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

Abstract

The present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions comprising an amphiphilic oligo ethylene glycol (OEG)-conjugated compound (as alternative to PEG lipids), to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy, to such amphiphilic OEG-conjugated compounds, and to conjugates of such amphiphilic OEG-conjugated compounds.

Description

[0001] NUCLEIC ACID COMPOSITIONS COMPRISING AMPHIPHILIC OLIGO ETHYLENE GLYCOL (OEG)-CONJUGATED COMPOUNDS AND METHODS OF USING SUCH COMPOUNDS AND COMPOSITIONS

[0002] Technical Field

[0003] The present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions comprising an amphiphilic oligo ethylene glycol (OEG)-conjugated compound (as alternative to PEG lipids), to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy, to such amphiphilic OEG-conjugated compounds, and to conjugates of such amphiphilic OEG-conjugated compounds.

[0004] Background

[0005] The use of a recombinant nucleic acid (such as DNA or RNA) for delivery of foreign genetic information into target cells is well known. A recombinant nucleic acid may be administered in naked form to a subject in need thereof; however, usually a recombinant nucleic acid is administered using a composition. For example, nucleic acid, such as RNA, may be delivered to a subject using different delivery vehicles, based mostly on cationic polymers or lipids which together with the nucleic acid form nanoparticles. The nanoparticles are intended to protect the nucleic acid, such as RNA, from degradation, enable delivery of the nucleic acid, such as RNA, to the target site and facilitate cellular uptake and processing by the target cells. The efficiency of the nucleic acid delivery depends, in part, on the molecular composition of the nanoparticle and can be influenced by numerous parameters, including particle size, formulation, and charge or grafting with molecular moieties, such as polyethylene glycol (PEG) or other ligands.

[0006] Grafting with PEG is considered to reduce serum interactions, to increase serum stability and to increase circulation time, which can be helpful for certain targeting approaches. Ligands which bind to receptors at the target site can help to improve targeting efficacy. Furthermore, PEGylation can be used for particle engineering. For example, if lipid nanoparticles (LNPs) are manufactured by mixing an aqueous phase of the nucleic acid, such as RNA, with an organic phase of the lipids a certain fraction of PEG- conjugated lipid (such PEG-conjugated lipids have at least 30 consecutive ethylene glycol repeating units) in the lipid mixture is required, otherwise the particles aggregate during or after the mixing step. It has been shown that by variation of the molar fraction of PEG-lipids comprising PEG at different molar masses the size of the particles can be adjusted. As well, the particle size may be adjusted by variation of the molar mass of the PEG moiety of the PEGylated lipids. Typical sizes which are accessible are in the range between 30 and 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). So-formed particles have additionally the advantage, that, due to the PEG fraction, they interact less with serum components, and have a longer circulation half-life, which is desirable in many drug delivery approaches. Without PEG-lipids, no particles with discrete size can be formed; the particles form large aggregates and precipitate. Thus, one of the primary roles of PEG-lipids is to facilitate particle self-assembly by providing a steric barrier at the surface of nascent particles formed when nucleic acids are rapidly mixed in ethanol solutions containing lipids to bind the nucleic acid, such as RNA. PEG steric hindrance prevents inter-particle fusion and promotes the formation of a homogeneous population of LNPs where diameters <100 nm can be achieved.

[0007] Despite these advantages, PEGylation of nanoparticles may lead as well to several effects which are detrimental to the intended use for drug delivery. PEGylation of liposomes and LNPs is known to reduce the cellular uptake and endosomal escape, thus reducing at the end the overall transfection efficiency. Indeed, the PEG shell provides a steric barrier to efficient binding of particles to the cell and also hinders endosomal release by preventing membrane fusion between the liposome and the endosomal membrane. This is why the type of PEG-lipid and the amount of PEG-lipid used must be always carefully adjusted. It should provide sufficient stealth effect for in vivo and stabilization aspects on the one hand, while not hindering transfection on the other. This phenomenon is known as the "PEG Dilemma".

[0008] Besides lowering transfection efficiency, PEGylation has been associated with accelerated blood clearance (ABC) phenomenon 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; S.M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection in rats of PEG-grafted liposomes may significantly alter the pharmacokinetic behavior of a second dose when this second dose is administered after an interval of several days (Laverman P et al., 2001, J. Pharmacol. Exp. Ther. 298(2), 607-12; Ishida et al., 2006, J. Control Release 115(3), 251-8). The phenomenon of "accelerated blood clearance" (ABC) appears to be related to the PEG content of liposomes. The presence of anti- PEG antibodies in the plasma induces a higher clearance of the particles by the Monophagocyte System (MPS) which at the end reduces the efficacy of the drug.

[0009] Due to broad use of PEGs as ingredient of food, cosmetic, hygienic products, and medicines, a certain percentage of the general population have "pre-existing" anti-PEG antibodies. The anti-PEG antibodies are associated with decreased efficacy of pegylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.

[0010] As PEG may induce immune responses there is a need to avoid it for certain applications where multiple injections are needed. Examples are therapies using nucleic acid (such as RNA, in particular mRNA), for example for protein replacement therapy. Here, the risk can be particularly high due to the potential intrinsic immunogenicity of nucleic acid (in particular RNA). Other examples are protein knock-down therapies using inhibitory RNA (such as siRNA), antisense oligonucleotides or DNA based therapies. Thus, there remains a need in the art for efficient compositions and methods for introducing nucleic acid, such as RNA, into cells which avoid the disadvantages accompanied by use of PEG. The present disclosure addresses this and other needs.

[0011] The inventors surprisingly found that the compositions, methods, polymer-conjugated compounds (which are also referred to as amphiphilic OEG-conjugated compounds herein), and conjugates described herein fulfill the above-mentioned requirements. In particular, it is demonstrated that the polymer-conjugated compounds described herein are not bound by antibodies raised against PEG structures and are stable under physiological conditions. The polymer component of the polymer- conjugated compounds as well as the polymer-conjugated compounds and their conjugates can be synthesized by well-known procedures, such as solid-phase peptide synthesis (SPPS). The polymer- conjugated compounds and conjugates can be end-group functionalized with different moieties to modulate charge or to introduce specific molecular moieties like ligands.

[0012] Summary

[0013] The present invention is defined by the appended claims.

[0014] In a first aspect, the present disclosure provides a composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains: wherein

[0015] X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester, preferably optionally substituted amide, optionally substituted thioamide, or ester;

[0016] Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100. This composition is also referred to as a nucleic acid composition herein.

[0017] As demonstrated in the present application, anti-PEG antibodies (polyclonal as well as IgG and IgM anti-PEG antibodies) which have been raised against PEG (i.e., having at least 30 consecutive ethylene glycol repeating units) and which bind to such PEG do not bind to a polymer comprising the structure of formula (I). Furthermore, the present application shows that a polymer comprising the structure of formula (I) is stable under physiological conditions. In some embodiments of the first aspect (in particular with respect to formula (I)), X2and X1taken together are an optionally substituted amide. Thus, in some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. In some embodiments, X1is -NR1- and X2is -C(O)-, wherein R1is hydrogen or Cus alkyl.

[0018] In some embodiments of the first aspect (in particular with respect to formula (I)), X2and X1taken together are an optionally substituted thioamide. Thus, in some embodiments, X1is -C(S)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. In some embodiments, X1is -NR1- and X2is -C(S)-, wherein R1is hydrogen or C1-8 alkyl.

[0019] In some embodiments of the first aspect (in particular with respect to formula (I)), X2and X1taken together are an ester. Thus, in some embodiments, X1is -C(O)- and X2is -O. In some embodiments, X1is -O- then X2is -C(O).

[0020] In some embodiments of the first aspect (in particular with respect to formula (I)), X2and X1taken together are a thioester. Thus, in some embodiments, X1is -C(S)- and X2is -O-. In some embodiments, X1is -O- then X2is -C(S)-. In some embodiments, X1is -C(O)- then X2is -S-. In some embodiments, X1is -S- then X2is -C(O)-.

[0021] In some embodiments of the first aspect (in particular with respect to formula (I)), X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. For example, R1may be hydrogen or methyl. In some embodiments, R1is hydrogen.

[0022] In some embodiments of the first aspect (in particular with respect to formula (I)), Y is -CH2- or -(CFFh-. In some embodiments, Y is -CH2-.

[0023] In some embodiments of the first aspect, the polymer comprises the following general formula (II): wherein R1is hydrogen or Cus alkyl. In some embodiments of formula (II), R1is hydrogen or methyl. For example, R1may be hydrogen. In some embodiments, R1at each occurrence (i.e., in each repeating unit) is the same (e.g., R1may be H or methyl in each repeating unit). In some embodiments, R1in at least one repeating unit differs from R1in another repeating unit (e.g., for at least one repeating unit R1 is one specific alkyl (such as H), and for at least one different repeating unit R1is a different specific alkyl (such as methyl)).

[0024] In some embodiments of the first aspect (in particular with respect to any one of formulas (I) and (II)), z is 2 to 20, such as 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.

[0025] In some embodiments of the first aspect, the polymer comprises the following general formula (III): wherein R1is hydrogen or Cus alkyl. In some embodiments of formula (III), R1is hydrogen or methyl. For example, R1may be hydrogen. In some embodiments, R1at each occurrence (i.e., in each repeating unit) is the same (e.g., R1may be H or methyl in each repeating unit). In some embodiments, R1in at least one repeating unit differs from R1in another repeating unit (e.g., for at least one repeating unit R1is one specific alkyl (such as H), and for at least one different repeating unit R1is a different specific alkyl (such as methyl)).

[0026] In some embodiments of the first aspect, the polymer comprises the following general formula (IV):

[0027] In some embodiments of the first aspect, the polymer comprises the following general formula (IVa):

[0028] (IVa).

[0029] In some embodiments of the first aspect (in particular with respect to any one of formulas (I), (II), (III), (IV), and (IVa)), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, such as 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.

[0030] In some embodiments of the first aspect (in particular with respect to any one of formulas (I), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are located at either the X1end or the X2end of the polymer.

[0031] In some embodiments of the first aspect (in particular with respect to any one of Formulas (I), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are independently selected from non-cyclic, preferably straight, hydrocarbyl groups, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the hydrocarbyl groups have at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl groups may 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 said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated.

[0032] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (V) or (V’): wherein

[0033] X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;

[0034] Y is -CH2-, -(CH2)2-, or -(CH2)3-;

[0035] R2is a moiety comprising the one or more hydrophobic chains;

[0036] R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100.

[0037] In some embodiments of formula (V) or (V’), X2and X1taken together are an optionally substituted amide. Thus, in some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. In some embodiments, X1is -NR1- and X2is -C(O)-, wherein R1is hydrogen or Cus alkyl.

[0038] In some embodiments of formula (V) or (V’), X2and X1taken together are an optionally substituted thioamide. Thus, in some embodiments, X1is -C(S)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. In some embodiments, X1is -NR1- and X2is -C(S)-, wherein R1is hydrogen or Cus alkyl.

[0039] In some embodiments of formula (V) or (V’), X2and X1taken together are an ester. Thus, in some embodiments, X1is -C(O)- and X2is -O. In some embodiments, X1is -O- then X2is -C(O).

[0040] In some embodiments of formula (V) or (V’), X2and X1taken together are a thioester. Thus, in some embodiments, X1is -C(S)- and X2is -O-. In some embodiments, X1is -O- then X2is -C(S). In some embodiments, X1is -C(O)- then X2is -S-. In some embodiments, X1is -S- then X2is -C(O)-.

[0041] In some embodiments of formula (V) or (V’), X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl. For example, R1may be hydrogen or methyl. In some embodiments, R1is hydrogen.

[0042] In some embodiments of formula (V) or (V’), Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-. In some embodiments of formula (V) or (V’), z is 2 to 20, such as 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.

[0043] In some embodiments of formula (V) or (V’), R1is hydrogen or methyl. For example, R1may be hydrogen. In some embodiments, R1at each occurrence (i.e., in each repeating unit) is the same (e.g., R1may be H or methyl in each repeating unit). In some embodiments, R1in at least one repeating unit differs from R1in another repeating unit (e.g., for at least one repeating unit R1is one specific alkyl (such as H), and for at least one different repeating unit R1is a different specific alkyl (such as methyl)).

[0044] In some embodiments of formula (V) or (V’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 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.

[0045] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VI) or (VI’): wherein z, n, R2, and R3are as defined for Formula (V) and (V’); and R1is hydrogen or Cus alkyl.

[0046] In some embodiments of formula (VI) or (VF), R1is hydrogen or methyl. For example, R1may be hydrogen. In some embodiments, R1at each occurrence (i.e., in each repeating unit) is the same (e.g., R1may be H or methyl in each repeating unit). In some embodiments, R1in at least one repeating unit differs from R1in another repeating unit (e.g., for at least one repeating unit R1is one specific alkyl (such as H), and for at least one different repeating unit R1is a different specific alkyl (such as methyl)).

[0047] In some embodiments of formula (VI) or (VF), z is 2 to 20, such as 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.

[0048] In some embodiments of formula (VI) or (VF), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 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. In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VII) or (VII’): wherein n, R2, and R3are as defined for formula (V) and (V’); and R1is hydrogen or Cus alkyl.

[0049] In some embodiments of formula (VII) or (VII’), R1is hydrogen or methyl. For example, R1may be hydrogen. In some embodiments, R1at each occurrence (i.e., in each repeating unit) is the same (e.g., R1may be H or methyl in each repeating unit). In some embodiments, R1in at least one repeating unit differs from R1in another repeating unit (e.g., for at least one repeating unit R1is one specific alkyl (such as H), and for at least one different repeating unit R1is a different specific alkyl (such as methyl)).

[0050] In some embodiments of formula (VII) or (VII’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 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.

[0051] In some embodiments of the first aspect, the polymer-conjugated compound comprises the following general formula (VIII), (Villa), (VIII’), or (Villa’): wherein n, R2, and R3are as defined for formula (V) and (V’).

[0052] In some embodiments of formula (VIII), (Villa), (VIII’), or (Villa’), n is 5 to 50, such as 5 to 45, 5 to 40, 5 to 35 or 5 to 30. In some embodiments, n is 5 to 25, such as 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.

[0053] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2is R4or -L^R^p, wherein each R4is independently a hydrophobic chain, such as a hydrocarbyl group; L1is a linker; and p is 1 or 2.

[0054] In some embodiments, L1comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, 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, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

[0055] In some embodiments, L1comprises a functional moiety selected from the group consisting of [*- C(O)O]p(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-, [*-NHC(O)]p(C1-6-alkylene)-, [*-C(O)NH]P(C1-6- alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6 -alkylene)-, [*-S(O)2]p(C1-6-alkylene)-, [(*- O)rC(OR25)3.r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*-C(R25)(=N-N(R26)C(O)-)]P(CI_6- alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-, [*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(OH))]p(Cw-alkylene)-, [*-OC(R25)(R26)O]P(C1-6- alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2.p, and [*-C(O)NH](CI-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6 -alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.

[0056] In some embodiments, L1further comprises at least one additional difunctional moiety via which R2is attached to either X1in formula (V) (or to the carbonyl group of any one of formulas (VI), (VII), (VIII), and (Villa)) or X2in formula (V’) (or the N atom of any one of formulas (VI’), (VII’), (VIII’), and (Villa’)). In some embodiments, the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6 -alkylene group from each other.

