optionally substituted with one, two or three substituents independently selected from the group consisting of: C 1-3 Alkyl, halogen, -CF3, -CN, azido, -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 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl;

[0287] R 81 、R 82 and R 83 Each of which is independently selected from H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 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 is optionally substituted with one, two or three substituents independently selected from the group consisting of: C 1-3 Alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, =O, -S(C 1-3alkyl), -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 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl; and

[0288] Each of X1 and X2 is independently selected from O, S and N (R 84 ), where R 84 Is H or C 1-3 alkyl.

[0289] Typical first-stage substituents are preferably selected from the following: 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, azido, -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(R72 )(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 , for example C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 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, 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 Alkyl is independently methyl, ethyl, propyl or isopropyl; wherein X 1 are independently selected from O, S, NH and N(CH3); and R 71 、R 72 and R 73 Each of which is as defined above, or preferably, independently selected from H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4alkynyl, 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 substituted with one, two, or three substituents independently selected from the group consisting of: C 1-3 Alkyl, halogen, -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 Alkyl is independently methyl, ethyl, propyl or isopropyl. In some embodiments, the first substituent is selected from C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH and -C(=O)OCH3, wherein z is 0, 1 or 2 and C 1-3 Alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, the first substituent is selected from methyl, ethyl, propyl or isopropyl, halogen (such as F, Cl or Br) and -CF3, such as halogen (such as F, Cl or Br) and -CF3.

[0290] Typical second-level substituents are preferably selected from C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 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 1-3Alkyl), -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 Alkyl is independently methyl, ethyl, propyl or isopropyl. Some specific examples of secondary substituents are independently selected from the following: 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 The alkyl group is independently methyl, ethyl, propyl or isopropyl. Particularly preferred secondary substituents are independently selected from methyl, ethyl, propyl, isopropyl, phenyl, =O and =S.

[0291] Typical 3rd-level substituents are preferably selected from C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH and -C(=O)OCH3, wherein z is 0, 1 or 2, and C 1-3 Alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred tertiary substituents are selected from methyl, ethyl, propyl, isopropyl, halogen (e.g. F, Cl or Br) and -CF3, such as halogen (e.g. F, Cl or Br) and -CF3.

[0292] As used herein, the term "tertiary amine moiety" refers to a moiety comprising a nitrogen atom substituted with three organic substituents (wherein the substituents may be the same or different from each other). In some embodiments, the organic substituents are selected from hydrocarbyl groups (e.g., alkyl groups, particularly C 2-3, C 3-4, C 5-6, C 7-8, C 9-10, C 10-11, C 12-13, C 13-14, C 14-15, C 15-16, C 16-17, C 17-18, C 18-19, C 19-20, C 19-21, C 19-22, C 19-23, C 19-24, C 19-25, C 19-26, C 19-27, C 19-28, C 19-29, C 20-30, C 21-31, C 22-32, C 23-33, C 24-34, C 25-35, C 26-36, C 27-37, C 28-38, C 29-40, C 29-41, C 29-42, C 29-43, C 29-44, C 29-45, C 29-46, C 29-47, C 29-48, C 29-50, C 29-51, C 29-52, C 29-53, C 29-54, C 29-55, C 29-56, C 1-6 alkyl).

[0293] 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 therein. Such separations may utilize micromolecularly permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis). Thus, examples of filtration include dialysis, tangential flow filtration, and diafiltration.

[0294] As used herein, the expression "substantially free of X" means that a mixture (e.g., a composition described herein or an aqueous phase thereof) is free of X in such a manner that it is practically possible and practicable to do so. For example, if the 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.

[0295] For example, as used herein, "substantially free of lipids containing polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive repeating units of ethylene glycol" means that the mixture (e.g., a composition described herein or an aqueous phase thereof) is free of lipids containing at least 30 consecutive repeating units of ethylene glycol in such a manner that it is practically possible and practicable to do so. For example, if the mixture is substantially free of lipids containing at least 30 consecutive ethylene glycol repeating units, the amount of lipids containing at least 30 consecutive ethylene glycol repeating 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 compositions comprising the phrase "substantially free" (e.g., "substantially free of sarcosinated lipids," "substantially free of (POX)-conjugated and / or poly(vinylpyrrolidone)." The invention also includes the following statements: "a POZ-conjugated lipid", "substantially free of any polymer-conjugated lipid other than the amphiphilic OEG-conjugated compound"; and "substantially free of any polymer-conjugated lipid other than the amphiphilic OEG-conjugated compound".

[0296] The expression "nucleic acid integrity" refers to the percentage of full-length (i.e., unfragmented) nucleic acids contained in a sample relative to the total amount of nucleic acids (i.e., unfragmented nucleic acids plus fragmented nucleic acids). Nucleic acid integrity can be determined by chromatographic separation of nucleic acids (e.g., separation 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 acids), determining the peak area of ​​the total nucleic acids, and dividing the peak area of ​​the main nucleic acid peak by the peak area of ​​the total nucleic acids. Similarly, the expression "RNA integrity" refers to the percentage of full-length (i.e., unfragmented) RNA contained in a sample relative to the total amount of RNA (i.e., unfragmented RNA plus fragmented RNA). RNA integrity can be determined by chromatographic separation of RNA (e.g., separation using capillary electrophoresis), determining the peak area of ​​the main RNA peak (i.e., the peak area of ​​the full-length (i.e., unfragmented) RNA), determining the peak area of ​​the total RNA, and dividing the peak area of ​​the main RNA peak by the peak area of ​​the total RNA.

[0297] The term "cryoprotectant" relates to a substance that is added to a preparation (eg a formulation or composition) to protect the active ingredients in the formulation during the freezing phase.

[0298] The term "lyoprotectant" relates to substances added to the formulation to protect the active ingredient during the drying phase.

[0299] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten 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 linked to each other by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide," "polypeptide," and "protein" are often used synonymously herein.

[0300] "Therapeutic peptides or proteins" when provided to a subject in a therapeutically effective amount, have a positive or beneficial effect on the subject's illness or disease state. In some embodiments, the therapeutic peptide or protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse a disease or condition, delay the onset of a disease or condition, or alleviate the severity of one or more symptoms of a disease or condition. Therapeutic peptides or proteins can have preventive properties and can be used to delay the onset of a disease or alleviate the severity of such a disease or pathological condition. The term "therapeutic peptide or protein" includes complete peptides or proteins and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of peptides or proteins. Some examples of therapeutically active peptides or proteins include, but are not limited to, antigens for vaccination and immunostimulants (e.g., cytokines). The terms "therapeutic peptide or protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

[0301] The term "portion" refers to a fraction. With respect to a specific structure, such as an amino acid sequence or a protein, the term "portion" thereof may refer to a continuous or discontinuous fraction of the structure.

[0302] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure (e.g., an amino acid sequence or protein) refers to a continuous element of the structure. When used in the context of a composition, the term "portion" means a portion of the composition. For example, a portion of a composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0303] With respect to an amino acid sequence (peptide, polypeptide or protein), a "fragment" refers to a portion of an amino acid sequence, i.e., a sequence representing an amino acid sequence that is shortened at the N-terminus and / or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A fragment shortened at the N-terminus (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 comprises a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises 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 comprise, for example, sequences of, for example, up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of an amino acid sequence.

[0304] According to the present disclosure, a part or fragment of a peptide, polypeptide or protein preferably has 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 has at least one pharmacological activity of the peptide, polypeptide or protein from which the fragment is derived. A part 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 part or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the peptide or protein.

[0305] As used herein, "variant" with respect to an amino acid sequence (peptide, polypeptide, or protein) means 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 may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified form of a wild-type amino acid sequence. In some 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 compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid differences.

[0306] "Wild-type" or "WT" or "native" with respect to an amino acid sequence means 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 means a nucleic acid sequence found in nature, including allelic variations. For example, a wild-type coding sequence means a coding sequence that is found in nature and has not been intentionally modified.

[0307] As used herein, "coding sequence" means the portion of a nucleic acid (eg, the DNA or RNA of a gene) that encodes a protein.

[0308] The expression "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 the DNA or RNA, or for the entire genome. When the G / C content refers to a portion, it can refer to the G / C content of a single gene or a portion (domain) of a gene, a group of genes or gene clusters, a noncoding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0309] For the purposes of this disclosure, "variants" of an amino acid sequence (peptide, protein, or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformers, isoforms, allelic variants, species variants, and species homologs, particularly those occurring in nature. The term "variant" particularly includes fragments of an amino acid sequence.

[0310] Amino acid insertion variants include insertions of single or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product is also possible. Amino acid addition variants include one or more amino acids, such as amino and / or carboxyl terminal fusions of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its place. Priority is given to modifications in positions in the amino acid sequence that are non-conserved between homologous proteins or peptides and / or replacing amino acids with other amino acids having similar properties. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of the families of amino acids to which their side chains are associated. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In one embodiment, conservative amino acid replacements include replacements within the following groups:

[0311] -Glycine, alanine;

[0312] -valine, isoleucine, leucine;

[0313] -Aspartic acid, glutamic acid;

[0314] -Asparagine, glutamine;

[0315] -serine, threonine;

[0316] - lysine, arginine; and

[0317] -Phenylalanine, tyrosine.

[0318] In some embodiments, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and the amino acid sequences of a variant of the given amino acid sequence will be 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 given 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 full 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 given 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, contiguous amino acids). In some embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignments for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0319] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences.

[0320] The terms "% identical" and "% identity" or similar terms are particularly intended to refer to the percentage of nucleotides or amino acids that are identical between the sequences to be compared in an optimal alignment. The percentages are purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences after optimal alignment over a segment or "comparison window" 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 Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the search similarity 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 some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) the expectation threshold is set to 10; (ii) the word length is set to 28; (iii) the maximum match within the query range is set to 0; (iv) the match / mismatch score is set to 1, -2; (v) the gap cost is set to linear; and (vi) a filter for low complexity regions is being used. In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10; (ii) word length set to 3; (iii) maximum match within query range set to 0; (iv) matrix set to BLOSUM62; (v) gap cost set to existence: 11 extension: 1; and (vi) condition composition scoring matrix adjustment.

[0321] Percent identity is calculated by determining the number of identical positions in the sequences being compared, dividing that number by the number of positions being compared (eg, the number of positions in the reference sequence), and multiplying the result by 100.

[0322] In some embodiments, the degree of similarity or identity is given for a region of 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 full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given 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, contiguous nucleotides). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0323] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly 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.

[0324] The amino acid sequence variants described herein can be readily prepared by a skilled artisan, for example, by recombinant DNA manipulation. DNA sequence manipulations for preparing peptides or proteins with substitutions, additions, insertions, or deletions are described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, as by solid phase synthesis and similar methods.

[0325] 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 relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to the functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent). With respect to antigens or antigenic sequences, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence (immunogenic fragment) comprising an amino acid sequence that has one or more amino acids changed compared to the amino acid sequence of the parent molecule or sequence and is still capable of achieving one or more functions of the parent molecule or sequence (e.g., inducing an immune response). In one embodiment, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the properties of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant can be reduced but still significantly present, for example, the immunogenicity of the functional variant can 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 can be enhanced compared to the parent molecule or sequence.

[0326] An amino acid sequence (peptide, protein or polypeptide) that is "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. In some embodiments, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence can be a variant of the specific sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that antigens suitable for use herein can be altered so that their sequences are different from the naturally occurring or natural sequences from which they are derived while retaining the desired activity of the native sequence.

[0327] In some embodiments, "isolated" means changed or removed (e.g., purified) from its natural state or from an artificial composition (e.g., a composition from a production process). For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from coexisting substances 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-natural environment, such as a host cell. In some embodiments, the RNA (e.g., mRNA) used in the present disclosure is in a substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of RNA (e.g., mRNA) in substantially purified form comprises a first buffer system.

[0328] The term "genetic modification" or simply "modification" includes transfecting cells with nucleic acid.

[0329] The term "transfection" refers to the introduction of a nucleic acid (particularly RNA) into a cell. For the purposes of this disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, wherein the cell may be present in a subject, such as a patient. Thus, according to the present disclosure, the cells used to transfect the nucleic acids described herein may be present in vitro (e.g., in cell culture) or in vivo, wherein, for example, the cells may form an organ, tissue, and / or part of the patient's body. According to the present disclosure, transfection may be transient or stable. For some applications of transfection, it may be sufficient if only the transfected genetic material is transiently expressed. RNA may be transfected into cells to transiently express the protein it encodes. Since the nucleic acids introduced during transfection generally do not integrate into the nuclear genome, the exogenous nucleic acids will be diluted or degraded by mitosis. Cells that allow for free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by transfection using viral-based systems or transposon-based systems. Typically, nucleic acid encoding an antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0330] The present disclosure includes analogs of peptides, polypeptides or proteins. According to the present disclosure, analogs of peptides, polypeptides or proteins are modified forms of the peptides, polypeptides or proteins from which they are derived, and have at least one functional property of the peptides, polypeptides or proteins. For example, a pharmacologically active analog of a peptide, polypeptide or protein has at least one pharmacological activity of the peptide, polypeptide or protein from which the analog is derived. Such modifications include any chemical modification and include single or multiple replacements, deletions and / or additions of any molecule (e.g., carbohydrates, lipids and / or proteins or peptides) associated with the protein, polypeptide or peptide. In one embodiment, "analogs" of proteins, polypeptides or peptides include those modified forms obtained by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protection / blocking groups, proteolytic cleavage or binding to antibodies or other cellular ligands. The term "analog" also extends to all functional chemical equivalents of the proteins, polypeptides and peptides.

