RNA LIPID NANOPARTICLES (LNPs) COMPRISING POLYAZOLINE AND / OR POLYAZINE POLYMERS

Abstract

The present disclosure relates to RNA nanoparticles (LNPs) for delivering RNA to a target tissue after administration, particularly after parenteral administration, such as intravenous, intramuscular, subcutaneous, intratumoral, intraarterial, intradermal, transdermal, intranasal, transrectal or oral administration, and compositions comprising such RNA LNPs. In some embodiments, the RNA LNP comprises a single-stranded RNA, such as mRNA, encoding a peptide or protein of interest, such as a pharmaceutically active peptide or protein. The RNA is absorbed by cells of the target tissue, and the RNA is translated into an encoded peptide or protein, which can exhibit its physiological activity. In addition, the present disclosure relates to certain conjugates of (a) a poly # imgabs0 # azoline (POX) and / or a poly # imgabs1 # azine (POZ) polymer and (b) one or more hydrophobic chains, compositions comprising such conjugates, and uses of such conjugates and compositions.

Description

Technical Field

[0001] The present invention relates to RNA nanoparticles (LNPs) for delivering RNA to target tissues after administration, particularly after parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intratumoral, intraarterial, intradermal, transdermal, intranasal, rectal or oral administration), and compositions comprising such RNA LNPs. In some embodiments, the RNA LNP particles comprise single-stranded RNA, such as mRNA encoding a target peptide or target protein (e.g., a pharmaceutically active peptide or protein). The RNA is absorbed by cells of the target tissue, and the RNA is translated into a coded peptide or protein that can exhibit its physiological activity. In addition, the present invention relates to (a) poly Oxazoline (POX) polymers and / or poly Certain conjugates of polyoxazine (POZ) polymers and (b) one or more hydrophobic chains, compositions comprising such conjugates, and uses of such conjugates and compositions. Background Art

[0002] The use of RNA to deliver foreign genetic information into target cells provides an attractive alternative to DNA. Advantages of using RNA include transient expression and non-transforming characteristics. RNA does not need to enter the cell nucleus for expression, and in addition, RNA will not integrate into the host genome, thereby eliminating the risk of tumorigenesis.

[0003] Different delivery vehicles can be used, mainly based on cationic polymers or lipids that form nanoparticles with RNA, and RNA is delivered to the object. Nanoparticles are intended to protect RNA from degradation, so that RNA can be delivered to the target site and promote cell absorption and processing of target cells. For delivery effectiveness, in addition to molecular composition, parameters such as particle size, charge or grafting with molecular parts (such as polyethylene glycol (PEG) or ligands) also play a role. It can be considered to reduce serum interactions with PEG grafting, improve serum stability and extend circulation time, which can help certain targeting methods. The ligand bound to the receptor at the target site can help improve the targeting effectiveness. In addition, PEGylation can be used for particle engineering. For example, if LNP is manufactured by mixing the aqueous phase of RNA with the organic phase of lipid, a certain fraction of PEG-conjugated lipids is required in the lipid mixture, otherwise particles will aggregate during the mixing step. It has been shown that by changing the molar fraction of the PEG-lipid containing PEG of different molar masses, the size of the particles can be adjusted. Similarly, the particle size can be adjusted by changing the molar mass of the PEG part of the PEGylated lipid. Typical sizes that can be obtained are 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles so formed have the additional advantage that, due to the PEG moiety, they interact less with serum components and have a longer circulation half-life, which is desirable in many drug delivery methods. Without PEG-lipid, particles with discrete sizes cannot be formed; the particles form large aggregates and precipitate.

[0004] Thus, for technologies where LNPs are formed from ethanol and aqueous phases, one of the primary roles of PEG-lipids is to facilitate particle self-assembly by providing a steric barrier at the surface of the nascent particles that form when nucleic acids are rapidly mixed in an ethanol solution containing lipids to bind RNA. PEG steric hindrance prevents inter-particle fusion and promotes the formation of a uniform LNP population, where LNPs with diameters <100 nm can be achieved.

[0005] PEG is the most widely used and gold standard "stealth" polymer in drug delivery. PEG-lipid is usually incorporated into the system to prepare a uniform and colloidally stable nanoparticle population due to its hydrophilic steric hindrance (PEG shell prevents electrostatic or van der Waals attraction that causes aggregation). PEGylation enables the water shell around the polymer to be attracted, protects RNA complexes from the opsonization of serum proteins, improves serum half-life and reduces rapid renal clearance, which improves pharmacokinetic behavior. The change in lipid acyl chain (C18, C16 or C14) length changes the stability of PEG-lipid incorporation in the particle, which leads to pharmacokinetic regulation. The use of PEG-lipids containing short (C14) acyl chains that dissociate from LNPs with a half-life of <30 minutes in vivo resulted in optimal hepatocyte gene silencing efficacy (Chen et al., 2014, J. Control Release 196: 106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669: 155-163). In addition, strict control of particle size can be obtained by varying PEG-lipid parameters: higher PEG MW or higher PEG-lipid mole fractions in the particles result in smaller particles.

[0006] In spite of these advantages, the PEGylation of nanoparticles can also lead to several effects that are unfavorable for the intended use of drug delivery. The PEGylation of known liposomes and LNPs reduces cellular uptake and endosomal escape, thereby ultimately reducing overall transfection efficiency. In fact, the PEG shell provides a spatial barrier for the effective combination of particles and cells, and also hinders endosomal release by preventing the membrane fusion between liposomes and the endosomal membrane. This is why the type of PEG-lipid and the amount of the PEG-lipid used must always be carefully adjusted. It should provide enough stealth effects in vivo and stable aspects on one hand, and do not hinder transfection on the other hand. This phenomenon is called "PEG dilemma (PEG Dilemma)".

[0007] In addition to reduced transfection efficiency, PEGylation is also associated with the accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and / or complement activation and storage diseases (Bendele et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; SM Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes in rats can significantly change the pharmacokinetic behavior of the second dose (when the 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 "accelerated blood clearance" (ABC) phenomenon was shown to be related to the PEG content of the liposomes. The presence of anti-PEG antibodies in plasma induces higher particle clearance through the mononuclear phagocyte system (MPS), which ultimately reduces the efficacy of the drug.

[0008] PEG is also thought to induce complement activation, which can lead to hypersensitivity reactions, also known as Complement-Activation Related Pseudo-Allergy (CARPA). It is still unclear from the literature whether the activation of complement is due to the presence of nanoparticles in general or to PEG in particular.

[0009] The presence of PEG in other lipid particles can also induce specific immune responses. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotides or plasmid DNA triggered a strong immune response, resulting in rapid blood clearance of subsequent doses in mice. The magnitude of this response is sufficient to induce significant morbidity and in some cases significant mortality. Rapid elimination of liposome-encapsulated oligodeoxynucleotides from the blood depends on the presence of PEG-lipids in the membrane, because the use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG-lipids eliminates this response. The generation of anti-PEG antibodies and assumed complement activation are possible explanations for the rapid elimination of vesicles from the blood (Semple et al., 2005, J. Pharmacol. Exp. Ther. 312 (3), 1020-6).

[0010] Since PEG can induce an immune response, it needs to be avoided for certain applications that require multiple injections. Some examples are therapies using mRNA, such as for protein replacement therapy. Here, the risk may be particularly high due to the potential inherent immunogenicity of RNA.

[0011] Therefore, there remains a need in the art for efficient methods and compositions for introducing RNA into cells that avoid the disadvantages associated with the use of PEG. The present disclosure addresses these and other needs.

[0012] The inventors unexpectedly found that the RNA LNP formulations described herein meet the above-mentioned requirements. In particular, it has been shown that the RNA LNP formulations comprising hydrophobic chains and poly Oxazoline (POX) polymers and / or poly Conjugates of oxazine (POZ) polymers are suitable components for assembling RNA LNPs. POX and POZ can be prepared by using unsubstituted or substituted 2- Oxazoline and 2- The POX / POZ conjugates enable RNALNPs to be manufactured using different techniques, resulting in defined surface properties and controlled size ranges. The preparation can be accomplished using robust methods that meet the requirements of pharmaceutical preparations. The particles can be end-functionalized with different moieties to adjust the charge or introduce specific molecular moieties such as ligands. Summary of the invention

[0013] In a first aspect, the present invention relates to a composition comprising lipid nanoparticles (LNPs), wherein the LNPs comprise: (i) RNA; (ii) a cationic lipid or a lipid that can ionize into a cation; and (iii) (a) a poly(vinylpyrrolidone) nanoparticle. Oxazoline polymer (POX) and / or poly Conjugates of polyoxazine (POZ) polymers and (b) one or more hydrophobic chains.

[0014] In some embodiments, the RNA LNP is a non-viral RNA particle.

[0015] In some embodiments, the total number of POX repeating units and / or POZ repeating units in the polymer is 2 to 200, e.g., 2 to 190, 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 ... In some embodiments, the total number of POX repeating units and / or POZ repeating units in the polymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120, or 10 to 100, such as 20 to 80, 30 to 70, or 40 to 50.

[0016] In some embodiments, the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains is a conjugate of (a) a POX polymer and / or a POZ polymer and (b) 1 or 2 hydrophobic chains.

[0017] In some embodiments, cationic lipids or lipids that can ionize cations are associated with RNA together with (a) POX polymers and / or POZ polymers and (b) conjugates of one or more hydrophobic chains to form particles. In some embodiments, lipids that can ionize cations are only positively charged at acidic pH and do not maintain cations at physiological pH. In some embodiments, LNPs include one or more additional lipids. In some embodiments, POX polymers and / or POZ polymers are conjugated to one or more hydrophobic chains through a linker. In some embodiments, one or more hydrophobic chains are one or more hydrocarbon groups, such as non-cyclic, preferably straight-chain hydrocarbon groups (e.g., straight-chain hydrocarbon groups with at least 10 carbon atoms), such as hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the linker includes a functional portion that connects one or more hydrophobic chains to POX polymers and / or POZ polymers, such as a cleavable portion (e.g., a portion that can be cleaved under physiological conditions). In some embodiments, the functional portion is neutral under physiological conditions; in certain embodiments, the complete linker is neutral under physiological conditions.

[0018] In some embodiments, the linker comprises at least one functional moiety. In some embodiments, the linker comprises an alkylene moiety (e.g., C ) substituted with at least one monovalent functional moiety and / or connected to a divalent functional moiety at the end where the alkylene is connected to one or more hydrophobic chains. 1-6 Alkylene moieties, such as C 1-3 The alkylene part or C 2-3In some embodiments, each monovalent functional moiety is independently selected from hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, aminosulfonate / ester, diselenide, sulfonamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate / ester, dithioate / ester, imidate / ester, imino, thioimidate / ester, sulfinamido, carbonate

[0043] In some embodiments, the present invention relates to an alkylene oxide, ... In some embodiments, each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioacid salt, imidate, imino, thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, ester, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties.

[0019] In some embodiments, the linker does not comprise a phosphate group. In some embodiments, the linker comprises at least one moiety selected from ester, thioether, disulfide, sulfone, orthoester, acylhydrazone, hydrazine, oxime, acetal, ketal, amino and amide moieties. In some embodiments, the linker comprises at least one divalent functional moiety selected from ester, thioether, sulfone, amino and amide moieties.

[0020] In some embodiments, the linker further comprises an alkylene moiety (preferably C 1-6 alkylene moiety).

[0021] In some embodiments, the conjugate may comprise the following structure (particularly if one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or POZ polymer, as shown, for example, in Formula (II) below): [(hydrophobic chain)-(divalent functional moiety)] 1-2 -(alkylene moiety)-(POX polymer and / or POZ polymer)).

[0022] In some embodiments, the conjugate may comprise the following structure (particularly if one or more hydrophobic chains are attached to the N-terminus (ie, the terminal N atom) of the POX polymer and / or POZ polymer, as shown, for example, in Formula (II) below): (hydrophobic chain) 1-2 -(alkylene moiety substituted with one or more monovalent functional moieties)-(POX polymer and / or POZ polymer).

[0023] In some embodiments, the conjugate may comprise the following structure (particularly if one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or the POZ polymer, as shown, for example, in Formula (II) below): [(hydrophobic chain) 2 -(trivalent functional moiety)]-(alkylene moiety)-(POX polymer and / or POZ polymer).

[0024] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or POZ polymer, such as, for example, embodiments shown in formula (II) below), the linker is selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)NH] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)-, [*-SS] p (C 1-6 Alkylene)-, [*-S(O) 2 ] p (C 1-6 Alkylene)-, [(*-O) r C(OR 25 ) 3-r ](C 1-6 Alkylene)-, [*-C(OR 25 ) 2 O] p(C 1-6 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-6 Alkylene)-, [*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-3 Alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-6 Alkylene)-, [*-N(R 26 )N(R 26 )] p -(C 1-6 alkylene)-, [*=C(=N(OH))] p (C 1-6 Alkylene)- and [*-OC(R 25 )(R 26 )O] p (C 1-6 Alkylene)-, where * represents the connection point between the linker and the hydrophobic chain; p is 1 or 2; C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); and r is an integer between 1 and 2. For example, the linker can be selected from the following: [*-NHC(O)] p (C 1-3 Alkylene)-, [*-C(O)NH] p (C 1-3 Alkylene)-, [*-C(O)O] p (C 1-3 Alkylene)-, [*-OC(O)] p (C 1-3 Alkylene)-, [*-S] p (C 1-3 Alkylene)-, [*-SS] p (C 1-3 Alkylene)-, [*-S(O) 2 ] p (C 1-3 Alkylene)-, [(*-O) r -C(OR25 ) 3-r ](C 1-3 Alkylene)-, [*-C(OR 25 ) 2 O] p (C 1-3 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-3 Alkylene)-, [*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-3 Alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-3 Alkylene)-, [*-N(R 26 )N(R 26 )] p -(C 1-3 alkylene)-, [*=C(=N(OH))] p (C 1-3 Alkylene)- and [*-OC(R 25 )(R 26 )O] p (C 1-3 Alkylene)-, where * represents the connection point between the linker and the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); and r is an integer between 1 and 2.

[0025] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or POZ polymer, such as, for example, embodiments shown in formula (II) below), the linker may be selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p(C 1-6 Alkylene)-, [*-SS] p (C 1-6 Alkylene)-, [*-S(O) 2 ] p (C 1-6 Alkylene)-, [(*-O) r C(OR 25 ) 3-r ](C 1-6 Alkylene)-, [*-C(OR 25 ) 2 O] p (C 1-6 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-6 Alkylene)-, [*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-6 Alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-6 Alkylene)-, [*-N(R 26 )N(R 26 )] p -(C 1-6 alkylene)-, [*=C(=N(OH))] p (C 1-6 Alkylene)- and [*-OC(R 25 )(R 26 )O] p (C 1-6 Alkylene)-, or selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)-, [*-SS] p (C 1-6 Alkylene)-, [*-S(O) 2 ] p (C 1-6 Alkylene)-, [(*-O) r C(OR 25) 3-r ](C 1-6 Alkylene)-, [*-C(OR 25 ) 2 O] p -(C 1-6 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-6 Alkylene)-, [*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-6 Alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-6 Alkylene)-, [*-N(R 26 )N(R 26 )] p (C 1-6 alkylene)-, [*=C(=N(OH))] p (C 1-6 Alkylene)- and [*-OC(R 25 )(R 26 )O] p (C 1-6 Alkylene)-, for example selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)- and [*-S(O) 2 ] p (C 1-6 Alkylene)-, for example selected from [*-NHC(O)] p (C 1-6 alkylene)- and [*-C(O)O] p (C 1-6 In some embodiments, the linker can be selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)NH] p (C 1-6 Alkylene)-, [*-C(O)O] p (C1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)- and [*-S(O) 2 ] p (C 1-6 Alkylene)-, preferably selected from the following: [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)- and [*-S(O) 2 ] p (C 1-6 In any of the above embodiments, preferably C 1-6 Alkylene is C 1-3 Alkylene, such as methylene, ethylene or propylene.

[0026] In some embodiments, R 25 Selected from C 1-3 Alkyl, phenyl and phenyl (C 1-3 Alkyl), for example selected from methyl, ethyl, phenyl, benzyl and phenethyl.

[0027] In some embodiments, R 26 Selected from H, C 1-3 Alkyl, phenyl and phenyl (C 1-3 Alkyl), for example selected from H, methyl, ethyl, phenyl, benzyl and phenethyl.

[0028] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or POZ polymer, such as, for example, those shown in Formula (II) below), the linker may be selected from the following: *-NHC(O)-(CH 2 )-, *-NHC(O)-(CH 2 ) 2 -, *-C(O)NH-(CH 2 )-, *-C(O)NH-(CH 2 ) 2 -、-(CH 2 )-CH(OC(O)-*)(CH 2 OC(O)-*)、-(CH 2)-CH(S-*) 2 、-(CH 2 )-CH(S-*)-CH 2 (S-*), *-S-(CH 2 ) 3 -,*-S(O) 2 -(CH 2 ) 3 -, and *-OC(O)-(CH 2 )-, the preferred linker is *-NHC(O)-(CH 2 )- or *-NHC(O)-(CH 2 ) 2 -, where * indicates the connection point between the linker and the hydrophobic chain.