[0057] In some embodiments, L1is selected from the group consisting of [*-C(O)O]p(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*- OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)O-

[0058] P(O)(OR27)O(C1-6-alkylene)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*- NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)O-

[0059] P(O)(OR27)O(C1-6-alkylene)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-NR26-, [*- C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6- alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*- C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C 1-6 -alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*- C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27is selected from the group consisting of H, Cu 6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH / ); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.

[0060] In some embodiments, L1is selected from the group consisting of [*-C(O)O]p(C 1-6 -alky lene)- OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(Cw-alkylene)-OP(O)(OR27)- O(C1-6-alkylene)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(Ci-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(0)]P(CI-6- alkylene)OP(O)(OR27)O(Ci-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6- alkylene)-, [*-C(O)NH]p(C 1-6 -alky lene)OP(O)(OR27)O(C 1-6 -alkylene)NH-, [*-C(O)NH]p(C1-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6- alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*- C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*-C(O)NH]p(Ci- 6-alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH / ); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen- 6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.

[0061] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)- O(Ci.3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(Ci.3-alkylene)C(O)-,

[0062] [R4NHC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4NHC(O)]p(C2-3-alkylene)-

[0063] OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)NH]p(C2-3-alkylene)-

[0064] OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3-alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O-, or R2is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH / ); or an organic cation, e.g., a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.

[0065] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VF), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R2is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2is a monoalkylamine moiety.

[0066] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VF), (VII), (VIE), (VIII), (Villa), (Vlir), and (Villa’)), each of the one or more hydrophobic chains (i.e., each of R4) is independently a non-cyclic, preferably straight, hydrocarbyl group, e.g., the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the 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. The one or more hydrocarbyl groups may 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 said two hydrophobic chains are the same. In some alternative embodiments, said two hydrophobic chains are different, e.g., one may be saturated and the other may be (mono)unsaturated and / or said two hydrophobic chains differ in their length. Examples of the one or more hydrophobic chains include the hydrocarbyl chains of fatty acids, in particular the hydrocarbyl chains of naturally occurring fatty acids, such as the hydrocarbyl chains of naturally occurring fatty acids and having at least 8 carbon atoms. Specific examples the one or more hydrophobic chains include the hydrocarbyl chains of caprylic alcohol, capric alcohol, lauric alcohol, myristic alcohol, palmitic alcohol, stearic alcohol, arachidic alcohol, behenic alcohol, lignoceric alcohol, cerotic alcohol, oleic alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

[0067] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (VIII’), and (Villa’)), R2is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2is a monomyristylamine moiety.

[0068] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair.

[0069] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair. In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(0)NH2, -C(0)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair.

[0070] In some embodiments of the first aspect (in particular with respect to any one of formulas (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (Vlir), and (Villa’)), the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody (including fragments or derivatives thereof) specific for said 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 - avf>3 integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor.

[0071] In some embodiments of the first aspect, the polymer-conjugated compound has one of the following wherein n is 5 to 25; R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH,

[0072] -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair; R27is H or a countercation (e.g., the countercation may be the cation of pharmaceutically acceptable salts, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH / ); or an organic cation, e.g., a quaternary ammonium or amine cation); and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)I6CH3(stearoyl), in each case -C(O)Ci5H3irefers to the moiety -C(O)(CH2)i4CH3(palmitoyl), in each case -C(O)CI3H22refers to the moiety -C(O)(CH2)I2CH3(myristoyl), in each case -Ci4H29 refers to the moiety -(CH2)I3CH3(myristyl), in each case -CI3H22refers to the moiety -(CH2)I2CH3, and in each case -C(O)CI?H33refers to the moiety -czs-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl). In some embodiments, n is 7 to 16, e.g., 7 to 14 (preferably 8, 10, 12, 14, or 16); and / or R3is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, C2-6 alkynyl, an antigen, and an antibody.

[0073] Further preferred embodiments of the polymer-conjugated compound (in particular with respect to any one of formulas (I), (II), (III), (IV), (IVa), (V), (V’), (VI), (VI’), (VII), (VII’), (VIII), (Villa), (VIII’), and (Villa’)) are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the 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 formulas (V-l), (V-5), (V-17), and (V-25).

[0074] In some embodiments of the first aspect, the composition is substantially free of a lipid or lipid-like material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.

[0075] 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 conjugate comprising hydrophobic chains and a polyoxazoline (POX) and / or polyoxazine (POZ) polymer.

[0076] In some embodiments of the first aspect, water is the main component in the composition and / or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v / v), such as less than about 0.5% (v / v). For example, the amount of water contained in the composition may be at least 50% (w / w), such as 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 contained in the composition may be at least 50% (w / w), such as at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition may be at least 95% (w / w). Additionally, or alternatively, the total amount of solvent(s) other than water contained in the composition may be less than about 0.5% (v / v), such as 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 respect, a cryoprotectant which is liquid under normal conditions will not be considered as a solvent other than water but as cryoprotectant. In other words, the above optional limitation that the total amount of solvent(s) other than water contained 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 which are liquids under normal conditions.

[0077] In some embodiments of the first aspect, the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg / 1 to about 500 mg / 1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg / 1 to about 100 mg / 1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg / 1 to about 500 mg / 1, such as about 10 mg / 1 to about 400 mg / 1, about 10 mg / 1 to about 300 mg / 1, about 10 mg / 1 to about 200 mg / 1, about 10 mg / 1 to about 150 mg / 1, or about 10 mg / 1 to about 100 mg / 1, preferably about 10 mg / 1 to about 140 mg / 1, more preferably about 20 mg / 1 to about 130 mg / 1, more preferably about 30 mg / 1 to about 120 mg / 1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is about 5 mg / 1 to about 150 mg / 1, such as about 10 mg / 1 to about 140 mg / 1, about 20 mg / 1 to about 130 mg / 1, about 25 mg / 1 to about 125 mg / 1, about 30 mg / 1 to about 120 mg / 1, about 35 mg / 1 to about 115 mg / 1, about 40 mg / 1 to about 110 mg / 1, about 45 mg / 1 to about 105 mg / 1, or about 50 mg / 1 to about 100 mg / 1. In some embodiments, the concentration of the nucleic acid (in particular RNA) in the composition is 1 mg / 1 to about 50 mg / 1 or about 10 mg / 1 to about 100 mg / 1.

[0078] 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.

[0079] In some embodiments of the first aspect, the cationically ionizable lipid comprises a head group which includes at least one tertiary amine moiety.

[0080] In some embodiments of the first aspect, the cationically ionizable lipid has the structure of Formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L10, L20, G1, G2, G3, R35, R36, and R37are as defined herein. In some embodiments, the cationically ionizable lipid is selected from the following: the structures X-l to X-36 (shown herein); or the structures A to G (shown herein). In some embodiments, the cationically ionizable lipid is the lipid having the structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., the lipid having the structure G). In some embodiments of the first aspect, the cationically ionizable lipid has the structure of Formula (XI): wherein Ri, R2 R3, R4, L2, G2, and m are as defined herein. In some embodiments, the cationically ionizable lipid is selected from the structures (XIV-1), (XIV-2), and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-1 In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-2. In some embodiments, the cationically ionizable lipid is the lipid having the structure XIV-3.

[0081] In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises 2,3- dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)- N,N,N -trimethylammonium chloride (DOTAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

[0082] In some embodiments of the first aspect, the cationically ionizable lipid is completely or partially replaced by a cationic lipid. In some embodiments, the cationic lipid is selected from the structures XV- 1 to XV -6 (shown herein).

[0083] In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol of the total lipid present in the composition.

[0084] 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 a phospholipid and a steroid.

[0085] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl- phosphatidylethanolamine (DPyPE).

[0086] In some embodiments, the phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid present in the composition.

[0087] In some embodiments, the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol.

[0088] In some embodiments, the steroid comprises from about 10 mol % to about 60 mol % of the total lipid present in the composition.

[0089] In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition; the polymer-conjugated compound (amphiphilic OEG-conjugated compound) comprises from about 0.5 mol % to about 15 mol % (such as from about 2 mol % to about 6 mol % or from about 2 mol % to about 5 mol %) of the total lipid present in the composition; the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition; and the steroid comprises from about 20 mol % to about 55 mol % of the total lipid present in the composition.

[0090] 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, where a cationic lipid is present, the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is used for calculations. E.g., if the amount of cationically ionizable lipid in a composition should be from about 20 mol % to about 70 mol % and the composition should also contain a cationic lipid, then the sum of (1) the amount the cationically ionizable lipid and (2) the amount of cationic lipid is to be from about 20 mol % to about 70 mol %.

[0091] In some embodiments of the first aspect, the only lipids contained in the composition are the cationic or cationically ionizable lipid, the steroid, the neutral lipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein (i.e., the amphiphilic OEG-conjugated compound), in particular the cationic or cationically ionizable lipid, the steroid, the phospholipid, and the polymer-conjugated compound comprising the polymer of formula (I) as defined herein.

[0092] 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 the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer-conjugated compound comprising the polymer of formula (I) as defined herein. In some embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In some embodiments, the particles comprise or are LNPs. In some embodiments, the particles comprise or are liposomes. In some embodiments, the particles comprise or are LPXs. In some embodiments, the particles comprise or are mixtures of LNPs and liposomes. In some embodiments, the particles comprise or are mixtures of LNPs and LPXs. In some embodiments, the particles comprise or are mixtures of liposomes and LPXs. In some embodiments, the particles comprise or are mixtures of LNPs, liposomes, and LPXs.

[0093] In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles comprise essentially all of lipids, (in particular all of the cationic or cationically ionizable lipid, the one or more additional lipids, if present, and the and the polymer- conjugated compound comprising the polymer of formula (I) as defined herein) present in the composition.

[0094] In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles comprise at least 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acid (in particular RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85% of the nucleic acid (in particular RNA) present in the composition.

[0095] In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of the nucleic acid.

[0096] In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the nucleic acid (such as RNA) is encapsulated within or associated with the particles. In some embodiments of the first aspect, where the composition comprises particles dispersed in an aqueous phase, the particles have a size of from about 30 nm to about 500 nm. In some embodiments, the particles have a size from about 50 nm to about 150 nm.

[0097] In some embodiments of the second aspect, the nucleic acid is DNA.

[0098] In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA, (e.g., siRNA).

[0099] In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and (i) comprises a modified nucleoside in place of uridine; (ii) has a coding sequence which is codon-optimized; and / or (iii) has a coding sequence whose G / C content is increased compared to the wild-type coding sequence. In some embodiments, the modified nucleoside is selected from pseudouridine (y), N1 -methyl - pseudouridine (ml\| / ), and 5 -methyl -uridine (m5U).

[0100] In some embodiments of the first aspect, the nucleic acid is RNA (such as 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 (such as 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 preferably is an interrupted sequence of A nucleotides. In some embodiments, the 5’ cap is a capl or cap2 structure.

[0101] In some embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and encodes one or more polypeptides. In some embodiments, the one or more polypeptides are pharmaceutically active polypeptides and / or comprise an epitope for inducing an immune response against an antigen in a subject.

[0102] In some embodiments of the first aspect, 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 the immunogenic variant thereof. In some embodiments, the pathogen is a pathogen causing an infectious disease.

[0103] In some embodiments of the first aspect, the nucleic acid is inhibitory RNA (such as 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, in particular a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.

[0104] In a second aspect, the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect. It is understood that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.

[0105] In a third aspect, the present disclosure relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first or second aspect may also apply to any embodiment of the third aspect.

[0106] In a fourth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. 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 disorder in a subject, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, or third aspect may also apply to any embodiment of the fourth aspect.

[0107] In a fifth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. 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 disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is understood that any embodiment described herein in the context of the first, second, third, or fourth aspect may also apply to any embodiment of the fifth aspect.

[0108] In some embodiments of the second to fifth aspect, the subject is a mammal, such as a human. In a sixth aspect, the present disclosure provides a polymer-conjugated compound (also referred to herein as amphiphilic OEG-conjugated compound) comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains. Preferred embodiments of the polymer- conjugated compound of the sixth aspect are specified in the first aspect and are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains".

[0109] In a seventh aspect, the present disclosure provides conjugate of (a) the polymer-conjugated compound of the sixth aspect containing a member of a targeting pair; 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 (such as an antigen or epitope), or an antibody. In some embodiments of the seventh aspect, the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen. In some embodiments of the seventh aspect, the conjugate has one of the following formulas: or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, r 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen. In some embodiments of the seventh aspect, the polymer-conjugated compound containing a member of a targeting pair has the following formula: wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety - C(O)(CH2)I6CH3 (stearoyl); 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 containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen.

[0110] It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspect may also apply to any embodiment of the seventh aspect.

[0111] In an eighth aspect, the present disclosure provides a composition comprising (i) nucleic acid (such as DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the seventh aspect.

[0112] It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, or seventh aspect may also apply to any embodiment of the eighth aspect.

[0113] In a ninth aspect, the present disclosure relates to a method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a composition of the eighth aspect. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect may also apply to any embodiment of the ninth aspect.

[0114] 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 a subject a composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect may also apply to any embodiment of the tenth aspect. In an eleventh aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. 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 disorder in a subj ect, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. It is 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 apply to any embodiment of the eleventh aspect.

[0115] In a twelfth aspect, the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. 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 disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. It is 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 apply to any embodiment of the twelfth aspect.

[0116] In a thirteenth aspect, the present disclosure provides a method of transfecting cells, comprising adding a composition of the first or eighth aspect to cells; and incubating the mixture of the composition and cells for a sufficient amount of time. In some embodiments, in particular those, where the nucleic acid is DNA or RNA (such as mRNA) and encodes a pharmaceutically active protein, the mixture of the composition and cells is incubated for a time sufficient to allow the expression of the pharmaceutically active protein. In some embodiments, in particular those, where the nucleic acid is inhibitory RNA (such as siRNA) directed against a target mRNA, the mixture of the composition and cells is incubated for a time sufficient to allow the inhibition of the transcription and / or translation of the target mRNA. In some embodiments, the sufficient amount of time is at least one hour (such 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 (such as up to about 36 or up to about 24 hours).

[0117] In some embodiments of the thirteenth aspect, the method is conducted in vivo (i.e., the cells form part of an organ, a tissue and / or an organism of a subject). In some embodiments of the thirteenth aspect, the method is conducted in vitro (i.e., the cells do not form part of an organ, a tissue and / or an organism of a subject, e.g., the cells are an ex vivo cell culture). It is 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 apply to any embodiment of the thirteenth aspect.

[0118] In a fourteenth aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate of the seventh aspect or a composition of the first or eighth aspect. In some embodiments, the pharmaceutical composition further comprises one or more of pharmaceutically acceptable carriers, diluents and excipients. It is 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 apply to any embodiment of the fourteenth aspect.

[0119] In a further aspect, the present disclosure provides a kit comprising a composition of the first or eighth aspect, a polymer-conjugated compound (amphiphilic OEG-conjugated compound) of the sixth aspect, a conjugate of the seventh aspect, or a pharmaceutical composition as described herein (such as a pharmaceutical composition of the fourteenth aspect). In some embodiments, the kit is for use in therapy, such as for inducing an immune response. In some embodiments, the kit is for use in inducing an immune response against a pathogen, such as for treating or preventing an infectious disease.