[0331] As used herein, the terms "linked," "fused," or "fusion / fusion" are used interchangeably. These terms refer to the joining together of two or more elements, components, or domains.

[0332] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide, polypeptide, or protein, such as RNA (e.g., mRNA), is taken up or introduced (i.e., transfected or transduced) into a cell, which results in expression of the peptide, polypeptide, or protein. The cell may be present in vitro or in a subject. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide, polypeptide, or protein, and / or may express the encoded peptide, polypeptide, or protein on its surface.

[0333] In accordance with the present disclosure, terms such as "nucleic acid that expresses..." and "nucleic acid that encodes..." or similar terms are used interchangeably herein and, in the context of a particular peptide, polypeptide or protein, mean that if the nucleic acid is present in a suitable environment, preferably within a cell, it can be expressed to produce the peptide, polypeptide or protein.

[0334] "Activation" or "stimulation" as used herein refers to the state of cells (e.g., immune effector cells such as T cells) that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" particularly refers to immune effector cells that are undergoing cell division.

[0335] The term "sensitization" refers to the process in which immune effector cells (eg, T cells) first come into contact with their specific antigen and result in differentiation into effector cells (eg, effector T cells).

[0336] The term "clonal expansion" or "amplification" refers to a process in which a specific entity multiplies. In some embodiments, the term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize the antigen are expanded. In some embodiments, the expansion results in the differentiation of the immune effector cells.

[0337] "Antigen" according to the present disclosure encompasses any substance that will cause an immune response and / or any substance to which an immune response or immune mechanism (e.g., cellular response and / or humoral response) is directed. This also includes situations in which the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present disclosure, the term "antigen" may include any molecule containing at least one epitope (e.g., a T cell epitope). In some embodiments, the antigen in the context of the present disclosure is a molecule that, optionally after processing, induces an immune response that is specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0338] According to the present disclosure, any suitable antigen as a candidate for an immune response can be used, wherein the immune response can be a humoral immune response or a cellular immune response or the two thereof. In the case of some embodiments of the present disclosure, the antigen is presented by a cell, preferably by an antigen presenting cell (in the case of an MHC molecule), which causes 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 can 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 disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein, or a part thereof.

[0339] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains an epitope that will stimulate the host's immune system to produce an antigen-specific cellular immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with an infection caused by a microorganism, typically a microbial antigen (e.g., a bacterial or viral antigen), or antigens associated with a cancer (typically a tumor), such as a tumor antigen.

[0340] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular, one that is primarily present intracellularly or as a surface antigen of a tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen from a virus, bacteria, unicellular organism, or parasite, for example, a viral antigen such as a viral ribonucleoprotein or envelope protein. In particular, the antigen should be presented by an MHC molecule, which leads to modulation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular activation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular by modulation of the activity of the T cell receptor.

[0341] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to those antigens produced intracellularly or produced as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetosulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer and that is characteristic of a tumor or cancer cell in terms of type and / or expression level.

[0342] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0343] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. Bacterial antigens may be derived from the bacterial cell wall or cytoplasmic membrane.

[0344] The term "epitope" refers to an antigenic determinant in a molecule (e.g., an antigen), i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by a T cell or B cell, particularly when presented in the context of an MHC molecule. The epitope of a protein can comprise a continuous or discontinuous portion of the protein and can be, for example, about 5 to about 100 amino acids, about 5 to about 50, about 8 to about 0, about 10 to about 25 amino acids in length, e.g., 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 some embodiments, the epitope in the context of the present disclosure is a T cell epitope.

[0345] Terms such as "epitope", "antigenic fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and, for example, may relate to an incomplete representation of an antigen that is, for example, capable of eliciting an immune response against the antigen or eliciting a cell that expresses or contains and presents the antigen. In some embodiments, the term relates to an immunogenic portion of an antigen. Preferably, it is a portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if 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 (e.g., 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. The epitope of an antigen can include a continuous or discontinuous portion of the antigen, and can be about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, most preferably about 8 to about 25 amino acids in length, such as an epitope length can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T cell epitopes.

[0346] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when present in the presence of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind to peptide epitopes and present them to be recognized by the T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invasive microorganisms) to T cells. In the case of class I MHC / peptide complexes, binding peptides are typically about 8 to about 10 amino acids long, but longer or shorter peptides may also be effective. In the case of class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, but longer and shorter peptides may also be effective.

[0347] The length of peptide and protein antigens can be 2 to 100 amino acids, including, for example, 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, the peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0348] The peptide or protein antigen can be any peptide or protein that can induce or enhance the ability of the immune system to produce antibodies and T cell responses against the peptide or protein.

[0349] In some embodiments, vaccine antigens (i.e., antigens that are inoculated into a subject to induce an immune response) are recognized by immune effector cells. In some embodiments, if vaccine antigens are recognized by immune effector cells, then the vaccine antigens can induce stimulation, sensitization and / or amplification of immune effector cells carrying antigen receptors that recognize the vaccine antigens in the presence of appropriate costimulatory signals. In the case of embodiments of the present disclosure, vaccine antigens are preferably presented or present on the surface of cells (preferably antigen presenting cells).

[0350] In some embodiments, the antigen is expressed in diseased cells (eg, tumor cells or infected cells).

[0351] In some embodiments, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the presence of MHC. In some embodiments, when expressed by a T cell and / or present on a T cell, the combination of TCR and an antigen presented by a cell (e.g., an antigen presenting cell) results in stimulation, sensitization, and / or amplification of the T cell. In some embodiments, when expressed by a T cell and / or present on a T cell, the combination of TCR and an antigen present on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0352] In some embodiments, antigen is expressed on the surface of diseased cells (such as tumor cells or infected cells).In some embodiments, antigen receptor is a CAR that is combined with an epitope in the extracellular domain of an antigen or the extracellular domain of an antigen.In some embodiments, CAR is combined with the natural epitope of an antigen present on the surface of a living cell.In some embodiments, when CAR is expressed by T cells and / or is present on T cells, CAR is combined with the antigen present on cells (such as antigen presenting cells) to stimulate, sensitize and / or expand the T cells.In some embodiments, when CAR is expressed by T cells and / or is present on T cells, CAR is combined with the antigen present on diseased cells to cause cell dissolution and / or apoptosis of diseased cells, wherein the T cells preferably release cytotoxic factors, such as perforin and granzyme.

[0353] In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B cell receptor binds to a native epitope of the antigen.

[0354] The term "expressed on the surface of a cell" or "associated with the cell surface" means that a molecule, such as an antigen, is associated with and located at the plasma membrane of a cell, wherein at least a portion of the molecule is oriented toward the extracellular space of the cell and is accessible from outside the cell, for example, 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 occur through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the cell surface can be a transmembrane protein with an extracellular portion, or can be a protein that is associated with the cell surface through interaction with another protein that is a transmembrane protein.

[0355] "Cell surface" or "surface of a cell" is used according to its normal meaning in the art and therefore includes the outside of the cell that can be bound by proteins and other molecules. An antigen is expressed on the cell surface if it is located on the surface of the cell and can be bound by, for example, an antigen-specific antibody added to the cell. In some embodiments, the antigen expressed on the cell surface is an integral membrane protein with an extracellular portion that can be recognized by a CAR.

[0356] In the context of the present disclosure, the term "extracellular portion" or "extracellular domain" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, for example, by a binding molecule such as an antibody (which is located outside the cell). In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0357] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL) (CD8+ T cells) including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen targeted by the T cell (particularly when presented on the surface of antigen-presenting cells or diseased cells such as cancer cells in the context of MHC molecules) and preferably exerts the effector function of the T cell. If the T cell kills a target cell expressing an antigen, it is considered that the cell is specific for the antigen. T cell specificity can be evaluated using any of a variety of standard techniques (e.g., in a chromium release assay or a proliferation assay). Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the present disclosure, RNA (particularly mRNA) encodes at least one epitope.

[0358] The term "target" shall mean a substance, such as a cell or tissue, that is the target of an immune response (e.g., a cellular immune response). Targets include cells that present an antigen or an antigenic epitope (i.e., a peptide fragment derived from an antigen). In one embodiment, a target cell is a cell that expresses an antigen and preferably presents class I MHC to the antigen.

[0359] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association of one or more of these fragments with an MHC molecule (e.g., association by binding) for presentation by a cell (preferably an antigen-presenting cell) to a specific T cell. Antigen-presenting cells can be divided into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0360] The term "professional antigen presenting cells" refers to antigen presenting cells that constitutively express the major histocompatibility complex class II (MHC class II) molecules required for interaction with naive T cells. If T cells interact with the MHC class II molecule complex on the membrane of antigen presenting cells, the antigen presenting cells produce costimulatory molecules that induce T cell activation. Professional antigen presenting cells include dendritic cells and macrophages.

[0361] The term "non-professional antigen-presenting cells" relates to antigen-presenting cells that do not constitutively express MHC class II molecules but express them in response to stimulation by certain cytokines (e.g., interferon-γ). Exemplarily, non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells, or vascular endothelial cells.

[0362] The term "dendritic cell" (DC) refers to a subtype of phagocytes belonging to the antigen-presenting cell class. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment, looking for pathogens such as viruses and bacteria. Once they come into contact with presentable antigens, they are activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small fragments, and after maturation, they use MHC molecules to present these fragments on their cell surface. At the same time, they raise cell surface receptors such as CD80, CD86, and CD40 that play a role as co-receptors in T cell activation, greatly enhancing their ability to activate T cells. They also raise CCR7, which is a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream or to reach lymph nodes through the lymphatic system. Here, they act as antigen presenting cells and activate helper T cells, killer T cells and B cells by presenting antigens and non-antigen specific co-stimulatory signals to 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, the dendritic cells are splenic dendritic cells.

[0363] The term "macrophage" refers to a subpopulation of phagocytes differentiated from monocytes. Macrophages activated by inflammation, immune cytokines or microbial products attack and non-specifically engulf and kill foreign pathogens in macrophages through hydrolysis and oxidation, resulting in pathogen degradation. Peptides from degraded proteins are displayed on the surface of macrophages, where they can be recognized by T cells, and they can directly interact with antibodies on the surface of B cells, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are spleen macrophages.

[0364] "Antigen-responsive CTL" refers to CD8 + T cells, the CD8 + T cells respond to antigens, or peptides derived from the antigens, which are presented using MHC class I on the surface of antigen-presenting cells.

[0365] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines (e.g., IFN-γ and TNF-α), upregulation of activation markers (e.g., CD44 and CD69), and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately indicate CTL responsiveness.

[0366] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the overall body response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against" with respect to a substance (e.g., an antigen, cell, or tissue) refer to an immune response, such as a cellular response, against the substance. An immune response may include one or more reactions selected from the group consisting of: the development of antibodies against one or more antigens, and the expansion of antigen-specific T lymphocytes, such as CD4 T lymphocytes. + and CD8 + T lymphocytes, such as CD8 + T lymphocytes, which can be detected in various in vitro proliferation or cytokine production assays.

[0367] In the context of the present disclosure, the terms "induce an immune response" and "cause an immune response" and similar terms refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / preventative / 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)). In this context, "induce" can mean that there is no immune response to a particular antigen or pathogen before induction, but it can also mean that there is a certain level of immune response to a particular antigen or pathogen before induction, and that after induction, the immune response is enhanced. Therefore, in this context, "induce an immune response" also includes "enhance an immune response." In some embodiments, after inducing 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 condition is improved by inducing an immune response.

[0368] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are intended to include cellular responses directed against cells characterized by expression of antigen and / or presentation of antigen by class I and / or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (also known as CD4 + T cells) play a key role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, such as diseased cells.

[0369] The term "humoral immune response" refers to the process in living organisms in which antibodies are produced in response to a substance or organism, which ultimately neutralize and / or eliminate it. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-associated receptors that bind a single specific antigen. After binding to the appropriate antigen and receiving a variety of other activation signals, B lymphocytes divide, which produces memory B cells and antibody-secreting plasma cell clones, each clone producing antibodies to the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. When re-exposed to the specific antigen, these lymphocytes provide the cellular basis of memory and lead to a gradual escalation of the antibody response.

[0370] The term "antibody" as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which can specifically bind to an epitope on an antigen under typical physiological conditions, preferably with a half-life of a significant period, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, or any other relevant functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or regulate the physiological response associated with the antibody bound to the antigen and / or a period sufficient to allow 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 interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The variable region and constant region are also referred to herein as the variable domain and constant domain, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The CDRs of VH are called HCDR1, HCDR2 and HCDR3, and the CDRs of VL are called LCDR1, LCDR2 and LCDR3. The heavy and light chain variable regions 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), wherein CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxyl terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins 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 can be complete immunoglobulins derived from natural or recombinant sources, and can be immunologically active portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can 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.