[0029] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the N-terminus (i.e., the terminal N atom) of the POX polymer and / or POZ polymer, such as, for example, embodiments shown in formula (II) below), the linker may be selected from the following: [*-] 2 NC(O)(C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-OP(O) 2 O(C 1-6 Alkylene)NHC(O)-(C 1-6 Alkylene)-, [*-OC(O)] p (C 1-6 Alkylene)-OP(O) 2 O(C 1-6 Alkylene)NHC(O)-(C 1-6 alkylene)- and [*-C(O)] p (C 1-6 alkylene)-C(O)-, wherein * represents the point of attachment of the linker to the hydrophobic chain; p is 1 or 2; and C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2). For example, the linker can be selected from the following: [*-] 2 NC(O)(C 1-2 Alkylene)-, [*-OC(O)] 2 (C 2-4 Alkylene)-OP(O) 2 O(C 2-3 Alkylene)NHC(O)-(C 1-2 Alkylene)- and *-C(O)(C 2-3 alkylene)-C(O)-, wherein * indicates the point of attachment of the linker to the hydrophobic chain.

[0030] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the C-terminus (i.e., the terminal C atom) of the POX polymer and / or POZ polymer, such as, for example, embodiments shown in Formula (II') below), the linker comprises at least one bifunctional moiety through which the one or more hydrophobic chains are attached to the POX polymer and / or POZ polymer. In some embodiments, the linker may additionally comprise an alkylene moiety (e.g., C 1-6 Alkylene moieties, such as C 1-3 Alkylene moiety), cycloalkylene moiety (preferably C 3-8 Cycloalkylene, such as C 3-6 Cycloalkylene moiety) or cycloalkenylene moiety (preferably C 3-8 Cycloalkenylene, such as C 3-6cycloalkenylene moieties), each of which connects a bifunctional moiety to the POX polymer and / or the POZ polymer (directly to a terminus of the POX polymer and / or the POZ polymer or, preferably, through another bifunctional moiety). For example, one hydrophobic chain can be linked to the end of the POX polymer and / or POZ polymer through one bifunctional moiety (directly or through an alkylene, cycloalkylene or cycloalkenylene moiety, or through an alkylene, cycloalkylene or cycloalkenylene moiety carrying another bifunctional moiety); two hydrophobic chains can be linked to the end of the POX polymer and / or POZ polymer through two bifunctional moieties (which in turn are preferably linked to an alkylene, cycloalkylene or cycloalkenylene moiety, or to an alkylene, cycloalkylene or cycloalkenylene moiety carrying another bifunctional moiety); or two hydrophobic chains can be linked to the end of the POX polymer and / or POZ polymer through the same bifunctional moiety (which is then followed by a trifunctional moiety and which can be linked to the end of the POX polymer and / or POZ polymer directly or through an alkylene, cycloalkylene or cycloalkenylene moiety, or to an alkylene, cycloalkylene or cycloalkenylene moiety carrying another bifunctional moiety). In some embodiments, each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioester, dithioester, imidoester, imino, thioimidoester, sulfamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamoyl ester, carbamoyl ester, carbamate, dithiocarbamate, dithiocarbamoyl ester, thiocarbamoyl ester, thiocarbamate, thiocarbamoyl ester, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. In some preferred embodiments (particularly those in which one or more hydrophobic chains are attached to the C-terminus (i.e., the terminal C atom) of the POX polymer and / or POZ polymer, such as, for example, the embodiment shown in Formula (II') below), the linker comprises at least one divalent functional portion selected from amide, thioether, sulfone and amino moieties.

[0031] In some embodiments, the cycloalkylene moiety is C 3-8 Cycloalkylene, such as C 3-6 Cycloalkylene, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, wherein the cycloalkylene portion is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents (e.g., independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl).

[0032] In some embodiments, the cycloalkenylene moiety is C 3-8 Cycloalkenylene, such as C 3-6 Cycloalkenylene, such as cyclopropene, cyclobutene, cyclopentene, cyclooctenene, wherein the cycloalkenylene portion is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents (e.g., independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl).

[0033] In some embodiments, the alkylene moiety is C 1-6 Alkylene, such as C 1-3 Alkylene, such as methylene, ethylene or trimethylene, or C 2-3 Alkylene.

[0034] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the C-terminus (i.e., the terminal C atom) of the POX polymer and / or POZ polymer, such as, for example, those shown in Formula (II') below), the conjugate comprises one of the following structures (and may have the general formula (II')):

[0035] (Hydrophobic chain)-(Divalent functional part)-(POX polymer and / or POZ polymer)

[0036] [(Hydrophobic chain)-(Divalent functional part)] 1-2 -(Alkylene moiety)-(Divalent functional moiety)-(POX polymer and / or POZ polymer)

[0037] (Hydrophobic chain)-(Divalent functional moiety)-(Cycloalkylene moiety)-(Divalent functional moiety)-(POX polymer and / or POZ polymer)

[0038] (Hydrophobic chain)-(Divalent functional moiety)-(Cycloalkenylene moiety)-(Divalent functional moiety)-(POX polymer and / or POZ polymer)

[0039] (Hydrophobic chain)-(Divalent functional part)-(Alkylene part)-(POX polymer and / or POZ polymer)

[0040] [(Hydrophobic chain) 2 -(trivalent functional moiety)]-(alkylene moiety)-(divalent functional moiety)-(POX polymer and / or POZ polymer)

[0041] In some embodiments (particularly those in which one or more hydrophobic chains are attached to the C-terminus (i.e., the terminal C atom) of the POX polymer and / or POZ polymer, such as, for example, those shown in Formula (II') below), the conjugate has one of the following formulae (and may fall within Formula (II')):

[0042] (Hydrophobic chain)-(Divalent functional part)-(POX polymer and / or POZ polymer)-(Terminal group)

[0043] [(Hydrophobic chain)-(Divalent functional part)] 1-2 -(Alkylene moiety)-(Divalent functional moiety)-(POX polymer and / or POZ polymer)-(Terminal group)

[0044] (Hydrophobic chain)-(Divalent functional part)-(Cycloalkylene part)-(Divalent functional part)-(POX polymer and / or POZ polymer)-(Terminal group)

[0045] (Hydrophobic chain)-(Divalent functional moiety)-(Cycloalkenylene moiety)-(Divalent functional moiety)-(POX polymer and / or POZ polymer)-(Terminal group)

[0046] (Hydrophobic chain)-(Divalent functional part)-(Alkylene part)-(POX polymer and / or POZ polymer)-(Terminal group)

[0047] [(Hydrophobic chain) 2 -(trivalent functional moiety)]-(alkylene moiety)-(divalent functional moiety)-(POX polymer and / or POZ polymer)-(terminal group)

[0048] The POX polymer and / or POZ polymer may comprise a neutral end group (e.g., H, alkyl, alkoxy, ester, or amide end group) or a functionalized end group (e.g., hydroxyl, thiol, cyano, azido, or amino end group). In some embodiments, the end group is as described herein (particularly with respect to any one of the following formulas:

[0049] (II), (II'), (IIa), (IIa'), (IIb), (IIb'), (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi'), (IIj'), (III), (III'), (IIIa), (I lIa'), (IIIb), (IIIb'), (VI), (VI'), (VIa), (VIa'), (VII), (VII'), (VIIa), (VIIa'), (VIII), (VIII'), (VIlla), (IX), (IX'), and (IXa)

[0050] Or about any of the following:

[0051] R is defined as (IIe), (IVc), (XV), (XV'), (XVI), (XVII), (XVII'), (XVIII), (XVIII'), (XIX), (XIX'), (XX'), (XXI'), (XXII'), or (XXIII')) 3 or as used herein (particularly with respect to any of the following formulae:

[0052] (IV), (IV'), (IVa), (IVb), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (V), and (V')

[0053] or R defined in formula (IVc) 6 .

[0054] In some embodiments, particularly those in which the hydrophobic chain / linker is attached to the C-terminus, the linker may be selected from the following: [*-Z] p (C 1-6 Alkylene)-Z-, *-Z-(C 3-8 Cycloalkylene)-Z-, *-Z-(C 3-8 Cycloalkenylene)-Z-, (*=N)(C 1-6 Alkylene)-Z-, *-Z-(C 1-6 alkylene)- and *-Z-, wherein * represents the point of attachment to the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-8 Cycloalkylene and C 3-8 Each of the cycloalkenylene groups is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z is independently selected from the following: -OP(O) 2 O(C 1-3 Alkylene) NH-, -NH(C 1-3 Alkylene)OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 -and-NR 22 -, where R 22 is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl. For example, the linker can be selected from the following: [*-C(O)O] p (C 1-6Alkylene)-Z-, (*-NH)(C 1-6 Alkylene)-Z-, (*=N)(C 1-6 Alkylene)-Z-, (*-NH)C(O)(C 1-6 Alkylene)-Z-, (*-C(O)NH(C 1-6 Alkylene)-Z-, (*-NH)C(O)(C 1-6 Alkylene)-, (*-C(O)NH(C 1-6 Alkylene)-, (*-NH)C(O)-, *-C(O)NH-, *-Z-(C 3-8 Cycloalkenylene)-Z-, -S- and -S(O) 2 -, where * indicates the connection point with the hydrophobic chain; p is 1 or 2; C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-8 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and Z is selected from the following: -OP(O) 2 O(C 1-3 Alkylene) NH-, -NH(C 1-3 Alkylene)OP(O) 2 O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0055] In some embodiments, particularly those in which the hydrophobic chain / linker is attached to the C-terminus, the linker may be selected from the following: [*-Z] p (C 1-3 Alkylene)-Z-, *-Z-(C 3-6 Cycloalkylene)-Z-, *-Z-(C 3-6 Cycloalkenylene)-Z-, (*=N)(C 1-3 Alkylene)-Z-, *-Z(C 1-3 alkylene)- and *-Z-, wherein * represents the point of attachment to the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-6 Cycloalkylene and C 3-6 Each of the cycloalkenylene groups is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z is independently selected from the following: -OP(O) 2O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 -、-N(C 1-3 For example, the linker can be selected from the following: [*-C(O)O] p (C 1-3 Alkylene)-Z-, (*-NH)(C 1-3 Alkylene)-Z-, (*=N)(C 1-3 Alkylene)-Z-, (*-NH)C(O)(C 1-3 Alkylene)-Z-, (*-C(O)NH(C 1-3 Alkylene)-Z-, (*-NH)C(O)(C 1-3 Alkylene)-, (*-C(O)NH(C 1-3 Alkylene)-, (*-NH)C(O)-, *-C(O)NH-, *-Z-(C 3-6 Cycloalkenylene)-Z-, -S- and -S(O) 2 -, where * indicates the connection point with the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and Z is selected from the following: -OP(O) 2 O(C 1-2 Alkylene) NH-, -NH(C 1-2 Alkylene)OP(O) 2 O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0056] In some embodiments, particularly those in which the hydrophobic chain / linker is attached to the C-terminus, the linker may be selected from the following: [*-Z] p (C 1-3 Alkylene)-Z-, *-Z-(C 3-6 Cycloalkylene)-Z-, *-Z-(C 3-6 Cycloalkenylene)-Z-, (*=N)(C 1-3 Alkylene)-Z-, *-Z(C1-3 alkylene)- and *-Z-, wherein * represents the point of attachment to the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-6 Cycloalkylene and C 3-6 Each of the cycloalkenylene groups is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z is independently selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 -、-N(C 1-3 For example, the linker can be selected from the following: [*-C(O)O] p (C 1-3 Alkylene)-Z-, (*-NH)(C 1-3 Alkylene)-Z-, (*=N)(C 1-3 Alkylene)-Z-, (*-NH)C(O)(C 1-3 Alkylene)-Z-, (*-C(O)NH(C 1-3 Alkylene)-Z-, (*-NH)C(O)(C 1-3 Alkylene)-, (*-C(O)NH(C 1-3 Alkylene)-, (*-NH)C(O)-, *-C(O)NH-, *-Z-(C 3-6 Cycloalkenylene)-Z-, -S- and -S(O) 2 -, where * indicates the connection point with the hydrophobic chain; p is 1 or 2; C 1-3 Alkylene is divalent (if p is 1) or trivalent (if p is 2); C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and Z is selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2) 2 OP(O) 2 O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0057] In some embodiments, particularly those in which the hydrophobic chain / linker is attached to the C-terminus, the linker may be selected from the following: [*-C(O)] p (C 1-6 alkylene)-C(O)NH- and [*-] 2 N-, where * indicates the point of connection to the hydrophobic chain; p is 1 or 2; and C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2). For example, the linker can be *-C(O)(C 2-3 Alkylene)-C(O)NH- or [*-] 2 N-, where * indicates the point of attachment to the hydrophobic chain.

[0058] In some embodiments, particularly those in which the hydrophobic chain / linker is attached to the C-terminus, the linker may be selected from the following: (*-C(O)O)(CH(OC(O)-*))(CH 2 )-Z-, (*=N)(C 1-3 Alkylene)-NHC(O)-, (*-Z)(C 1-3 Alkylene)-Z-, *-Z-(C 3-6 Cycloalkenylene)-Z- and *-Z-, wherein * indicates the point of attachment to the hydrophobic chain; C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z is independently selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 - and -NH-. For example, the linker can be selected from the following: (*-C(O)O)(CH(OC(O)-*))(CH 2 )-Z-, (*=N)(C 1-3 Alkylene)-NHC(O)-, (*-NH)(C 1-3alkylene)-NHC(O)-, *-C(O)NH-, *-NHC(O)-, *-Z-(C 3-6 Cycloalkenylene)-Z-, -S- and -S(O) 2 -, where * indicates the connection point with the hydrophobic chain; C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and Z is selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0059] In some embodiments, the terminal groups of the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains on one side of a POX polymer and / or a POZ polymer are selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 、-SR 20 (e.g. SH), halogen, -CN, -N 3 、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs, wherein C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -COOCH 3 、-NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 , sugars, amino acids, peptides, and members of targeting pairs.

[0060] In some embodiments, the targeting pair is selected from the following pairs: antigen-antibody specific for the antigen; avidin-streptavidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α v β 3 Integrin; Asparagine-Glycine-Arginine (NGR) peptide-aminopeptidase N; Galactose-Asialoglycoprotein receptor. Thus, in some embodiments, the members of the targeting pair include one of the following: antigen, antibody, avidin, streptavidin, folic acid, transferrin, aptamer; RGD peptide; NGR peptide; and galactose.

[0061] In some embodiments, the terminal groups of the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains on one side of a POX polymer and / or a POZ polymer are selected from the following: H, C 1-3 Alkyl, -OR 20 、-N 3 、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , and members of a targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 、-COOH、-COOCH 3 、-NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , and members of the targeting pair; R 20 Selected from H, C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 、-COOH、-NR 22 R 23 , and members of the targeting pair; R 21 Selected from C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 、-COOH、-NR 22 R 23 , and members of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl, or R 22 and R 23 may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein C1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , -COOH, -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 , and members of a targeting pair.

[0062] In some embodiments, the end group of the conjugate on one side of the POX polymer and / or POZ polymer is selected from the group consisting of: H, C 1-3 Alkyl, -OR 20 、-N 3 、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , and members of the targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from -OH, -N 3 、-COOH、-COOCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , and members of the targeting pair; R 20 Selected from H and C 1-3 Alkyl; R 21 is C optionally substituted by one or more substituents 1-3 Alkyl, the substituents are independently selected from -OH, SH, halogen, -CN, -N 3 、-COOH、-NR 22 R 23 , and a member of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Alkynyl, where C 1-3 Alkyl, C 2-3Alkenyl and C 2-3 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkenyl, -COOH, -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 , and the member of the targeting pair, or R 22 and R 23 They may be linked together with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclic group.

[0063] In some embodiments, the end group of the conjugate on one side of the POX polymer and / or POZ polymer is selected from the group consisting of: H, C 1-3 Alkyl, -OH, -N 3 , C 2-6 Alkynyl, -COOH, -NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-NH(CH 2 CH 3 )、-NHC(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)CH 3 、-C(O)NH 2 、-C(O)NHCH 3 、-OC(O)(CH 2 ) 2 COOH, and a member of a targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from the group consisting of: -OH, -N 3 , C 2-6 Alkynyl, -COOH, -COOCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)NH(CH 2 )2 NH 2 , and members of the targeting pair.

[0064] In some embodiments, the POX polymer and / or POZ polymer comprises the following general formula (I):

[0065]

[0066] wherein a is an integer from 1 to 2; R 1 is an alkyl group, especially C 1-3 alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; and m is 2 to 200. In some embodiments, R 1 is the same at each occurrence (ie, in each repeating unit) (e.g., R 1 In each repeating unit, R may be methyl. In some embodiments, R in at least one repeating unit 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., ethyl), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl).

[0067] In some embodiments, the POX polymer and / or POZ polymer is a POX polymer and comprises repeating units of the following general formula (Ia):

[0068]

[0069] Where R 1 is the above definition of formula (I).

[0070] In some embodiments, the POX polymer and / or POZ polymer is a POZ polymer and comprises repeating units of the following general formula (Ib):

[0071]

[0072] Where R 1 is the above definition of formula (I).