[0120] In a further aspect, the present disclosure provides a method for preparing an amphiphilic OEG- conjugated compound comprising the following steps: (a) providing an intermediate compound having the formula (VII) or (VIE) as disclosed herein, wherein, for formula (VII), R2is OH, and R3is H, acetyl, or Fmoc; and, for formula (VIE), R2is H, acetyl, or Fmoc, and R3is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, in particular a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, thereby obtaining the amphiphilic OEG-conjugated compound. Preferred embodiments of this aspect are given herein under the heading "Polymer-conjugated compound comprising (a) a polymer which comprises the structure of formula (I); and (b) one or more hydrophobic chains". It is 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 apply to any embodiment of this aspect of a method for preparing an amphiphilic OEG-conjugated compound. Brief description of the Figures

[0121] Figure 1: Scheme of the anti-PEG ELISA assay used 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-NEE-pSAR) were synthesized and captured on Neutravidin-coated plates in wash buffer (IxPBS, 0.1% CHAPS) for 2 h at RT and under slow shaking. After washing with wash buffer (4x), the plates were incubated with different amounts of rabbit anti-PEG polyclonal sera (7 ng / ml, 3 ng / ml, 167 ng / ml, or 830 ng / ml) in assay buffer (IxPBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT and under slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (diluted at 1:5000) and the plates were incubated for 1 h at RT and under slow shaking. After washing with wash buffer (5x), HRP substrate was added and the fluorescence signal was measured.

[0122] Figure 2: Results of the anti-PEG ELISA assay of Figure 1 for different biotin-labeled antigens (biotin- PEG36K, biotin-capped-AEEA14, biotin-NH2-AEEA14, biotin-capped-pSAR, or biotin-NH2-pSAR).

[0123] Figure 3: Results of a further anti-PEG ELISA assay using monoclonal anti PEG IgG (A) or monoclonal anti PEG IgM (B) and different antigens (PEG36, capped-AEEA14, NH2-AEEA14). The PEG36 had a molecular weight of about 1.6 kDa. The capped AEEA14 was Ac-AEEA14.

[0124] Figure 4: Stability of pAEEA under physiological conditions. Ac-AEEA14 (A) and NH2-AEEAI4 (B) were incubated in mouse or human plasma for 0 h to 72 h.

[0125] Figure 5: Particle size and PDI of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.

[0126] Figure 6: Zeta potential of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.

[0127] Figure 7: Accessible RNA of DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0128] Figure 8: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.

[0129] Figure 9: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Figure 10: Viability of all tested formulations after transfection with DSPE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. The vability of BM_2 and other PEG containing formulations are also included. Plotted are mean values (n=3) + / - StDev.

[0130] Figure 11: Particle size and PDI of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Particle size and PDI of BM LNPs formulation are also included.

[0131] Figure 12: Zeta potential of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. Zeta potential of BM formulations is also included.

[0132] Figure 13: Accessible RNA of VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0133] Figure 14: Quantification of SI protein expression after transfection with VE-AEEA14-AC containing LNPs using HY501 as cationically ionizable lipid. (A) Variation in VE-AEEA14-AC composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function. (B) Viability of all tested formulations. The MFI and viability of BM_2 and other PEG containing formulations are also included. Plotted are mean values (n=3) + / - StDev.

[0134] Figure 15: Yield and purity of peptide intermediates. Peptide intermediates Ac-(AEEA)8-OH (A-D) and Ac-(AEEA)i4-OH (E-H) were synthesized and their yields and purities were determined using UPLC (A, B, E, F) and mass spectrometry (C, D, G, H). Samples for UPLC and mass spectrometry were taken either directly after cleavage from the resin, i.e., prior to the QC method (crude purities; Figures 15 A, C, E, G), of after the QC method (final purities; Figures 15 B, D, F, H).

[0135] Figure 16: Monitoring of the synthesis of Ac-(AEEA)8-a-tocopherol. Samples of the reaction were taken and analyzed using the methods described herein. UPLC chromatograms are shown for samples taken (A) after 5 minutes and (B) at the end of the reaction time (4 h).

[0136] Figure 17: Purity of the amphiphilic compounds Ac-(AEEA)8-a-tocopherol (A, B), Ac-(AEEA)i4-a- tocopherol (C, D), AC-(AEEA)I4-DMA (E, F), Ac-(AEEA)8-DMG (G, H), and AC-(AEEA)I4-DSPE (I, J) as shown by exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), respectively. Figure 18: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Particle size and PDI of BM LNPs formulation are also included.

[0137] Figure 19: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). Zeta potential of BM formulations is also included.

[0138] Figure 20: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0139] Figure 21: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.

[0140] Figure 22: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0141] Figure 23: Quantification of SI protein expression after transfection with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid). (A) Variation in composition vs. MFI (Mean Fluorescence Intensity) of the overall cell population. Data was fitted using a quadratic polynomial function. (B) Viability of all tested formulations. The MFI and viability of BM_1 and BM_2 and other control formulations are also included. Plotted are mean values (n=3) + / - StDev.

[0142] Figure 24: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0143] Figure 25: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0144] Figure 26: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0145] Figure 27: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 27A, B), a murine macrophage cell line (Raw) (Figures 27C, D), and a hepatocarcinoma cell line (HepG2) (Figures 27E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) + / - StDev.

[0146] Figure 28: T-cell targeting using LNPs with different stealth lipids and Alfa lipids. LNPs were formulated with different lipid compositions (Cargo: Thy 1.1 RNA / Luc RNA / Np proxy Venus 1: 1:2 w / w; N / P ratio: 6; Lipid mix: HY501 / Cholesterol / DSPC / stealth lipid / Alfa lipid. The lipid ratio has been selected as [47.5 / 40.5 / 10 / 1.8 / 0.2] for the following combinations of stealth lipid and Alfa lipid: C16 PEG2k Ceramide / DSPE PEG2k Alfa, DSPE PEG2k / DSPE PEG2k Alfa, DSPE-AEEA14 / DSPE- AEEA14-Alfa or VE-AEEA8 / DSPE-AEEA14-Alfa. The lipid ratio has been selected as [47.5 / 38.5 / 10 / 3.8 / 0.2] for the following combinations of stealth lipid and Alfa lipid: VE-PEGlk / DSPE- PEG2k-Alfa or VE-AEEA8 / DSPE-AEEA14-Alfa. The LNPs were equipped with aCD3 VHH X NbAlfa ligand via post functionalization [w / w* = ligand to cargo ratio 0.48]; RNA concentration: 0.1 pg / pl). Diameter of all LNPs is between 100 to 170 nm with a PDI below 0.4 as determined via DLS measurement.

[0147] For transfection studies, 10 pl (1000 ng dose) of respective formulations were pre-diluted in 50 pl X- Vivo 15 in an ultra-low adhesion 96 well plate. 106thawed human PBMC were diluted in 50 pl of 100% clotted PHS and added to nanoparticle dilution. After 30 min of incubation (37°C, 5% CO2) 30 pl of each transfection reaction was transferred to second ultra-low adhesion 96 well plate and 170 pl of X- Vivo 15 medium + 100 U / ml IL2 was added per well. Cell dilutions were cultivated for additional 18 h (37°C, 5% CO2). In the following cell-type specific transfection (Thy 1.1) was analyzed via flowcytometry. Depicted are the percentages of transfected cell (CD2 negative cells, CD 19+ B cells, CD4+ T cells and CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation condition.

[0148] Figure 29: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0149] Figure 30: Zeta potential of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0150] Figure 31: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B). Figure 32: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.

[0151] Figure 33: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0152] Figure 34: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 34A, B), a murine macrophage cell line (Raw) (Figures 34C, D), and a hepatocarcinoma cell line (HepG2) (Figures 34E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) + / - StDev.

[0153] Figure 35: Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).

[0154] Figure 36: Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).

[0155] Figure 37: Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0156] Figure 38: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.

[0157] Figure 39: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds and HY501 (as cationically ionizable lipid).

[0158] Figure 40: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic VE-(AEEA)n-AC-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 40A, B), a murine macrophage cell line (Raw) (Figures 40C, D), and a hepatocarcinoma cell line (HepG2) (Figures 40E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA- loaded LNPs. Plotted are mean values (n=3) + / - StDev. Figure 41: Particle size and PDI of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0159] Figure 42: Accessible RNA of LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0160] Figure 43: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS.

[0161] Figure 44: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing various amphiphilic OEG-conjugated compounds and HY501 (as cationically ionizable lipid).

[0162] Figure 45: In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG-conjugated compounds in skeletal muscle cell line (C2C12) (Figures 45A, B), a murine macrophage cell line (Raw) (Figures 45C, D), and a hepatocarcinoma cell line (HepG2) (Figures 45E, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) + / - StDev.

[0163] Figure 46: Particle size and PDI of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

[0164] Figure 47: Zeta potential of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

[0165] Figure 48: Accessible RNA of LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) measured via RiboGreen Assay (A) or agarose gel electrophoresis (B).

[0166] Figure 49: Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and control items. The horizontal dashed line shows the level of SC5b-9 formation for PBS. Figure 50: Hemolysis analysis after incubation (in neutral pH condition) of whole human blood with LNPs containing an amphiphilic OEG-conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

[0167] Figure 51 : In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing an amphiphilic OEG- conjugated compound and various cationical or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) in skeletal muscle cell line (C2C12) (Figures 51A, B), a murine macrophage cell line (Raw) (Figures 51C, D), and a hepatocarcinoma cell line (HepG2) (Figures 5 IE, F). Firefly luciferase expression 24h post-incubation with 12.5, 25 and 50 ng per well of mRNA-loaded LNPs. Plotted are mean values (n=3) + / - StDev.

[0168] Figure 52: Particle size and PDI of functionalized LNPs prepared with 5 different OEG-conjugated compounds as stealth lipids.

[0169] Figure 53: Agarose gel electrophoresis of controls, untreated functionalized LNPs (upper row) and functionalized LNPs treated with release solution (lower row).

[0170] Figure 54: Particle size and PDI of functionalized LNPs prepared respectively 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.

[0171] Figure 55: In vitro transfection with LNPs. A) The percentages of transfected cells (CD 14+ Monocytes, CD 19+ B cells, CD4+ T cells or CD8+ T cells) within all transfected PBMCs (Transfection, y-axes) per tested formulation. B) Total cell counts acquired by flow cytometry within 20 seconds. Numbers are corrected relative to counting beads. BD FACS Lyric was used for sample acquisition. FlowJo was used for data analysis.

[0172] Figure 56: Ligand-mediated transfection of T cells in vivo. Intramuscular injection of naked or LNP- formulated luciferase- and Thy 1.1 -encoding RNA mixtures (1 : 1 weight-to-weight mix) in B6- hCD3EDG transgeneic mice (1 pg RNA dose per injection side; 2 pg total RNA dose per mouse). Analysis performed 18h after injection. (A) Drainage analyzed via ex vivo bioluminescence imaging of popliteal, inguinal, axially and brachial lymph nodes, and spleens. (B) Cell-type specific transfection analyzed by flow-cytometry via detection of delivered Thy 1. 1 RNA expression in immune cell subtypes within the popliteal and inguinal lymph nodes, and spleens. (C) T-cell activation status analyzed via mean fluorescent intensity of CD69 surface expression within depicted organ (following staining with anti-CD69-APC antibody). LN, lymph nodes; LNP, lipid nanoparticle; NK, natural killer; PMN, polymorphonuclear cells.

[0173] Description of the sequences

[0174] The following table provides a listing of certain sequences referenced herein.

[0175] Table 1 : Description of the sequences

[0176] Detailed Description

[0177] Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0178] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0179] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0180] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0181] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0182] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0183] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0184] Definitions

[0185] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0186] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of' means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of'. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of' or "consisting of'. Likewise, at each occurrence in the present application, the term "consisting essentially of' may be replaced with the term "consisting of'.

[0187] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0188] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein. In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±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 for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0189] Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.

[0190] Terms such as "enhance" and "increase" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. In some embodiments, these terms relate to an increase or enhancement by 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%.

[0191] "Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0192] "Physiological conditions" as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and / or a temperature of about 37°C.

[0193] As used in the present disclosure, "% (w / v)" (or "% w / v") refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (ml).

[0194] As used in the present disclosure, "% by 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 percent of the total volume of solution in milliliters (ml). As used in the present disclosure, "% by 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 percent of the total weight of the total composition in grams (g).

[0195] As used in the present disclosure, "mol %" 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.

[0196] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like material.

[0197] The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum E is taken over all the different kinds of ions (i) in solution.

[0198] According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions.

[0199] Regarding the presence of divalent inorganic ions, in particular divalent inorganic cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is in one embodiment sufficiently low so as to prevent degradation of the RNA. In one embodiment, the concentration or effective concentration of divalent inorganic ions is below the catalytic level for hydrolysis of the phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent inorganic ions is 20 pM or less. In one embodiment, there are no or essentially no free divalent inorganic ions.

[0200] A "monovalent" compound relates to a compound having only one functional group of interest. For example, a monovalent anion relates to a compound having only one negatively charged group, preferably under physiological conditions. A "divalent" or "dibasic" compound relates 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 Ca2+.

[0201] A "polyvalent" or "polybasic" compound relates to a compound having three or more functional groups of interest. For example, a polybasic organic acid has three or more acid groups.

[0202] A "monovalent moiety" relates to a monoradical, i.e., a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, etc.

[0203] A "divalent moiety" or "bivalent moiety" relates to a diradical, i.e., a moiety having a valence of 2. Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, etc. A further example of a divalent moiety is the C1-6-alkylene moiety in the group [* -S]p(C1-6 -alkylene)-, if p is 1 (resulting in the group *-S(C1-6-alkylene)-, such as *-S-(CH2)g- or *-S-CH2-, wherein * represents the attachment point to R4).

[0204] A "polyvalent moiety" relates to a polyradical, i.e., a moiety having a valence of at least 3. E.g., a "trivalent moiety" relates to a triradical, i. e., a moiety having a valence of 3. For example, by removing a further H atom of an alkylene group the resulting alkylene is trivalent. A further example of a trivalent moiety is the C1-6-alkylene moiety in the group [*-S]p(C1-6-alkylene)-, if p is 2 (resulting in the group [*-S]2(C1-6-alkylene)-, such as *-S-CH(S-*)(CH2)s- or *-S-CH(S-*)(CH2)-, wherein * represents the attachment point to R4). Another example of a trivalent moiety is the C1-6 -alkyltriyl moiety in the group [*-C(O)NH](C1-6-alkyltriyl)- (here the [*-C(O)NH] moiety as well as a further hydrophobic chain are bound to the C1-6 -alkyltriyl moiety which, in turn, is attached to X1(for formula (V)) or to X2(for formula (V’)), either directly or through at least one additional difunctional moiety). Thus, a [*-C(O)NH](C1-6- alkyltriyl) moiety as an example for L1, wherein C1-6 -alkyltriyl is directly attached to another hydrophobic chain R4, may comprise at least the following structures: wherein one of ww represents the bond by which the C1-6 -alkyltriyl is attached to [*-C(O)NH] and the other ww represents the bond by which the C1-6-alkyltriyl is attached (either directly or via an additional difunctional moiety) to the remainder of the compound (e.g., the compound of formula (V)). In case the C1-6 -alkyltriyl is also substituted with one -OH moiety, at least the following structures are encompassed: "Molar ratio", as used herein, refers to the ratio between the amounts in moles 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 millimole (mmol), the molar ratio of the first substance to the second substance is 1:2 or 0.5.

[0205] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0206] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably.

[0207] The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0208] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0209] The term "freezing" relates to the solidification of a liquid, usually with the removal of heat. In some embodiments, freezing is reverse action to thawing.

[0210] The term "thawing" relates to the liquification of a solid, usually with the addition of heat. In some embodiments, thawing is reverse action to freezing.