[0371] The variable regions of the heavy and light chains of the immunoglobulin molecules contain binding domains that interact with antigens. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to regions that interact with antigens and contain both the VH region and the VL region. Antibodies as used herein 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.

[0372] As mentioned above, unless otherwise indicated or clearly contradicted by the context, the term antibody herein includes fragments of antibodies that are antigen-binding fragments, i.e., that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Some examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, or a monovalent antibody as described in WO 2007 / 059782 (Genmab); (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 2006). 341 , 544-546 (1989)), which are essentially composed of VH domains and are also called 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). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using recombinant methods via a synthetic linker, enabling them to become a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242 , 423-426(1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988)). Unless otherwise indicated or the context clearly indicates, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of antibody, they are collectively and independently unique features of the present disclosure, exhibiting different biological properties and utilities. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific forms of such fragments, are further discussed herein. It should also be understood that, unless otherwise indicated, the term antibody also includes 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 an antigen provided by any known technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques).

[0373] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, for example to antigen receptors such as antibodies or B cell receptors (B cell receptors, BCRs). Immunoglobulins are characterized by structural domains (i.e., immunoglobulin domains) that have a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are typically part of the BCR. Soluble immunoglobulins are often called antibodies. Immunoglobulins typically comprise several chains, typically two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V and L chains. L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domain and C H (Constant heavy chain) domain C H1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which make up the different classes of antibodies, namely 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 their carboxyl termini. In mammals, there are two types of light chains, namely λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved among different immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0374] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the administration to the individual of one or more immunogens or antigens as described herein, or derivatives thereof, particularly in the form of RNA (especially mRNA) encoding the same, and the stimulation of an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens.

[0375] By "cell characterized by presenting an antigen" or "cell presenting an antigen" or "MHC molecules presenting an antigen on the surface of an antigen-presenting cell" or similar expressions is meant a cell, such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen-presenting cell, which presents an antigen or an antigenic peptide directly or after processing in the presence of an MHC molecule (preferably MHC class I and / or MHC class II molecule, most preferably MHC class I molecule).

[0376] In the context of this disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA). Subsequently, the RNA (particularly mRNA) can be translated into a peptide, polypeptide or protein.

[0377] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence.

[0378] With respect to RNA, the terms "expression" or "translation" refer to the process in the cell's ribosomes by which a strand of mRNA directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0379] In the context of this disclosure, the term "RNA encoding" means that, if present in a suitable environment, such as within a cell of a target tissue, the RNA can direct the assembly of amino acids during the process of translation to produce its encoded peptide or protein.

[0380] As used herein, the term "serum" refers to a fluid obtained by removing cells and coagulation factors from whole blood, such as whole blood obtained from humans or mice. In some embodiments, the serum is human serum or mouse serum.

[0381] The pharmaceutical preparations described herein, particularly kits, may include instructional materials or instructions. As used herein, "instructional materials" or "instructions" include publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present disclosure. For example, the instructional materials of a kit of the present disclosure may be attached to a container containing the composition of the present disclosure or shipped together with the container containing the composition. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the recipient uses the instructional materials and the composition in conjunction.

[0382] As used herein, the terms "optional" or "optionally" mean that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance or condition occurs and instances where it does not.

[0383] The prodrugs of the specific compounds described herein are those compounds that undergo chemical transformation under physiological conditions to provide the specific compound after being applied to an individual. In addition, the prodrug can be converted into the specific compound by chemical or biochemical methods in an ex vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir together with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into the specific compound. Exemplary prodrugs are esters (using the alcohol or carboxyl group contained in the specific compound) or amides (using the amino or carboxyl group contained in the specific compound) that can be hydrolyzed in vivo. Specifically, any amino group contained in the specific compound and having at least one hydrogen atom can be converted into a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines and enaminones.

[0384] In this specification, the structural formula of a compound can represent a certain isomer of the compound. However, it should be understood that the disclosure includes all isomers, such as geometric isomers, isomers and isomer mixtures present in structures such as optical isomers, stereoisomers, tautomers based on asymmetric carbons, and is not limited to the description of formula. In addition, in this specification, the structural formula of a compound can represent a specific salt and / or solvate of the compound. However, it should be understood that the disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of specific salts and / or solvates.

[0385] "Isomers" are compounds that have the same molecular formula but different structures ("structural isomers") or different geometric (spatial) positions of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are pairs of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is denoted by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly convert into each other due to the migration of a single atom or group of atoms, even when pure; that is, the tautomers are in dynamic chemical equilibrium with each other. Examples of tautomers are keto-enol-tautomeric isomers. "Conformational isomers" are stereoisomers that are interconvertible only by rotation about a formal single bond, and in particular include those that result in different 3-dimensional forms of (hetero)rings, e.g., chair, half-chair, boat and twist-boat stereoisomers of cyclohexane.

[0386] As used herein, the term "solvate" refers to an addition complex of a dissolved material in a solvent (e.g., an organic solvent (e.g., aliphatic alcohols (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, diethyl ether, etc.), water, or a mixture of two or more of these liquids), wherein the addition complex exists in the form of crystals or mixed crystals. The amount of solvent included in the addition complex may be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solvent is water.

[0387] In an isotopically labeled compound, one or more atoms are replaced by corresponding atoms having the same number of protons but a different number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes that can be used in the compounds of the present disclosure include deuterium, tritium, 11 C. 13 C. 14 C. 15 N. 18 F. 32 P. 32 S. 35 S. 36 Cl and 125 I.

[0388] The term "mean diameter" refers to the mean hydrodynamic diameter of the particles as measured by dynamic light scattering (DLS) and data analysis using the so-called cumulant algorithm, which provides the so-called Z diameter with a length dimension. 平均值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 the particles is the same as Z. 平均值 .

[0389] In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis mentioned in the definition of "mean diameter". Under certain conditions, it can be used as a measure of the size distribution of the nanoparticle ensemble.

[0390] The radius of gyration of the particle around the axis of rotation (abbreviated as R in this paper) g ) refers to the radial distance of a point from the axis of rotation. Here, if the entire mass of the particle is assumed to be concentrated, the moment of inertia about a given axis is the same as its actual mass distribution. Mathematically speaking, R g is the root mean square distance of a particle component from its center of mass or a given axis. For example, for a particle consisting of n mass elements, mass m i (i=1, 2, 3, ..., n) and located at a fixed distance s from the center of mass i The macromolecule at R g Is all the mass elements on s i 2 The square root of the mass mean of and can be calculated as follows:

[0391]

[0392] The radius of gyration can be determined experimentally (e.g. by using light scattering) or calculated. In particular, for small scattering vectors The structure function S is defined as follows:

[0393]

[0394] where N is the number of components (Guinier's law).

[0395] The "D10 value" (particularly with respect to a quantitative size distribution of particles) is the diameter below which 10% of the particles have a diameter smaller than this value. The D10 value is a way of describing the proportion of the smallest particles within a population of particles (e.g., within a particle peak obtained from field-flow fractionation).

[0396] The "D50 value" (particularly with respect to a quantitative size distribution of particles) is the diameter below which 50% of the particles have a diameter. The D50 value is a way of describing the average particle size within a population of particles (e.g., within a particle peak obtained from field flow fractionation).

[0397] The "D90 value" (particularly with respect to the quantitative size distribution of particles) is the diameter at which 90% of the particles have a diameter smaller than this value. The "D95," "D99," and "D100" values ​​have corresponding meanings. The D90, D95, D99, and D100 values ​​are a way of describing the proportion of larger particles within a particle population (e.g., within a particle peak obtained from field flow fractionation).

[0398] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") 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 and takes into account not only size but also solvent effects. For example, a smaller, more strongly hydrated particle can have a larger hydrodynamic radius than a larger, less hydrated particle. This is because smaller particles carry more water molecules with them as they move through a solution. Since the actual size of a particle in a solvent cannot be directly measured, the hydrodynamic radius is defined by the Stokes-Einstein equation:

[0399]

[0400] where k B is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally (e.g., by using dynamic light scattering (DLS)). Therefore, a procedure for determining the hydrodynamic radius of a particle or a population of particles (e.g., the hydrodynamic radius of a particle (e.g., LNP) contained in a formulation or composition as disclosed herein, or the hydrodynamic radius of a particle peak obtained by subjecting such a formulation or composition to field flow fractionation) is to measure the DLS signal of the particle or population of particles (e.g., the DLS signal of a particle (e.g., LNP) contained in a formulation or composition as disclosed herein, or the DLS signal of a particle peak obtained by subjecting such a formulation or composition to field flow fractionation).

[0401] The term "aggregate" as used herein relates to a cluster of particles in which the particles are identical or very similar and attached to each other in a non-covalent manner (e.g., through ionic interactions, H-bridge interactions, dipole interactions and / or van der Waals interactions).

[0402] As used herein, the expression "light scattering" refers to the physical process by which light is forced to deviate from a straight-line trajectory by one or more paths due to local inhomogeneities in a medium through which the light passes.

[0403] The term "UV" means ultraviolet and refers to a band of the electromagnetic spectrum with wavelengths from 10 nm to 400 nm, ie, shorter than visible light but longer than X-rays.

[0404] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered by a sample into a plurality of angles. In this context, "multi-angle" means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector that is moved within a range comprising a selected specific angle or by an array of detectors that is fixed at specific angular positions. 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 comprising particles and by using a suitable format (e.g. a Zimm plot, a Berry plot or a Debye plot), it is possible to determine the radius of gyration (R g ) and thus the size of the particles can be determined. Preferably, the Zim diagram is a graphical representation using the following equation (or its inverse):

[0405]

[0406] Where c is the mass concentration of particles in the solvent (g / mL); A2 is the second virial coefficient (mol·mL / g 2 ); P(θ) is the shape factor related to the angular dependence of the scattered light intensity; R θ is the Rayleigh ratio (cm -1 ); and K* is equal to 4π 2 η o (dn / dc) 2 λ0 -4 N A -1 The optical constants, where η o is the refractive index of the solvent at the wavelength of the incident radiation (vacuum), λ0 is the wavelength of the incident radiation (vacuum) (nm), N A is Avogadro's number (mol -1 ), and dn / dc is the differential refractive index increment (mL / g) (see, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry diagram is calculated as follows (or its inverse):

[0407]

[0408] Where c, R θ and K* are as defined above. Preferably, the Debye graph is calculated as follows (or its inverse):

[0409]

[0410] Where c, R θ and K* are as defined above.

[0411] As used herein, the expression "dynamic light scattering" or "DLS" refers to a technique for determining the size and size distribution spectrum of particles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source (usually a laser) is directed into 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 the solution are struck by the light and diffract it in various directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated at short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each point over time.

[0412] The expression "static light scattering" or "SLS" used in this article refers to a technique for determining particle size and size distribution spectrum, particularly about the radius of gyration of particles and / or the molar mass of particles. High-intensity monochromatic light, typically a laser, is emitted in a solution containing particles. One or more detectors are used to measure the scattered intensity at one or more angles. In order to obtain accurate measurements of both the molar mass and size of macromolecules of all radii, angle dependence is required. Therefore, the simultaneous measurement results at multiple angles relative to the direction of incident light (referred to as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS)) are generally regarded as standard implementations of static light scattering.

[0413] "Immunogenicity" refers to the ability of a foreign substance (such as RNA) to elicit an immune response in humans or other animals. The innate immune system is a relatively nonspecific and immediate component of the immune system. Along with the adaptive immune system, it is one of the two main components of the vertebrate immune system.

[0414] As used herein, "endogenous" refers to any substance originating from or produced within an organism, cell, tissue, or system.

[0415] As used herein, the term "exogenous" refers to any substance introduced from or originating outside an organism, cell, tissue, or system.

[0416] The term "repeat unit" relates to a basic unit that repeats itself periodically along the polymer chain of a polymer and that is derived from one monomer. Although the structure of a repeat unit and the structure of its corresponding monomer are generally identical, they may differ from each other.

[0417] As used herein, the term "functional moiety" refers to a group of atoms in a molecule having unique chemical properties, wherein the atoms of the functional moiety are connected to each other and to the rest of the molecule by covalent bonds. Preferably, the atoms of the functional moiety include at least one atom selected from O, N, and S. The functional moiety can be monovalent (e.g., hydroxyl, cyano, nitro, or amide (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 some embodiments, the functional moiety provides hydrophilicity to the group to which the functional moiety is bound, for example, by providing at least one hydrogen bond acceptor / donor and / or at least one charge (positive or negative) to the group to which the functional moiety is bound. In certain embodiments, the functional moiety comprises a hydrogen bond acceptor (e.g., a carbonyl moiety), a hydrogen bond donor (e.g., a hydroxyl moiety, -NH- (e.g., an amide moiety), or a thiol moiety) or both (e.g., an amide moiety), and / or is charged (e.g., a phosphate, an amino, or an ammonium moiety). Some examples of monovalent functional moieties include hydroxy, ether, halogen, cyano, azide, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfonamide, sulfuramide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), thio Imidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamoyl imide, carboimidate, carbamate, dithiocarbamate, dithiocarboimidate, thiocarbamate, thiocarbamate, thiocarboimidate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties. Some examples of divalent functional moieties include ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfonamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), thioimidate, sulfinamido, carbonate / ester, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamoyl imide, carboimidate, carbamate, dithiocarbamate, dithiocarboimidate, thiocarbamate, thiocarbamate, thiocarboimidate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

[0418]

[0046] The term "hydroxyl" or "hydroxy" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -OH.