[0073] In any of the above embodiments of formula (I), (Ia) and (Ib), m (i.e. the number of repeating units of formula (Ia) or formula (Ib) in the polymer) is preferably 2 to 180, for example 4 to 160, 6 to 140, 8 to 120 or 10 to 100.

[0074] In some embodiments, the POX polymer and / or POZ polymer is a copolymer comprising repeating units of the following general formulas (Ia) and (Ib):

[0075]

[0076] Where R 1 is the above definition of formula (I); the number of repeating units of formula (Ia) in the copolymer is 1 to 199; the number of repeating units of formula (Ib) in the copolymer is 1 to 199; and the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 200.

[0077] Some preferred embodiments of formula (I), (Ia) and (Ib) are given herein under the heading "Conjugates of POX and / or POZ polymers with one or more hydrophobic chains".

[0078] In some embodiments, the conjugate has the following general formula (II) or (II'):

[0079]

[0080] in:

[0081] a is an integer from 1 to 2;

[0082] R 1 is an alkyl group, especially C 1-3 Alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit;

[0083] m is 2 to 200;

[0084] R 2 YesR 4 or -L 1 (R 4 ) p , where each R 4 are independently a hydrocarbon group; L 1 is a linker; and p is 1 or 2; and

[0085] R 3 Selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 、-SR 20 , halogen, -CN, -N 3 、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs, wherein C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -COOCH 3 、-NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted with one or more (e.g., 1, 2, 3 or 4) substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 , sugars, amino acids, peptides, and members of targeting pairs.

[0086] In some embodiments, R 1 is the same at each occurrence (ie, in each repeating unit) (e.g., R 1 In each repeating unit, R may be methyl. In some embodiments, R in at least one repeating unit 1 Different from R in another repeating unit 1 (For example, for at least one repeating unit, R 1 is a specific alkyl group (e.g., ethyl), and for at least one different repeating unit, R 1 are different specific alkyl groups (eg, methyl groups).

[0087] In some embodiments, a is 1, ie, the conjugate has the following general formula (IIa) or (IIa'):

[0088]

[0089] In some embodiments, a is 2, ie, the conjugate has the following general formula (IIb) or (IIb'):

[0090]

[0091] In any of the above embodiments of Formula (IIa), (IIa'), (IIb) and (IIb'), R 1 , R 2 , R 3 and m as defined for formula (II) or (II'). Some preferred embodiments of formula (II), (II'), (IIa), (IIa'), (IIb) and (IIb') (e.g., formula (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi') and (IIj') and formula (IIe)) are given herein under the heading "Conjugates of POX and / or POZ polymers with one or more hydrophobic chains".

[0092] In certain embodiments, the conjugate has the following general formula (III) or (III'):

[0093]

[0094] in:

[0095] a is an integer from 1 to 2;

[0096] R 1 is methyl or ethyl and is independently selected for each repeat unit;

[0097] m is 10 to 100 (preferably 20 to 80, 30 to 70 or 40 to 50);

[0098] For formula (III), R 2 Selected from -L 1 R 4 、-(CH 2 )-CH(OC(O)R 4 )(CH 2 OC(O)R 4 )、-(CH 2 )-CH(SR 4 ) 2 and -(CH 2 )-CH(SR 4 )-CH 2 (SR 4 ), where each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; and L 1 Selected from *-NHC(O)-(CH 2 )-, *-NHC(O)-(CH 2 ) 2 -, *-C(O)NH-(CH 2 )-, *-C(O)NH-(CH 2 ) 2 -, *-S-(CH 2 ) 3 -,*-S(O) 2 -(CH 2 ) 3 - and *-OC(O)-(CH 2 )-(preferably L 1 Selected from *-NHC(O)-(CH 2 )-, *-NHC(O)-(CH 2 ) 2 -, *-C(O)NH-(CH 2 )- and *-C(O)NH-(CH 2 ) 2 -, for example *-NHC(O)-(CH 2 )- or *-NHC(O)-(CH 2 ) 2 -), where * indicates the same as R 4 or for formula (III'), R 2 Selected from (R 4 C(O)O)(CH(OC(O)R 4 ))(CH 2 )-Z-、(R 4) 2 N(C 1-3 Alkylene)-Z-, R 4 Z(C 1-3 Alkylene)-Z-, R 4 Z-(C 3-6 Cycloalkenylene)-Z- and R 4 Z-, where C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z is independently selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 - and -NH-; and R 3 Selected from the following: H, C 1-3 Alkyl, -OH, -N 3 , C 2-6 Alkynyl, -COOH, -NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-NH(CH 2 CH 3 )、-NHC(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)CH 3 、-C(O)NH 2 、-C(O)NHCH 3 、-OC(O)(CH 2 ) 2 COOH, and a member of a targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from the group consisting of: -OH, -N 3 , C 2-6Alkynyl, -COOH, -COOCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)NH(CH 2 ) 2 NH 2 , and members of the targeting pair.

[0099] In certain embodiments, the conjugate has the general formula (III) or (III'), wherein: a is an integer from 1 to 2;

[0100] R 1 is methyl or ethyl and is independently selected for each repeat unit;

[0101] m is 10 to 100;

[0102] For formula (III), R 2 Selected from (R 4 ) 2 NC(O)(C 1-2 Alkylene)-, R 4 C(O)OCH 2 CH(OC(O)R 4 )-CH 2 -OP(O) 2 O-(CH 2 ) 2 -NHC(O)-CH 2 - and R 4 -C(O)(CH 2 ) 2 -C(O)-, where each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; or for formula (III'), R 2 YesR 4 -C(O)(CH 2 ) 2 C(O)NH- or R 4 N-, where each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms, or a tocopherol moiety, wherein the same R 2 An R in part 4 is a tocopherol moiety; and

[0103] R 3 Selected from the following: H, C 1-3 Alkyl, -OH, -N 3 , C2-6 Alkynyl, -COOH, -NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-NH(CH 2 CH 3 )、-NHC(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)CH 3 、-C(O)NH 2 、-C(O)NHCH 3 、-OC(O)(CH 2 ) 2 COOH, and a member of a targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from the group consisting of: -OH, -N 3 , C 2-6 Alkynyl, -COOH, -COOCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)NH(CH 2 ) 2 NH 2 , and members of the targeting pair.

[0104] In some embodiments, a is 1, ie, the conjugate has the following general formula (IIIa) or (IIIa'):

[0105]

[0106] In some embodiments, a is 2, ie, the conjugate has the following general formula (IIIb) or (IIIb'):

[0107]

[0108] In any of the above embodiments of Formula (IIIa), (IIIa'), (IIIb) and (IIIb'), R 1 , R 2 , R 3and m are as defined for formula (III) or (III').

[0109] Some preferred embodiments of formula (III), (III'), (IIIa), (IIIa'), (IIIb) and (IIIb') are given herein under the heading "Conjugates of POX and / or POZ polymers with one or more hydrophobic chains".

[0110] In some embodiments, the conjugate has the following general formula (IV):

[0111] R 5 -POXZ-R 6

[0112] in:

[0113] R 5 YesR 7 or -L 2 (R 7 ) q , where each R 7 are independently a hydrocarbon group; L 2 is a linker; and q is 1 or 2;

[0114] POXZ is a copolymer containing repeating units of the following general formulae (Ia) and (Ib):

[0115]

[0116] Where R 1 Each of the 1-3 alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 199; the number of repeating units of formula (Ib) in the copolymer is 1 to 199; the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 200; and the repeating units of formula (Ia) and (Ib) are arranged randomly, periodically, alternatingly or in blocks; and

[0117] R 6 Selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 、-SR 20 , halogen, -CN, -N 3 、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs, wherein C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -COOCH 3 、-NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 Alkyl and 3 to 6 membered heterocyclic groups, wherein C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is optionally substituted with one or more substituents independently selected from -OH, SH, halogen, -CN, -N 3 , C 2-6 Alkynyl, -COOH, -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2, sugars, amino acids, peptides, and members of targeting pairs.

[0118] In certain embodiments, the conjugate has the following general formula (V):

[0119] R 5 -POXZ-R 6

[0120] in:

[0121] When attached to the N-terminus of the POXZ copolymer, R 5 Select from the following: -L 2 R 7 、-(CH 2 )-CH(OC(O)R 7 )(CH 2 OC(O)R 7 )、-(CH 2 )-CH(SR 7 ) 2 and -(CH 2 )-CH(SR 7 )-CH 2 (SR 7 ), where each R 7 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; and L 2 Selected from the following: *-NHC(O)-(CH 2 )-, *-NHC(O)-(CH 2 ) 2 -, *-C(O)NH-(CH 2 )-, *-C(O)NH-(CH 2 ) 2 -, *-S-(CH 2 ) 3 -,*-S(O) 2 -(CH 2 ) 3 - and *-OC(O)-(CH 2 )-(preferably L 2 Selected from *-NHC(O)-(CH 2 )-, *-NHC(O)-(CH 2 ) 2 -, *-C(O)NH-(CH 2 )- and *-C(O)NH-(CH 2 ) 2 -, for example *-NHC(O)-(CH 2 )- or *-NHC(O)-(CH 2 ) 2 -) where * indicates the same as R7 or when attached to the C-terminus of the POXZ copolymer, R 5 Selected from the following: (R 7 C(O)O)(CH(OC(O)R 7 ))(CH 2 )-Z 1 -、(R 7 ) 2 N(C 1-3 Alkylene)-Z 1 -、R 7 Z 1 (C 1-3 Alkylene)-Z 1 -、R 7 Z 1 -(C 3-6 Cycloalkenylene)-Z 1 - and R 7 Z 1 -, where C 3-6 The cycloalkenylene group is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from -OH, =O, -SH, halogen, -CN, -N 3 and C 1-3 alkyl; and each Z 1 Independently selected from the following: -OP(O) 2 O(CH 2 ) 2 NH-、-NH(CH 2 ) 2 OP(O) 2 O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O) 2 - and -NH-;

[0122] POXZ is a copolymer containing repeating units of the following general formulae (Ia) and (Ib):

[0123]

[0124] Where R 1Each of is independently methyl or ethyl and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 99; the number of repeating units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 10 to 100 (preferably 20 to 80, 30 to 70 or 40 to 50); and the repeating units of formula (Ia) and (Ib) are arranged randomly, periodically, alternatingly or in blocks; and R 6 Selected from the following: H, C 1-3 Alkyl, -OH, -N 3 , -COOH, -NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-NH(CH 2 CH 3 )、-NHC(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)(CH 2 ) 2 COOH, -N(CH 2 CH 3 )C(O)CH 3 、-C(O)NH 2 、-C(O)NHCH 3 、-OC(O)(CH 2 ) 2 COOH, and a member of a targeting pair, where C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from the group consisting of: -OH, -N 3 、-COOH、-COOCH 3 、-NH 2 、-NHCH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)NH(CH 2 ) 2 NH 2 , , and members of the targeting pair.

[0125] Some preferred embodiments of formula (IV) and (V) and conjugates of POX polymers and / or POZ polymers with one or more hydrophobic chains (as well as other preferred embodiments of formula (IV'), (V'), (VI), (VI'), (VIa), (VIa'), (VII), (VII'), (VIIa'), (VIII), (VIII'), (VIIIa), (IX), (IX'), and (IXa) and formula (IVc), (XV), (XV'), (XVI), (XVII), (XVII'), (XVIII), (XVIII'), (XIX), (XIX'), (XX'), (XXI'), (XXII'), and (XXIII')) and specific examples thereof are given herein under the heading "Conjugates of POX and / or POZ polymers with one or more hydrophobic chains".

[0126] In some embodiments, the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains inhibits aggregation of LNPs.

[0127] In some embodiments, the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains comprises about 0.25 mol % to about 50 mol % of the total lipids present in the LNP.

[0128] In some embodiments, the composition comprises substantially no lipids containing polyethylene glycol (PEG), and preferably comprises substantially no PEG.

[0129] In some embodiments, the lipid that is ionizable to release a cation comprises a head group comprising at least one nitrogen atom that is capable of being protonated under physiological conditions.

[0130] In some embodiments, the lipid that can ionize into a cation has the structure of Formula (X):

[0131]

[0132] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0133] L 10 and L 20 One is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR aC(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 10 and L 20 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or direct bond; G 1 and G 2 Each is independently unsubstituted C 1 -C 12 Alkylene or C 2-12 alkenylene;

[0134] G 3 It is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 Cycloalkenylene;

[0135] R a Is H or C 1-12 alkyl;

[0136] R 35 and R 36 Each independently is C 6-24 Alkyl or C 6-24 alkenyl;

[0137] R 37 Yes H, OR 50 、CN、-C(=O)OR 40 、-OC(=O)R 40 or -NR 50 C(=O)R 40 ;

[0138] R 40 It is C 1-12 alkyl;

[0139] R 50 Is H or C 1-6 Alkyl; and

[0140] x is 0, 1, or 2.

[0141] In some embodiments, the lipid that can ionize to give a cation is selected from structures X-1 to X-36 disclosed herein. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure X-3.

[0142] In some embodiments, the lipid that can ionize into a cation is selected from Structures A to G disclosed herein.

[0143] In some embodiments, the lipid that can ionize into a cation has the structure of Formula (XI):

[0144]

[0145] in

[0146] R 1 and R 2 Each of which is independently R 5 or -G 1 -L 1 -R 6 , where R 1 and R 2 At least one of them is -G 1 -L 1 -R 6 ;

[0147] R 3 and R 4 Each of which is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 Cycloalkyl;

[0148] R 5 and R 6 Each of is independently a non-cyclic hydrocarbon group having at least 10 carbon atoms;

[0149] G 1 and G 2 Each of which is independently unsubstituted C 1-12 Alkylene or C 2-12 alkenylene;

[0150] L 1 and L 2 Each of the following is independently selected from -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;

[0151] Ra is H or C1-12 alkyl;

[0152] m is 0, 1, 2, 3 or 4; and

[0153] x is 0, 1, or 2.

[0154] In some embodiments, the lipid that ionizes into a cation has structure (XIV-1), (XIV-2), or (XIV-3) disclosed herein.

[0155] In some embodiments, the cationic lipid or the lipid that can ionize to release cations comprises: 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N, N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

[0156] In some embodiments, the cationic lipid or lipid that can ionize into a cation comprises about 20 mol % to about 80 mol % of the total lipid present in the LNP.

[0157] In some embodiments, the LNP further comprises one or more additional lipids, preferably selected from phospholipids, steroids, and combinations thereof, more preferably the LNP comprises lipids that can ionize into cations, conjugates, phospholipids, and steroids.

[0158] In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, preferably selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), dioctano ... In some embodiments, the phospholipid is DOPE.

[0159] In some embodiments, the phospholipids comprise about 1 mol % to about 30 mol % of the total lipids present in the LNP.

[0160] In some embodiments, the steroid comprises a sterol, such as cholesterol.

[0161] In some embodiments, the steroid comprises about 10 mol % to about 60 mol % of the total lipids present in the LNP.

[0162] In some embodiments, cationic lipids or lipids that can ionize into cations account for about 20 mol% to about 70 mol% of the total lipids present in the LNP; the conjugate of (a) a POX polymer and / or a POZ polymer and (b) one or more hydrophobic chains accounts for about 0.5 mol% to about 15 mol% of the total lipids present in the LNP; the phospholipids account for about 5 mol% to about 25 mol% of the total lipids present in the LNP; and the steroids account for about 25 mol% to about 55 mol% of the total lipids present in the LNP.

[0163] In some embodiments, the LNPs have a size of about 30 nm to about 500 nm.

[0164] In some embodiments, the RNA is encapsulated within or associated with the LNP.

[0165] In some embodiments, the RNA is single-stranded RNA, such as mRNA.

[0166] In some embodiments, the RNA comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0167] In some embodiments, 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.

[0168] In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably interrupted by A nucleotides.

[0169] In some embodiments, the 5' cap is a Cap 1 or Cap 2 structure.

[0170] In some embodiments, the RNA encodes one or more peptides or proteins, wherein preferably, the one or more peptides or proteins are therapeutic peptides or proteins and / or comprise epitopes for inducing an immune response to an antigen in a subject.

[0171] In a second aspect, the invention relates to a method for delivering RNA to cells of a subject, the method comprising administering to the subject a composition of the first aspect.

[0172] In a third aspect, the invention relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject the composition of the first aspect, wherein the RNA encodes the therapeutic peptide or protein.

[0173] In a fourth aspect, the invention relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject the composition of the first aspect, wherein delivery of RNA to cells of the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the invention relates to a composition of the first aspect for use in a method for treating or preventing a disease or condition in a subject, wherein delivery of RNA to cells of the subject is beneficial for treating or preventing the disease or condition.

[0174] In a fifth aspect, the invention relates to a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject the composition of the first aspect, wherein the RNA encodes a therapeutic peptide or protein and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition. In a related aspect, the invention relates to a composition of the first aspect for use in a method for treating or preventing a disease or condition in a subject, wherein the RNA encodes a therapeutic peptide or protein and wherein delivery of the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition.

[0175] In some embodiments of the second to fifth aspects, the subject is a mammal, such as a human.