[0211] The term "aqueous phase" as used herein in relation to a composition / formulation comprising particles, in particular LNPs, liposomes, and / or lipoplexes, means the mobile or liquid phase, i.e., the continuous water phase including all components dissolved therein but (formally) excluding the particles. Thus, if particles, such as LNPs, are dispersed in an aqueous phase and the aqueous phase is to be substantially free of compound X, the aqueous phase is free of X is such manner as it is practically and realistically feasible, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, it is possible that, at the same time, the particles dispersed in the aqueous phase may comprise compound X in an amount of more than 1% by weight. The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0212] The term "naturally occurring" as used herein 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 viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source.

[0213] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, preferably from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[0214] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, abbreviated as C1-12 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C1-10 alkyl), 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, iso-propyl (also called 2-propyl or 1- methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n- decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0215] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. 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), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2- butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1 -pentylene, 1,2-pentylene, 1,3- pentylene, 1,4-pentylene, 1,5 -pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the 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), the 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), the 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), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0216] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is 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 uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 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 comprises from 2 to 12, abbreviated as C2-12 alkenyl, (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, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1- propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5 -hexenyl, 1 -heptenyl, 2-heptenyl, 3 -heptenyl, 4- heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7- octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1- decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1- undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8- undecenyl, 9-undecenyl, 10-undecenyl, 1 -dodecenyl, 2-dodecenyl, 3 -dodecenyl, 4-dodecenyl, 5- dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0217] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is 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 uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carboncarbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0218] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is 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 uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 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 from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2,

[0219] 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen- 1,2-diyl, vinylidene (also called ethenylidene), 1 -propen- 1,2-diyl, 1 -propen- 1,3 -diyl, 1 -propen-2,3 -diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, 1- buten-3,4-diyl, 2-buten- 1,2-diyl, 2-buten-l,3-diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0220] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with 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 (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to

[0221] 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0222] The term "cycloalkylene" represents cyclic non-aromatic versions of "alkylene" and is a geminal, vicinal or isolated diradical. In certain embodiments, the cycloalkylene (i) is monocyclic or polycyclic (such as bi- 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, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkylene is a mono-, bi- or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkylene. Generally, instead of using the ending "ylene" for cycloalkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-cyclopropylene can also be called cyclopropan-l,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3. 1. l3’7]decan-2,2- diyl). A "substituted cycloalkylene " means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0223] The term "cycloalkenylene" represents cyclic non-aromatic versions of "alkenylene" and is a geminal, vicinal or isolated diradical. Generally, the maximal number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer which is 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 uneven, rounding the result of the division down to the next integer. For example, for an cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. In certain embodiments, the cycloalkenylene (i) is monocyclic or polycyclic (such as bi- 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, such as 3- to 12-membered or 3- to 10-membered). 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. Exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. A "substituted cycloalkenylene " means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0224] The term "aryl" refers to a monoradical 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 can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein. Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4-methoxyphenyl).

[0225] 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 heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. 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 fiiranyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0226] The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 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 completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein.

[0227] The term "heterocycloalkylene" as used herein means a heterocyclyl group as defined above which contains at least one ring heteroatom (such as those selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed resulting in a geminal, vicinal or isolated diradical. In some embodiments, the heteroatoms of the heterocycloalkylene group are selected from the group consisting of O, S, and N. For example, the heterocycloalkylene may be O / S-heterocycloalkylene, such as O-heterocycloalkylene. In some embodiments, in each ring of the heterocycloalkylene group the maximum number of O atoms is 1 , the maximum number of S atoms is 1 , and the maximum total number of O and S atoms is 2. The heterocycloalkylene may be monocyclic or polycyclic (such as bi- or tricyclic). In some embodiments, the heterocycloalkylene is a mono-, bi- or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 4- to 12-membered or 4- to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups (preferably partially or completely 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 resulting in a geminal diradical. Thus, in some embodiments, a heterocycloalkylene is saturated or unsaturated (i.e., it contains one or more double bonds within the ring) but cannot be aromatic. A "substituted heterocycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene 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 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as specified herein. The expression "partially hydrogenated form" of an unsaturated compound or group as used herein means that part of the unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group without removing all unsaturated moieties. The phrase "completely hydrogenated form" of an unsaturated compound or group is used herein interchangeably with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group. For example, partially hydrogenated forms of a 5-membered heteroaryl group (containing 2 double bonds in the ring, such as furan) include dihydro forms of said 5- membered heteroaryl group (such as 2,3 -dihydrofuran or 2,5 -dihydrofuran), whereas the tetrahydro form of said 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a completely hydrogenated (or perhydro) form of said 5 -membered heteroaryl group. Likewise, for a 6-membered heteroaryl group having 3 double bonds in the ring (such as pyridyl), partially hydrogenated forms include di- and tetrahydro forms (such as di- and tetrahydropyridyl), whereas the hexahydro form (such as piperidinyl in case of the heteroaryl pyridyl) is the completely hydrogenated (or perhydro) derivative of said 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl can only be considered a partially hydrogenated form according to the present disclosure if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms.

[0228] The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic). A similar distinction is made within the present application between heteroaryl and heterocyclyl. For example, indolinyl, i.e., a dihydro variant of indolyl, is classified as heterocyclyl for the purposes of the present disclosure, since only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0229] The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are C1-30 alkyl (such as Cg-30 alkyl, Cx-24 alkyl, or C 10-20 alkyl), C2-30 alkenyl (such as Cg-30 alkenyl, Cx-24 alkenyl, or C 10-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(C1-6 alkyl). In some embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more 1stlevel substituents, one or more 2ndlevel substituents, or one or more 3rdlevel substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively nonpolar. In some embodiments, the hydrocarbyl group is the hydrocarbyl chain of naturally occurring fatty acids and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) C8-20 alkyl chain, e.g., a (preferably linear) Cio-is alkyl chain).

[0230] The term "optionally substituted" indicates that one or more (such as 1 to the maximum number of hydrogen atoms bound to a group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atom(s) may be replaced with a group (i.e., a 1stlevel substituent) different from hydrogen such as alkyl (preferably, C1-6 alkyl), alkenyl (preferably, C2-6 alkenyl), alkynyl (preferably, C2-6 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, -OR71, -N(R72)(R73), -S(0)o-2R71, -S(O)I.2OR71, -OS(O)I.2R71, -OS(O)I.2OR71, -S(O)I.2N(R72)(R73), -OS(O)I.2N(R72)(R73),

[0231] -N(R71)S(O)I-2R71, -NR71S(O)I-2OR71, -NR71S(O)I-2N(R72)(R73), -OP(O)(OR71)2, -C(=X1)R71, -C(=XI)XIR71, -XiC(=Xi)R71, and -XiC(=Xi)X1R71, and / or any two 1stlevel substituents which are bound to the same carbon atom of a cycloalkyl or heterocyclyl group may join together to form =Xi, wherein each of the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups of the 1stlevel substituent may themselves be substituted by one or more (e.g., one, two or three) substituents (i.e., a 2ndlevel substituent) selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 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, -OR81, -N(R82)(R83), -S(0)o-2R81, -S(O)I-2OR81, -OS(O)I-2R81, -OS(O)I-2OR81, -S(O)I-2N(R82)(R83), -OS(O)I-2N(R82)(R83),

[0232] -N(R81)S(O)I-2R81, -NR81S(O)I-2OR81, -NR81S(O)I-2N(R82)(R83), -OP(O)(OR81)2, -C(=X2)R81, -C(=X2)X2R81, -X2C(=X2)R81, and -X2C(=X2)X2R81, and / or any two 2ndlevel substituents which are bound to the same carbon atom of a cycloalkyl or heterocyclyl group being a 1stlevel substituent may join together to form =X2, wherein each of the C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl groups of the 2ndlevel substituent is optionally substituted with one or more (e.g., one, two or three) substituents (i.e., a 3rdlevel substituent) independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(CI.3 alkyl)2, -NHS(O)2(CI_3 alkyl), -S(O)2NH2.Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=O)(C1-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl) C(=NH)NH2-Z(C 1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl, and / or any two 3rdlevel substituents which are bound to the same carbon atom of a 3- to 14-membered cycloalkyl or heterocyclyl group being a 2ndlevel substituent may join together to form =0, =S, =NH, or =N(CI-3 alkyl); wherein each of R71, R72, and R73is independently selected from the group consisting of H, CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl, wherein each of the CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 7- membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, =0, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=0)(CM alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; each of R81, R82, and R83is independently selected from the group consisting of H, CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 3- to 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl, wherein each of the CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 3- to 6- membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -OCF3, =0, -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=0)(CM alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; and each of Xi and X2 is independently selected from O, S, and N(R84), wherein R84is H or C1-3 alkyl.

[0233] Typical 1stlevel substituents are preferably selected from the group consisting of CM alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR71, -N(R72)(R73), -S(0)o-2R71, -S(O)I.2OR71, -OS(O)I.2R71, -OS(O)I.2OR71, -S(O)I.2N(R72)(R73), -OS(O)I.2N(R72)(R73),

[0234] -N(R71)S(O)I.2R71, -NR71S(O)I.2OR71, -C(=XI)R71, -C(=XI)XIR71, -XIC(=XI)R71, and -XiC(=Xi)XiR71, such as CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7- membered cycloalkyl, 3- to 7-membered (such as 5- or 6-membered) heterocyclyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CI-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl; wherein Xi is independently selected from O, S, NH and N(CHs); and each of R71, R72, and R73is as defined above or, preferably, is independently selected from the group consisting of H, CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=O)(C1-3 alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. In some embodiments, 1stlevel substituents are selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -0CH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and Ci. 3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, 1stlevel substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.

[0235] Typical 2ndlevel substituents are preferably selected from the group consisting of CM alkyl, C2-4 alkenyl, C2-4 alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =0, =S, -CF3, -CN, azido, -NO2, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -S(O)2NH2-Z(C1-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CM alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. Particular examples of 2ndlevel substituents are independently selected from the group consisting of C1-3 alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =0, =S, -CF3, -CN, -OH, -O(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), -C(=0)0H, -C(=O)O(C1-3 alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(CM alkyl), -NHC(=NH)NHZ.2(CI-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. Particularly preferred 2ndlevel substituents are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, =0, and =S.

[0236] Typical 3rdlevel substituents are preferably selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=0)0H, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred 3rdlevel substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3. The term "tertiary amine moiety" as used herein relates to a moiety containing a nitrogen atom which is 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 (such as alkyl groups, in particular C1-6 alkyl groups) which are optionally substituted (e.g., with one or more 1stlevel substituents, one or more 2ndlevel substituents, or one or more 3rdlevel substituents as defined herein).

[0237] The term "filtrating" as used herein relates to any process that involves removal or separation of at least one component (such as permeable molecules like salts, small proteins, solvents etc.,) of a liquid composition based on the molecular size of the components contained in the composition. This separation may use micro-molecule permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis). Thus, examples of filtrating comprise dialyzing, tangential flow filtrating and diafiltrating.

[0238] The expression "substantially free of X", as used herein, means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of X in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture.

[0239] For example, "substantially free of a lipid comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units" as used herein, means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of a lipid comprising at least 30 consecutive ethylene glycol repeating units in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of a lipid comprising at least 30 consecutive ethylene glycol repeating units, the amount of a lipid comprising at least 30 consecutive ethylene glycol repeating units in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Similar considerations apply to expressions containing the phrase "substantially free of (such as "substantially free of a sarcosinylated lipid", "substantially free of a (POX) -conjugated and / or polyoxazine (POZ)-conjugated lipid ", and "substantially free of any polymer- conjugated lipid other than the amphiphilic OEG-conjugated compound").

[0240] The expression "nucleic acid integrity" means the percentage of the full-length (i.e., non-fragmented) nucleic acid to the total amount of nucleic acid (i.e., non-fragmented plus fragmented nucleic acid) contained in a sample. The nucleic acid integrity may be determined by chromatographically separating the nucleic acid (e.g., using capillary electrophoresis), determining the peak area of the main nucleic acid peak (i. e. , the peak area of the full-length (z. e., non-fragmented) nucleic acid), determining the peak area of the total nucleic acid, and dividing the peak area of the main nucleic acid peak by the peak area of the total nucleic acid. Likewise, the expression "RNA integrity" means the percentage of the full- length (i.e., non-fragmented) RNA to the total amount of RNA (i.e., non-fragmented plus fragmented RNA) contained in a sample. The RNA integrity may be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of the main RNA peak (i.e., the peak area of the full-length (i. e . , non-fragmented) 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.

[0241] The term "cryoprotectant" relates to a substance that is added to a preparation (e.g., formulation or composition) in order to protect the active ingredients of the preparation during the freezing stages.

[0242] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0243] According to the present disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" or "polypeptide " refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide", "polypeptide", and "protein" are used herein usually as synonyms.

[0244] A "therapeutic peptide or protein" has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In some embodiments, a therapeutic peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term "therapeutic peptide or protein" includes entire peptides or proteins, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a peptide or protein. Examples of therapeutically active peptides or proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines. The terms "therapeutic peptide or protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

[0245] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.

[0246] The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of the composition. For example, a part of a composition may any portion from 0. 1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0247] "Fragment", with reference to an amino acid sequence (peptide, polypeptide or protein), relates to apart of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular 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 an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of 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 the amino acid sequence.

[0248] 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 has been derived. Such functional properties comprise a pharmacological activity, the interaction with other peptides, polypeptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids. E.g., a pharmacological active fragment of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the fragment has been derived. A part or fragment of a peptide, polypeptide or protein preferably comprises a sequence of at least 6, in particular 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, 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 the peptide or protein.

[0249] "Variant", as used herein and with reference to an amino acid sequence (peptide, polypeptide, or protein), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of 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 version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, and preferably from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.

[0250] By "wild type" or "WT" or "native" with respect to an amino acid sequence is meant an amino acid sequence that is 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. Likewise by "wild type" or "WT" or "native" with respect to a nucleic acid sequence is meant a nucleic acid sequence that is found in nature, including allelic variations. For example, a wild type coding sequence is meant to be a coding sequence that is found in nature and that has not been intentionally modified.

[0251] A "coding sequence", as sued herein means the portion of a nucleic acid (e.g., a gene's DNA or RNA) that codes for protein.

[0252] The expression "guanosine / cytosine (G / C) content" or "G / C content" means the percentage of bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). The G / C content may be given for a specific portion of DNA or RNA or for an entire genome. When the G / C content refers to a portion, it may denote the G / C content of an individual gene or portion of a gene (domain), a group of genes or gene clusters, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0253] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise 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, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0254] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy -terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:

[0255] - glycine, alanine;

[0256] - valine, isoleucine, leucine;

[0257] - aspartic acid, glutamic acid;

[0258] - asparagine, glutamine;

[0259] - serine, threonine;

[0260] - lysine, arginine; and

[0261] - phenylalanine, tyrosine.

[0262] In some embodiments, the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said 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 given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably 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 continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0263] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0264] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States 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 used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0265] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is 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 continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0266] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0267] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0268] 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 exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response (immunogenic fragment). In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0269] An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0270] In some embodiments, "isolated" means altered or removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In some embodiments, the RNA (such as mRNA) used in the present disclosure is in substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of RNA (such as mRNA) in substantially purified form contains a first buffer system.

[0271] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid.