[0419] The term "halogen" as used herein with respect to a functional moiety, particularly as a component of a linker, means fluorine, chlorine, bromine, or iodine.

[0420] The term "cyano" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -CN.

[0421] The term "azido" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group N3.

[0422] The term "nitro" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -NO2.

[0423] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "amino" includes unsubstituted amino groups (i.e., -NH2 groups) and substituted amino groups (i.e., mono- or di-substituted amino groups in which one or both hydrogen atoms have been replaced by groups other than hydrogen). The amino group can be monovalent (e.g., -NRR, wherein each R is independently H or an organic group, such as R as defined below). 72 or R 73 ) or a divalent (e.g. -NR-, wherein R is H or an organic group, such as R as defined below 72 In some embodiments, the term "amino" refers to the group -N(R 72 )(R 73 ), where R 72 and R 73 is independently selected at each occurrence from -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 72 and R 73 can be linked together with the nitrogen atom to which they are attached to form the group -N=CR 75 R 76 , wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally replaced by one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups, 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 Replacement; R 75 and R 76independently selected from -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and -NH y R 80 2-y , or R 75 and R 76 The atoms to which they are attached may be linked together to form a ring, which is optionally substituted by one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the ring, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 substituted, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally replaced by one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups, 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 Substitution; y is an integer from 0 to 2; R 80 R 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 is optionally replaced by one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 Replace; and R 70 is a first-stage substituent, a second-stage substituent, or a third-stage substituent other than H, preferably as disclosed herein. In some embodiments, R 72 and R 73 Each of them is independently H or a hydrocarbon group, for example, selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl), wherein each of the hydrocarbon groups (e.g., C 1-6 Alkyl, aryl and aryl (C 1-6 Each of the alkyl) groups is optionally replaced by one or more (eg, 1 to 2) hydrocarbon groups (eg, C 1-6 Alkyl, aryl or aryl (C 1-6 The maximum number of hydrogen atoms to which the alkyl) group is bound, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 replace.

[0424] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "ammonium" refers to the group -N+ (R 72 )2(R 73 ), where R 72 and R 73 As defined for the term "amino".

[0425]

[0046] The term "thiol" or "sulfanyl" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -SH.

[0426] The term "disulfanyl" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to the group -SSH.

[0427]

[0046] The term "carboxyl" or "carboxy" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -COOH.

[0428] The term "amide" or "amido" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -C(O)NH- (including the structure -NHC(O)- of its isomeric arrangement, unless specified to the contrary). Preferably, each of the two ends of the amide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker) (if both ends are linked to the same organic group, the amide moiety is also referred to as a lactam). The amide group can be monovalent (e.g., -C(O)NRR or -NRC(O)R, wherein each R is independently H or an organic group such as the R shown above in the definition of the term "amino"). 72 or a divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0429] The term "ester" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -C(O)O- (including the structure -OC(O)- of its isomeric arrangement, unless specified to the contrary). Preferably, each of the two ends of the ester structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker) (if both ends are linked to the same organic group, the ester moiety is also referred to as a lactone). The ester group can be monovalent (e.g., -C(O)OR or -OC(O)R, where R is independently an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -C(O)O- or -OC(O)-).

[0430] The term "ether" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -O-, wherein each of the two ends of the ether structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The ether group can be monovalent (e.g., -OR, wherein R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -O-).

[0431] The term "sulfide" or "thioether" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -S-, wherein each of the two ends of the thioether structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The thioether group can be monovalent (e.g., -SR, wherein R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -S-).

[0432] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "disulfide" refers to a group comprising the structure -SS-, wherein each of the two ends of the disulfide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The disulfide group can be monovalent (e.g., -SSR, where R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -SS-).

[0433] The term "diselenide" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -SeSe-, wherein each of the two ends of the diselenide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The diselenide group can be monovalent (e.g., -SeSeR, wherein R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -SeSe-).

[0434] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfoxide" refers to a group comprising a sulfinyl structure -S(O)-, wherein each of the two ends of the sulfoxide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The sulfoxide group can be monovalent (e.g., -S(O)R, wherein R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (such as, for example, -S(O)-).

[0435] The term "sulfone" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising a sulfonyl structure -S(O)2-, wherein each of the two ends of the sulfone structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The sulfone group can be monovalent (e.g., -S(O)2R, wherein R is an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (such as, for example, -S(O)2-).

[0436] The term "sulfite" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OS(O)O-, wherein one of the two ends of the sulfite structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or a C atom of the same or another organic group (e.g., an alkylene group as another component of the linker). The sulfite group can be monovalent (e.g., -OS(O)OR, wherein R is H or an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -OS(O)O-).

[0437] The term "sulfate" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -OS(O)2O-, wherein one of the two ends of the sulfate structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or a C atom of the same or another organic group (e.g., an alkylene group as another component of the linker). The sulfate group can be monovalent (e.g., -OS(O)2OR, wherein R is H or an organic group such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -OS(O)2O-).

[0438] The term "phosphate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OP(O)(OR)O-, wherein one of the two ends of the phosphate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as the other component of the linker) and the other end is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino" 72 The phosphate group may be monovalent (e.g., -OP(O)(OR)2, wherein each R is independently H or an organic group such as R shown above in the definition of the term "amino"). 72 ) or a divalent (e.g., -OP(O)(OR)O-, wherein R is independently H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0439] The term "sulfenamide" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -S(O)N(R)-, wherein the S terminus of the sulfenamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the N terminus is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino"). 72 The sulfonamide group can be monovalent (e.g., -S(O)N(R)2, wherein each R is independently H or an organic group, such as R shown above in the definition of the term "amino"). 72 ) or a divalent (e.g., -S(O)N(R)-, wherein R is H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0440] The term "sulfonamide" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -S(O)2N(R)-, wherein the S terminus of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the N terminus is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino"). 72 The sulfonamide group can be monovalent (e.g., -S(O)2N(R)2, wherein each R is independently H or an organic group such as R shown above in the definition of the term "amino").72 ) or a divalent (e.g., -S(O)2N(R)-, wherein R is H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0441] The term "sulfamate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OS(O)2N(R)- (including, unless otherwise stated, the structure -N(R)S(O)2O- of isomeric arrangements thereof), wherein one of the two termini of the sulfamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other terminus is covalently linked to H or to a C atom of the same or another organic group (R is H or an organic group, such as R as described above in the definition of the term "amino"). 72 The sulfamate group may be monovalent (e.g., -OS(O)2N(R)2 or -N(R)S(O)2OR, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 ) or a divalent (e.g., -OS(O)2N(R)- or -N(R)S(O)2O-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0442] The term "sulfonamide" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)S(O)N(R)-, wherein one of the two termini of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The sulfonamide group may be monovalent (e.g., -N(R)S(O)N(R)2, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 ) or a divalent (e.g., -N(R)S(O)N(R)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0443] The term "sulfuric acid diamide" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)S(O)2N(R)-, wherein one of the two termini of the sulfuric acid diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The sulfuric acid diamide group can be monovalent (e.g., -N(R)S(O)2N(R)2, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 ) or is divalent (e.g., -N(R)S(O)2N(R)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0444] The term "urea" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -N(R)C(O)N(R)-, wherein one of the two termini of the urea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The urea group may be monovalent (e.g., -N(R)C(O)N(R)2, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 or a divalent (e.g., -N(R)C(O)N(R)-, wherein each R is H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0445] The term "thiourea" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)C(S)N(R)-, wherein one of the two termini of the thiourea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72The thiourea group may be monovalent (e.g., -N(R)C(S)N(R)2, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 or a divalent (e.g., -N(R)C(S)N(R)-, wherein each R is H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0446] The term "carbonyl" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(O)-, wherein one of the two termini of the carbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (if both termini are linked to C atoms of organic groups, the carbonyl moiety is also referred to as a "keto" moiety). The carbonyl group can be monovalent (e.g., -C(O)R, wherein R is H or an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -C(O)-).

[0447] The term "thiocarbonyl" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(S)-, wherein one of the two termini of the thiocarbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group. The thiocarbonyl group may be monovalent (e.g., -C(S)R, wherein R is H or an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -C(S)-).

[0448] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "orthoester" refers to a group comprising an alkylene group (i.e., -OR, wherein R is an organic group (e.g., an alkylene group as a further component of the linker), such as R described above in the definition of the term "amino" 72 The moiety of a C atom to which an orthoester is attached is one of the organic groups specified in the definition of . Exemplary formulas of orthoesters include the structure (-O) r C(OR) 3-r -, wherein each R is independently an organic group, for example independently selected from the R described in the definition of the term "amino" above 72wherein the orthoester comprises an organic group specified in the definition of ; r is 1 or 2; and each of the two ends of the orthoester structure is covalently linked to a C atom of another organic group or two additional separate organic groups. In some embodiments, the orthoester comprises the structure (—O) r C(OR 25 ) 3-r -, where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more primary, secondary, or tertiary substituents as defined herein); r is 1 or 2; and each of the two termini of the orthoester structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The orthoester group can be monovalent (e.g., -C(OR)3 or -OC(OR)2R, wherein each R is an organic group, such as independently selected from the R groups described above in the definition of the term "amino" 72 ) or a divalent (e.g., -(O)2C(OR)(R) or -OC(OR)2-, wherein each R is an organic group, such as independently selected from the R groups described above in the definition of the term "amino" 72 (organic groups as specified in the definition of ).

[0449] The term "thioate" or "thioester" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -SC(O)- (including, unless otherwise indicated, the structures -C(O)S-, -OC(S), and -C(S)O-, which are isomeric arrangements thereof), wherein each of the two termini of the thioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The thioate group can be monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).

[0450] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "dithioate" refers to a group comprising the structure -SC(S)- (including, unless otherwise indicated, the structure -C(S)S- of isomeric arrangements thereof), wherein each of the two termini of the dithioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The dithioate group can be monovalent (e.g., -SC(S)R or -C(S)SR, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -SC(S)- or -C(S)S-).

[0451] The term "imidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)- (including, unless otherwise stated, the structure -C(=NR)O-) wherein each of the two termini of the imidate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as a further component of the linker) (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The imidate / ester group may be monovalent (e.g., -OC(=NR)R' or -C(=NR)OR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -OC(=NR)- or -C(=NR)O-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0452] The term "imino" or "imine" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(=NR)-, wherein one of the two termini of the imino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino"). 72The -C(=NR)H moiety is also referred to as an "aldimine," and the -C(=NR)R' moiety (wherein R' is an organic group, such as R as described above in the definition of the term "amino" 72 The imino group may be monovalent (e.g., -C(=NR)R, wherein each R is independently H or an organic group such as R described above in the definition of the term "amino"). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -C(=NR)-, wherein R is H or an organic group such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0453] The term "thioimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(=NR)S- (including, unless otherwise indicated, the structure -SC(=NR)- of isomeric arrangements thereof), wherein one of the two termini of the thioimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The thioimidate group may be monovalent (e.g., -C(=NR)SR or -SC(=NR)R, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 or a divalent (e.g., -C(=NR)S- or -SC(=NR)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0454] The term "sulfinamido" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(S)NR- (including, unless otherwise indicated, the structure -N(R)C(S)- of isomeric arrangements thereof), wherein one of the two termini of the sulfinamido structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The sulfinamido group can be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 or a divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0455] The term "carbonate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(O)O-, wherein each of the two ends of the carbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonate group may be monovalent (e.g., -OC(O)OR', wherein R' is an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -OC(O)O-).

[0456] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "thiocarbonate" refers to a group comprising the structure -OC(S)O- or -OC(O)S- (including the structure -SC(O)O- of isomeric arrangements thereof, unless otherwise indicated), wherein each of the two termini of the thiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The thiocarbonate group may be monovalent (e.g., -OC(S)OR' or -OC(O)SR' or -SC(O)OR', wherein each R' is independently an organic group, such as R' as described above in the definition of the term "amino" 72 ) or divalent (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).

[0457] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "dithiocarbonate" refers to a group comprising the structure -SC(O)S- or -OC(S)S- (including the structure -SC(S)O- of isomeric arrangements thereof, unless otherwise indicated), wherein each of the two termini of the dithiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The dithiocarbonate group may be monovalent (e.g., -SC(O)SR', -OC(S)SR', or -SC(S)OR', wherein each R' is independently an organic group, such as R' as described above in the definition of the term "amino" 72 ) or divalent (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).