[0176] In a sixth aspect, the present invention provides a conjugate of (a) a POX polymer or a POZ polymer and (b) one or more hydrophobic chains, wherein the conjugate has the general formula (II) or (II') disclosed herein. In some embodiments, the conjugate of the sixth aspect has any one of the following formulae as disclosed herein:

[0177] (IIa), (IIa'), (IIb), (IIb'), (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi'), (I Ij'), (III), (III'), (IIIIIa), (IIIa'), (IIIb), (IIIb'), (VI), (VI'), (VIa), (VIa'), (V II), (VII'), (VIIa), (VIIa'), (VIII), (VIII'), (VIIIa), (IX), (IX'), (IXa), (II-1), (II -2),(II-3),(II-4),(II-5),(II-6),(II-7),(II-8),(II-9),(II-10),(II-11),(II-12) , (II-13), (II-14), (II-15), (II-16), (II-17), (II-18), (II-19), (II-20), (II-21), (I I-22),(II-23),(II-24),(II-25),(II-26),(II-27),(II-28),(II-29),(II-30),(II-31 ), (II-32), (II-33), (II-34), (II'-35), (II'-36), (II'-37), (II'-38), (II'-39), (II'- 40), (II'-41), (II'-42), (II'-43), (II'-44), (II'-45), (II'-46), (II'-47), and (II'-48)

[0178] Or any of the following:

[0179] (IIe), (IVc), (XV), (XV'), (XVI), (XVII), (XVIr), (XVIII), (XVIII'), (XIX), (XIX'), (XX'), (XXI') , (XXII'), (XXIII'), (II-49), (II-50), (II'-51), (II'-52), (II-53), (II-54), (II-55), and (II-56).

[0180] In a seventh aspect, the present invention provides conjugates of (a) a POX polymer and a POZ polymer and (b) one or more hydrophobic chains, wherein the POX polymer and the POZ polymer are copolymers comprising repeating units of the following general formulae (Ia) and (Ib):

[0181]

[0182] Where R 1 Each of the 1-3 alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 199; the number of repeating units of formula (Ib) in the copolymer is 1 to 199; and the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 200. In some embodiments, the conjugate of the seventh aspect has any one of formula (IV), (IV'), (IVa), (IVb), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (V), and (V') as disclosed herein or formula (IVc) as disclosed herein.

[0183] In an eighth aspect, the present invention provides a composition comprising (i) a nucleic acid (eg, DNA or RNA); (ii) a cationic lipid or a lipid that can ionize into a cation; and (iii) a conjugate of the sixth or seventh aspect.

[0184] In some embodiments of the eighth aspect, the conjugate comprises about 0.25 mol % to about 50 mol % of the total lipids present in the composition.

[0185] In some embodiments of the eighth aspect, the composition comprises essentially no lipids comprising polyethylene glycol (PEG), preferably essentially no PEG.

[0186] In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations comprises a head group, the head group comprising at least one nitrogen atom, the nitrogen atom being capable of being protonated under physiological conditions. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations has the structure of formula (X) shown herein. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations has the structure of formula (X) disclosed herein. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations is selected from structures X-1 to X-36 disclosed herein. In some embodiments of the eighth aspect, the lipid that can ionize out cations is a lipid with structure X-3. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations is selected from structures A to G disclosed herein. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations has the structure of formula (XI) disclosed herein. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out cations has structures (XIV-1), (XIV-2) or (XIV-3) disclosed herein. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize to release cations comprises: 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof. In some embodiments of the eighth aspect, the cationic lipid or the lipid that can ionize out a cation accounts for about 20 mol% to about 80 mol% of the total lipids present in the composition.

[0187] In some embodiments of the eighth aspect, the composition further comprises one or more additional lipids, preferably selected from phospholipids, steroids, and combinations thereof, more preferably the composition comprises lipids, conjugates, phospholipids and steroids that can ionize cations. In some embodiments of the eighth aspect, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, preferably selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), dioctanol ... Phosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), ditricosanoylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE) and diphytanoyl-phosphatidylethanolamine (DPyPE). In some embodiments, the phospholipid is DOPE. In some embodiments of the eighth aspect, the phospholipids account for about 1 mol% to about 30mol% of the total lipids present in the composition. In some embodiments of the eighth aspect, the steroid comprises a sterol, such as cholesterol. In some embodiments of the eighth aspect, the steroid accounts for about 10mol% to about 60mol% of the total lipids present in the composition.

[0188] In some embodiments of the eighth aspect, the cationic lipid or lipid that can ionize into a cation accounts for about 20 mol% to about 70 mol% of the total lipids present in the composition; the conjugate accounts for about 0.5 mol% to about 15 mol% of the total lipids present in the composition; the phospholipid accounts for about 5 mol% to about 25 mol% of the total lipids present in the composition; and the steroid accounts for about 25 mol% to about 55 mol% of the total lipids present in the composition.

[0189] In some embodiments of the eighth 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 lipid or the lipid that can ionize the cation and at least a portion of the conjugate, and, if present, at least a portion of one or more additional lipids. In some embodiments of the eighth aspect, the particles comprise or are selected from lipid nanoparticles (LNP), liposomes, lipid complexes (lipoplex, LPX), and mixtures thereof. In some embodiments of the eighth aspect, the particles comprise LNP or are LNP. In some embodiments of the eighth aspect, the particles comprise liposomes or are liposomes. In some embodiments of the eighth aspect, the particles comprise LPX or are LPX. In some embodiments of the eighth aspect, the particles comprise a mixture of LNP and liposomes or are a mixture of LNP and liposomes. In some embodiments of the eighth aspect, the particles comprise a mixture of LNP and LPX or are a mixture of LNP and LPX. In some embodiments of the eighth aspect, the particles comprise a mixture of liposomes and LPX or are a mixture of liposomes and LPX. In some embodiments of the eighth aspect, the particles comprise a mixture of LNP, liposomes and LPX or are a mixture of LNP, liposomes and LPX. In some embodiments of the eighth aspect, the particles comprise a mixture of LNP, liposomes and LPX or are a mixture of LNP, liposomes and LPX. In some embodiments of the eighth aspect, the particle has a size of about 30 nm to about 500 nm. In some embodiments of the eighth aspect, the nucleic acid is encapsulated within or associated with the particle.

[0190] In some embodiments of the eighth aspect, the nucleic acid is DNA or RNA, in particular RNA, such as single-stranded RNA, especially mRNA. In some embodiments of the eighth aspect, the RNA comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments of the eighth aspect, the RNA comprises at least one of the following, preferably all of the following: 5' cap; 5'UTR; 3'UTR; and poly-A sequence. In some embodiments of the eighth aspect, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted sequence of A nucleotides. In some embodiments of the eighth aspect, the 5' cap is a cap 1 or cap 2 structure.

[0191] In some embodiments of the eighth aspect, the nucleic acid (e.g., DNA or RNA, particularly mRNA) encodes one or more peptides or proteins, wherein preferably, the one or more peptides or proteins are therapeutic peptides or proteins and / or comprise epitopes for inducing an immune response to the antigen in a subject.

[0192] The composition of the eighth aspect can be used for: (i) a method for delivering a nucleic acid (e.g., DNA or RNA) to a subject's cells (the method comprises administering the composition of the eighth aspect to a subject); (ii) a method for delivering a therapeutic peptide or protein to a subject (the method comprises administering the composition of the eighth aspect to a subject, wherein the nucleic acid (e.g., DNA or RNA) encodes the therapeutic peptide or protein); (iii) a method for treating or preventing a disease or condition in a subject (the method comprises administering the composition of the eighth aspect to a subject, wherein delivering the nucleic acid (e.g., DNA or RNA) to a subject's cells is beneficial for treating or preventing the disease or condition); and / or (iv) a method for treating or preventing a disease or condition in a subject (the method comprises administering the composition of the eighth aspect to a subject, wherein the nucleic acid (e.g., DNA or RNA) encodes the therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition).

[0193] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth or seventh aspect may also be applicable to any embodiment of the eighth aspect (including the use of any embodiment of the eighth aspect).

[0194] In the ninth aspect, the present invention provides a method for transfecting cells, which includes adding the composition of the first or eighth aspect to the cell; and incubating the mixture of the composition and the cell for a sufficient amount of time. In some embodiments, particularly in those embodiments in which nucleic acid (such as DNA or RNA) encodes a pharmaceutically active protein, the mixture of the composition and the cell is incubated for a sufficient amount of time to allow the pharmaceutically active protein to be expressed. In some embodiments, the sufficient amount of time is at least one hour (for example, 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 (for example, up to about 36 hours or up to about 24 hours). In some embodiments of the ninth aspect, the method is performed in vivo (that is, the cell forms a part of an organ, tissue and / or organism of the object). In some embodiments of the ninth aspect, the method is performed in vitro (that is, the cell does not form a part of an organ, tissue and / or organism of the object, for example, the cell is an in vitro cell culture).

[0195] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect may also be applicable to any embodiment of the ninth aspect.

[0196] In a tenth aspect, the present disclosure provides the use of the composition of the first or eighth aspect for transfecting cells. In some embodiments of the tenth aspect, the use is an in vivo use (i.e., the cell forms a part of an organ, tissue, and / or organism of a subject). In some embodiments of the tenth aspect, the use is an in vitro use (i.e., the cell does not form a part of an organ, tissue, and / or organism of a subject, e.g., the cell is an ex vivo cell culture).

[0197] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect may also be applicable to any embodiment of the tenth aspect.

[0198] In another aspect, the disclosure provides a kit comprising the composition of the first or eighth aspect, the conjugate of the sixth or seventh aspect, or a pharmaceutical composition as described herein. In some embodiments, the kit is used for treatment, such as for inducing an immune response. In some embodiments, the kit is used for inducing an immune response against a pathogen, such as for treating or preventing an infectious disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] Figure 1 : Use unsubstituted (R = H) or substituted (R ≠ H) 2- A simplified protocol for the synthesis of POX via living cationic ring-opening polymerization of oxazoline compounds.

[0200] Figure 2 : Exemplary preparation and storage of RNA-LNP compositions.

[0201] Figure 3 :Poly-(2- Evaluation of the grafting percentage of four different poly-(2- The C14-PMeOx (C14-PMeOx 45-50 -OH; tetradecyl alkyl chain followed by poly-2-methyl-2- oxazoline); C14-PEtOx(C14-PEtOx 45-50 -OH; tetradecyl alkyl chain followed by poly-2-ethyl-2- oxazoline); C14-NHCO-PMeOx (C14-NHCO-PMeOx 45-50 -N 3 ; a tetradecyl alkyl chain connected to the poly-2-methyl-2- oxazoline); and bis-C14-COO-PMeOX (bis-C14-COO-PMeOx 45-50 -N 3 ; two tetradecyl alkyl chains, each linked by an ester bond to poly-2-methyl-2- LNPs (using different amounts of POX-lipid) were prepared and tested for the following parameters: (A) size; (B) accessible mRNA; and (C) in vitro luciferase expression.

[0202] Figure 4 : Effect of introducing a polar linker between the hydrophilic and hydrophobic blocks in POX lipids. Three different stealth grafted lipids were used to form LNPs: POX-lipid C14-PMeOx (C14-PMeOx 45-50 -OH; tetradecyl alkyl chain followed by poly-2-methyl-2- oxazoline); POX-lipid C14-NHCO-PMeOx (C14-NHCO-PMeOx 45-50 -N 3 ; a tetradecyl alkyl chain connected to the poly-2-methyl-2- and a reference stealth lipid C14-PSar (a tetradecyl alkyl chain followed by 23 units of poly(N-methyl-glycine)). The LNP formulations were analyzed for size, RNA accessibility, zeta potential, and in vitro luciferase expression.

[0203] Figure 5 : Determining the role of the end groups in POX-lipids. Four different POX-lipids were used to form lipid nanoparticles: C14-NHCO-PMeOx-N3 (a tetradecyl alkyl chain linked via an amide bond to an azide-terminated poly-2-methyl-2- C14-NHCO-PMeOx-NH (a tetradecyl alkyl chain connected by an amide bond to a poly-2-methyl-2- oxazoline blocks of 45 to 50 units); C14-NHCO-PMeOx-COOH (a tetradecyl alkyl chain connected by an amide bond to a carboxyl-terminated poly-2-methyl-2- blocks of 45 to 50 units of oxazoline); and the size, RNA accessibility, zeta potential, and in vitro luciferase expression of C14-NHCO-PMeOx-COOH / NH (a physical mixture of C14-NHCO-PMeOx-COOH and C14-NHCO-PMeOx-NH in a 1:1 ratio) P preparations were analyzed.

[0204] Figure 6 : Particle size and PDI of LNPs containing C14-PMeOX25-NH2 using HY501 as the cationic lipid. Also included is the particle size and PDI of BM_2 LNP formulation.

[0205] Figure 7 : Zeta potential of LNPs containing C14-PMeOX25-NH2 using HY501 as the cationic lipid. Also included is the zeta potential of the BM_2 LNP formulation.

[0206] Figure 8 : Accessible RNA of LNPs containing C14-PMeOX25-NH2 using HY501 as cationic lipid measured by Ribogreen assay.

[0207] Fig. 9 : Agarose gel electrophoresis images of LNPs containing C14-PMeOX25-NH2 using HY501 as the cationic lipid.

[0208] Fig.10 : Formation of terminal complement complex (SC5b-9) after incubation of human serum with LNP formulations containing C14-PMeOX25-NH2 and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0209] Fig.11 : Hemolytic analysis of human whole blood after incubation (under neutral pH conditions) with LNPs containing C14-PMeOX25-NH2 using HY501 as ionizable lipid.

[0210] Fig.12 : BM_2 and LNPs containing C14-PMeOX25-NH2 in skeletal muscle cell lines (C2C12) ( Fig.12 A, D), liver cancer cell line (HepG2) ( Fig.12 B, E) and mouse macrophage cell line (Raw cells) ( Fig.12 C, F) in vitro expression ( Fig.12 A to C) and viability ( Fig.12 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0211] Fig.13 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX25-COCH3 using HY501 as ionizable lipid.

[0212] Fig.14 : Zeta potential of BM_2 and LNPs containing C14-PMeOX25-COCH3 using HY501 as ionizable lipid.

[0213] Fig.15 : Accessible RNA of BM_2 and LNPs containing C14-PMeOX25-COCH3 using HY501 as ionizable lipid measured by Ribogreen assay.

[0214] Fig.16 : Agarose gel electrophoresis images of LNPs containing C14-PMeOX25-COCH3 using HY501 as ionizable lipid.

[0215] Fig.17 : Formation of terminal complement complex (SC5b-9) after incubation of human serum with LNP formulations containing C14-PMeOX25-COCH3 and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0216] Fig.18 : Hemolytic analysis of human whole blood after incubation (under neutral pH conditions) with BM_2 and LNPs containing C14-PMeOX25-COCH3 using HY501 as ionizable lipid.

[0217] Fig.19 : BM_2 and LNPs containing C14-PMeOX25-COCH3 in skeletal muscle cell lines (C2C12) ( Fig.19 A, D), liver cancer cell line (HepG2) ( Fig.19 B, E) and mouse macrophage cell line (Raw cells) ( Fig.19 In vitro mRNA expression in C and E Fig.19 A to C) and cell viability ( Fig.19 D to F). Firefly luciferase mRNA expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0218] Fig. 20 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX25-COOH using HY501 as ionizable lipid.

[0219] Fig.21 : Zeta potential of BM_2 and LNPs containing C14-PMeOX25-COOH using HY501 as ionizable lipid.

[0220] Fig. 22: Accessible RNA of LNPs containing C14-PMeOX25-COOH using HY501 as cationic lipid measured by Ribogreen assay.

[0221] Fig.23 : Agarose gel electrophoresis images of BM_2 and LNPs containing C14-PMeOX25-COOH using HY501 as ionizable lipid.

[0222] Fig.24 : Formation of terminal complement complex (SC5b-9) after incubation of human serum with LNP formulations containing C14-PMeOX25-COOH and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0223] Fig.25 : Hemolytic analysis of human whole blood after incubation (under neutral pH conditions) with BM_2 and LNPs containing C14-PMeOX25-COOH using HY501 as ionizable lipid.

[0224] Fig.26 : BM_2 and LNPs containing C14-PMeOX25-COOH in skeletal muscle cell lines (C2C12) ( Fig.26 A, D), liver cancer cell line (HepG2) ( Fig.26 B, E) and mouse macrophage cell line (Raw cells) ( Fig.26 C, F) in vitro expression ( Fig.26 A to C) and viability ( Fig.26 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0225] Fig. 27 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX25-OH using HY501 as ionizable lipid.

[0226] Fig.28 : Zeta potential of BM_2 and LNPs containing C14-PMeOX25-OH using HY501 as ionizable lipid.

[0227] Fig.29 : Accessible RNA of BM_2 and LNPs containing C14-PMeOX25-OH.

[0228] Fig.30 : Agarose gel electrophoresis images of BM_2 and LNPs containing C14-PMeOX25-OH.