[0272] The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro (e.g., in cell culture) or in vivo, e.g., the cell can form part of an organ, a tissue and / or an organism of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

[0273] The disclosure includes analogs of a peptide, polypeptide or protein. According to the present disclosure, an analog of a peptide, polypeptide or protein is a modified form of said peptide, polypeptide or protein from which it has been derived and has at least one functional property of said peptide, polypeptide or protein. E.g., a pharmacological active analog of a peptide, polypeptide or protein has at least one of the pharmacological activities of the peptide, polypeptide or protein from which the analog has been derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and / or additions of any molecules associated with the protein, polypeptide or peptide, such as carbohydrates, lipids and / or proteins or peptides. In one embodiment, "analogs" of proteins, polypeptides or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective / blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term "analog" also extends to all functional chemical equivalents of said proteins, polypeptides and peptides.

[0274] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0275] According to various embodiments of the present disclosure, a nucleic acid such as RNA (e.g., mRNA) encoding a peptide, polypeptide or protein is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide, polypeptide or protein. 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 it on the surface.

[0276] According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide, polypeptide or protein mean that the nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce said peptide, polypeptide or protein.

[0277] "Activation" or "stimulation", as used herein, refers to the state of a cell (e.g., an immune effector cell such as T cell) that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.

[0278] The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

[0279] The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In some embodiments, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. In some embodiments, expansion leads to differentiation of the immune effector cells.

[0280] An "antigen" according to the present disclosure covers any substance that will elicit an immune response and / or any substance against which an immune response or an immune mechanism such as a cellular response and / or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells). According to the present disclosure, the term "antigen" may comprise any molecule which comprises at least one epitope, such as a T cell epitope. In some embodiments, an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen). In some embodiments, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.

[0281] According to the present disclosure, any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may be a humoral or cellular immune response or both. In the context of some embodiments of the present disclosure, the antigen is presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.

[0282] 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 which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0283] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, in particular via the modulation of the activity of a T-cell receptor.

[0284] The term "tumor antigen" or "tumor-associated antigen" refers to a constituent of cancer cells which may be derived from the cytoplasm, the cell surface or the cell nucleus. In particular, it refers to those antigens which are produced intracellularly or as surface antigens on tumor cells. For example, tumor antigens include the carcinoembryonal antigen, al -fetoprotein, isoferritin, and fetal sulphoglycoprotein, a2-H-ferroprotein and y-fetoprotein, as well as various virus tumor antigens. According to some embodiments of the present disclosure, a tumor antigen comprises any antigen which is characteristic for tumors or cancers as well as for tumor or cancer cells with respect to type and / or expression level.

[0285] The term "viral antigen" refers to any viral component having antigenic properties, i.e., being able to provoke an immune response in an individual. The viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0286] The term "bacterial antigen" refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of the bacterium.

[0287] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies T cells or B cells, in particular when presented in the context of MHC molecules. An epitope of a protein may comprise a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 0, between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 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. Terms such as "epitope", "fragment of an antigen", "immunogenic peptide" and "antigen peptide" are used interchangeably herein and, e.g., may relate to an incomplete representation of an antigen which is, e.g., capable of eliciting an immune response against the antigen or a cell expressing or comprising and presenting the antigen. In some embodiments, the terms relate to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 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, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0288] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both selfantigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

[0289] The peptide and protein antigen 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 length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0290] The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein. In some embodiments, vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. In some embodiments, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell.

[0291] In some embodiments, an antigen is expressed in a diseased cell (such as tumor cell or an infected cell).

[0292] In some embodiments, an antigen is presented by a diseased cell (such as tumor cell or an infected cell). In some embodiments, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In some embodiments, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In some embodiments, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.

[0293] In some embodiments, an antigen is expressed on the surface of a diseased cell (such as tumor cell or an infected cell). In some embodiments, an antigen receptor is a CAR which binds to an extracellular domain or to an epitope in an extracellular domain of an antigen. In some embodiments, a CAR binds to native epitopes of an antigen present on the surface of living cells. In some embodiments, binding of a CAR when expressed by T cells and / or present on T cells to an antigen present on cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In some embodiments, binding of a CAR when expressed by T cells and / or present on T cells to an antigen present on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.

[0294] In some embodiments, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In some embodiments, an antibody or B cell receptor binds to native epitopes of an antigen.

[0295] The term "expressed on the cell surface" 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 part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part may be, e.g., at least 4, at least 8, at least 12, or at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, 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 surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0296] "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by, e.g., antigen-specific antibodies added to the cells. In some embodiments, an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion which may be recognized by a CAR.

[0297] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. In some embodiments, the term refers to one or more extracellular loops or domains or a fragment thereof.

[0298] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells. The term "antigen-specific T cell" or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular 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 effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured. In certain embodiments of the present disclosure, the RNA (in particular mRNA) encodes at least one epitope.

[0299] The term "target" shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell expressing an antigen and preferably presenting said antigen with class I MHC.

[0300] "Antigen processing" refers to the degradation of an antigen into processing products which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T-cells. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.

[0301] The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.

[0302] The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.

[0303] The term "dendritic cell" (DC) refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells. 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 for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.

[0304] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, 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 splenic macrophages.

[0305] By "antigen-responsive CTL" is meant a CD8+T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.

[0306] According to the disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-y and TNF-a, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.

[0307] The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4+and CD8+T-lymphocytes, e.g., CD8+T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.

[0308] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure 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 may be protective / preventive / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigen peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV or RSV)). "Inducing" in this context may mean that there was no immune response against a particular antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced. Thus, "inducing the immune response" in this context also includes "enhancing the immune response". In some embodiments, after inducing an immune response in an individual, said individual is protected from developing a disease such as an infectious disease or a cancerous disease or the disease condition is ameliorated by inducing an immune response. The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen and / or presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes which act as either "helpers" or "killers". The helper T cells (also termed CD4+T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+T cells or CTLs) kill cells such as diseased cells.

[0309] The term "humoral immune response" refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and / or eliminate. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind antigen of a single specificity. Following binding of an appropriate antigen and receipt of various other activating signals, B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen.

[0310] The term "antibody" as used herein, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which is able to specifically bind to an epitope on an antigen under typical physiological conditions, preferably with a half-life of significant periods of time, 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, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or time sufficient for the antibody to recruit an effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3, the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

[0311] The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms "binding region" and "antigen-binding region" are used herein interchangeably and refer to the region which interacts with the antigen and comprises both a VH region and a VL region. An antibody as used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.

[0312] As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. 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 CHI domains, or a monovalent antibody as described in WO 2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a 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 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(l 1):484- 90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l): 111-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al. , Science 242. 423-426 (1988) and Huston et al. , PNAS USA 85. 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0313] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, such as to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain, and the CH (constant heavy chain) domains CHI, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e., a, 8, a, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

[0314] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s). By "cell characterized by presentation of an antigen" or "cell presenting an antigen" or "MHC molecules which present 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 presenting the antigen or an antigen peptide, either directly or following processing, in the context of MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0315] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) may be translated into peptide, polypeptide or protein.

[0316] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0317] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0318] In the context of the present disclosure, the term "RNA encodes" means that the RNA, if present in the appropriate environment, such as within cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the process of translation.

[0319] The term "serum" as used herein means the fluid resulting from the removal of cells and clotting factors from whole blood, such as whole blood obtained from humans or mice. In some embodiments, serum is human serum or mouse serum.

[0320] A medical preparation, in particular kit, described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions of the present disclosure or be shipped together with a container which contains the compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient. The term "optional" or "optionally" as used herein means 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 in which it does not occur.

[0321] Prodrugs of a particular compound described herein are those compounds that upon administration to an individual undergo chemical conversion under physiological conditions to provide the particular compound. Additionally, prodrugs can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the particular compound when, for example, placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Exemplary prodrugs are esters (using an alcohol or a carboxy group contained in the particular compound) or amides (using an amino or a carboxy group contained in the particular compound) which are hydrolyzable in vivo. Specifically, any amino group which is contained in the particular compound and which bears at least one hydrogen atom can be converted into a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0322] In the present specification, a structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present disclosure includes all isomers such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures and is not limited to the description of the formula. Furthermore, in the present specification, a structural formula of a compound may represent a specific salt and / or solvate of said compound. It is to be understood, however, that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates) and is not limited to the description of the specific salt and / or solvate.

[0323] "Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical (spatial) positioning of the functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers which are non-superimposable mirrorimages of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is denoted by the prefix (±). "Diastereomers" are stereoisomers which are non- superimposable and which are not mirror-images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; i.e., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol- tautomerism. "Conformers" are stereoisomers that can be interconverted just by rotations about formally single bonds, and include - in particular - those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane. The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A "hydrate" is a solvate wherein the solvent is water.

[0324] In isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes which can be used in the present disclosure include deuterium, tritium,nC,13C,14C,15N,18F,32P,32S,35S,36C1, and125I.

[0325] The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage-

[0326] In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0327] The "radius of gyration" (abbreviated herein as Rg) of a particle about an axis of rotation is the radial distance of a point from the axis of rotation at which, if the whole mass of the particle is assumed to be concentrated, its moment of inertia about the given axis would be the same as with its actual distribution of mass. Mathematically, Rgis the root mean square distance of the particle's components from either its center of mass or a given axis. For example, for a macromolecule composed of n mass elements, of masses m, (i = 1, 2, 3, ... , n), located at fixed distances s, from the center of mass, Rgis the square-root of the mass average of Si2over all mass elements and can be calculated as follows:

[0328] The radius of gyration can be determined or calculated experimentally, e.g., by using light scattering. In particular, for small scattering vectors q the structure function S is defined as follows: wherein N is the number of components (Guinier's law). The "DIO value", in particular regarding a quantitative size distribution of particles, is the diameter at which 10% of the particles have a diameter less than this value. The DIO value is a means to describe the proportion of the smallest particles within a population of particles (such as within a particle peak obtained from a field-flow fractionation).

[0329] "D50 value", in particular regarding a quantitative size distribution of particles, is the diameter at which 50% of the particles have a diameter less than this value. The D50 value is a means to describe the mean particle size of a population of particles (such as within a particle peak obtained from a field-flow fractionation).

[0330] The "D90 value", in particular regarding a quantitative size distribution of particles, is the diameter at which 90% of the particles have a diameter less than this value. The "D95", "D99", and "DI 00" values have corresponding meanings. The D90, D95, D99, and DI 00 values are means to describe the proportion of the larger particles within a population of particles (such as within a particle peak obtained from a field-flow fractionation).

[0331] The "hydrodynamic radius" (which is sometimes called "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as said particle. The hydrodynamic radius is related to the mobility of the particle, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a greater hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags a greater number of water molecules with it as it moves through the solution. Since the actual dimensions of the particle in a solvent are not directly measurable, the hydrodynamic radius may be defined by the Stokes-Einstein equation:

[0332] R — kBD_ - T h 6 ■ n ■ r / ■ D wherein kBis the Boltzmann constant; T is the temperature; 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). Thus, one procedure to determine the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of particles such as LNPs contained in a formulation or composition as disclosed herein or the hydrodynamic radius of a particle peak obtained from subjecting such a formulation or composition to field-flow fractionation) is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles such as LNPs contained in a formulation or composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a formulation or composition to field-flow fractionation). The term "aggregate" as used herein relates to a cluster of particles, wherein the particles are identical or very similar and adhere to each other in a non-covalently manner (e.g., via ionic interactions, H bridge interactions, dipole interactions, and / or van der Waals interactions).

[0333] The expression "light scattering" as used herein refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized non-uniformities in the medium through which the light passes.

[0334] The term "UV" means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e., shorter than that of visible light but longer than X-rays.

[0335] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles. "Multi -angle" means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations. In one 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 an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), it is possible to determine the radius of gyration (Rg) and, thus, the size of said particles. Preferably, the Zimm plot is a graphical presentation using the following equation (or the reciprocal thereof): wherein c is the mass concentration of the particles in the solvent (g / mL); A ? is the second virial coefficient (mol-mL / g2); P(3) is a form factor relating to the dependence of scattered light intensity on angle; Re is the excess Rayleigh ratio (cm1); and K* is an optical constant that is equal to 4TI2T|O (dw / dc)2Z(iUAA"1, where r|0is the refractive index of the solvent at the incident radiation (vacuum) wavelength, Xo is the incident radiation (vacuum) wavelength (nm), NA is Avogadro’s number (mol1), and dn / dc is the differential refractive index increment (mL / g) (cf., e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B.H. 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 plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above. Preferably, the Debye plot is calculated the following term or the reciprocal thereof: wherein c, Re and K* are as defined above. The expression "dynamic light scattering" or "DLS" as used herein refers to a technique to determine the size and size distribution profde of particles, in particular with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shot through a polarizer and into a sample. The scattered light then goes through a second polarizer where it is detected and the resulting image is projected onto a screen. The particles in the solution are being hit with the light and diffract the light in all directions. The diffracted light from the particles can either interfere constructively (light regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares the intensity of light at each spot over time.

[0336] The expression "static light scattering" or "SLS" as used herein refers to a technique to determine the size and size distribution profde of particles, in particular with respect to the radius of gyration of the particles, and / or the molar mass of particles. A high-intensity monochromatic light, usually a laser, is launched in a solution containing the particles. One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius. Hence simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multiangle laser light scattering (MALLS), is generally regarded as the standard implementation of static light scattering.

[0337] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

[0338] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0339] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0340] The term "repeating unit" relates to an elementary unit which periodically repeats itself along the polymeric chain of a polymer and which is derived from one monomer. Although the structures of the repeating unit and its corresponding monomer are often coincident, they may differ from each other. The term "functional moiety" as used herein relates to a group of atoms in a molecule with distinctive chemical properties, wherein the atoms of the functional moiety are linked to each other and to the rest of the molecule by covalent bonds. Preferably, the atoms of the functional moiety comprise at least one atom selected from the group consisting of O, N, and S. Functional moieties may be monovalent (such as hydroxy, cyano, nitro, or amide (e.g., -C(0)NHCH3)) or divalent (such as 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, e.g., 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 (such as a carbonyl moiety), a hydrogen bond donor (such as a hydroxyl moiety, -NH- (of, e.g., an amide moiety), or thiol moiety) or both (e.g., an amide moiety), and / or is charged (e.g., phosphate, amino, or ammonium moiety). Examples of monovalent functional moieties include hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties. Examples of divalent functional moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

[0341] The term "hydroxyl" or "hydroxy" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -OH.

[0342] The term "halogen" as used herein with respect to a functional moiety, in particular as component of a linker, means fluoro, choloro, bromo, or iodo.

[0343] The term "cyano" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -CN. The term "azido" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group N3.

[0344] The term "nitro" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -NO2.

[0345] The term "amino" as used herein with respect to a functional moiety, in particular as component of a linker, includes unsubstituted amino (i.e., the group -NH2) and substituted amino (i.e., mono- or disubstituted amino, wherein one or two of the hydrogen atoms have been replaced with a group other than hydrogen). An amino group may be monovalent (e.g., -NRR, wherein each R is independently H or an organic group, such as R72or R73as defined below) or divalent (e.g., -NR-, wherein R is H or an organic group, such as R72as defined below). In some embodiments, the term "amino" means the group -N(R72)(R73), wherein R72and R73are, in each case, independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R72and R73may join together with the nitrogen atom to which they are attached to form the group -N=CR75R76, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; R75and R76are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NHyR802-y, or R75and R76may join together with the atom to which they are attached to form a ring which is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; y is an integer from 0 to 2; R80is 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 substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; and R70is other than H, preferably a 1stlevel substituent, a 2ndlevel substituent, or a 3rdlevel substituent as disclosed herein. In some embodiments, each of R72and R73is independently H or a hydrocarbyl group, such as selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl), wherein each of the hydrocarbyl groups (such as each of the C1-6 alkyl, aryl, and aryl(C1-6 alkyl) groups) is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the hydrocarbyl group (such as C1-6 alkyl, aryl, or aryl(C1-6 alkyl) group)), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70.