[0458] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "trithiocarbonate" refers to a group comprising the structure -SC(S)S-, wherein each of the two termini of the trithiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker). The trithiocarbonate group can be monovalent (e.g., -SC(S)SR, wherein R is an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -SC(S)S-).

[0459] The term "guanidinyl" or "imidamido" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -N(R)C(=NR)NR- (wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 The guanidinyl group may be a monovalent group (e.g., -N(R)C(=NR)NRR, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 or a divalent (e.g., -N(R)C(=NR)NR-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0460] The term "carbamidite" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)NR- (including, unless otherwise indicated, the structure -N(R)C(=NR)O-, isomeric arrangements thereof), wherein the O terminus of the carbamidite structure is covalently linked 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 linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The carbamoyl imide group may be monovalent (e.g., -OC(=NR)NRR or -N(R)C(=NR)OR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -OC(=NR)NR- or -N(R)C(=NR)O-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0461] The term "carboimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)O-, wherein each end of the carboimidate structure is covalently linked 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 organic group, such as the R groups described above in the definition of the term "amino"). 72 The carboimidate group may be monovalent (e.g., -OC(=NR)OR', wherein R is H or an organic group such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -OC(=NR)O-, wherein R is H or an organic group such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0462] The term "carbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -OC(O)NR- (including, unless otherwise indicated, the structure -N(R)C(O)O- of isomeric arrangements thereof), wherein the O terminus of the carbamate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other terminus (the N terminus) is covalently bonded to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The carbamate / ester group can be monovalent (e.g., -OC(O)NRR or -N(R)C(O)OR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0463] The term "dithiocarbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -SC(S)NR- (including, unless otherwise indicated, the structure -N(R)C(S)S- of isomeric arrangements thereof), wherein the S terminus of the dithiocarbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The dithiocarbamate group may be monovalent (e.g., -SC(S)NRR or -N(R)C(S)SR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -SC(S)NR- or -N(R)C(S)S-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0464] The term "dithiocarboimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(=NR)S-, wherein each end of the dithiocarboimidate structure is covalently linked 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 organic group, such as the R groups described above in the definition of the term "amino"). 72 The dithiocarbimidate group may be monovalent (e.g., -SC(=NR)SR', where R is H or an organic group such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -SC(=NR)S-, wherein R is H or an organic group such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0465] The term "thiocarbamyl ester" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(=NR)NR- (including, unless otherwise indicated, the structure -N(R)C(=NR)S-, isomeric arrangements thereof), wherein the S terminus of the thiocarbamyl ester structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The thiocarbamyl imide group may be monovalent (e.g., -SC(=NR)NRR or -N(R)C(=NR)SR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -SC(=NR)NR- or -N(R)C(=NR)S-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0466] The term "thiocarbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including, unless otherwise indicated, the structures -SC(O)NR- or -OC(S)NR- of isomeric arrangements thereof), wherein the O / S terminus of the thiocarbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other terminus (the N terminus) is covalently linked to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The thiocarbamate group may be monovalent (e.g., -N(R)C(O)SR' or -N(R)C(S)OR' or -SC(O)NRR or -OC(S)NRR, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -N(R)C(O)S- or -N(R)C(S)O- or -SC(O)NR- or -OC(S)NR-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0467] The term "thiocarboimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)S- (including, unless otherwise stated, the structure -SC(=NR)O- of isomeric arrangements thereof), wherein the O / S terminus of the thiocarboimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the Rs described above in the definition of the term "amino"). 72 The thiocarbimidate group may be monovalent (e.g., -OC(=NR)SR' or -SC(=NR)OR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72or a divalent (e.g., -OC(=NR)S- or -SC(=NR)O-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0468] The term "acylhydrazone" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(R')(=NN(R)C(O)-) (including, unless otherwise indicated, isomeric arrangements thereof (-C(O)(N(R)-N=)C(R')-) and / or =C(=NN(R)C(O)R'), wherein each R' is independently an organic group, such as R as described above in the definition of the term "amino" 72 One of the organic groups specified in the definition of "amino"; each R is H or an organic group, such as R described in the definition of the term "amino" above 72 and each of the two ends of the acylhydrazone structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the acylhydrazone comprises the structure -C(R 25 )(=NN(R 26 )C(O)-)(Unless otherwise specified, structures including isomeric arrangements thereof (-C(O)(N(R 26 )-N=)C(R 25 )-) and / or =C(=NN(R 26 )C(O)R 25 ), where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more 1st, 2nd, or 3rd substituents as defined herein); each R 26 are independently H or hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more primary 1, secondary, or tertiary substituents as defined herein); and each of the two termini of the acylhydrazone structure is covalently linked to a C atom of another organic group or two additional separate organic groups. An exemplary chemical structure of an acylhydrazone is shown below:

[0469]

[0470] Each of these represents a bond by which the acylhydrazone is covalently linked to another organic group (e.g., an alkylene group as another component of the linker). The acylhydrazone group can be monovalent (e.g., -C(R')(=NN(R)C(O)R') or -C(O)(N(R)-N=)C(R')2, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino". 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 one of the organic groups specified in the definition of ) or a divalent (e.g., -C(R')(=NN(R)C(O)-, -C(O)(N(R)-N=)C(R')-, or =C(=NN(R 26 )C(O)R 25 ), wherein each R' is independently an organic group, such as R described above in the definition of the term "amino" 72 and each R is H or an organic group, such as R described in the definition of the term "amino" above 72 (one of the organic groups specified in the definition of ).

[0471] The term "hydrazine" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -N(R)N(R)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino". 72 and each of the two ends of the hydrazine structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the hydrazine comprises the structure -N(R 26 )N(R 26 )-, where each R 26 are independently H or hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more primary substituents, secondary substituents, or tertiary substituents as defined herein); and each of the two termini of the hydrazine structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The hydrazine group may be monovalent (e.g., -N(R)N(R)2, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 ) or a divalent (e.g., -N(R)N(R)-, wherein each R' is independently an organic group, such as R as described above in the definition of the term "amino" 72(one of the organic groups specified in the definition of ).

[0472] The term "oxime," as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure =C(=N(OH)), wherein each of the two termini of the oxime structure are covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). An exemplary chemical formula for an oxime is shown below:

[0473]

[0474] Each of these represents a bond through which the oxime is covalently linked to another organic group. The oxime group may be monovalent (e.g., -C(=N(OH))(R), wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 ) or divalent (e.g., ═C(═N(OH))).

[0475] The term "acetal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCH(R')O-, wherein R' is an organic group, such as R described above in the definition of the term "amino". 72 and each of the two O atoms of the acetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the acetal comprises the structure -OCH(R 25 )O-, where R 25 Is a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more 1st, 2nd, or 3rd substituents as defined herein); and each of the two O atoms of the acetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The acetal group can be monovalent (e.g., -OCH(R')OR', wherein each R' is independently an organic group, such as independently selected from the R groups described above in the definition of the term "amino"). 72 ) or a divalent (e.g., -OCH(R')O-, wherein R' is an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0476] The term "hemiacetal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCH(OH)-, wherein each of the two termini of the hemiacetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). The hemiacetal group may be monovalent (e.g., -OCH(OH)OR', wherein R' is an organic group, such as R described above in the definition of the term "amino"). 72 ) or divalent (e.g., -OCH(OH)-).

[0477] The term "ketal" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -OC(R')(R')O-, wherein each R' is independently an organic group, for example independently selected from the R groups described above in the definition of the term "amino". 72 and each of the two O atoms of the ketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the ketal comprises the structure -OC(R 25 )(R 25 )O-, where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more 1st, 2nd, or 3rd substituents as defined herein); and each of the two O atoms of the ketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The ketal group can be monovalent (e.g., -OC(R')(R')OR', wherein each R' is independently an organic group, such as independently selected from the R's described above in the definition of the term "amino" 72 ) or a divalent (e.g., -OC(R')(R')O-, wherein each R' is independently an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0478] The term "himeketal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCR'(OH)-, wherein R' is an organic group, such as R described above in the definition of the term "amino". 72and each of the two ends of the hemiketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the hemiketal comprises the structure -OCR 25 (OH)-, where R 25 Is a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more primary, secondary, or tertiary substituents as defined herein); and each of the two termini of the hemiketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The hemiketal group can be monovalent (e.g., -OC(R')2(OH), wherein each R' is independently an organic group, such as R' independently selected from the group described above in the definition of the term "amino" 72 ) or a divalent (e.g., -OCR'(OH)-, wherein R' is an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0479] The term "imide" as used herein with respect to a functional moiety, particularly as a component thereof, relates to a group comprising the structure -C(O)N(R)C(O)-, wherein R is an organic group, such as R as described above in the definition of the term "amino". 72 and each of the two ends of the imide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as an additional component of the linker). The imide group can be monovalent (e.g., -C(O)N(R)C(O)R', where R is independently H or an organic group, such as R described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -C(O)N(R)C(O)-, wherein R is H or an organic group such as R as described above in the definition of the term "amino" 72 (one of the organic groups specified in the definition of ).

[0480] As used herein, the term "acyclic" in the context of an organic group relates to an open-chain organic group that does not contain a ring. "Open-chain" or "acyclic" organic groups can be linear (i.e., they contain only one unbranched chain without any side chains) or branched (i.e., the main chain contains one or more side chains).

[0481] An organic group that is "substituted with one or more substituents" means that one or more hydrogen atoms of the organic group (e.g., 1 to the maximum number of hydrogen atoms bound to the organic group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the one or more substituents may be selected from the first-level substituents, second-level substituents, or third-level substituents described herein.

[0482] As used herein, the expression "hydrogen bond" or "H-bond" means a non-covalent bond (in some embodiments, primarily an electrostatic attraction) between (i) a hydrogen atom covalently bonded to a more electronegative atom or group and (ii) a lone pair of electrons of another electronegative atom. In some embodiments, the more electronegative atom or group includes a nitrogen atom and an oxygen atom; thus, some examples of groups in which a hydrogen atom is covalently bonded to a more electronegative atom or group include an amino group with at least one covalently attached hydrogen atom, an -NH- group of an amide group, a hydroxyl group (as such (e.g., in the corresponding alcohol) or as part of another functional group (e.g., as part of a carboxyl group (-COOH)), and a sulfanyl group (as such (e.g., in the corresponding thiol) or as part of another functional group (e.g., as part of a disulfanyl (-SSH) or thioester (-C(OSH) group)). In some embodiments, the lone pair of electrons of the other electronegative atom is the lone pair of oxygen atoms present in a carbonyl group or the lone pair of nitrogen atoms present in a primary, secondary, or tertiary amino group.

[0483] As used herein, the expression "hydrogen bond donor" means an atom, ion, or molecular component that provides a hydrogen bond bridging (shared) hydrogen atoms. In some embodiments, hydrogen bond donors include amino groups, -NH- groups of amide groups, hydroxyl groups (either as such (e.g., in corresponding alcohols) or as part of other functional groups (e.g., as part of carboxyl groups (-COOH)), and sulfanyl groups (either as such (e.g., in corresponding thiols) or as part of other functional groups (e.g., as part of disulfanyl groups (-SSH) or thioester groups (-C(OSH) groups))) with at least one covalently attached hydrogen atom.

[0484] As used herein, the expression "hydrogen bond acceptor" means an atom, ion, or molecular component that does not provide a hydrogen bond bridging (shared) hydrogen atoms. In some embodiments, the hydrogen bond acceptor comprises at least one lone pair of electrons. Some examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary, and tertiary amino groups.

[0485] The term "stealth" is used herein to describe the ability of the particles described herein to be undetected and subsequently sequestered and / or degraded, or barely detected and subsequently sequestered and / or degraded, and / or detected and subsequently sequestered and / or degraded relatively late by the immune system of a host to which they are administered.

[0486] The term "phosphatidylethanolamine" means a diacylphosphatidylethanolamine having the formula:

[0487]

[0488] or a salt thereof, wherein acyl in each case refers to an acyl moiety (e.g., a -C(O)-alkyl moiety, wherein the alkyl group is preferably a linear moiety). In some embodiments, each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (e.g., monounsaturated). Thus, the two acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (e.g., monounsaturated). Some examples of acyl moieties include -C(O)(CH2) 16 CH3(stearyl), -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" means a monovalent radical of phosphatidylethanolamine, preferably one in which the hydrogen atom of the amino group has been removed.

[0489] The term "DSPE" means distearoylphosphatidylethanolamine having the formula:

[0490]

[0491] or a salt thereof, wherein in each case -C(O)C 17 H 35 Refers to -C(O)(CH2) 16 CH3 (stearoyl) moiety. The term "distearoylphosphatidylethanolamine moiety" means a monovalent radical of DSPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0492] The term "DPPE" means dipalmitoylphosphatidylethanolamine having the formula:

[0493]

[0494] or a salt thereof, wherein in each case -C(O)C 15 H31 Refers to -C(O)(CH2) 14 CH3 (palmitoyl) moiety. The term "dipalmitoylphosphatidylethanolamine moiety" means a monovalent radical of DPPE, preferably one in which the hydrogen atom of the amino group has been removed.