[0229] Fig.31 : Formation of terminal complement complex (SC5b-9) after incubation of human serum with LNP formulations containing C14-PMeOX25-OH and controls. The horizontal dashed line shows the level of SC5b-9 formation with PBS.

[0230] Fig.32 : Hemolytic analysis of human whole blood after incubation with BM_2 and LNPs containing C14-PMeOX25-OH (under neutral pH conditions).

[0231] Fig.33 : BM_2 and LNPs containing C14-PMeOX25-OH in skeletal muscle cell lines (C2C12) ( Fig.33 A, D), liver cancer cell line (HepG2) ( Fig.33 B, E) and mouse macrophage cell line (Raw cells) ( Fig.33 C, F) in vitro expression ( Fig.33 A to C) and viability ( Fig.33 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0232] Fig.34 : (A) Accessible RNA measured by Ribogreen assay and (B) agarose gel electrophoresis images of LNPs containing POX-grafted lipids used for in vivo experiments.

[0233] Fig.35 : BM_3 and LNPs containing POX grafted lipids in skeletal muscle cell lines (C2C12) ( Fig.35 A, D), liver cancer cell line (HepG2) ( Fig.35 B, E) and mouse macrophage cell line (Raw cells) ( Fig.35 C, F) in vitro expression ( Fig.35 A to C) and viability ( Fig.35 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0234] Fig.36 : In vivo whole body bioluminescence image (BLI) at 6 hours (upper row), 24 hours (middle row) and ex vivo BLI at 6 hours (lower row) of animals (n=3) treated iv with the test formulation.

[0235] Fig.37: (A) In vivo luciferase expression 6 hours and 24 hours after iv injection of LNPs (from in vivo imaging). (B) Ex vivo luciferase expression 6 hours after iv injection of LNPs (from organ imaging).

[0236] Fig.38 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX45-NH2.

[0237] Fig.39 : Zeta potential of BM_2 and LNPs containing C14-PMeOX45-NH2.

[0238] Fig.40 : Accessible RNA of LNPs containing C14-PMeOX45-NH2 using HY501 as cationic lipid measured by Ribogreen assay.

[0239] Fig.41 : Agarose gel electrophoresis images of BM_2 and LNPs containing C14-PMeOX45-NH2.

[0240] Fig.42 : BM_2 and LNPs containing C14-PMeOX45-NH2 in skeletal muscle cell lines (C2C12) ( Fig.42 A, D), liver cancer cell line (HepG2) ( Fig.42 B, E) and mouse macrophage cell line (Raw cells) ( Fig.42 C, F) in vitro expression ( Fig.42 A to C) and viability ( Fig.42 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0241] Fig.43 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX45-COOH.

[0242] Fig.44 : Zeta potential of BM_2 and LNPs containing C14-PMeOX45-COOH.

[0243] Fig.45 : Accessible RNA of LNPs containing C14-PMeOX45-COOH using HY501 as ionizable lipid measured by Ribogreen assay.

[0244] Fig.46: Agarose gel electrophoresis images of LNPs containing C14-PMeOX45-COOH using HY501 as ionizable lipid.

[0245] Fig.47 : BM_2 and LNPs containing C14-PMeOX45-COOH in skeletal muscle cell lines (C2C12) ( Fig.47 A, D), liver cancer cell line (HepG2) ( Fig.47 B, E) and mouse macrophage cell line (Raw cells) ( Fig.47 C, F) in vitro expression ( Fig.47 A to C) and viability ( Fig.47 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0246] Fig.48 : Particle size and PDI of BM_2 and LNPs containing C14-PMeOX45-OH.

[0247] Fig.49 : Zeta potential of BM_2 and LNPs containing C14-PMeOX45-OH using HY501 as cationic lipid.

[0248] Fig.50 : Accessible RNA of LNPs containing C14-PMeOX45-OH using HY501 as cationic lipid measured by Ribogreen assay.

[0249] Fig.51 : Agarose gel electrophoresis images of LNPs containing C14-PMeOX25-OH using HY501 as ionizable lipid.

[0250] Fig.52 : BM_2 and LNPs containing C14-PMeOX45-OH in skeletal muscle cell lines (C2C12) ( Fig.52 A, D), liver cancer cell line (HepG2) ( Fig.52 B, E) and mouse macrophage cell line (Raw cells) ( Fig.52 C, F) in vitro expression ( Fig.52 A to C) and viability ( Fig.52 D to F). Firefly luciferase expression 24 hours after incubation with 12.5 ng (black bars), 25 ng (grey bars) and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0251] Fig.53: Particle size and PDI of BM_2 and LNPs containing C14-PMeOX45-NH-AC.

[0252] Fig.54 : Zeta potential of BM_2 and LNPs containing C14-PMeOX45-NH-AC using HY501 as cationic lipid.

[0253] Fig.55 : Accessible RNA of LNPs containing C14-PMeOX45-NH-AC using HY501 as cationic lipid measured by Ribogreen assay.

[0254] Fig.56 : Agarose gel electrophoresis images of LNPs containing C14-PMeOX45-NH-AC using HY501 as the ionizable lipid.

[0255] Fig.57 : Particle size and PDI of BM_2 and LNPs containing VE-PMeOX50-CH3.

[0256] Fig.58 : Zeta potential of BM_2 and LNPs containing VE-PMeOX50-CH3 using HY501 as cationic lipid.

[0257] Fig.59 : Accessible RNA of LNPs containing VE-PMeOX50-CH3 using HY501 as cationic lipid measured by Ribogreen assay.

[0258] Fig.60 : Agarose gel electrophoresis images of LNPs containing VE-PMeOX50-CH3 using HY501 as the ionizable lipid.

[0259] Sequence Description

[0260] The following table provides a listing of certain sequences referenced herein.

[0261] DETAILED DESCRIPTION

[0262] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing some specific embodiments, and are not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0263] Hereinafter, the elements of the present disclosure will be described in more detail. These elements are listed together with some specific embodiments, however, it should be understood that it can be combined in any way and in any number to produce other embodiments. The examples and preferred embodiments of the various descriptions should not be construed as limiting the present disclosure to only some embodiments clearly described. The description should be understood as supporting and covering the embodiments that clearly describe the embodiments and any number of disclosed and / or preferred element combinations. In addition, unless the context otherwise indicates, any arrangement and combination of all elements described in the application should be considered as disclosed by the specification of the application. For example, if in one embodiment the composition comprises the conjugate of formula (III) and in another embodiment the lipid that can ionize cations has one of structures XIV-1, XIV-2 and XIV-3, then in another embodiment the composition comprises the conjugate of formula (III) and has the lipid that can ionize cations of one of structures XIV-1, XIV-2 and XIV-3.

[0264] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel and H. ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0265] Unless otherwise indicated, the practice of the present disclosure will employ methods of conventional chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the art (see, 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; 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).

[0266] Unless otherwise specified herein or clearly contradictory to the context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure and is not intended to limit the scope of the additional protection of the present disclosure. The language in this specification should not be interpreted as indicating any unclaimed elements necessary for practicing the present disclosure.

[0267] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0268] Several documents are cited throughout the text of this specification. Whether above or below, each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, guidelines, etc.) is hereby incorporated by reference in its entirety. None of the content herein should be interpreted as admitting that the present disclosure is not entitled to predate such disclosure.

[0269] definition

[0270] Definitions applicable to all aspects of the present disclosure are provided below. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0271] Throughout this specification and the appended claims, unless the context requires otherwise, the words "comprises / comprising" and variations will be understood to imply the inclusion of the stated members, integers or steps or groups of members, integers or steps, but not the exclusion of any other members, integers or steps or groups of members, integers or steps. The term "consisting essentially of" is meant to exclude other members, integers or steps with any substantive meaning. The term "comprises / comprising" encompasses the term "consisting essentially of", which in turn encompasses the term "consisting of". Therefore, the term "comprises / comprising" may be replaced with the term "consisting essentially of" or "consisting of" at each occurrence in this application. Similarly, the term "consisting essentially of" may be replaced with the term "consisting of" at each occurrence in this application.

[0272] Unless otherwise indicated herein or clearly contradicted by context, nouns used in the context of describing the present disclosure (especially in the context of the claims) should be interpreted to cover one and / or more.

[0273] As used herein, "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is considered to be a specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each were individually listed herein.

[0274] In the context of the present disclosure, the term "about" means that ordinary technicians will understand to still ensure the accuracy interval of the technical effect of the feature in question. The term generally means ± 5%, ± 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 understood by those of ordinary skill in the art, the specific such deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have greater such deviations than artificial or engineered technical effects.

[0275] As used herein, terms such as "reduce / reduced" or "inhibit" mean the ability to cause an overall decrease in levels, such as a decrease of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

[0276] In some embodiments, terms such as "increase" or "enhance" refer to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0277] As used herein, "physiological pH" refers to a pH of about 7.4. In some embodiments, as used herein, "physiological pH" refers to neutral pH, ie, a pH of about 7.0.

[0278] As used herein, "physiological conditions" refers to conditions (particularly pH and temperature) of living subjects (particularly humans). Preferably, physiological conditions means physiological pH and / or temperature of about 37°C.

[0279] As used in this disclosure, "% w / v" refers to weight percent by volume, which is a concentration unit measuring the amount of solute in grams (g) as a percentage of the total volume of a solution in milliliters (mL).

[0280] "Weight %" or "% (w / w)" (or "% w / w") as used in this disclosure refers to weight percentage, which is a concentration unit measuring the amount of a substance (in grams (g)) expressed as a percentage of the total weight (in grams (g)) of the total composition.

[0281] "Mol %" as used in this disclosure is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0282] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Therefore, the ionic strength I is mathematically represented by the formula

[0283]

[0284] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is taken over all the different species of ions (i) in the solution.

[0285] According to the present disclosure, in some embodiments, the term "ionic strength" refers to the presence of monovalent ions. Regarding divalent ions, particularly the presence of divalent cations, in some embodiments, their concentration or effective concentration (presence of free ions) is low enough to prevent RNA degradation due to the presence of chelating agents. In some embodiments, the concentration or effective concentration of divalent ions is lower than the catalytic level for hydrolyzing the phosphodiester bonds between RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or substantially absent.

[0286] A "monovalent" compound refers to a compound having only one functional group of interest. For example, a monovalent anion refers to a compound having only one negatively charged group, preferably under physiological conditions.

[0287] A "divalent" or "dibasic" compound refers to a compound that has two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is Ca 2+ .

[0288] A "multivalent" or "polyvalent" compound refers to a compound having three or more functional groups of interest. For example, a polyvalent organic acid has three or more acid groups.

[0289] "Monovalent moiety" refers to a monovalent group, ie a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, and the like.

[0290] "Divalent moiety" or "bivalent moiety" refers to a diradical, i.e. a moiety having a valence of 2. Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, and the like. Another example of a divalent moiety is the group [*-S] p (C 1-6 Alkylene)-C 1-6 Alkylene moiety, if p is 1 (yielding group *-S(C 1-6 Alkylene)-, such as *-S-(CH 2 ) 6 -or*-S-CH 2 -, where * indicates the same as R 4 or R 7 connection point).

[0291] A "polyvalent moiety" refers to a polyradical, i.e. a moiety having a valence of at least 3. For example, a "trivalent moiety" refers to a triradical, i.e. a moiety having a valence of 3. For example, by removing an additional H atom of an alkylene group, the resulting alkylene group is trivalent. Another example of a trivalent moiety is the group [*-S] p (C 1-6 Alkylene)-C 1-6 Alkylene moiety, if p is 2 (yielding the group [*-S] 2 (C 1-6 alkylene)-, for example *-S-CH(S-*)(CH 2 ) 5 - or *-S-CH(S-*)(CH 2 )-, where * indicates the same as R 4 or R 7Another example of a trivalent moiety is the group (*=N)(C 1-3 Alkylene)-Z- in the N atom (where two hydrophobic chains are bonded to the N atom, which is in turn bonded to the C 1-3 alkylene moiety).

[0292] "Osmolarity" refers to the concentration of a solute, expressed as osmoles of solute per kilogram of solvent.

[0293] The term "freezing" relates to a phase change from liquid to solid. It usually occurs when the temperature of a system is lowered below the critical temperature and is accompanied by a characteristic change in the enthalpy of the system.

[0294] The term "lyophilization" or variations thereof refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure to allow the freezing medium in the substance to sublime directly from the solid phase to the gas phase.

[0295] The term "spray drying" refers to spray drying of a substance by mixing a (heated) gas with a fluid which is atomized (sprayed) in a vessel (spray dryer), wherein the solvent from the formed droplets evaporates, producing a dry powder.

[0296] The term "cryoprotectant" relates to substances added to the formulation to protect the active ingredient during the freezing phase.

[0297] The term "lyoprotectant" relates to substances added to the formulation to protect the active ingredient during the drying phase.

[0298] The term "reconstitution" involves adding a solvent (eg, water) to a dry product to return it to a liquid state, eg, its original liquid state.

[0299] The term "recombinant" in the context of the present disclosure means "prepared by genetic engineering." In some embodiments, a "recombinant object" in the context of the present disclosure does not occur in nature.

[0300] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0301] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15° C., preferably about 15° C. to about 35° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 17° C. to about 22° C. Such temperatures would include 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., and 22° C.

[0302] The term "alkyl" refers to a monovalent group of a saturated straight or branched hydrocarbon. Preferably, the alkyl group contains 1 to 14 (e.g., 1 to 12 or 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms, abbreviated as C 1-14 Alkyl, (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, abbreviated as C 1-10 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, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. "Substituted alkyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bonded to the alkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced by a substituent other than hydrogen (when replacing more than one hydrogen atom, the substituent may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent, a secondary substituent, or a tertiary substituent, as specified herein.Some examples of substituted alkyl groups include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0303] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Some exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH 3 )CH 2 -), 2,2-propylene (-C(CH 3 ) 2-) and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-isopentylene, 1,1-sec-pentylene, 1,1-neopentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2 The isomers of the alkylene radicals are 1,1-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene and 1,1-isohexylene), 1,1-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), 1,1-octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene and 1,1-isooctylene), etc. A straight-chain alkylene moiety having at least 3 carbon atoms and one free valence at each end may also be referred to as a multiple methylene group (e.g., 1,4-butylene may also be referred to as a tetramethylene group). In general, as an alternative to using the ending "ylene" for the alkylene moieties described above, the ending "diyl" can also be used (e.g., 1,2-butylene can also be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bonded to the alkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the alkylene 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 primary substituent, a secondary substituent, or a tertiary substituent, as specified herein.

[0304] The term "alkenyl" refers to a monovalent group of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is an odd number, the division result is rounded to the next integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. Preferably, an alkenyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in certain embodiments, an alkenyl group contains from 2 to 12 carbon atoms (abbreviated as C 2-12The term "alkenyl" refers to an alkylene group (e.g., 2 to 10) carbon atoms, and 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, 4 or 5) carbon-carbon double bonds, more preferably, it contains 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 bonds may be in cis (Z) or trans (E) configurations. Some exemplary alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 9-nonenyl, 10-nonenyl, 11-nonenyl, 12-nonenyl, 13-nonenyl, 14-nonenyl, 15-nonenyl, 16-nonenyl, 17-nonenyl, 18-nonenyl, 19-nonenyl, 20-nonenyl, 21-nonenyl, 22-nonenyl, 23-nonenyl, 24-nonenyl, 25-nonenyl, 26-nonenyl, 27-nonenyl, 28-nonenyl The double bond may be alkyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, etc. If the alkenyl group is attached to a nitrogen atom, the double bond may not be in the alpha position to the nitrogen atom. "Substituted alkenyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a tertiary substituent, as specified herein.

[0305] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in alkenylene can be equal to the integer calculated by dividing the number of carbon atoms in alkenylene by 2, and if the number of carbon atoms in alkenylene is an odd number, the division result is rounded to the next integer. For example, for alkenylene with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, alkenylene has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. Preferably, alkenylene comprises 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in certain embodiments, alkenylene comprises 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, 4 or 5) carbon-carbon double bonds, more preferably, it comprises 2 to 8 carbon atoms and 1, 2, 3 or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in cis (Z) or trans (E) configurations. Some exemplary alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propylene-1,2-diyl, 1-propylene-1,3-diyl, 1-propylene-2,3-diyl, allylene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. If the alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkenylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituent may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a tertiary substituent, as specified herein.

[0306] The term "alkynyl" refers to a monovalent group of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. In general, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to an integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is an odd number, the division result is rounded to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in certain embodiments, the alkynyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, 4 or 5 (preferably 1, 2 or 3)) carbon-carbon triple bonds, more preferably, it comprises 2 to 8 carbon atoms and 1, 2, 3 or 4 (preferably 1 or 2) carbon-carbon triple bonds, e.g., 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Some exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, etc. If the alkynyl group is attached to a nitrogen atom, the triple bond cannot be in the alpha position to the nitrogen atom. "Substituted alkynyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when replacing more than one hydrogen atom, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent, a secondary substituent, or a tertiary substituent, as specified herein.