[0346] The term "ammonium" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -N+(R72)2(R73), wherein R72and R73are as defined for the term "amino".

[0347] The term "thiol" or "sulfanyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SH.

[0348] The term "disulfanyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -SSH.

[0349] The term "carboxyl" or "carboxy" as used herein with respect to a functional moiety, in particular as component of a linker, relates to the group -COOH.

[0350] The term "amide" or "amido" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)NH- (including its isomerically arranged structure -NHC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the amide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the amide moiety is also referred to as lactam). An amide group may be monovalent (e.g., -C(O)NRR or -NRC(O)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0351] The term "ester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)O- (including its isomerically arranged structure -OC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (if both ends are linked to the same organic group the ester moiety is also referred to as lactone). An ester group may be monovalent (e.g., -C(O)OR or -OC(O)R, wherein R is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(O)O- or -OC(O)-)). The term "ether" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -O-, wherein each of both ends of the ether structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An ether group may be monovalent (e.g., -OR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -O-).

[0352] The term "sulfide" or "thioether" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S-, wherein each of both ends of the sulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfide group may be monovalent (e.g., -SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -S-).

[0353] The term "disulfide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SS-, wherein each of both ends of the disulfide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A disulfide group may be monovalent (e.g., -SSR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SS-).

[0354] The term "diselenide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SeSe-, wherein each of both ends of the diselenide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A diselenide group may be monovalent (e.g., -SeSeR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SeSe-).

[0355] The term "sulfoxide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfinyl structure -S(O)-, wherein each of both ends of the sulfoxide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfoxide group may be monovalent (e.g., -S(O)R, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)-).

[0356] The term "sulfone" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the sulfonyl structure -S(O)2-, wherein each of both ends of the sulfone structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A sulfone group may be monovalent (e.g., -S(O)2R, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (as, e.g., -S(O)2-).

[0357] The term "sulfite" as used herein with respect to a functional moiety, in particular as 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 to a C atom of the same or another organic group (e.g., an alkylene group as further component of the linker). A sulfite group may be monovalent (e.g., -OS(O)OR, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OS(O)O-).

[0358] The term "sulfate" as used herein with respect to a functional moiety, in particular as component of a linker, relates 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 to a C atom of the same or another organic group (e.g., an alkylene group as further component of the linker). A sulfate group may be monovalent (e.g., -OS(O)2OR, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OS(O)2O-).

[0359] The term "phosphate" as used herein with respect to a functional moiety, in particular as 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 further component of the linker) and the other end 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A phosphate group may be monovalent (e.g., -OP(O)(OR)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OP(O)(OR)O-, wherein R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0360] The term "sulfmamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S(O)N(R)-, wherein the S end of the sulfmamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the N end 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A sulfmamide group may be monovalent (e.g., -S(O)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -S(O)N(R)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0361] The term "sulfonamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -S(O)2N(R)-, wherein the S end of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the N end 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A sulfonamide group may be monovalent (e.g., -S(O)2N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -S(0)2N(R)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0362] The term "sulfamate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -0S(0)2N(R)- (including its isomerically arranged structure -N(R)S(0)20-, unless it is specified to the contrary), wherein one of both ends of the sulfamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A sulfamate group may be monovalent (e.g., -OS(O)2N(R)2 or -N(R)S(0)20R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -0S(0)2N(R)- or -N(R)S(0)20-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0363] The term "sulfiirous diamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)S(O)N(R)-, wherein one of both ends of the sulfiirous diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A sulfiirous diamide 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)S(O)N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0364] The term "sulfuric diamide" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)S(0)2N(R)-, wherein one of both ends of the sulfuric diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A sulfuric diamide group may be monovalent (e.g., -N(R)S(O)2N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)S(0)2N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0365] The term "urea" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(O)N(R)-, wherein one of both ends of the urea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). An urea group may be monovalent (e.g., -N(R)C(0)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0366] The term "thiourea" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(S)N(R)-, wherein one of both ends of the thiourea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(S)N(R)-, wherein each R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0367] The term "carbonyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(O)-, wherein one of both ends of the carbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (if both ends are linked to C atoms of organic groups the carbonyl moiety is also referred to as "keto" moiety). A carbonyl group may be monovalent (e.g., -C(O)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(O)-).

[0368] The term "thiocarbonyl" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)-, wherein one of both ends of the thiocarbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group. A thiocarbonyl group may be monovalent (e.g., -C(S)R, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(S)-).

[0369] The term "orthoester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a moiety comprising a C atom to which three alkoxy groups (i.e., -OR, wherein R is an organic group (e.g., an alkylene group as further component of the linker), such one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") are attached. An exemplary formula of an orthoester comprises the structure (-0)rC(0R)3-r-, wherein each R is independently an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. In some embodiments, an orthoester comprises the structure (-O)rC(OR25)3 r-, wherein each R25is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl) which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); r is 1 or 2; and each of both ends of the orthoester structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An orthoester group may be monovalent (e.g., -C(0R)3 or -0C(0R)2R, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R72 indicated above in the definition of the term "amino") or divalent (e.g., (-0)2C(0R)(R) or -OC(OR)2-, wherein each R is an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0370] The term "thioate" or "thioester" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(O)- (including its isomerically arranged structures -C(O)S-, _-OC(S), and -C(S)O-, unless it is specified to the contrary), wherein each of both ends of the thioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A thioate group may 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).

[0371] The term "dithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)- (including its isomerically arranged structure -C(S)S-, unless it is specified to the contrary), wherein each of both ends of the dithioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). A dithioate group may be monovalent (e.g., -SC(S)R or -C(S)SR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)- or -C(S)S-).

[0372] The term "imidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)- (including its isomerically arranged structure -C(=NR)O-, unless it is specified to the contrary), wherein each of both ends of the imidate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). An imidate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)- or -C(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). The term "imino" or "imine" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(=NR)-, wherein one of both ends of the imino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). The moiety -C(=NR)H is also called "aldimine", and the moiety -C(=NR)R’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"), is also called "ketimine". An imino group may be monovalent (e.g., -C(=NR)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(=NR)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0373] The term "imidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(=NR)S- (including its isomerically arranged structure -SC(=NR)-, unless it is specified to the contrary), wherein one of both ends of the imidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). An imidothioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(=NR)S- or -SC(=NR)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0374] The term "thionylamino" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(S)NR- (including its isomerically arranged structure -N(R)C(S)-, unless it is specified to the contrary), wherein one of both ends of the thionylamino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A thionylamino group may 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0375] The term "carbonate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(O)O-, wherein each of both ends of the carbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonate group may be monovalent (e.g., -OC(O)OR’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(O)O-).

[0376] The term "carbonothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(S)O- or -OC(O)S- (including its isomerically arranged structure -SC(O)O-, unless it is specified to the contrary), wherein each of both ends of the carbonothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonothioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).

[0377] The term "carbonodithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(O)S- or -OC(S)S- (including its isomerically arranged structure -SC(S)O-, unless it is specified to the contrary), wherein each of both ends of the carbonodithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonodithioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).

[0378] The term "carbonotrithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)S-, wherein each of both ends of the carbonotrithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker). A carbonotrithioate group may be monovalent (e.g., -SC(S)SR, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)S-). The term "guanidino" or "imidamido" as used herein with respect to a functional moiety, in particular as 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"), wherein one of both ends of the guanidino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end is covalently linked to H or to a C atom of the same or another organic group. A guanidino group may be monovalent (e.g., -N(R)C(=NR)NRR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)C(=NR)NR-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0379] The term "carbamimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)NR- (including its isomerically arranged structure -N(R)C(=NR)O-, unless it is specified to the contrary), wherein the O end of the carbamimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other (N) end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbamimidate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)NR- or -N(R)C(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0380] The term "carbonimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)O-, wherein each of the ends of the carbonimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbonimidate group may be monovalent (e.g., -OC(=NR)OR’, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)O-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0381] The term "carbamate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(O)NR- (including its isomerically arranged structure -N(R)C(O)O-, unless it is specified to the contrary), wherein the O end of the carbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbamate group may 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0382] The term "carbamodithioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(S)NR- (including its isomerically arranged structure -N(R)C(S)S-, unless it is specified to the contrary), wherein the S end of the carbamodithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbamodithioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(S)NR- or -N(R)C(S)S-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0383] The term "carbonodithioimidate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(=NR)S-, wherein each of the ends of the carbonodithioimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbonodithioimidate group may be monovalent (e.g., -SC(=NR)SR’, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(=NR)S-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0384] The term "carbamimidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -SC(=NR)NR- (including its isomerically arranged structure -N(R)C(=NR)S-, unless it is specified to the contrary), wherein the S end of the carbamimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbamimidothioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(=NR)NR- or -N(R)C(=NR)S-, wherein each R is independently H or an organic group, such one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0385] The term "carbamothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including their isomerically arranged structures -SC(O)NR- or -OC(S)NR-, unless it is specified to the contrary), wherein the O / S end of the carbamothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbamothioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0386] The term "carbonimidothioate" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(=NR)S- (including its isomerically arranged structure -SC(=NR)O-, unless it is specified to the contrary), wherein the O / S end of the carbonimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of the linker) and the other end (N end) is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A carbonimidothioate 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 one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(=NR)S- or -SC(=NR)O-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0387] The term "acylhydrazone" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -C(R’)(=N-N(R)C(O)-) (including its isomerically arranged structure (-C(O)(N(R)-N=)C(R’)-, unless it is specified to the contrary) and / or =C(=N- N(R)C(O)R’), wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; each R is H or an organic group, such as such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both ends of the acylhydrazone structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, an acylhydrazone comprises the structure -C(R25)(=N-N(R26)C(O)-) (including its isomerically arranged structure (-C(O)(N(R26)-N=)C(R25)-, unless it is specified to the contrary) and / or =C(=N-N(R26)C(O)R25), wherein each R25is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl) which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); each R26is independently H or a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); and each of both ends of the acylhydrazone structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. Exemplary chemical structures of an acylhydrazone are shown below: wherein each 'ww represents the bond by which the acylhydrazone is covalently linked to the further organic group(s) (e.g., an alkylene group as further component of the linker). A acylhydrazone group may be monovalent (e.g., -C(R’)(=N-N(R)C(O)R’) or -C(0)(N(R)-N=)C(R’)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(R’)(=N-N(R)C(O)-, -C(O)(N(R)-N=)C(R’)-, or =C(=N- N(R26)C(O)R25), wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each R is H an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino".

[0388] The term "hydrazine" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -N(R)N(R)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a hydrazine comprises the structure -N(R26)N(R26)-, wherein each R26is independently H or a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl), which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); and each of both ends of the hydrazine structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. A hydrazine group may be monovalent (e.g., -N(R)N(R)2, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -N(R)N(R)-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0389] The term "oxime" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure =C(=N(OH)), wherein each of both ends of the oxime structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An exemplary chemical formula of an oxime is shown below: wherein each AAAAV represents the bond by which the oxime is covalently linked to the further organic group(s). An oxime group may be monovalent (e.g., -C(=N(OH))(R), wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., =C(=N(OH))).

[0390] The term "acetal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, an acetal comprises the structure -OCH(R25)O-, wherein R25is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); and each of both O atoms of the acetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An acetal group may be monovalent (e.g., -OCH(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OCH(R’)O-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0391] The term "hemiacetal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCH(OH)-, wherein each of both ends of the hemiacetal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). A hemiacetal group may be monovalent (e.g., -OCH(OH)OR’, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OCH(OH)-).

[0392] The term "ketal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a ketal comprises the structure -OC(R25)(R25)O -, wherein each R25is independently a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); and each of both O atoms of the ketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. An ketal group may be monovalent (e.g., -OC(R’)(R’)OR’, wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OC(R’)(R’)O-, wherein each R’ is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0393] The term "hemiketal" as used herein with respect to a functional moiety, in particular as component of a linker, relates to a group comprising the structure -OCR’ (OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups (e.g., an alkylene group as further component of the linker). In some embodiments, a hemiketal comprises the structure -OCR25(OH)-, wherein R25is a hydrocarbyl group, such as C1-6 alkyl, aryl, and aryl(C1-6 alkyl)), which is optionally substituted (e.g., with one or more 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents as defined herein); and each of both ends of the hemiketal structure is covalently linked to a C atom of a further organic group or of two further separate organic groups. A hemiketal group may be monovalent (e.g., -0C(R’)2(0H), wherein each R’ is independently an organic group, such as independently selected from the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -OCR’(OH)-, wherein R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0394] The term "imide" as used herein with respect to a functional moiety, in particular within a as component of, relates to a group comprising the structure -C(O)N(R)C(O)-, wherein R is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and each of both ends of the imide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). An imide group may be monovalent (e.g., -C(O)N(R)C(O)R’, wherein R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"; and R’ is an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(O)N(R)C(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). The term "non-cyclic" as used herein in the context of organic groups relates to open-chain organic groups which contain no rings. "Open-chain" or "acyclic" organic groups may be straight (i.e., they contain only one unbranched chain without any sidechain) or branched (i.e., the main chain comprises one or more sidechains).

[0395] An organic group which is "substituted with one or more substituents" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the organic 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 one or more substituents may be selected from the 1stlevel substituents, 2ndlevel substituents, or 3rdlevel substituents described herein.

[0396] The expression "hydrogen bond" or "H-bond" as used herein means a non-covalent bond (in some embodiments a primarily electrostatic force of attraction) between (i) a hydrogen atom which is covalently bound 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 nitrogen atoms and oxygen atoms; thus, examples of groups in which a hydrogen atom is covalently bound to a more electronegative atom or group include amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))). In some embodiments, the lone pair of electrons of another electronegative atom is a lone pair of an oxygen atom present in a carbonyl group or a lone pair of a nitrogen atom present in a primary, secondary or tertiary amino group.

[0397] The expression "hydrogen bond donor" as used herein means an atom, ion, or a molecule component of a hydrogen bond which supplies the bridging (shared) hydrogen atom. In some embodiments, a hydrogen bond donor includes amino groups bearing at least one covalently attached hydrogen atom, the -NH- group of amide groups, hydroxyl groups (as such (as in respective alcohols) or as part of other functional groups (e.g., as part of carboxyl (-COOH) groups)), and sulfanyl groups (as such (as in respective thiols) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) groups))).

[0398] The expression "hydrogen bond acceptor" as used herein means an atom, ion, or a molecule component of a hydrogen bond which does not supply the bridging (shared) hydrogen atom. In some embodiments, a hydrogen bond acceptor comprises at least one lone pair of electrons. Examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary and tertiary amino groups. The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and / or degraded, or to be hardly detected and then sequestered and / or degraded, and / or to be detected and then sequestered and / or degraded late, by the immune system of the host to which they are administered.

[0399] The term "phosphatidylethanolamine" means diacylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). 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 (such as monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (such as monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)I6CH3(stearoyl), -C(O)(CH2)i4CH3(palmitoyl), -C(O)(CH2)I2CH3(myristoyl), and -CZ5-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl). The term "phosphatidylethanolamine moiety" means a monovalent radical of phosphatidylethanolamine, preferably that in which a hydrogen atom of the amino group has been removed.

[0400] The term "DSPE" means distearoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3(stearoyl). The term "distearoylphosphatidylethanolamine moiety" means a monovalent radical of DSPE, preferably that in which a hydrogen atom of the amino group has been removed.