[0495] The term "DOPE" means dioleoylphosphatidylethanolamine having the following formula:

[0496]

[0497] or a salt thereof, wherein in each case -C(O)C 17 H 33 It refers to the -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl) moiety. The term "dioleoylphosphatidylethanolamine moiety" means a monovalent group of DOPE, preferably a group in which the hydrogen atom of the amino group has been removed.

[0498] The term "POPE" means palmitoyloleoylphosphatidylethanolamine having the formula:

[0499]

[0500] or a salt thereof, wherein -C(O)C 15 H 31 Refers to -C(O)(CH2) 14 CH3(palmitoyl) moiety; and -C(O)C 17 H 33 It refers to the -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl) moiety. The term "palmitoyloleoylphosphatidylethanolamine moiety" means a monovalent group of POPE, preferably a group in which the hydrogen atom of the amino group has been removed.

[0501] The term "tocopherol" refers to a group of four compounds, namely, α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, which have the following formula:

[0502]

[0503] where R t1 and R t2 Each of R is independently hydrogen or methyl. In α-tocopherol, R t1 and R t2 Both are methyl groups; in β-tocopherol, R t1 is a methyl group, and R t2 is H; in γ-tocopherol, R t1 is H, and R t2 is a methyl group; and in delta-tocopherol, Rt1 and R t2 Both are H. The term "tocopherol moiety" or "tocopheryl moiety" means a monovalent radical of tocopherol, preferably a radical in which the hydrogen atom of the hydroxyl group has been removed.

[0504] The term "DAG" means diacylglycerides having the formula:

[0505]

[0506] or a salt thereof, wherein acyl in each case refers to an acyl moiety (e.g., a -C(O)-alkyl moiety, wherein the alkyl group is preferably a linear moiety). In some embodiments, each acyl moiety is an acyl moiety of a fatty acid, more preferably an acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (e.g., monounsaturated). Thus, the two acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (e.g., monounsaturated). Some examples of acyl moieties include -C(O)(CH2) 16 CH3(stearyl), -C(O)(CH2) 14 CH3(palmitoyl), -C(O)(CH2) 12 CH3 (myristoyl) and -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). For example, DMG means 1,2-dimyristoylglycerol, a diacylglycerol of the above formula in which both acyl groups are -C(O)(CH2) 12 CH3 (myristoyl). The term "diacylglyceride moiety" means a monovalent group of diacylglyceride, preferably a group in which the hydrogen atom of the hydroxyl group has been removed.

[0507] The term "DAA" means a dialkylamine having the formula HN(alkyl)2 or a salt thereof, 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. Some examples of alkyl moieties include -(CH2) 17 CH3(stearyl), -(CH2) 15 CH3(palmityl) and -(CH2) 13 CH3(myristyl). For example, DMA means 1,2-dimyristamide, which is a dialkylamine of the above formula where both alkyl groups are -(CH2) 13 CH3 (myristyl). The term "dialkylamine moiety" means a monovalent radical of a dialkylamine, preferably a radical in which the hydrogen atom of the amino group has been removed.

[0508] The term "ceramide" means a ceramide having the formula:

[0509]

[0510] or a salt thereof, wherein -C 13 H 27 refers to -(CH2) 12 A CH3 moiety; and acyl refers to an acyl moiety (e.g., a -C(O)-alkyl moiety, wherein the alkyl 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). Some examples of acyl moieties include -C(O)(CH2) 16 CH3(stearyl), -C(O)(CH2) 14 CH3(palmitoyl), -C(O)(CH2) 12 CH3 (myristoyl) and -cis-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). For example, palmitoylceramide refers to the ceramide of the above formula, wherein the acyl group is -C(O)(CH2) 14 CH3 (palmitoyl). The term "ceramide moiety" means a monovalent radical of ceramide, preferably a radical in which a hydrogen atom of a hydroxyl group, preferably a hydrogen of a terminal (primary) hydroxyl group, has been removed.

[0511] The term "MAA" means a monoalkylamine or salt thereof having the formula H2N(alkyl), wherein the alkyl portion is preferably linear. In some embodiments, the alkyl portion has at least 8 carbon atoms. Preferably, the alkyl portion is the alkyl portion of a fatty acid alcohol, more preferably the alkyl portion is the alkyl portion of a fatty acid alcohol having at least 8 carbon atoms. Some examples of alkyl portions include -(CH2) 17 CH3(stearyl), -(CH2) 15 CH3(palmityl) and -(CH2) 13 CH3 (myristyl). For example, MMA means myristylamine, which is a monoalkylamine of the above formula, wherein the alkyl group is -(CH2) 13 CH3 (myristyl). The term "monoalkylamine moiety" means a monovalent radical of a monoalkylamine, preferably a radical in which one of the hydrogen atoms of the amino group has been removed.

[0512] Nucleic Acids

[0513] 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 and linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to the present disclosure, the term "isolated nucleic acid" means a nucleic acid that (i) is amplified in vitro, for example, by polymerase chain reaction (PCR) for DNA, or is transcribed in vitro (using, for example, RNA polymerase) for RNA, (ii) is produced by cloning and recombination, (iii) is purified, for example, by cleavage and separation by gel electrophoresis, or (iv) is synthesized, for example, by chemical synthesis.

[0514] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide consists of a nucleoside and one or more phosphate groups. Some examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0515] The five standard nucleosides that usually constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are usually abbreviated as their single-letter codes, namely U, A, T, C and G. However, thymidine is more usually written as "dT" ("d" represents "deoxy") because it contains 2'-deoxyribofuranose moiety, rather than the ribofuranose ring present in uridine. This is because thymidine is present in deoxyribonucleic acid (DNA) rather than ribonucleic acid (RNA). On the contrary, uridine is present in RNA rather than DNA. The remaining three nucleosides can be present in both RNA and DNA. In RNA, it will be expressed as A, C and G, and in DNA, it will be expressed as dA, dC and dG.

[0516] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably replaced by one or more alkyl groups, more preferably one or more C 1-4 Alkyl, even more preferably one or more methyl modifications. Some specific 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, such as N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C1-4 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.

[0517] In some embodiments of all aspects of the present disclosure, the nucleic acid is DNA.

[0518] As used herein, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In some preferred embodiments, DNA comprises all or most deoxyribonucleotide residues. "Deoxyribonucleotides" as used herein refer to nucleotides lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA (e.g., partially purified DNA), substantially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such alterations can refer to the addition of non-nucleotide substances to internal DNA nucleotides or to the ends of DNA. It is also contemplated herein that the nucleotides in the DNA can be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered to be analogs of naturally occurring DNA. A molecule contains "mostly 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 the 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).

[0519] The DNA may be recombinant DNA and may be obtained by cloning nucleic acids, particularly cDNA. cDNA may be obtained by reverse transcription of RNA.

[0520] RNA

[0521] In some embodiments of all aspects of the present disclosure, the nucleic acid is RNA.

[0522] According to the present disclosure, the term "RNA" means a nucleic acid molecule comprising ribonucleotide residues. In some preferred embodiments, RNA comprises all or most ribonucleotide residues. "Ribonucleotide" as used herein refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such changes can refer to the addition of non-nucleotide substances to internal RNA nucleotides or to the ends of RNA. It is also contemplated herein that the nucleotides in RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA comprising such altered / modified nucleotides or nucleosides (i.e., altered / modified RNA) can 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 the 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).

[0523] "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.

[0524] In a preferred embodiment, RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. The RNA can express the encoded peptide, polypeptide or protein. For example, the RNA can be an RNA that encodes and expresses a pharmaceutically active peptide or protein. In some embodiments, RNA can interact with the cell translation mechanism, thereby allowing the translation of the peptide or protein. The cell can produce the encoded peptide or protein in the cell (for example, in the cytoplasm), can secrete the encoded peptide or protein, or can produce the encoded peptide or protein on the surface. Alternatively, RNA can be a non-coding RNA, such as antisense RNA, microRNA (miRNA) or siRNA.

[0525] As used herein, the term "in vitro transcription" or "IVT" means performing transcription (i.e., producing RNA) in a cell-free manner. That is, IVT does not utilize living / cultured cells, but rather utilizes transcription machinery extracted from cells (e.g., cell lysate or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0526] mRNA

[0527] In some embodiments of all aspects of the present disclosure, the nucleic acid is mRNA.

[0528] According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced using a DNA template. Generally speaking, mRNA encodes a peptide, polypeptide, or protein. Generally speaking, mRNA comprises a 5'-UTR, a peptide / protein coding region, and a 3'-UTR. In the context of the present disclosure, mRNA is preferably produced by in vitro transcription (IVT) from a DNA template. As described above, in vitro transcription methods are known to those skilled in the art, and a variety of in vitro transcription kits are commercially available.

[0529] mRNA is single-stranded but may contain self-complementary sequences that allow portions of the mRNA to fold and pair with themselves to form a double helix.

[0530] According to the present disclosure, "dsRNA" means double-stranded RNA, and is RNA having two partially or fully complementary strands.

[0531] In some preferred embodiments of the present disclosure, mRNA refers to an RNA transcript encoding a peptide, polypeptide, or protein.

[0532] In some embodiments, preferably the length of the RNA encoding the peptide, polypeptide or protein is 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, 00, 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 nucleotides, 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.

[0533] As is well established in the art, RNA (e.g., mRNA) typically 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 one embodiment, RNA (e.g., mRNA) is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those of skill in the art; see, for example, Molecular Cloning: A Laboratory Manual, Second Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, a variety of in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (e.g., TranscriptAid TM T7 test kit, T7 test kit, ), New England BioLabs Inc. (e.g. HiScribe TM T7 kit, HiScribe TM T7 ARCA mRNA kit), Promega (such as RiboMAX TM 、 systems), Jena Bioscience (e.g., SP6 or T7 transcription kits), and Epicentre (e.g., AmpliScribe TMTo provide modified RNA (e.g., mRNA), corresponding 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 the mRNA can be modified and / or modified after transcription.

[0534] In some embodiments, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter used to control transcription can be any promoter of 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 cDNA) and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0535] In some embodiments of the present disclosure, the RNA (e.g., mRNA) is a "replicon RNA" (e.g., "replicon mRNA") or simply a "replicon," in particular a "self-replicating RNA" (e.g., "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (e.g., self-replicating mRNA) is derived from or comprises elements derived from ssRNA viruses (in particular, positive-strand ssRNA viruses such as alphaviruses). Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many alphaviruses is typically 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 the transcription, modification and...

Claims

1. A composition comprising (i) a nucleic acid; (ii) a cationic lipid or a lipid that can ionize into a cation; and (iii) a polymer-conjugated compound, wherein the polymer-conjugated compound comprises (a) a polymer comprising the following general formula (I); and (b) one or more hydrophobic chains: in X 2 and X 1 taken together is an optionally substituted amide, optionally substituted thioamide, ester or thioester; Y is -CH2-, -(CH2)2- or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

2. The composition of claim 1, wherein: (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is-O-; (vi) When X 1 If it is -O-, then X 2 is -C(O)-; (vii) When X 1 If it is -C(S)-, then X 2 is-O-; (viii) When X 1 If it is -O-, then X 2 is -C(S)-; (ix) When X 1 When it is -C(O)-, then X 2 is -S-; or (x) When X 1 If it is -S-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 Alkyl; preferably (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is -O-; or (vi) When X 1 If it is -O-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 alkyl.

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

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

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

6. The composition of any one of claims 1 to 5, wherein Y is -CH2- or -(CH2)2-.

7. The composition of any one of claims 1 to 6, wherein Y is -CH2-.

8. The composition of any one of claims 1 to 7, wherein the polymer comprises the following general formula (II): Where R 1 is hydrogen or C 1-8 alkyl.

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

10. The composition of any one of claims 1 to 9, wherein z is 2 to 5.

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

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

13. The composition of any one of claims 1 to 12, wherein the polymer comprises the following general formula (III): Where R 1 is hydrogen or C 1-8 alkyl.

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

15. The composition of any one of claims 8 to 14, wherein R 1 It's hydrogen.

16. The composition of any one of claims 1 to 15, wherein the polymer comprises the following general formula (IV):

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

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

19. The composition of any one of claims 1 to 18, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

20. The composition of any one of claims 1 to 19, wherein the one or more hydrophobic chains are located at X of the polymer. 1 End or X 2 The end.

21. The composition of any one of claims 1 to 20, wherein the one or more hydrophobic chains are acyclic hydrocarbon groups, preferably linear hydrocarbon groups, more preferably hydrocarbon groups having at least 8 carbon atoms, for example hydrocarbon groups having at least 10 carbon atoms or at least 12 carbon atoms.

22. The composition of any one of claims 1 to 21, wherein the polymer-conjugated compound comprises the following general formula (V) or (V'): in X 2 and X 1 taken together is an optionally substituted amide, optionally substituted thioamide, ester or thioester; Y is -CH2-, -(CH2)2- or -(CH2)3-; R 2 is a moiety containing one or more hydrophobic chains; R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of: -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be joined together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and members of targeting pairs; z is 2 to 24; and n is 1 to 100.