[0307] The term "cycloalkyl" or "cycloaliphatic" refers to a cyclic non-aromatic form of "alkyl" and "alkenyl" having preferably 3 to 14 carbon atoms, for example 3 to 10 carbon atoms, i.e. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 3 to 7 carbon atoms. In some embodiments, the cycloalkyl group has 1, 2 or more (preferably 1 or 2) double bonds. Some exemplary cycloalkyls include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl and adamantyl. The term "cycloalkyl" is also intended to include its dicyclic and tricyclic forms. If a dicyclic is formed, it is preferred that each ring is connected to each other on two adjacent carbon atoms, but, or, two rings are connected by the same carbon atom, that is, they form a spirocyclic system or they form a "bridged" ring system. Some preferred examples of cycloalkyl include C 3 -C 8 Cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[4,5]decyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl (i.e. norbornyl), bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, bicyclo[4.2.0]octyl, bicyclo[4.3.0]nonyl, 1,2,3,4-tetrahydronaphthyl (i.e. tetralinyl) and bicyclo[4.4.0]decyl (i.e. decahydronaphthyl). "Substituted cycloalkyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when replacing more than one hydrogen atom, the substituent may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent, a secondary substituent, or a tertiary substituent, as specified herein.

[0308] The term "cycloalkylene" represents a cyclic non-aromatic form of "alkylene", and is a paired, ortho or separated diradical. In certain embodiments, cycloalkylene (i) is monocyclic or polycyclic (e.g., bicyclic or tricyclic) and / or (ii) is 3 to 14 yuan (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 yuan, e.g., 3 to 12 yuan or 3 to 10 yuan). In some embodiments, cycloalkylene is a mono-, bi- or tricyclic 3 to 14 yuan (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 yuan, e.g., 3 to 12 yuan or 3 to 10 yuan) cycloalkylene. Typically, as an alternative to using the ending "subunit" of the above-mentioned cycloalkylene moiety, the ending "diyl" (e.g., 1,2-cyclopropylene may also be referred to as cyclopropane-1,2-diyl) may also be used. Some exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantylene (e.g., tricyclo[3.3.1.1 3,7 ] decane-2,2-diyl). "Substituted cycloalkylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the cycloalkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene 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 primary substituent, a secondary substituent, or a tertiary substituent, as specified herein.

[0309] The term "cycloalkenylene" represents a cyclic non-aromatic form of "alkenylene", and is a paired, ortho or separated diradical. In general, the maximum number of carbon-carbon double bonds in cycloalkenylene can be equal to the integer calculated by dividing the number of carbon atoms in cycloalkenylene by 2, and if the number of carbon atoms in cycloalkenylene is an odd number, the division result is rounded to the next integer. For example, for cycloalkenylene with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, cycloalkenylene has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. In certain embodiments, cycloalkenylene (i) is monocyclic or polycyclic (e.g., bicyclic or tricyclic) and / or (ii) is 3 to 14 yuan (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 yuan, such as 3 to 12 yuan or 3 to 10 yuan). In some embodiments, cycloalkenylene is a mono-, bi- or tricyclic 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14-membered, such as 3 to 12-membered or 3 to 10-membered) cycloalkenylene. Some exemplary cycloalkenylenes include cyclohexene, cycloheptene, cyclopropene, cyclobutene, cyclopentene and cyclooctenes. "Substituted cycloalkenylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the cycloalkenylene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of cycloalkenylene are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-level substituents, second-level substituents or third-level substituents, as specified herein.

[0310] The term "aryl" or "aromatic ring" refers to a monovalent group of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9 or 10, such as 5, 6 or 10) carbon atoms which may be arranged in one ring (e.g., phenyl) or in two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropane, The term "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Some preferred examples are phenyl and naphthyl. Aryl does not include fullerenes. "Substituted aryl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the aryl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituent may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent or a third-level substituent as specified herein. Some examples of substituted aryl groups include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilino, 4-hydroxyphenyl, and methoxyphenyl (ie, 2-, 3-, or 4-methoxyphenyl).

[0311] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by O, S or N heteroatoms. Preferably, heteroaryl refers to a five- or six-membered aromatic monocyclic ring in which 1, 2 or 3 carbon atoms are replaced by the same or different O, N or S heteroatoms. Alternatively, it means an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4 or 5 carbon atoms are replaced by the same or different O, N or S heteroatoms. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, Azolyl, iso Azolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzo Azolyl, indole Indoxazinyl, Benzene oxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolyl, isoquinolyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxyl Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophene, pyridinyl, 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-membered heteroaryl groups include furanyl, thiophene, Azolyl, iso Azolyl, The term "substituted heteroaryl" refers to a group consisting of oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the heteroaryl, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituent may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, a second-level substituent, or a third-level substituent, as specified herein.

[0312] The term "heterocyclyl" or "heterocycle" means a cycloalkyl group as defined above in which 1, 2, 3 or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus or sulfur, preferably O, S or N. The heterocyclyl group preferably has 1 or 2 rings containing 3 to 10, for example 3, 4, 5, 6 or 7 ring atoms. Preferably, in each ring of the 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. The term "heterocyclyl" is also intended to encompass partially or fully hydrogenated forms (e.g. dihydro, tetrahydro or perhydro forms) of the above heteroaryl groups. Some exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also known as piperidyl), piperazinyl, dihydrofuranyl and tetrahydrofuranyl, dihydrothiophenyl and tetrahydrothiophenyl, dihydropyranyl and tetrahydropyranyl, hexamethylenetetramine (urotropinyl), lactones, lactams, cyclic imides, and cyclic anhydrides. "Substituted heterocyclyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the heterocyclyl, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl are replaced by a substituent other than hydrogen (when replacing more than one hydrogen atom, the substituent may be the same or different). Preferably, a substituent other than hydrogen is a primary substituent, a secondary substituent or a tertiary substituent, as specified herein.

[0313] The expression "partially hydrogenated form" of an unsaturated compound or group used herein means removing a portion of the unsaturation while not removing all the unsaturated portions by formally adding hydrogen to the initial unsaturated compound or group. The phrase "fully hydrogenated form" of an unsaturated compound or group used herein is interchangeable with the term "perhydro", and means removing all unsaturation by formally adding hydrogen to the initial unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl (comprising 2 double bonds in the ring, such as furan) includes a dihydro form of the 5-membered heteroaryl (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while a tetrahydro form of the 5-membered heteroaryl (e.g., tetrahydrofuran, i.e., THF) is a fully hydrogenated (or perhydro) form of the 5-membered heteroaryl. Likewise, for a 6-membered heteroaryl having 3 double bonds in the ring (e.g., pyridyl), partially hydrogenated forms include dihydro and tetrahydro forms (e.g., dihydropyridyl and tetrahydropyridyl), while the hexahydro form (in the case of heteroaryl pyridyl, e.g., piperidyl) is a fully hydrogenated (or perhydro) derivative of the 6-membered heteroaryl. Thus, if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms, the hexahydro form of the aryl or heteroaryl can only be considered as a partially hydrogenated form according to the present disclosure.

[0314] The term "aromatic" used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aromatic group is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as a cycloalkyl for the purposes of this disclosure. Similarly, if a bicyclic or polycyclic aromatic group (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for the purposes of this disclosure (even if one ring (e.g., in 1,2-dihydronaphthyl) is still aromatic). In the present application, there is a similar distinction between heteroaryl and heterocyclic groups. For example, for the purposes of this disclosure, dihydroindole, i.e., a dihydro variant of indolyl, is classified as a heterocyclic group because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0315] The term "optionally substituted" indicates that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms may be replaced by a group other than hydrogen (i.e., a primary substituent), such as an alkyl group (preferably C 1-6 Alkyl), alkenyl (preferably C 2-6 alkenyl), alkynyl (preferably C 2-6 alkynyl), aryl (preferably 6- to 14-membered aryl), heteroaryl (preferably 3- to 14-membered heteroaryl), cycloalkyl (preferably 3- to 14-membered cycloalkyl), heterocyclyl (preferably 3- to 14-membered heterocyclyl), halogen, -CN, azido, -NO 2 、-OR 71 、-N(R 72 )(R 73 )、-S(O) 0-2 R 71 、-S(O) 1-2 OR 71 、-OS(O) 1-2 R 71 、-OS(O) 1-2 OR 71 、-S(O) 1-2 N(R 72 )(R 73 )、-OS(O) 1-2 N(R 72 )(R 73 )、-N(R 71 )S(O) 1-2 R 71 、-NR 71 S(O) 1-2 OR 71 、-NR 71 S(O) 1-2N(R 72 )(R 73 )、-OP(O)(OR 71 ) 2 ,-C(=X 1 )R 71 ,-C(=X 1 )X 1 R 71 , -X 1 C(=X 1 )R 71 and -X 1 C(=X 1 )X 1 R 71 , and / or any two first-level substituents bound to the same carbon atom of a cycloalkyl or heterocyclic group may be linked together to form =X 1 , wherein each of the first-level substituents of alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl may itself be substituted with one or more (e.g., one, two or three) substituents (i.e., second-level substituents) selected from the following: C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl, halogen, -CF 3 , -CN, azido, -NO 2 、-OR 81 、-N(R 82 )(R 83 )、-S(O) 0-2 R 81 、-S(O) 1-2 OR 81 、-OS(O) 1-2 R 81 、-OS(O) 1-2 OR 81 、-S(O) 1- 2 N(R 82 )(R 83 )、-OS(O) 1-2 N(R 82 )(R 83 )、-N(R 81 )S(O) 1-2 R 81 、-NR 81 S(O) 1-2 OR 81 、-NR 81 S(O) 1-2 N(R 82 )(R 83)、-OP(O)(OR 81 ) 2 ,-C(=X 2 )R 81 ,-C(=X 2 )X 2 R 81 , -X 2 C(=X 2 )R 81 and -X 2 C(=X 2 )X 2 R 81 , and / or any two second-level substituents bonded to the same carbon atom of the cycloalkyl or heterocyclic group as the first-level substituent may be linked together to form =X 2 , where the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, and 3- to 14-membered heterocyclyl is optionally substituted with one or more (e.g., one, two, or three) substituents (i.e., third-level substituents) independently selected from the following: 1-3 Alkyl, halogen, -CF 3 , -CN, azido, -NO 2 、-OH、-O(C 1-3 Alkyl), -OCF 3 、-S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O) 2 NH 2-z (C 1-3 alkyl) z 、-C(=O)OH、-C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z, wherein each z is independently 0, 1 or 2 and each C 1-3 The alkyl group is independently methyl, ethyl, propyl or isopropyl, and any two 3rd-level substituents bonded to the same carbon atom of the 3- to 14-membered cycloalkyl or heterocyclyl group as the 2nd-level substituent may be linked together to form =O, =S, =NH or =N(C 1-3 alkyl);

[0316] in

[0317] R 71 , R 72 and R 73 Each independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, 3 to 7 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 7 membered heterocyclyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 3 to 7 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 7 membered heterocyclyl is optionally substituted with one, two or three substituents independently selected from the following: 1-3 Alkyl, halogen, -CF 3 , -CN, azido, -NO 2 、-OH、-O(C 1-3 Alkyl), -OCF 3 , =O, -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O) 2 NH 2-z (C 1-3 alkyl) z , -C(=O)(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl)z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl;

[0318] R 81 , R 82 and R 83 Each independently selected from H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 alkynyl, 3- to 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 6-membered heterocyclyl, wherein C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Each of the alkynyl, 3 to 6 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 6 membered heterocyclyl is optionally substituted with one, two or three substituents independently selected from the following: 1-3 Alkyl, halogen, -CF 3 , -CN, azido, -NO 2 、-OH、-O(C 1-3 Alkyl), -OCF 3 , =O, -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O) 2 NH 2-z (C 1-3 alkyl) z , -C(=O)(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl; and

[0319] X1 and X 2 Each independently selected from O, S and N (R 84 ), where R 84 Is H or C 1-3 alkyl.

[0320] Typical first-stage substituents are preferably selected from the following: C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, 6- to 14-membered (e.g., 6- to 10-membered) aryl, 3- to 14-membered (e.g., 5- or 6-membered) heteroaryl, 3- to 14-membered (e.g., 3- to 7-membered) cycloalkyl, 3- to 14-membered (e.g., 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO 2 、-OR 71 、-N(R 72 )(R 73 )、-S(O) 0-2 R 71 、-S(O) 1-2 OR 71 、-OS(O) 1-2 R 71 、-OS(O) 1-2 OR 71 、-S(O) 1-2 N(R 72 )(R 73 )、-OS(O) 1-2 N(R 72 )(R 73 )、-N(R 71 )S(O) 1-2 R 71 、-NR 71 S(O) 1-2 OR 71 ,-C(=X 1 )R 71 ,-C(=X 1 )X 1 R 71 , -X 1 C(=X 1 )R 71 and -X 1 C(=X 1 )X 1 R 71 , for example C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7-membered cycloalkyl, 3- to 7-membered (e.g., 5- or 6-membered) heterocyclyl, halogen, -CF 3 , -CN, azido, -NO 2、-OH、-O(C 1-3 Alkyl), -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O) 2 NH 2-z (C 1-3 alkyl) z 、-C(=O)OH、-C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl; wherein X 1 independently selected from O, S, NH and N(CH 3 ); and R 71 , R 72 and R 73 Each of which is as defined above, or preferably, independently selected from H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 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 is optionally substituted with one, two or three substituents independently selected from the following: 1-3 Alkyl, halogen, -CF 3 , -CN, azido, -NO 2 、-OH、-O(C 1-3 Alkyl), -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O)2 NH 2-z (C 1-3 alkyl) z 、-C(=O)OH、-C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl. In some embodiments, the first substituent is selected from C 1-3 Alkyl, phenyl, halogen, -CF 3 、-OH、-OCH 3 、-SCH 3 、-NH 2-z (CH 3 ) z , -C(=O)OH and -C(=O)OCH 3 , where z is 0, 1, or 2 and C 1-3 Alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, the first-level substituent is selected from methyl, ethyl, propyl or isopropyl, halogen (such as F, Cl or Br) and -CF 3 , such as halogen (such as F, Cl or Br) and -CF 3 .

[0321] Typical second-level substituents are preferably selected from C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =O, =S, -CF 3 , -CN, azido, -NO 2 、-OH、-O(C 1-3 Alkyl), -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 Alkyl), -S(O)2 NH 2-z (C 1-3 alkyl) z 、-C(=O)OH、-C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 Alkyl is independently methyl, ethyl, propyl or isopropyl. Some specific examples of second-level substituents are independently selected from the following: C 1-3 Alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, =O, =S, -CF 3 、-CN、-OH、-O(C 1-3 Alkyl), -S(C 1-3 Alkyl), -NH 2 、-NH(C 1-3 Alkyl), -N(C 1-3 alkyl) 2 、-NHS(O) 2 (C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C 1-3 Alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 Alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2 and each C 1-3 The alkyl group is independently methyl, ethyl, propyl or isopropyl. Particularly preferred secondary substituents are independently selected from methyl, ethyl, propyl, isopropyl, phenyl, =O and =S.

[0322] Typical tertiary substituents are preferably selected from C 1-3Alkyl, phenyl, halogen, -CF 3 、-OH、-OCH 3 、-SCH 3 、-NH 2-z (CH 3 ) z , -C(=O)OH and -C(=O)OCH 3 , where z is 0, 1, or 2, and C 1-3 The alkyl group is methyl, ethyl, propyl or isopropyl. Particularly preferred tertiary substituents are selected from methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl or Br) and -CF 3 , such as halogen (such as F, Cl or Br) and -CF 3 .

[0323] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising 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 connected to each other by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide", "polypeptide" and "protein" are often used as synonyms herein.

[0324] "Therapeutic peptide or protein" means a peptide or protein that can be used to treat an individual, the expression of which will be beneficial, such as in improving the symptoms of a disease. In some embodiments, the therapeutic peptide or protein, when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or disease state. In some embodiments, the therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, mitigate, reverse a disease or condition, delay the onset of a disease or condition, or mitigate the severity of one or more symptoms of a disease or condition. The therapeutic protein may have preventive properties and may be used to delay the onset of a disease or mitigate the severity of such a disease or pathological condition. The term "therapeutic peptide or protein" includes complete peptides or proteins, and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Some examples of therapeutically active proteins include, but are not limited to, antigens used for vaccination and immunostimulants (e.g., cytokines).

[0325] The term "portion" refers to a fraction. With respect to a specific structure, such as an amino acid sequence or a protein, the term "portion" thereof may refer to a continuous or discontinuous fraction of the structure.

[0326] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure (e.g., an amino acid sequence or a protein) refers to a continuous element of the structure. When used in the context of a composition, the term "portion" means a portion of a composition. For example, a portion of a composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0327] With respect to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of an amino acid sequence, i.e. a sequence representing an amino acid sequence shortened at the N-terminus and / or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of an open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of an open reading frame, as long as the truncated open reading frame comprises a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, 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. Fragments of an amino acid sequence comprise, for example, a sequence of, for example, up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of an amino acid sequence.

[0328] According to the present disclosure, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which the fragment is derived. A part or fragment of a peptide or protein preferably comprises at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 continuous amino acid sequences of a peptide or protein. A part or fragment of a peptide or protein preferably comprises at most 8, particularly at most 10, at most 12, at most 15, at most 20, at most 30 or at most 55 continuous amino acid sequences of a peptide or protein.