[0401] The term "DPPE" means dipalmitoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(0)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl). The term "dipalmitoylphosphatidylethanolamine moiety" means a monovalent radical of DPPE, preferably that in which a hydrogen atom of the amino group has been removed.

[0402] The term "DOPE" means dioleoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein in each case -C(O)CI7H33 refers to the moiety -cz5-C(O)(CH2)7-CH=CH- (CH2)7CH3(oleoyl). The term "dioleoylphosphatidylethanolamine moiety" means a monovalent radical of DOPE, preferably that in which a hydrogen atom of the amino group has been removed.

[0403] The term "POPE" means palmitoyloleoylphosphatidylethanolamine having the following formula: or a salt thereof, wherein -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl); and -C(O)CI7H33 refers to the moiety -cz5-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The term "palmitoyloleoylphosphatidylethanolamine moiety" means a monovalent radical of POPE, preferably that in which a hydrogen atom of the amino group has been removed.

[0404] The term "tocopherol" means a group of four compounds (i.e., a-tocopherol, P-tocopherol, y-tocopherol, and 5-tocopherol) having the following formula: wherein each of Ru and R2 is independently H or methyl. In a-tocopherol, Ru and Rt2 are both methyl; in P-tocopherol, Ri is methyl, and Rt2 is H; in y-tocopherol, Ru is H, and Rt2 is methyl; and in 5- tocopherol, Rti and Rt2 are both H. The term "tocopherol moiety" or "tocopheryl moiety" means a monovalent radical of tocopherol, preferably that in which the hydrogen atom of the hydroxy group has been removed.

[0405] The term "DAG" means diacylglyceride having the following formula: or a salt thereof, wherein in each case acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). 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 (such as monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (such as monounsaturated). In some embodiments, one acyl moiety is saturated and the other is unsaturated (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)i6CH3(stearoyl), -C(O)(CH2)i4CH3(palmitoyl), -C(O)(CH2)i2CH3(myristoyl), and -CA-C(O)(CH2)7-CH=CH-(CH2)7CH3(oleoyl). For example, DMG means 1,2-dimyristoylglycerol, i.e., a diacylglyceride of the above formula, wherein both acyl groups are -C(O)(CH2)i2CH3(myristoyl). The term "diacylglyceride moiety" means a monovalent radical of diacylglyceride, preferably that in which a hydrogen atom of the hydroxy group has been removed.

[0406] The term "DAA" means dialkylamine having the formula HN(alkyl)2 or a salt thereof, wherein each alkyl moiety preferably is straight. In some embodiments, each alkyl moiety has at least 8 carbon atoms. Preferably, each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties include -(CFDnCFE (stearyl), -(CFDisCFE (palmityl), and -(CH2)I3CH3(myristyl). For example, DMA means 1,2-dimyristylamine, i.e., a dialkylamine of the above formula, wherein both alkyl groups are -(CH2)I3CH3(myristyl). The term "dialkylamine moiety" means a monovalent radical of dialkylamine, preferably that in which the hydrogen atom of the amino group has been removed.

[0407] The term "ceramide" means acyl sphingosine having the following formula: or a salt thereof, wherein -C13H27 refers to the moiety -(CFDnCFE; and acyl refers to an acyl moiety (such as a -C(O)-hydrocarbyl moiety, wherein the hydrocarbyl group preferably is straight). In some embodiments, the 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 (such as monounsaturated). Examples of acyl moieties include -C(O)(CH2)ieCH3 (stearoyl), -C(O)(CH2)i4CH3 (palmitoyl), -C(O)(CH2)i2CH3 (myristoyl), and -cA-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). For example, palmitoyl ceramide means a ceramide of the above formula, wherein acyl is -C(O)(CH2)i4CH3 (palmitoyl). The term "ceramide moiety" means a monovalent radical of ceramide, preferably that in which the hydrogen atom of a hydroxy group (preferably the hydrogen of the terminal (primary) hydroxy group) has been removed.

[0408] The term "MAA" means monoalkylamine having the formula H2N(alkyl) or a salt thereof, wherein the alkyl moiety preferably is straight. In some embodiments, the alkyl moiety has at least 8 carbon atoms. Preferably, the alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties include -(CIDnCFE (stearyl), -(CIDisCFE (palmityl), and -(CTDBCTE (myristyl). For example, MMA means myristylamine, i.e., a monoalkylamine of the above formula, wherein the alkyl group is - (CH2)i3CH3(myristyl). The term "monoalkylamine moiety" means a monovalent radical of monoalkylamine, preferably that in which one of the hydrogen atoms of the amino group has been removed.

[0409] Nucleic Acids

[0410] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0411] The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0412] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0413] A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is preferably modified by one or more alkyl groups, more preferably one or more CM alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl- guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N’-CM alkyl- guanine, N6-CM alkyl-adenine, 5-CM alkyl-cytosine, 5-CM alkyl-uracil, and N(1)-CM alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)- methyl -uracil.

[0414] In some embodiments of all aspects of the disclosure, the nucleic acid is DNA.

[0415] Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0416] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0417] RNA In some embodiments of all aspects of the disclosure, the nucleic acid is RNA.

[0418] According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may 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 RNAs containing such altered / modified nucleotides or nucleosides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule . The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0419] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0420] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and expressing a pharmaceutically active peptide or protein. In some embodiments, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface. Alternatively, the RNA can be non-coding RNA such as antisense -RNA, micro RNA (miRNA) or siRNA. The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). mRNA

[0421] In some embodiments of all aspects of the disclosure, the nucleic acid is mRNA.

[0422] According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. Typically, an mRNA comprises a 5'-UTR, a peptide / protein coding region, and a 3'-UTR. In the context of the present disclosure, mRNA is preferably generated by in vitro transcription (IVT) from a DNA template. As set forth above, the in vitro transcription methodology is known to the skilled person, and a variety of in vitro transcription kits is commercially available. mRNA is single -stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0423] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0424] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

[0425] In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0426] As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non- naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0427] In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0428] In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (such as self-replicating mRNA) is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5 ’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould etal., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non- structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame e...

Claims

CLAIMS A composition comprising (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains:whereinX2and X1taken together are 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. The composition of claim 1, wherein:(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-;(vi) when X1is -O- then X2is -C(O)-;(vii) when X1is -C(S)- then X2is -O-;(viii) when X1is -O- then X2is -C(S)-;(ix) when X1is -C(O)- then X2is -S-; or(x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or Cus alkyl; preferably(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-; or(vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl.

3. The composition of claim 1 or 2, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Ci-8 alkyl.

4. The composition of any one of claims 1 to 3, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl.

5. The composition of any one of claims 1 to 4, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen.

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

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):wherein R1is hydrogen or C1-8 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):wherein R1is hydrogen or C1-8 alkyl.

14. The composition of any one of claims 8 to 13, wherein R1is hydrogen or methyl.

15. The composition of any one of claims 8 to 14, wherein R1is 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.

0. The composition of any one of claims 1 to 19, wherein the one or more hydrophobic chains are located at either the X1end or the X2end of the polymer.

1. The composition of any one of claims 1 to 20, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms.

2. The composition of any one of claims 1 to 21, wherein the polymer-conjugated compound cwhereinX2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester;Y is -CH2-, -(CH2)2-, or -(CH2)3-;R2is a moiety comprising the one or more hydrophobic chains;R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100. The composition of claim 22, wherein:(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-;(vi) when X1is -O- then X2is -C(O)-;(vii) when X1is -C(S)- then X2is -O-;(viii) when X1is -O- then X2is -C(S)-;(ix) when X1is -C(O)- then X2is -S-; or(x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl; preferably(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-; or(vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl. The composition of claim 22 or 23, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or C1-8 alkyl. The composition of any one of claims 22 to 24, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl. The composition of any one of claims 22 to 25, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen. The composition of any one of claims 22 to 26, wherein Y is -CH2- or -(CTh^-. The composition of any one of claims 22 to 27, wherein Y is -CH2-. The composition of any one of claims 22 to 28, wherein the polymer-conjugated compound comprises the following general formula (VI)wherein R1is hydrogen or C1-8 alkyl. The composition of any one of claims 22 to 29, wherein z is 2 to 10, such as 2 to 7. The composition of any one of claims 22 to 30, wherein z is 2 to 5.The composition of any one of claims 22 to 31, wherein z is 2 or 3. The composition of any one of claims 22 to 32, wherein z is 2. The composition of any one of claims 22 to 33, which comprises the following general formulawherein R1is hydrogen or Cus alkyl. The composition of any one of claims 29 to 34, wherein R1is hydrogen or methyl. The composition of any one of claims 29 to 35, wherein R1is hydrogen. The composition of any one of claims 22 to 36, wherein the polymer-conjugated compound cThe composition of any one of claims 22 to 37, wherein n is 5 to 50. The composition of any one of claims 22 to 38, wherein n is 5 to 25. 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. The composition of any one of claims 22 to 40, wherein R2is R4or -L1(R4)P, wherein each R4is independently a hydrophobic chain, such as a hydrocarbyl group; L1is a linker; and p is 1 or 2.The composition of claim 41, wherein L1comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, 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, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. The composition of claim 41 or 42, wherein L1comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)-, [*-OC(O)]p(C1-6 -alkylene)-, [*-NHC(0)]P(CI-6- alkylene)-, [*-C(O)NH]p(C1-6-alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6 -alkylene)-, [*- S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*- C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)-N=)C(R25)-]p(C1-6-alkylene)-,[*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-, [*-N(R26)N(R26)]p(C1-6-alkylene)-,[*=C(=N(OH))]p(C1-6-alkylene)-, [*-OC(R25)(R26)O]p(C1-6-alkylene)-, *-(3,4-dihydro-2H- chromen-6-yl)-, (*-)pN(R26)2.p, and [*-C(0)NH](CI-6 -alkyltriyl)-, wherein * represents the attachment point to R4; p is 1 or 2; C1-6-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.The composition of any one of claims 41 to 43, wherein L1further comprises at least one additional difunctional moiety via which R2is attached to either X1in formula (V) or X2in formula (V’). The composition of claim 44, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6 -alkylene group from each other. The composition of any one of claims 41 to 45, wherein L1is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(Ci-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(Ci- 6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6 -alkylene)©-, [*- OC(O)]p(C1-6-alkylene)O-, (*-)pN(R26)2-p, and [*-C(O)NH](C1-6-alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C1-6-alkylene), [*-OC(O)]p(C i-6 -alkylene), [*-NHC(O)]p(C1-6-alkylene), and [*-C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6-alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.The composition of any one of claims 41 to 46, wherein L1is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(Ci-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6- alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)-, [*-OC(O)]p(Ci-6- alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-NHC(O)]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene), [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)-O(Cw- alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O]p(C1-6-alkylene)O-, [*- OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6-alkyltriyl)O- or L1is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(Ci- 6-alkylene), [*-OC(O)]p(C1-6 -alkylene), [*-NHC(0)]P(CI-6 -alkylene), and [*-C(O)NH]p(C1-6- alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The composition of any one of claims 22 to 47, wherein R2is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4NHC(O)]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3- alkylene)C(O)-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(Ci.3-alkylene), [R4C(O)NH]P(C2-3- alkylene)OP(O)(OR27)O(Ci.3-alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(Ci.3- alkylene)C(O)-, (2-R4-3,4-dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*- OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2is (R4)(R26)N-, wherein p is 1 or 2; the C2-3-alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and - OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4.The composition of any one of claims 22 to 48, wherein R2is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2is a monoalkylamine moiety. The composition of any one of claims 41 to 49, wherein each R4is independently a non-cyclic, preferably straight, hydrocarbyl group. The composition of any one of claims 41 to 50, wherein each R4is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The composition of any one of claims 22 to 51, wherein R2is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE(palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2is a monomyristylamine moiety. The composition of any one of claims 22 to 52, wherein R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2. -NH(CI-3 alkyl), -N(CI-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair.The composition of any one of claims 22 to 53, wherein R3is selected from the group consisting ofH, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2. -NH(CI-3 alkyl), -N(CI-3 alkyl )2. and a member of a targeting pair. The composition of any one of claims 22 to 54, wherein R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair. The composition of any one of claims 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide - thiol; thiol - halogenated (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said 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 - av[T integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor. The composition of any one of claims 1 to 56, wherein the polymer-conjugated compound has one of the following formulas:wherein n is 5 to 25;R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;R27is H or a countercation; and in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(CH2)i2CH3, and in each case -C(O)Ci7H33 refers to the moiety -cw-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). The composition of claim 57, wherein n is 7 to 16, such as 7 to 14, preferably 8, 10, 12, 14, or 16. The composition of claim 57 or 58, wherein R3is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, C2-6 alkynyl, an antigen, or an antibody. The composition of any one of claims 1 to 59, wherein the polymer-conjugated compound has one of the following formulas:wherein in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(CfDnCHs, and in each case n is 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 formulas:wherein n is 14, and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl);wherein n is 14, and in each case -C14H29 refers to the moiety -(CFDBCFE (myristyl);wherein 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 material comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeating units.

63. The composition of any one of claims 1 to 62, wherein water is the main component in the composition and / or the total amount of solvent(s) 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 the nucleic acid in the composition is about 1 mg / 1 to about 500 mg / 1, such as about 1 mg / 1 to about 100 mg / 1, about 5 mg / 1 to about 100 mg / 1, or about 10 mg / 1 to about 100 mg / 1.

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

66. The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (X)or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L10and L20is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L10and L20is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;G1and G2are each independently unsubstituted C1-C12 alkylene or C2-12 alkenylene;G3is C 1-24 alkylene, C2-24 alkenylene, C3-8 cycloalkylene, or C3-8 cycloalkenylene;Rais H or C1-12 alkyl;R35and R36are each independently C6-24 alkyl or C6-24 alkenyl;R37is H, OR50, CN, -C(=O)OR40, -OC(=O)R40or -NR50C(=O)R40;R40is C1-12 alkyl;R50is H or C1-6 alkyl; and x is 0, 1 or 2.The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid has the structure of Formula (XI):wherein each of Ri and R2 is independently R5 or -G1-L1-R5, wherein at least one of Ri and R2 is -G1-L1- each of R3and R4 is independently selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, aryl, and C3-10 cycloalkyl; each of R5and Rs is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; each of Gi and G2 is independently unsubstituted C1-12 alkylene or C2-12 alkenylene; each of Li and L2 is independently selected from the group consisting of -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 C1-12 alkyl; m is 0, 1, 2, 3, or 4; andx is 0, 1 or 2. The composition of any one of claims 1 to 65, wherein the cationic or cationically ionizable lipid comprises 2,3-dioleyloxy-l-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(l- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof. The composition of any one of claims 1 to 68, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol % of the total lipid present in the composition. The composition of any one of claims 1 to 69, further comprising one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid. The composition of claim 70, wherein the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3 - phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 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 phospholipid comprises from about 5 mol % to about 30 mol % of the total lipid 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 from about 10 mol % to about 60 mol % of the total lipid present in the composition.

75. The composition of any one of claims 70 to 74, wherein the cationic or cationically ionizable lipid comprises from about 20 mol % to about 70 mol % of the total lipid present in the composition; the polymer-conjugated compound comprises from about 0.5 mol % to about 15 mol % of the total lipid present in the composition; the phospholipid comprises from about 5 mol % to about 25 mol % of the total lipid present in the composition; and the steroid comprises from about 20 mol % to about 55 mol % of the total lipid 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 the nucleic acid, at least a portion of the cationic or cationically ionizable lipid, and at least a portion of the polymer-conjugated compound.