23. The composition of claim 22, wherein: (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is-O-; (vi) When X 1 If it is -O-, then X 2 is -C(O)-; (vii) When X 1 If it is -C(S)-, then X 2 is-O-; (viii) When X 1 If it is -O-, then X 2 is -C(S)-; (ix) When X 1 When it is -C(O)-, then X 2 is -S-; or (x) When X 1 If it is -S-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 Alkyl; preferably (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is -O-; or (vi) When X 1 If it is -O-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 alkyl.

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

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

26. The composition of any one of claims 22 to 25, wherein X 1 is -C(O)- and X 2 Yes - NR 1 -, where R 1 It's hydrogen.

27. The composition of any one of claims 22 to 26, wherein Y is -CH2- or -(CH2)2-.

28. The composition of any one of claims 22 to 27, wherein Y is -CH2-.

29. The composition of any one of claims 22 to 28, wherein the polymer-conjugated compound comprises the following general formula (VI) or (VI'): Where R 1 is hydrogen or C 1-8 alkyl.

30. The composition of any one of claims 22 to 29, wherein z is 2 to 10, such as 2 to 7.

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

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

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

34. The composition of any one of claims 22 to 33, comprising the following general formula (VII) or (VII'): Where R 1 is hydrogen or C 1-8 alkyl.

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

36. The composition of any one of claims 29 to 35, wherein R 1 It's hydrogen.

37. The composition of any one of claims 22 to 36, wherein the polymer-conjugated compound comprises the following general formula (VIII) or (VIII'):

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

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

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

41. The composition of any one of claims 22 to 40, wherein R 2 YesR 4 or -L 1 (R 4 ) p , where each R 4 L is independently a hydrophobic chain, such as a hydrocarbon group; 1 is a linker; and p is 1 or 2.

42. The composition of claim 41, wherein L 1 Contains at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or between the alkylene and R 4 The alkylene moiety is connected to a divalent functional moiety at the terminal end of the connection, wherein preferably each monovalent functional moiety is independently selected from hydroxyl, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, sulfamic acid salt / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioester salts / esters, dithioates / esters, imidates / esters, imino, thioimidates / esters, sulfinamido, carbonates / esters, thiocarbonates / esters, dithiocarbonates / esters, trithiocarbonates / esters, guanidino (imidamido), carbamoylimidates / esters, carboimidates / esters, carbamates / esters, dithiocarbamates / esters, dithiocarboimidates / esters, thiocarbamoylimidates / esters, thiocarbamates / esters, thiocarboimidates / esters, acylhydrazones , hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties; and / or each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, aminosulfonate / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester, imino, thioimidate / ester [0063] The invention also includes but is not limited to, but is not limited to, ester, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, guanidine (imidamido), carbamoyl imide, carboimidate, carbamate, dithiocarbamate, dithiocarbamate, thiocarbamate, thiocarbamate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties.

43. The composition of claim 41 or 42, wherein L 1 Contains a functional moiety selected from the following [*-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)-, I*-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 * indicates the same as R 4 The connection point of 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from 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 substituted by one or more selected from halogen, C 1-3 alkyl, -OH, -CN and -OC1-3 alkyl; and C 1-6 The alkyltriyl radical is optionally substituted with one or more -OH substituents and is bonded to another hydrophobic chain R 4 Direct connection.

44. The composition of any one of claims 41 to 43, wherein L 1 Also comprising at least one additional bifunctional moiety, R 2 By the additional bifunctional part and X in formula (V) 1 Or X in formula (V') 2 connect.

45. The composition of claim 44, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamic acid salt, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioacid salt, imidate, imino, thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate The moieties are preferably selected from the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioacid salt, dithioacid salt, carbonyl and thiocarbonyl, wherein if L 1 further comprises at least two additional bifunctional moieties, the at least two additional bifunctional moieties are optionally C 1-6 The alkylene groups are separated from each other.

46. ​​The composition of any one of claims 41 to 45, wherein L 1 Selected from [*-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 * indicates the same as R 4 The attachment point of; p is 1 or 2; [*-C(O)O] 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 C in alkylene 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 27 Selected from H, C 1-6 Alkyl, aryl, aryl (C 1-6 alkyl) and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl group is optionally substituted with one or more -OH substituents and is directly linked to another hydrophobic chain R4.

47. The composition of any one of claims 41 to 46, wherein L 1 Selected from [*-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 Yes(*-)(R 26 )N-, where * indicates 4 The attachment point of; p is 1 or 2; [*-C(O)O] 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 C in alkylene 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl group is optionally substituted with one or more -OH substituents and is directly linked to another hydrophobic chain R4.

48. The composition of any one of claims 22 to 47, wherein R 2 Selected from [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 (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 )2N- and [R 4 C(O)NH](C 2-3 Alkyltriyl)O- or R 2 Yes (R 4 )(R 26 )N-, where p is 1 or 2; C 2-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 2-3 The alkyltriyl radical is optionally substituted with one or more -OH substituents and is bonded to another hydrophobic chain R 4 Direct connection.

49. The composition of any one of claims 22 to 48, wherein R 2 is selected from a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglycerol moiety, a dialkylamino moiety and a ceramide moiety, or R 2 It is a monoalkylamine moiety.

50. The composition of any one of claims 41 to 49, wherein each R4 is independently a non-cyclic hydrocarbon group, preferably a linear hydrocarbon group.

51. The composition of any one of claims 41 to 50, wherein each R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as a hydrocarbyl group having at least 10 carbon atoms or at least 12 carbon atoms.

52. The composition of any one of claims 22 to 51, wherein R 2 Selected from: DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopherol, DMG (1,2-dimyristoylglycerol), DMA (dimyristamide) and palmitoylceramide moieties, or R 2 It is the monomyristamide part.

53. The composition of any one of claims 22 to 52, wherein R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 , -NR 22 C(O)R 21 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and substituents of members of targeting pairs.

54. The composition of any one of claims 22 to 53, wherein R 3 Selected from 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 members of a targeting pair, wherein the C 1-3 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 and a substituent group substituted with a member of a targeting pair; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a substituent that is substituted with a member of a targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and substituted with a substituent targeting a member of the pair.

55. The composition of any one of claims 22 to 54, wherein R 3 Selected from 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 the C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of: -OH, -SH, halogen, -CN, -N3, C 2-6 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.

56. The composition of any one of claims 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper (I) catalyzed reactions); conjugated diene-substituted olefin (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-α v β3 integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

57. The composition of any one of claims 1 to 56, wherein the polymer-conjugated compound has one of the following formulae: in n is 5 to 25; R 3 Selected from H, -C(O)(C 1-3 alkyl) and a member of a targeting pair, wherein the C 1-3 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and substitutions of substituents of members of a targeted pair; R 27 is H or a counter cation; and In each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3(stearoyl) moieties, in each case -C(O)C 15 H 31 means -C(O)(CH2) 14 CH3(palmitoyl) moieties, in each case -C(O)C 13 H 27 means -C(O)(CH2) 12 CH3(myristoyl) moieties, in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moieties, in each case -C 13 H 27 refers to -(CH2) 12 CH3 moiety, and in each case -C(O)C 17 H 33 It refers to the cis-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl) moiety.

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

59. The composition of claim 57 or 58, wherein R 3 is H or -C(O)(C 1-3 alkyl), wherein the C 1-3 The alkyl group is optionally substituted with a substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimido), -SH, -Br, -N3, C 2-6 Alkynyl, antigen or antibody.

60. The composition of any one of claims 1 to 59, wherein the polymer-conjugated compound has one of the following formulae: In each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3(stearoyl) moieties, in each case -C(O)C 15 H 31 means -C(O)(CH2) 14 CH3(palmitoyl) moieties, in each case -C(O)C 13 H 27 means -C(O)(CH2) 12 CH3(myristoyl) moieties, in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moieties, in each case -C 13 H 27 refers to -(CH2) 12 A CH3 moiety and n is in each case 8, 10 or 14.

61. The composition of any one of claims 1 to 60, wherein the polymer-conjugated compound has one of the following formulae: (i) where n is 14 and in each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3 (stearoyl) moiety; (ii) where n is 14, and in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moiety; (iii) Where n is 8, 12, 14, or 16.

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

63. The composition of any one 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. The composition of any one of claims 1 to 63, wherein the concentration of nucleic acid in the composition is from about 1 mg / l to about 500 mg / l, such as 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. The composition of any one of claims 1 to 64, wherein the cationic ionizable lipid comprises a head group comprising at least one tertiary amine moiety.

66. The composition of any one of claims 1 to 65, wherein the cationic lipid or the lipid that can ionize into a cation has the structure of formula (X): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L 10 and L 20 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, And L 10 and L 20 The other one 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 C1-C 12 Alkylene or C 2-12 alkenylene; G 3 It is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 Cycloalkenylene; R a Is H or C 1-12 alkyl; R 35 and R 36 Each independently is C 6-24 Alkyl or C 6-24 alkenyl; R 37 is H, OR 50 , CN, -C(=O)OR 40 , -OC(=O)R 40 or -NR 50 C(=O)R 40 ; R 40 It is C 1-12 alkyl; R 50 Is H or C 1-6 Alkyl; and x is 0, 1, or 2.

67. The composition of any one of claims 1 to 65, wherein the cationic lipid or the lipid that can ionize into a cation has the structure of formula (XI): in Each of R1 and R2 is independently R5 or -G1-L1-R6, wherein at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 Cycloalkyl; Each of R5 and R6 is independently a non-cyclic hydrocarbon group having at least 10 carbon atoms; Each of G1 and G2 is independently an unsubstituted C 1-12 Alkylene or C 2-12 alkenylene; Each of L1 and L2 is independently selected from -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-; Ra is H or C 1-12 alkyl; m is 0, 1, 2, 3 or 4; and x is 0, 1, or 2.

68. The composition of any one of claims 1 to 65, wherein the cationic lipid or cationic lipid that can be ionized to give off cations comprises 2,3-dioleoyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleoyl-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-diole ... propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-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. The composition of any one of claims 1 to 68, wherein the cationic lipid or lipid that can ionize into a cation comprises about 20 mol% to about 80 mol% of the total lipids present in the composition.

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

71. The composition of claim 70, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DMPC), dioleo ... The following are the phosphatidylcholine compounds: dioleoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), ditetracosanoylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-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).

72. The composition of claim 70 or 71, wherein the phospholipids comprise about 5 mol% to about 30 mol% of the total lipids present in the composition.

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

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

75. The composition of any one of claims 70 to 74, wherein the cationic lipid or lipid that can ionize into a cation accounts for about 20 mol % to about 70 mol % of the total lipids present in the composition; the polymer-conjugated compound accounts for about 0.5 mol % to about 15 mol % of the total lipids present in the composition; the phospholipid accounts for about 5 mol % to about 25 mol % of the total lipids present in the composition; and the steroid accounts for about 20 mol % to about 55 mol % of the total lipids present in the composition.

76. The composition of any one of claims 1 to 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 lipid or a lipid that can ionize out a cation, and at least a portion of a polymer-conjugated compound.

77. The composition of claim 76, wherein the particle is selected from the group consisting of lipid nanoparticles (LNP), liposomes, lipid complexes (LPX), and mixtures thereof.

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

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

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

81. The composition of claim 80, wherein the RNA (1) comprises modified nucleosides replacing 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. The composition of claim 80 or 81, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

83. The composition of claim 82, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A 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. The composition of any one 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 to an antigen in a subject.

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

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

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

89. A method for treating or preventing a disease or condition in a subject, the method comprising administering to a subject a composition as described in any one of claims 1 to 86, wherein delivery of the nucleic acid to cells of the subject is beneficial for treating or preventing the disease or condition.

90. A method for treating or preventing a disease or condition in a subject, the method comprising administering to a subject a composition as described in any one of claims 1 to 86, wherein the nucleic acid encodes a therapeutic peptide or protein, and wherein delivering the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition.

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

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

93. A polymer-conjugated compound comprising (a) a polymer comprising the following general formula (I); and (b) one or more hydrophobic chains: in X 2 and X 1 taken together is an optionally substituted amide, optionally substituted thioamide, ester or thioester; Y is -CH2-, -(CH2)2- or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

94. The polymer-conjugated compound of claim 93, wherein: (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is-O-; (vi) When X 1 If it is -O-, then X 2 is -C(O)-; (vii) When X 1 If it is -C(S)-, then X 2 is-O-; (viii) When X 1 If it is -O-, then X 2 is -C(S)-; (ix) When X 1 When it is -C(O)-, then X 2 is -S-; or (x) When X 1 If it is -S-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 Alkyl; preferably (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is-O-; (vi) When X 1 If it is -O-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 alkyl.

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

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

97. The polymer-conjugated compound of any one of claims 93 to 96, wherein X 1 is -C(O)- and X 2 Yes - NR 1 -, where R 1 It's hydrogen.