[0329] As used herein and with reference to an amino acid sequence (peptide or protein), a "variant" is intended to mean an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified form of a wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid differences.

[0330] "Wild type" or "WT" or "native" herein means an amino acid sequence that occurs in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

[0331] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0332] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of an amino acid sequence variant with insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product are also possible. Amino acid addition variants include one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids of amino and / or carboxyl terminal fusions. Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be in any position of the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its position. It is preferred to replace amino acids in positions in non-conserved amino acid sequences between homologous proteins or peptides and / or replace amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacements of one of the families of amino acids to which their side chains are associated. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid replacements include replacements within the following groups:

[0333] -Glycine, alanine;

[0334] - valine, isoleucine, leucine;

[0335] -Aspartic acid, glutamic acid;

[0336] - Asparagine, glutamine;

[0337] -serine, threonine;

[0338] - lysine, arginine; and

[0339] -Phenylalanine, tyrosine.

[0340] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids (contiguous amino acids in some embodiments). In some embodiments, the degree of similarity or identity is given for the total length of the reference amino acid sequence. Comparisons for determining sequence similarity, preferably sequence identity, can use tools known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap open (Gap Open) 10.0, gap extension (Gap Extend) 0.5 to complete.

[0341] "Sequence similarity" indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences.

[0342] The terms "same (%)" and "identity (%)" or similar terms are particularly intended to refer to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 28; (iii) the maximum match within the query range is set to 0; (iv) the match / mismatch score is set to 1, -2; (v) the gap cost is set to linear; and (vi) the filter for low complexity regions is being used.In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum match within the query range is set to 0; (iv) the matrix is ​​set to BLOSUM62; (v) the gap cost is set to existence 11: extension: 1; and (vi) the condition composition scoring matrix is ​​adjusted.

[0343] The percent identity is calculated by determining the number of identical positions in the sequences being compared, dividing that number by the number of positions being compared (eg, the number of positions in the reference sequence), and multiplying the result by 100.

[0344] In some embodiments, the degree of similarity or identity is given for a region that 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 full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (contiguous nucleotides in some embodiments). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0345] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0346] The amino acid sequence variants described herein can be easily prepared by a technician, for example, by recombinant DNA manipulation. DNA sequence manipulations for preparing peptides or proteins with substitutions, additions, insertions or deletions are described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be easily prepared with the help of known peptide synthesis techniques (e.g., such as by solid phase synthesis and similar methods).

[0347] In some embodiments, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to the functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent). With respect to antigens or antigenic sequences, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence comprising an amino acid sequence that has one or more amino acids changed compared to the amino acid sequence of a parent molecule or sequence and is still capable of achieving one or more functions of the parent molecule or sequence (e.g., inducing an immune response). In some embodiments, the modification in the amino acid sequence of a parent molecule or sequence does not significantly affect or change the properties of the molecule or sequence. In different embodiments, the function of a functional fragment or functional variant can be reduced but still significantly present, for example, the immunogenicity of a functional variant can be at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of a parent molecule or sequence. However, in other embodiments, the immunogenicity of a functional fragment or functional variant can be enhanced compared to a parent molecule or sequence.

[0348] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that antigens suitable for use herein may be altered so that their sequences are different from the naturally occurring or native sequences from which they are derived while retaining the desired activity of the native sequence.

[0349] In some embodiments, "isolated" means changed or removed (e.g., purified) from a natural state or from an artificial composition (e.g., a composition from a production process). For example, a nucleic acid (e.g., RNA), a peptide, or a polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide, or polypeptide partially or completely separated from coexisting substances in its natural state is "isolated". An isolated nucleic acid, peptide, or polypeptide may exist in a substantially purified form, or may exist in a non-natural environment, such as a host cell. In certain embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) used in the present disclosure exist in a substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of a nucleic acid (e.g., DNA or RNA, particularly mRNA) in a substantially purified form comprises a first buffer system.

[0350] The term "genetic modification" or simply "modification" includes transfecting cells with nucleic acids.

[0351] The term "transfection" refers to the introduction of nucleic acid (particularly RNA) into a cell. For the purposes of the present disclosure, the term "transfection" also includes the introduction of nucleic acid into a cell or the uptake of nucleic acid by such a cell, wherein the cell may be present in a subject (e.g., a patient). Therefore, according to the present disclosure, the cell used to transfect the nucleic acid described herein may be present in vitro or in vivo, for example, the cell may form a part of an organ, tissue, and / or organism of a patient. According to the present disclosure, transfection may be transient or stable. For some applications of transfection, it is sufficient if only the transfected genetic material is transiently expressed. RNA may be transfected into a cell to transiently express its encoded protein. Since the nucleic acid introduced during the transfection process is usually not integrated into the nuclear genome, the exogenous nucleic acid will be diluted or degraded by mitosis. Cells that allow nucleic acid to be amplified free greatly reduce the dilution rate. If it is desired that the transfected nucleic acid is actually retained in the genome of the cell and its daughter cells, a stable transfection must occur. Such a stable transfection can be achieved by transfecting using a virus-based system or a transposon-based system. Generally speaking, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express its encoded protein.

[0352] The present disclosure includes analogs of peptides, polypeptides or proteins. According to the present disclosure, analogs of peptides, polypeptides or proteins are modified forms of the peptides, polypeptides or proteins from which they are derived, and have at least one functional property of the peptides, polypeptides or proteins. For example, pharmacologically active analogs of peptides, polypeptides or proteins have at least one pharmacological activity of the peptides, polypeptides or proteins from which the analogs are derived. Such modifications include any chemical modification, and include single or multiple replacements, deletions and / or additions of any molecule (e.g., carbohydrates, lipids and / or proteins or peptides) associated with the peptides, polypeptides or proteins. In some embodiments, "analogs" of peptides, polypeptides or proteins include those modified forms obtained by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protection / blocking groups, proteolytic cleavage or binding to antibodies or other cell ligands. The term "analog" also extends to all functional chemical equivalents of the peptides, polypeptides or proteins.

[0353] As used herein, the terms "linked", "fused" or "fusion / fusion" are used interchangeably. These terms refer to two or more elements or components or domains being linked together.

[0354] According to the present disclosure, it is preferred that a nucleic acid (e.g., DNA or RNA) (e.g., mRNA) encoding a peptide, polypeptide, or protein, once taken up by a cell or introduced (i.e., transfected or transduced) into a cell, results in the expression of the peptide, polypeptide, or protein, which cell may be present in vitro or in a subject. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide, polypeptide, or protein, or may express the encoded peptide, polypeptide, or protein on the surface.

[0355] According to the present disclosure, terms such as "nucleic acid expressing..." and "nucleic acid encoding..." or similar terms (e.g., "RNA encoding...") are used interchangeably herein, and in the case of a particular peptide, polypeptide or protein, mean that the nucleic acid can be expressed to produce the peptide, polypeptide or protein if present in a suitable environment, such as within a cell.

[0356] As used herein, "activation" or "stimulation" refers to the state of immune effector cells (e.g., T cells) that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cell" refers especially to an immune effector cell that is undergoing cell division.

[0357] The term "sensitization" refers to the process in which an immune effector cell (eg, a T cell) first comes into contact with its specific antigen and results in differentiation into an effector cell (eg, an effector T cell).

[0358] The term "amplification" refers to a process in which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize the antigen are amplified. In some embodiments, amplification results in differentiation of immune effector cells.

[0359] "Antigen" according to the present disclosure encompasses any substance that will cause an immune response and / or any substance to which an immune response or immune mechanism (e.g., a cellular response) is directed. This also includes situations in which the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed to one or more antigenic peptides, particularly if presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present disclosure, the term "antigen" includes any molecule comprising at least one epitope (e.g., a T cell epitope). In certain embodiments, an antigen in the context of the present disclosure is a molecule that induces an immune response, optionally after processing, that can be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0360] In some embodiments, antigen is presented on the surface of immune system cell or is present on the surface of immune system cell, and described immune system cell is for example antigen presenting cell, such as dendritic cell or macrophage.In some embodiments, antigen or its processed product such as T cell epitope is combined with antigen receptor.Therefore, antigen or its processed product can react specifically with immune effector cell such as T lymphocyte (T cell).

[0361] According to the present disclosure, any suitable antigen as a candidate for an immune response can be used, wherein the immune response can be both a humoral immune response and a cellular immune response. In the case of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably by an antigen presenting cell (in the case of an MHC molecule), which results in an immune response to the antigen. The antigen is preferably a product corresponding to or 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 the antigen may also be a tumor antigen. According to the present disclosure, the antigen may correspond to a naturally occurring product, such as a viral protein, or a portion thereof.

[0362] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains an epitope that will stimulate the host's immune system to produce an antigen-specific cellular immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with an infection caused by a microorganism, typically a microbial antigen (e.g., a bacterial or viral antigen), or antigens associated with a cancer (typically a tumor), such as a tumor antigen.

[0363] In certain embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular, those that are primarily present in the cell or as surface antigens of a tumor cell. In some embodiments, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen from a virus, bacteria, a unicellular organism, or a parasite, such as a viral antigen such as a viral ribonucleoprotein or a coating protein. In particular, the antigen should be presented by an MHC molecule, which leads to the regulation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular, the activation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular, by the regulation of the activity of the T cell receptor.

[0364] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface or cell nucleus. In particular, it refers to those antigens produced intracellularly or as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetosulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, and a variety of viral tumor antigens. According to the present disclosure, tumor antigens preferably include any antigens that are characteristic of a tumor or cancer and that are characteristic of a tumor or cancer cell in terms of type and / or expression level.

[0365] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0366] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. Bacterial antigens may be derived from the cell wall or cytoplasmic membrane of the bacteria.

[0367] The term "epitope" refers to an antigenic determinant in a molecule (e.g., an antigen), i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by a T cell or B cell, particularly when presented in the context of an MHC molecule. The epitope of a protein preferably comprises a continuous or discontinuous portion of the protein and is preferably about 5 to about 100 amino acids in length, preferably about 5 to about 50, more preferably about 8 to about 0, most preferably about 10 to about 25 amino acids in length, e.g., the length of the epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope.

[0368] Terms such as "epitope", "antigenic fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen, which is preferably capable of eliciting an immune response against the antigen or eliciting a cell expressing or comprising and preferably presenting the antigen. Preferably, the term relates 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, particularly if presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen that is recognized by the immune system). For example, an epitope can be recognized by a T cell, a B cell or an antibody. The epitope of an antigen may include a continuous or discontinuous portion of the antigen, and may be about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, most preferably about 8 to about 25 amino acids in length, such as 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 length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T cell epitopes.

[0369] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when present in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules, and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind peptide epitopes and present them to be recognized by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display both autoantigens (peptide fragments from the cell itself) and non-autoantigens (e.g., fragments of invasive microorganisms) to T cells. In the case of class I MHC / peptide complexes, binding peptides are generally about 8 to about 10 amino acids long, but longer or shorter peptides may also be effective. In the case of class II MHC / peptide complexes, binding peptides are generally about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, but longer and shorter peptides may also be effective.

[0370] The length of 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 some embodiments, the peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0371] The peptide or protein antigen may be any peptide or protein that can induce or enhance the ability of the immune system to produce antibodies and T cell responses against the peptide or protein.

[0372] In some embodiments, vaccine antigens (i.e., antigens that induce an immune response in a subject when inoculated) are recognized by immune effector cells. Preferably, if vaccine antigens are recognized by immune effector cells, the antigens can induce stimulation, sensitization and / or amplification of immune effector cells carrying antigen receptors that recognize the vaccine antigens in the presence of appropriate co-stimulatory signals. In the case of embodiments of the present disclosure, vaccine antigens are preferably presented or present on the surface of cells (preferably antigen presenting cells).

[0373] In some embodiments, the antigen is expressed in a diseased cell (eg, a tumor cell or an infected cell).

[0374] In some embodiments, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of MHC. In some embodiments, when expressed by a T cell and / or present on a T cell, the combination of a TCR with an antigen presented by a cell (e.g., an antigen presenting cell) results in stimulation, sensitization, and / or amplification of the T cell. In some embodiments, when expressed by a T cell and / or present on a T cell, the combination of a TCR with an antigen present on a diseased cell results in cell lysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0375] In some embodiments, the antigen is expressed on the surface of diseased cells (eg, tumor cells or infected cells). In some embodiments, the antigen receptor is a CAR that binds to an epitope in the extracellular domain of an antigen or an extracellular domain of an antigen. In some embodiments, CAR binds to the natural epitope of an antigen present on the surface of a living cell. In some embodiments, when CAR is expressed by T cells and / or present on T cells, CAR binds to an antigen present on a cell (eg, an antigen presenting cell) resulting in stimulation, sensitization and / or amplification of the T cell. In some embodiments, when CAR is expressed by T cells and / or present on T cells, CAR binds to an antigen present on diseased cells resulting in cell lysis and / or apoptosis of diseased cells, wherein the T cells preferably release cytotoxic factors, such as perforin and granzyme.

[0376] In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B cell receptor binds to a native epitope of the antigen.

[0377] 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 the cell, wherein at least a portion of the molecule is oriented toward the extracellular space of the cell and is accessible from the outside of the cell, for example, by an antibody located outside the cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably 20 amino acids. The association may be direct or indirect. For example, the association may be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure that may be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the cell surface may be a transmembrane protein with an extracellular portion, or may be a protein associated with the cell surface by interacting with another protein that is a transmembrane protein.

[0378] "Cell surface" or "surface of a cell" is used according to its normal meaning in the art, and therefore includes the outside of the cell that can be bound by proteins and other molecules. If an antigen is located on the surface of a cell and can be bound by, for example, an antigen-specific antibody added to the cell, then the antigen is expressed on the cell surface. In some embodiments, the antigen expressed on the cell surface is an integral membrane protein with an extracellular portion that can be recognized by a CAR.

[0379] In the context of the present disclosure, the term "extracellular part" or "extracellular domain" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, for example by binding molecules such as antibodies (which are located outside the cell). Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0380] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTL) (CD8+T cells) including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes an antigen targeted by the T cell (particularly when presented on the surface of an antigen presenting cell or a diseased cell such as a cancer cell in the context of an MHC molecule) and preferably exerts the effector function of the T cell. If a T cell kills a target cell expressing an antigen, the cell is considered to be specific for the antigen. T cell specificity can be evaluated using any of a variety of standard techniques (e.g., in a chromium release assay or a proliferation assay). Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the present disclosure, a nucleic acid (e.g., DNA or RNA, particularly mRNA) encodes at least one epitope.

[0381] The term "target" shall mean a substance, such as a cell or tissue, that is a target for an immune response (e.g., a cellular immune response). Targets include cells that present antigens or antigenic epitopes (i.e., peptide fragments derived from antigens). In some embodiments, target cells are cells that express antigens and preferably present class I MHC to the antigen.

[0382] "Antigen processing" refers to the degradation of antigens into processing products that are fragments of the antigen (e.g., degradation of proteins into peptides), and one or more of these fragments are associated with MHC molecules (e.g., associated by binding) for presentation to specific T cells by cells (preferably antigen presenting cells). Antigen presenting cells can be divided into professional antigen presenting cells and non-professional antigen presenting cells.

[0383] The term "professional antigen presenting cells" refers to antigen presenting cells that constitutively express the major histocompatibility complex class II (MHC class II) molecules required for interaction with naive T cells. If T cells interact with the MHC class II molecule complex on the membrane of antigen presenting cells, the antigen presenting cells produce co-stimulatory molecules that induce activation of T cells. Professional antigen presenting cells include dendritic cells and macrophages.

[0384] The term "non-professional antigen presenting cells" relates to antigen presenting cells that do not constitutively express class II molecules but express them under the stimulation of certain cytokines (e.g., interferon-γ). Exemplary non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells, or vascular endothelial cells.

[0385] The term "dendritic cell" (DC) refers to a phagocytic cell subtype belonging to the class of antigen presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially converted into immature dendritic cells. The characteristics of these immature cells are high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample from the surrounding environment, looking for pathogens such as viruses and bacteria. Once they come into contact with presentable antigens, they will be activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small fragments, and after maturation, these fragments are presented on their cell surfaces using MHC molecules. At the same time, they raise cell surface receptors such as CD80, CD86 and CD40 that play the role of co-receptors in T cell activation, greatly enhancing their ability to activate T cells. They also raise CCR7, which is a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream or to reach lymph nodes through the lymphatic system. Here, they act as antigen presenting cells and activate helper T cells and killer T cells and B cells by presenting antigens and non-antigen specific co-stimulatory signals to helper T cells and killer T cells and B cells. Therefore, dendritic cells can actively induce T cell-related immune responses or B cell-related immune responses. In some embodiments, the dendritic cells are spleen dendritic cells.

[0386] The term "macrophage" refers to a subpopulation of phagocytes differentiated from monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf and kill foreign pathogens in macrophages by hydrolysis and oxidative attack, resulting in pathogen degradation. Peptides from degraded proteins are displayed on the surface of macrophages, where they can be recognized by T cells, and they can directly interact with antibodies on the surface of B cells, resulting in T and B cell activation and further stimulating immune response. Macrophages belong to antigen presenting cells. In some embodiments, macrophages are spleen macrophages.