77. The composition of claim 76, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), 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 from 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 a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (y), Nl- methyl-pseudouridine (ml\| / ), and 5 -methyl -uridine (m5U); (2) has a coding sequence which iscodon-optimized; and / or (3) has a coding sequence whose G / C content is increased compared to the 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 preferably is an interrupted sequence of A nucleotides.

84. The composition of claim 82 or 83, wherein the 5’ cap is a capl or cap2 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 against 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 the immunogenic variant thereof.

87. A method for delivering nucleic acid to cells of a subject, the method comprising administering to a subject a 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 a subject a 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 disorder in a subject, the method comprising administering to a subject a composition of any one of claims 1 to 86, wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.

90. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject a composition of 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 in treating or preventing the disease or disorder.

91. The method of any one of claims 87 to 90, wherein the subject is a mammal.The method of claim 91, wherein the mammal is a human. A polymer-conjugated compound comprising (a) a polymer which comprises the following general formula (I); and (b) one or more hydrophobic chains:whereinX2and X1taken together are 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. The polymer-conjugated compound of claim 93, wherein:(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-;(vi) when X1is -O- then X2is -C(O)-;(vii) when X1is -C(S)- then X2is -O-;(viii) when X1is -O- then X2is -C(S)-;(ix) when X1is -C(O)- then X2is -S-; or(x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or Cus alkyl; preferably(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-;(vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl.

95. The polymer-conjugated compound of claim 93 or 94, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Cus alkyl.

96. The polymer-conjugated compound of any one of claims 93 to 95, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl.

97. The polymer-conjugated compound of any one of claims 93 to 96, wherein X1is -C(O)- and X2is -NR1-, wherein R1is 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):wherein R1is hydrogen or C1-8 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):wherein R1is hydrogen or Cus alkyl. The polymer-conjugated compound of any one of claims 100 to 105, wherein R1is hydrogen or methyl. The polymer-conjugated compound of any one of claims 100 to 106, wherein R1is hydrogen. The polymer-conjugated compound of any one of claims 93 to 107, wherein the polymer comprises the following general formula (IV):The polymer-conjugated compound of any one of claims 93 to 108, wherein n is 5 to 50. The polymer-conjugated compound of any one of claims 93 to 109, wherein n is 5 to 25. 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. The polymer-conjugated compound of any one of claims 93 to 111, wherein the one or more hydrophobic chains are located at either the X1end or the X2end of the polymer. The polymer-conjugated compound of any one of claims 93 to 112, wherein the one or more hydrophobic chains are non-cyclic, preferably straight, hydrocarbyl groups, more preferably those having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The polymer-conjugated compound of any one of claims 93 to 113, comprising the following general formula (V) or (V’):whereinX2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, thioester;Y is -CH2-, -(CH2)2-, or -(CH2)3-;R2is a moiety comprising the one or more hydrophobic chains;R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -OR20, -SR20, halogen, -CN, -N3, -OC(O)R21, -C(O)R21, -NR22R23, -COOH, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R20is selected from the group consisting of H, C1-3 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-3 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, C2.g alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, a sugar, an amino acid, a peptide, and a member of a targeting pair; z is 2 to 24; and n is 1 to 100. The polymer-conjugated compound of claim 114, wherein:(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(0)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(0)- then X2is -0-;(vi) when X1is -O- then X2is -C(O)-;(vii) when X1is -C(S)- then X2is -O-;(viii) when X1is -O- then X2is -C(S)-;(ix) when X1is -C(O)- then X2is -S-; or(x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl; preferably(i) when X1is -C(O)- then X2is -NR1-;(ii) when X1is -NR1- then X2is -C(O)-;(iii) when X1is -C(S)- then X2is -NR1-;(iv) when X1is -NR1- then X2is -C(S)-;(v) when X1is -C(O)- then X2is -O-; or(vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl. The polymer-conjugated compound of claim 114 or 115, wherein X1is -C(O)- and X2is -NR1- , wherein R1is hydrogen or C1-8 alkyl. The polymer-conjugated compound of any one of claims 114 to 116, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl. The polymer-conjugated compound of any one of claims 114 to 117, wherein X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen. The polymer-conjugated compound of any one of claims 114 to 118, wherein Y is -CH2- or -(CH2)2-. The polymer-conjugated compound of any one of claims 114 to 119, wherein Y is -CH2-. The polymer-conjugated compound of any one of claims 114 to 120, comprising the following general formula (VI) or (VI’):wherein R1is hydrogen or C1-8 alkyl. The polymer-conjugated compound of any one of claims 114 to 121, wherein z is 2 to 10, such as 2 to 7. The polymer-conjugated compound of any one of claims 114 to 122, wherein z is 2 to 5. The polymer-conjugated compound of any one of claims 114 to 123, wherein z is 2 or 3. The polymer-conjugated compound of any one of claims 114 to 124, wherein z is 2. The polymer-conjugated compound of any one of claims 114 to 125, comprising the following general formula (VII) or (VIT):wherein R1is hydrogen or Cus alkyl. The polymer-conjugated compound of any one of claims 121 to 126, wherein R1is hydrogen or methyl. The polymer-conjugated compound of any one of claims 121 to 127, wherein R1is hydrogen. The polymer-conjugated compound of any one of claims 114 to 128, comprising the following general formula (VIII) or (VIIT):The polymer-conjugated compound of any one of claims 114 to 129, wherein n is 5 to 50. The polymer-conjugated compound of any one of claims 114 to 130, wherein n is 5 to 25. 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. The polymer-conjugated compound of any one of claims 114 to 132, wherein R2is R4or -L1(R4)P, wherein each R4is independently a hydrophobic chain, such as a hydrocarbyl group; L1is a linker; and p is 1 or 2. The polymer-conjugated compound of claim 133, wherein L1comprises at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or linked, at the end by which the alkylene group is attached to R4, to a divalent functional moiety, wherein preferably each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, 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, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. The polymer-conjugated compound of claim 133 or 134, wherein L1comprises a functional moiety selected from the group consisting of [*-C(O)O]p(C1-6 -alkylene)-, [*-OC(O)]p(C1-6- alkylene)-, [*-NHC(O)]P(CI-6 -alkylene)-, [*-C(O)NH]P(CI-6 -alkylene)-, [*-S]p(C1-6 -alkylene)-, [*-SS]p(C1-6-alkylene)-, [*-S(O)2]P(C1-6-alkylene)-, [(*-O)rC(OR25)3-r](C1-6-alkylene)-, [*-C(OR25)2O]p(C1-6-alkylene)-, [*-C(R25)(=N-N(R26)C(O)-)]p(C1-6-alkylene)-, [*-C(O)(N(R26)- N=)C(R25)-]p(C1-6-alkylene)-, [*=C(=N-N(R26)C(O)(R25))]p(C1-6-alkylene)-,[*-N(R26)N(R26)]p(C1-6-alkylene)-, [*=C(=N(0H))]p(C1-6-alkylene)-, [*-OC(R25)(R26)O]P(C1-6- alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-)pN(R26)2-p, and [*-C(0)NH](C1-6-alkyltriyl)- , wherein * represents the attachment point to R4; p is 1 or 2; C1-6 -alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R25is selected from the group consisting of C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); r is an integer between 1 and 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, - CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 133 to 135, wherein L1further comprises at least one additional difunctional moiety, via which R2is attached to either X1in formula (V) or X2in formula (V’). The polymer-conjugated compound of claim 136, wherein the at least one additional difunctional moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfmamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamido, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino (imidamido), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein if L1further comprises at least two additional difunctional moieties, these at least two additional difunctional moieties are optionally separated by a C1-6-alkylene group from each other. The polymer-conjugated compound of any one of claims 133 to 137, wherein L1is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- , [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-NHC(O)]P(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NR26-, [*-C(O)NH]P(C1-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*- C(0)0]p(C1-6-alkylene)0-, [*-0C(0)]p(C1-6-alkylene)0-, (*-)pN(R26)2.p, and [*-C(O)NH](C1-6- alkyltriyl)O-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C 1-6 -alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(0)]P(CI-6 -alkylene), and [*- C(0)NH]P(CI-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H, C1-6 alkyl, aryl, and aryl(C1-6 alkyl); R27is selected from the group consisting of H, C1-6 alkyl, aryl, aryl(C1-6 alkyl), and a countercation; 3,4-dihydro-2H-chromen- 6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 133 to 138, wherein L1is selected from the group consisting of [*-C(O)O]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)-, [*- C(O)O]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(O)O]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)- , [*-OC(O)]p(C1-6-alkylene)-OP(O)(OR27)O(C1-6-alkylene)NH-, [*-OC(O)]p(C1-6-alkylene)- OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-NHC(O)]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-NHC(0)]P(CI-6- alkylene)OP(O)(OR27)O(C1-6-alkylene)C(O)-, [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(Cw- alkylene), [*-C(O)NH]p(C1-6-alkylene)OP(O)(OR27)O(C1-6-alkylene)NH-, [*-C(0)NH]P(CI-6- alkylene)OP(O)(OR27)-O(C1-6-alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*- C(O)O]p(C1-6-alkylene)O-, [*-OC(O)]p(C1-6-alkylene)O-, (*-)2N-, and [*-C(O)NH](C1-6- alkyltriyl)O- or L1is (*-)(R26)N-, wherein * represents the attachment point to R4; p is 1 or 2; the C1-6-alkylene in [*-C(O)O]p(C 1-6 -alkylene), [*-OC(O)]p(C1-6-alkylene), [*-NHC(0)]P(CI-6- alkylene), and [*-C(O)NH]p(C 1-6 -alkylene) is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C 1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 114 to 139, wherein R2is selected from the group consisting of [R4C(O)O]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)-, [R4C(O)O]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4C(O)O]p(C2-3-alkylene)- OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)-, [R4OC(O)]p(C2-3-alkylene)-OP(O)(OR27)O(C1-3-alkylene)NH-, [R4OC(O)]p(C2-3- alkylene)-OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O- (C1-3-alkylene), [R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)NH-,[R4NHC(O)]p(C2-3-alkylene)OP(O)(OR27)O(C1-3-alkylene)C(O)-, [R4C(O)NH]p(C2-3- alkylene)OP(O)(OR27)O(C1-3-alkylene), [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)O(C1-3- alkylene)NH-, [R4C(O)NH]p(C2-3-alkylene)OP(O)(OR27)-O(C1-3-alkylene)C(O)-, (2-R4-3,4- dihydro-2H-chromen-6-yl)O-, [R4C(O)O]p(C2-3-alkylene)O-, [*-OC(O)]p(C2-3-alkylene)O-, (R4)2N-, and [R4C(O)NH](C2-3-alkyltriyl)O- or R2is (R4)(R26)N-, wherein p is 1 or 2; the C2-3- alkylene is either bivalent (if p is 1) or trivalent (if p is 2); R26is selected from the group consisting of H and C1-6 alkyl; R27is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-3 alkyl, -OH, -CN, and -OC1-3 alkyl; and C2-3 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R4. The polymer-conjugated compound of any one of claims 114 to 140, wherein R2is selected from a phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety or R2is a monoalkylamine moiety. The polymer-conjugated compound of any one of claims 133 to 141, wherein each R4is independently a non-cyclic, preferably straight, hydrocarbyl group. The polymer-conjugated compound of any one of claims 133 to 142, wherein each R4is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. The polymer-conjugated compound of any one of claims 114 to 143, wherein R2is selected from DSPE (distearoylphosphatidylethanolamine), DPPE(dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoyl ceramide moieties or R2is a monomyristylamine moiety. The polymer-conjugated compound of any one of claims 114 to 144, wherein R3is selected from the group consisting of H, C1-6 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C1-6 alkyl group is optionally substituted with one or more substituents independently selectedfrom the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, a sugar, an amino acid, a peptide, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. a sugar, an amino acid, a peptide, and a member of a targeting pair. The polymer-conjugated compound of any one of claims 114 to 145, wherein R3is selected from the group consisting of H, C1-3 alkyl, C2-6 alkynyl, -C(O)R21, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NR22R23, -C(O)NR22R23, -NR22C(O)R21, and a member of a targeting pair; R21is selected from the group consisting of C1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C1-6 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, C2-6 alkynyl, -COOH, -NR22R23, and a member of a targeting pair; and each of R22and R23is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R22and R23may join together with the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl )2. and a member of a targeting pair. The polymer-conjugated compound of any one of claims 114 to 146, wherein R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), -NH(CI-3 alkyl), and -N(CI-3 alkyl)2, and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN,-N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair. 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 (in particular, brominated) alkyl; azide - alkyne (especially in a copper(I)-catalyzed reaction); conjugated diene - substituted alkene (dienophile) (especially in a Diels-Alder reaction); antigen - antibody specific for said 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 - avp3 integrin; asparagine-glycine- arginine (NGR) peptide - aminopeptidase N; galactose - asialoglyco-protein receptor. The polymer-conjugated compound of any one of claims 93 to 148, having one of the following formulas:wherein n is 5 to 25;R3is selected from the group consisting of H, -C(O)(C1-3 alkyl), and a member of a targeting pair, wherein the C1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N3, C2-6 alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and a member of a targeting pair;R27is H or a countercation; and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CisH3i refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)I2CH3 (myristoyl), in each case -CUHM refers to the moiety-(CIDBCHS (myristyl), in each case -C13H27 refers to the moiety -(CThjnClT, and in each case -C(O)CI7H33refers to the moiety -cw-C(O)(CH2)7-CH=CH-(CH2)7CH3 (oleoyl). 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. The polymer-conjugated compound of claim 149 or 150, wherein R3is H or -C(O)(C1-3 alkyl), wherein the C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl (maleimidyl), -SH, -Br, -N3, and C2-6 alkynyl. The polymer-conjugated compound of any one of claims 93 to 151, having one of the following formulas:wherein in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl), in each case -C(O)CI5H3I refers to the moiety -C(O)(CH2)i4CH3 (palmitoyl), in each case -C(O)Ci3H27 refers to the moiety -C(O)(CH2)i2CH3 (myristoyl), in each case -C14H29 refers to the moiety -(CH2)i3CH3 (myristyl), in each case -C13H27 refers to the moiety -(ClDnCTL, and in each case n is 8, 10, 12, 14, or 16.

153. The polymer-conjugated compound of any one of claims 93 to 152, having one of the following formulas:wherein n is 14, and in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl);wherein n is 14, and in each case -C14H29 refers to the moiety -(CIDBCIT (myristyl);wherein n is 8, 12, 14, or 16.A conjugate of (a) the polymer-conjugated compound of any one of claims 93 to 153 containing a member of a targeting pair; and (b) a compound comprising the other member of the targeting pair. 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 (such as an antigen or epitope), or an antibody. The conjugate of claim 154 or 155, having one of the following formulas:or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)ieCH3 (stearoyl); each of ml and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen. The conjugate of claim 156, having one of the following formulas:wherein ml is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2;wherein each of ml and m2 is independently 1, 2, or 3, preferably ml is 1 is and m2 is 2 or ml is 2 and m2 is 1. The conjugate of any one of claims 154 to 157, having one of the following formulas:or a salt thereof, wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)Ci7H35 refers to the moiety -C(O)(CH2)igCH3 (stearoyl); and Pept is an antigen or an antibody specific for said antigen. The conjugate of claim 154, wherein the polymer-conjugated compound containing a member of a targeting pair has the following formula:wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case -C(O)CI?H35 refers to the moiety -C(O)(CH2)i6CH3 (stearoyl); 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 containing a member of a targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) said antibody specific for said antigen and (2) said antigen.

Images