98. The polymer-conjugated compound of any one of claims 93 to 97, wherein Y is -CH2- or -(CH2)2-.

99. The polymer-conjugated compound of any one of claims 93 to 98, wherein Y is -CH2-.

100. The polymer-conjugated compound of any one of claims 93 to 99, wherein the polymer comprises the following general formula (II): Where R 1 is hydrogen or C 1-8 alkyl.

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

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

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

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

105. The polymer-conjugated compound of any one of claims 93 to 104, wherein the polymer comprises the following general formula (III): Where R 1 is hydrogen or C 1-8 alkyl.

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

107. The polymer-conjugated compound of any one of claims 100 to 106, wherein R 1 It's hydrogen.

108. The polymer-conjugated compound of any one of claims 93 to 107, wherein the polymer comprises the following general formula (IV):

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

110. The polymer-conjugated compound of any one of claims 93 to 109, wherein n is 5 to 25. 111 . The polymer-conjugated compound of any one of claims 93 to 110 , wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14 or 16.

112. The polymer-conjugated compound of any one of claims 93 to 111, wherein the one or more hydrophobic chains are located at X of the polymer. 1 End or X 2 The end.

113. The polymer-conjugated compound of any one of claims 93 to 112, wherein the one or more hydrophobic chains are non-cyclic hydrocarbon groups, preferably linear hydrocarbon groups, more preferably hydrocarbon groups having at least 8 carbon atoms, for example hydrocarbon groups having at least 10 carbon atoms or at least 12 carbon atoms.

114. The polymer-conjugated compound of any one of claims 93 to 113, comprising the following general formula (V) or (V'): in X 2 and X 1 Taken together are optionally substituted amides, optionally substituted thioamides, esters, thioesters; Y is -CH2-, -(CH2)2- or -(CH2)3-; R 2 is a moiety containing one or more hydrophobic chains; R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 , -NR 22 C(O)R 21 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and substituent substitutions of members of targeting pairs; z is 2 to 24; and n is 1 to 100.

115. The polymer-conjugated compound of claim 114, wherein: (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is-O-; (vi) When X 1 If it is -O-, then X 2 is -C(O)-; (vii) When X 1 If it is -C(S)-, then X 2 is-O-; (viii) When X 1 If it is -O-, then X 2 is -C(S)-; (ix) When X 1 When it is -C(O)-, then X 2 is -S-; or (x) When X 1 If it is -S-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 Alkyl; preferably (i) When X 1 When it is -C(O)-, then X 2 Yes - NR 1 -; (ii) When X 1 Yes - NR 1 -, then X 2 is -C(O)-; (iii) When X 1 If it is -C(S)-, then X 2 Yes - NR 1 -; (iv) When X 1 Yes - NR 1 -, then X 2 is -C(S)-; (v) When X 1 When it is -C(O)-, then X 2 is -O-; or (vi) When X 1 If it is -O-, then X 2 is -C(O)-; Where R 1 is hydrogen or C 1-8 alkyl.

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

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

118. The polymer-conjugated compound of any one of claims 114 to 117, wherein X 1 is -C(O)- and X 2 Yes - NR 1 -, where R 1 It's hydrogen.

119. The polymer-conjugated compound of any one of claims 114 to 118, wherein Y is -CH2- or -(CH2)2-.

120. The polymer-conjugated compound of any one of claims 114 to 119, wherein Y is -CH2-.

121. The polymer-conjugated compound of any one of claims 114 to 120, comprising the following general formula (VI) or (VI'): Where R 1 is hydrogen or C 1-8 alkyl.

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

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

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

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

126. The polymer-conjugated compound of any one of claims 114 to 125, comprising the following general formula (VII) or (VII'): Where R 1 is hydrogen or C 1-8 alkyl.

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

128. The polymer-conjugated compound of any one of claims 121 to 127, wherein R 1 It's hydrogen.

129. The polymer-conjugated compound of any one of claims 114 to 128, comprising the following general formula (VIII) or (VIII'):

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

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

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

133. The polymer-conjugated compound of any one of claims 114 to 132, wherein R 2 YesR 4 or -L 1 (R 4 ) p , where each R 4 L is independently a hydrophobic chain, such as a hydrocarbon group; 1 is a linker; and p is 1 or 2.

134. The polymer-conjugated compound of claim 133, wherein L 1 The alkylene group comprises at least one functional moiety, for example an alkylene moiety substituted with at least one monovalent functional moiety and / or an alkylene moiety connected to a divalent functional moiety at the terminal end of the alkylene group connected to R4, wherein preferably each monovalent functional moiety is independently selected from hydroxyl, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, sulfamic acid salt / ester, diselenide, sulfonate, sulfonamide, sulfamate, diselenide, sulfonate ... sulfuric acid diamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester, imino, thioimidate / ester, sulfinamido, carbonate / ester, thiocarbonate / ester, dithiocarbonate / ester, trithiocarbonate / ester, guanidine (imidamido), carbamoylimidate / ester, carboimidate / ester, carbamate / ester, dithiocarbamate / ester, dithiocarboimidate / ester, thiocarbamoylimidate / ester, sulfur and / or each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, aminosulfonate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester, imino, thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamoylimidate, carboimidate, carbamate, dithiocarbamate, dithiocarbamate, thiocarbamate, thiocarbamate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

135. The polymer-conjugated compound of claim 133 or 134, wherein L 1 Contains a functional moiety selected from: [*-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 * represents 4 The connection point of 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from 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 substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl radical is optionally substituted with one or more -OH substituents and is bonded to another hydrophobic chain R 4 Direct connection.

136. The polymer-conjugated compound of any one of claims 133 to 135, wherein L 1 Also comprising at least one additional bifunctional moiety, R 2 By the additional bifunctional part and X in formula (V) 1 Or X in formula (V') 2 connect.

137. The polymer-conjugated compound of claim 136, wherein the at least one additional bifunctional moiety is selected from the group consisting of ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioacid salt, imidate, imino, thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate , trithiocarbonate, imidamido, carbamoyl imide, carboimidate, carbamate, dithiocarbamate, dithiocarbamate, thiocarbamate, thiocarbamate, thiocarbamate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties are preferably selected from phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioacid salt, dithioacid salt, carbonyl and thiocarbonyl, wherein if L 1 further comprises at least two additional bifunctional moieties, the at least two additional bifunctional moieties are optionally C 1-6 The alkylene groups are separated from each other.

138. The polymer-conjugated compound of any one of claims 133 to 137, wherein L 1 Selected from [*-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 * indicates the same as R 4 The attachment point of; p is 1 or 2; [*-C(O)O] 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 C in alkylene 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 27 Selected from H, C 1-6 Alkyl, aryl, aryl (C 1-6 alkyl) and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The C1-6 alkyl triyl is optionally substituted by one or more -OH substituents and is directly connected to another hydrophobic chain R4.

139. The polymer-conjugated compound of any one of claims 133 to 138, wherein L 1 Selected from [*-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 Yes(*-)(R 26 )N-, Where * indicates the same as R 4 The attachment point of; p is 1 or 2; [*-C(O)O] 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 C in alkylene 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 1-6 The alkyltriyl radical is optionally substituted with one or more -OH substituents and is bonded to another hydrophobic chain R 4 Direct connection.

140. The polymer-conjugated compound of any one of claims 114 to 139, wherein R 2 Selected from [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 (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 )2N- and [R 4 C(O)NH](C 2-3 Alkyltriyl)O- or R 2 Yes (R 4 )(R 26 )N-, wherein p is 1 or 2; said C 2-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 26 Selected from H and C 1-6 Alkyl; R 27 3,4-dihydro-2H-chromen-6-yl is optionally substituted by one or more selected from halogen, C 1-3 Alkyl, -OH, -CN and -OC 1-3 The alkyl group is substituted with a substituent; and C 2-3 The alkyltriyl radical is optionally substituted with one or more -OH substituents and is bonded to another hydrophobic chain R 4 Direct connection.

141. The polymer-conjugated compound of any one of claims 114 to 140, wherein R2 is selected from phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or R2 is a monoalkylamine moiety.

142. The polymer-conjugated compound of any one of claims 133 to 141, wherein each R4 is independently a non-cyclic hydrocarbon group, preferably a linear hydrocarbon group.

143. The polymer-conjugated compound of any one of claims 133 to 142, wherein each R4 is independently a hydrocarbon group having at least 8 carbon atoms, such as a hydrocarbon group having at least 10 carbon atoms or at least 12 carbon atoms.

144. The polymer-conjugated compound of any one of claims 114 to 143, wherein R2 is selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), or POPE (palmitoyloleoylphosphatidylethanolamine), tocopherol, DMG (1,2-dimyristoylglycerol), DMA (dimyristamide) and palmitoylceramide moieties, or R2 is a monomyristamide moiety.

145. The polymer-conjugated compound of any one of claims 114 to 144, wherein R 3 Selected from 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 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and substituents of members of the targeting pair; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and substituents of members of the targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and substituents of members of targeting pairs.

146. The polymer-conjugated compound of any one of claims 114 to 145, wherein R 3 Selected from 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 members of a targeting pair, wherein the C 1-3 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 and a substituent group substituted with a member of a targeting pair; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a substituent that is substituted with a member of a targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and substituted with a substituent targeting a member of the pair.

147. The polymer-conjugated compound of any one of claims 114 to 146, wherein R 3 Selected from 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 the C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, -SH, halogen, -CN, -N3, C 2-6 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.

148. The polymer-conjugated compound of any one of claims 114 to 147, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper (I) catalyzed reactions); conjugated diene-substituted olefin (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-α v β3 integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

149. The polymer-conjugated compound of any one of claims 93 to 148, having one of the following formulae: in n is 5 to 25; R 3 Selected from H, -C(O)(C 1-3 alkyl) and a member of a targeting pair, wherein the C 1-3 The alkyl group is optionally substituted by one or more independently selected from -OH, -SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and substitutions of substituents of members of a targeted pair; R 27 is H or a counter cation; and In each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3(stearoyl) moieties, in each case -C(O)C 15 H 31 means -C(O)(CH2) 14 CH3(palmitoyl) moieties, in each case -C(O)C 13 H 27 means -C(O)(CH2) 12 CH3(myristoyl) moieties, in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moieties, in each case -C 13 H 27 refers to -(CH2) 12 CH3 moiety, and in each case -C(O)C 17 H 33 It refers to the cis-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl) moiety.

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

151. The polymer-conjugated compound of claim 149 or 150, wherein R 3 is H or -C(O)(C 1-3 alkyl), wherein the C 1-3 The alkyl group is optionally substituted with a substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimido), -SH, -Br, -N3, and C 2-6 Alkynyl.

152. The polymer-conjugated compound of any one of claims 93 to 151, having one of the following formulae: In each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3(stearoyl) moieties, in each case -C(O)C 15 H 31 means -C(O)(CH2) 14 CH3(palmitoyl) moieties, in each case -C(O)C 13 H 27 means -C(O)(CH2) 12 CH3(myristoyl) moieties, in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moieties, in each case -C 13 H 27 refers to -(CH2) 12 A CH3 moiety and n is in each case 8, 10, 12, 14 or 16.

153. The polymer-conjugated compound of any one of claims 93 to 152, having one of the following formulae: （i) where n is 14 and in each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3 (stearoyl) moiety; (ii) where n is 14, and in each case -C 14 H 29 refers to -(CH2) 13 CH3(myristyl) moiety; (iii) Where n is 8, 12, 14, or 16.

154. A conjugate of (a) a polymer-conjugated compound of any one of claims 93 to 153 comprising a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair.

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 (eg, an antigen or epitope), or an antibody.

156. The conjugate of claim 154 or 155, which has one of the following formulae: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3 (stearoyl) moiety; each of m1 and m2 is independently 1, 2, 3, 4 or 5; and Pept is an antigen or an antibody specific for the antigen.

157. The conjugate of claim 156, which has one of the following formulae: (i) wherein m1 is 2, 3 or 4, preferably 2; and m2 is 2, 3 or 4, preferably 2; (ii) wherein each of m1 and m2 is independently 1, 2 or 3, preferably m1 is 1 and m2 is 2, or m1 is 2 and m2 is 1.

158. The conjugate of any one of claims 154 to 157, which has one of the following formulae: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3 (stearoyl) moiety; and Pept is an antigen or an antibody specific for the antigen.

159. The conjugate of claim 154, wherein the polymer-conjugated compound comprising a member of a targeting pair has the formula: wherein n is 5 to 25, preferably 8, 10, 12, 14 or 16; in each case -C(O)C 17 H 35 means -C(O)(CH2) 16 CH3 (stearoyl) moiety; and Pept is an antigen or an antibody specific for the antigen; The compound comprising the other member of the targeting pair is a compound comprising: (i) if Pept is the antigen, an antibody specific for the antigen; or (ii) if Pept is an antibody specific for the antigen, the antigen, and The polymer-conjugated compound comprising one member of a targeting pair is conjugated to a compound comprising the other member of the targeting pair through the interaction between (1) the antibody specific for the antigen and (2) the antigen.

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