[0387] "Antigen-responsive CTL" means CD8 + T cells, the CD8 + T cells respond to antigens, or peptides derived from the antigen, which are presented using class I MHC on the surface of antigen-presenting cells.

[0388] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines (e.g., IFN-γ and TNF-α), upregulation of activation markers (e.g., CD44 and CD69), and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately indicate CTL responsiveness.

[0389] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the overall body response to an antigen, and preferably refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the term "immune response to" or "immune response against" with respect to a substance (e.g., an antigen, a cell, or a tissue) relates to an immune response, such as a cellular response, against the substance. The immune response may include one or more reactions selected from the group consisting of the appearance of antibodies against one or more antigens, and the expansion of antigen-specific T lymphocytes, preferably CD4 + and CD8 + T lymphocytes, more preferably CD8 + T lymphocytes, which can be detected in various in vitro proliferation or cytokine production assays.

[0390] In the context of the present disclosure, the terms "induce an immune response" and "cause an immune response" and similar terms refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response or both. The immune response may be protective / preventative / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B or C), CMV or RSV)). In this context, "induce" may mean that there is no immune response to a particular antigen or pathogen before induction, but it may also mean that there is a certain level of immune response to a particular antigen or pathogen before induction, and after induction, the immune response is enhanced. Therefore, in this context, "induce an immune response" also includes "enhance an immune response". Preferably, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease condition is improved by inducing an immune response.

[0391] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are intended to include cellular responses directed against cells characterized by expression of antigen and / or presentation of antigen by class I and / or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (also known as CD4 + T cells) play a key role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, such as diseased cells.

[0392] The term "humoral immune response" refers to a process in living organisms in which antibodies are produced in response to a substance or organism, which will ultimately neutralize and / or eliminate it. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-associated receptors that bind a single specificity to an antigen. After binding to the appropriate antigen and receiving a variety of other activation signals, B lymphocytes divide, which produces memory B cells and antibody-secreting plasma cell clones, each clone producing antibodies to the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. When exposed to the specific antigen again, these lymphocytes provide the cellular basis of memory and lead to a step-wise escalation of the antibody response.

[0393] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable region and constant region are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2 and HCDR3, and the CDRs of VL are called LCDR1, LCDR2 and LCDR3. The heavy and light chain variable regions contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxyl terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. An antibody can be a complete immunoglobulin derived from a natural source or a recombinant source, and can be an immunologically active portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single-chain antibodies and humanized antibodies.

[0394] The term "immunoglobulin" refers to a protein of the immunoglobulin superfamily, preferably to an antigen receptor, such as an antibody or a B cell receptor (B cell receptor, BCR). Immunoglobulins are characterized by structural domains (i.e., immunoglobulin domains) with characteristic immunoglobulin (Ig) folds. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are typically part of the BCR. Soluble immunoglobulins are typically referred to as antibodies. Immunoglobulins typically comprise several chains, typically two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V and L chains. L(Variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domain and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ and μ, which constitute the different classes of antibodies, namely IgA, IgD, IgE, IgG and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. In mammals, there are two types of light chains, namely λ and κ. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within different isotypes of immunoglobulins, wherein the variable part is highly diverse and leads to antigen recognition.

[0395] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the administration to the individual of one or more immunogens or antigens as described herein, or derivatives thereof, particularly in the form of DNA or RNA (particularly mRNA) encoding the same, and the stimulation of an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens.

[0396] "Cell characterized by presenting an antigen" or "cell presenting an antigen" or "MHC molecules presenting an antigen on the surface of an antigen-presenting cell" or similar expressions refer to cells, such as diseased cells, in particular tumor cells or infected cells, or antigen-presenting cells, which present antigens or antigenic peptides directly or after processing in the context of MHC molecules (preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules).

[0397] In the context of the present disclosure, the term "transcription" relates to the process in which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA). Subsequently, the RNA (particularly mRNA) can be translated into a peptide, polypeptide or protein.

[0398] As used herein, the term "expression" includes transcription and / or translation of a specific nucleotide sequence.

[0399] With respect to RNA, the terms "expression" or "translation" refer to the process in the ribosomes of the cell by which a strand of mRNA directs the assembly of a sequence of amino acids to produce a peptide, polypeptide, or protein.

[0400] In the context of the present disclosure, the term "RNA encoding" means that, if present in a suitable environment, such as within a cell of a target tissue, the RNA can direct the assembly of amino acids during translation to produce the peptide or protein it encodes.

[0401] The terms "optional" or "optionally" as used herein mean that the subsequently described event, condition or circumstance may or may not occur, and that the description includes instances where said event, condition or circumstance occurs and instances where said event, condition or circumstance does not occur.

[0402] The pharmaceutical preparations described herein, particularly medicine boxes, may include instructional materials or instructions. "Instructional materials" or "instructions" as used herein include publications, recordings, diagrams, or any other medium of expression that can be used to convey the usability of the compositions and methods of the present disclosure. For example, the instructional materials of the kit of the present disclosure may be attached to a container containing the composition / preparation of the present disclosure, or transported together with a container containing the composition / preparation. Alternatively, the instructional materials may be transported separately from the container, with the purpose that the instructional materials and the composition are used in coordination by the recipient.

[0403] The prodrugs of specific compounds described herein are those compounds that undergo chemical transformation under physiological conditions to provide specific compounds after being applied to individuals. In addition, prodrugs can be converted into specific compounds by chemical or biochemical methods in an ex vivo environment. For example, when, for example, prodrugs are placed in a transdermal patch reservoir together with suitable enzymes or chemical reagents, prodrugs can be slowly converted into specific compounds. Exemplary prodrugs are hydrolyzable esters (using alcohol or carboxyl groups included in specific compounds) or amides (using amino or carboxyl groups included in specific compounds) in vivo. Specifically, any amino group contained in a specific compound and with at least one hydrogen atom can be converted into a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines and enamine ketones.

[0404] In this specification, the structural formula of a compound may represent a certain isomer of the compound. However, it should be understood that the disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers and other structural isomers and isomer mixtures, and is not limited to the description of the formula. In addition, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it should be understood that the disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of specific salts and / or solvates.

[0405] "Isomers" are compounds with the same molecular formula but different structures ("structural isomers") or different geometric (spatial) positions of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are pairs of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is indicated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly convert into each other due to the migration of single atoms or groups of atoms, even when pure; that is, the tautomers are in dynamic chemical equilibrium with each other. Examples of tautomers are keto-enol-tautomeric isomers. "Conformational isomers" are stereoisomers that are interconvertible only by rotation about a formal single bond, and in particular include those that result in different 3-dimensional forms of (hetero)rings, e.g. chair, half-chair, boat and twist-boat stereoisomers of cyclohexane.

[0406] The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (e.g., an organic solvent (e.g., aliphatic alcohols (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, diethyl ether, etc.), water, or a mixture of two or more of these liquids), wherein the addition complex exists in the form of crystals or mixed crystals. The amount of solvent included in the addition complex may be stoichiometric or non-stoichiometric. "Hydrate" is a solvate in which the solvent is water.

[0407] In an isotopically labeled compound, one or more atoms are replaced by corresponding atoms having the same number of protons but a different number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes that can be used in compounds of the present disclosure include deuterium, tritium, 11 C. 13 C. 14 C. 15 N. 18 F. 32 P. 32 S. 35 S. 36 Cl and 125 I.

[0408] The term "mean diameter" refers to the mean hydrodynamic diameter of the particles as measured by dynamic light scattering (DLS) and data analysis using the so-called cumulant algorithm, which provides the so-called Z having a length dimension. 平均值and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of the particles is related to Z. 平均值 .

[0409] In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis mentioned in the definition of "mean diameter". Under certain conditions, it can be used as a measure of the size distribution of the nanoparticle ensemble.

[0410] The radius of gyration of the particle around the axis of rotation (abbreviated as R in this paper) g ) refers to the radial distance of a point from the axis of rotation. If the entire mass of the particle is assumed to be concentrated, the moment of inertia about a given axis is the same as its actual mass distribution. Mathematically speaking, R g is the root mean square distance of the particle components from their center of mass or a given axis. For example, for a particle consisting of n mass elements and mass m i (i=1, 2, 3, ..., n) and located at a fixed distance s from the center of mass i The macromolecule at R g is all the mass elements i 2 The square root of the mass mean of

[0411]

[0412] The radius of gyration can be determined experimentally (e.g. by using light scattering) or calculated. In particular, for small scattering vectors The structure function S is defined as follows:

[0413]

[0414] where N is the number of components (Guinier's law).

[0415] The "D10 value" (particularly with respect to a quantitative size distribution of particles) is the diameter below which 10% of the particles have a diameter. The D10 value is a way of describing the proportion of the smallest particles within a population of particles (e.g. within a particle peak obtained from field-flow fractionation).

[0416] The "D50 value" (particularly with respect to a quantitative size distribution of particles) is the diameter below which 50% of the particles have a diameter. The D50 value is a way of describing the average particle size within a population of particles (e.g., within a particle peak obtained from field flow fractionation).

[0417] The "D90 value" (particularly with respect to the quantitative size distribution of particles) is the diameter at which 90% of the particles have a diameter less than this value. The "D95", "D99" and "D100" values ​​have corresponding meanings. The D90, D95, D99 and D100 values ​​are a way of describing the proportion of larger particles within a particle population (e.g., within a particle peak obtained from field flow fractionation).

[0418] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle. The hydrodynamic radius is related to the mobility of the particle and takes into account not only size but also solvent effects. For example, a smaller, more strongly hydrated particle can have a larger hydrodynamic radius than a larger, less hydrated particle. This is because when a smaller particle moves in a solution, it carries more water molecules with it. Since the actual size of a particle in a solvent cannot be measured directly, the hydrodynamic radius can be defined by the Stokes-Einstein equation:

[0419]

[0420] where k B is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally (e.g., by using dynamic light scattering (DLS)). Therefore, a procedure for determining the hydrodynamic radius of a particle or a population of particles (e.g., the hydrodynamic radius of a particle (e.g., LNP) included in a formulation or composition as disclosed herein, or the hydrodynamic radius of a particle peak obtained by subjecting such a formulation or composition to field flow fractionation) is to measure the DLS signal of the particle or population of particles (e.g., the DLS signal of a particle (e.g., LNP) included in a formulation or composition as disclosed herein, or the DLS signal of a particle peak obtained by subjecting such a formulation or composition to field flow fractionation).

[0421] The term "aggregate" as used herein relates to a cluster of particles in which the particles are identical or very similar and attached to each other in a non-covalent manner (eg, by ionic interactions, H-bridge interactions, dipole interactions and / or van der Waals interactions).

[0422] As used herein, the expression "light scattering" refers to the physical process by which light is forced to deviate from a straight-line trajectory by one or more paths due to local inhomogeneities in a medium through which the light passes.

[0423] The term "UV" means ultraviolet and refers to a band of the electromagnetic spectrum with wavelengths from 10 nm to 400 nm, ie, shorter than visible light but longer than X-rays.

[0424] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered by a sample into multiple angles. In this regard, "multi-angle" means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector moving within a range including a selected specific angle or an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser source (MALLS: Multi-angle Laser Scattering). Based on the MALS signal of a composition comprising particles and by using a suitable format (e.g., a Zimm plot, a Berry plot, or a Debye plot), it is possible to determine the radius of gyration (R g ) and thus the size of the particle can be determined. Preferably, the Zim diagram is a graphical representation using the following equation (or its inverse):

[0425]

[0426] Where c is the mass concentration of particles in the solvent (g / mL); A 2 is the second virial coefficient (mol·mL / g 2 ), P(θ) is the shape factor related to the angular dependence of the scattered light intensity; R θ is the Rayleigh ratio (cm -1 ); and K* is equal to 4π 2 η o (dn / dc) 2 λ 0 -4 N A -1 The optical constants of o is the refractive index of the solvent at the wavelength of the incident radiation (vacuum), λ 0 is the wavelength of the incident radiation (vacuum) (nm), N A is Avogadro's number (mol -1), and dn / dc is the differential refractive index increment (mL / g) (see, e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry diagram is calculated as follows (or its inverse):

[0427]

[0428] Where c, R θ and K* are defined as above. Preferably, the Debye graph is calculated as follows (or its inverse):

[0429]

[0430] Where c, R θ and K* are as defined above.

[0431] The expression "dynamic light scattering" or "DLS" as used herein refers to a technique for determining the size and size distribution spectrum of particles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is directed into a sample through a polarizer. The scattered light then passes through a second polarizer where it is detected and the resulting image is projected onto a screen. Particles in the solution are hit by the light and diffract the light in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). The process is repeated at short time intervals and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each point over time.

[0432] The expression "static light scattering" or "SLS" used in this article refers to a technique for determining particle size and size distribution spectrum, particularly about the radius of gyration of particles and / or the molar mass of particles. High-intensity monochromatic light, typically a laser, is emitted in a solution containing particles. One or more detectors are used to measure the scattering intensity at one or more angles. In order to obtain accurate measurements of both the molar mass and size of macromolecules of all radii, angle dependence is required. Therefore, the simultaneous measurement results at multiple angles relative to the direction of the incident light (referred to as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS)) are generally regarded as standard implementations of static light scattering.

[0433] "Immunogenicity" is the ability of a foreign substance (e.g., RNA) to elicit an immune response in humans or other animals. The innate immune system is a relatively nonspecific and immediate component of the immune system. Along with the adaptive immune system, it is one of the two major components of the vertebrate immune system.

[0434] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue or system.

[0435] As used herein, the term "exogenous" refers to any substance introduced or generated from outside an organism, cell, tissue or system.

[0436] As used herein, the terms "linked", "fused" or "fusion / fusion" are used interchangeably. These terms refer to two or more elements or components or domains being linked together.

[0437] The term "colloid" as used herein refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0438] In the context of the present disclosure, the term "nucleic acid particle" relates to a particle containing a nucleic acid (e.g., DNA or RNA). Thus, the term "RNA particle" relates to a particle containing RNA.

[0439] "Polymer" as used herein has its common meaning, i.e. a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may be all the same, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is biologically derived, i.e. a biopolymer (e.g., a protein). In some cases, additional moieties may also be present in the polymer, such as targeting moieties, such as those described herein.

[0440] The term "repeating unit" relates to a basic unit which repeats itself periodically along the polymer chain of a polymer and which is derived from one monomer. Although the structure of a repeating unit and the structure of its corresponding monomer are generally identical, they may differ from each other.

[0441] If more than one type of repeating unit is present in a polymer, the polymer is referred to as a "copolymer". It should be understood that in any embodiment using a polymer, the polymer used may be a copolymer in some cases. The repeating units that form the copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random manner, a periodic manner, an alternating manner, or a block manner. In this regard, the term "arranged in a random manner" means that the sequence of repeating units in the copolymer follows a statistical rule (such copolymers are called statistical copolymers). The term "arranged in a periodic manner" means that the repeating units appear in the copolymer in a repeating pattern (for example, a periodic copolymer composed of repeating units A and B may have the formula (-ABAAABBB-) n The term "arranged in an alternating manner" means that the corresponding copolymer has regular alternating repeating units, for example in the following formula -ABABABABAB- or -(-AB-) n -. The term "arranged in a blocky manner" means that the corresponding copolymer comprises at least two homopolymer subunits (i.e. blocks) connected by covalent bonds. Block copolymers can have two (diblock copolymers), three (triblock copolymers) or more different blocks. Examples of sequences in triblock copolymers composed of A and B repeating units are as follows:- (A) a -(B) b -(A) c -, where a, b and c represent the number of repeating units in the corresponding block.

[0442] The term "protamine" refers to any of a variety of relatively low molecular weight strongly basic proteins, which are rich in arginine and are found to be particularly associated with DNA to replace the somatic histones in the sperm cells of different animals (e.g., fish). In particular, the term "protamine" refers to a protein present in fish semen, which is strongly alkaline, soluble in water, not heat-condensed and mainly produces arginine after hydrolysis. In purified form, it is used for long-acting preparations of insulin and for neutralizing the anticoagulant effect of heparin. According to the present disclosure, the term "protamine" used herein means any protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and polymer forms of the amino acid sequence or its fragments and (synthetic) polypeptides, which are artificial and specially designed for a specific purpose and cannot be separated from a natural or biological source.

[0443] The term "hydrophobicity" as used herein for compounds, groups or parts means that the compounds, groups and parts are not attracted by water molecules, and when present in an aqueous solution, gather and exclude water molecules. In some embodiments, the term "hydrophobicity" refers to any compound, group or part that is substantially immiscible or insoluble in an aqueous solution. In some embodiments, hydrophobic compounds, groups or parts are substantially non-polar. Some examples of hydrophobic groups are alkyl and fluorinated (e.g., perfluorinated) alkyl. In some embodiments, hydrophobic compounds, groups or parts are lipophilic. In this regard, the term "lipophilicity" as used herein for compounds, groups or parts means that the compounds, groups or parts are soluble in non-polar solvents (e.g., hexane, tetrahydrofuran (THF) and / or chloroform). In some embodiments, the term "lipophilicity" refers to any compound, group or part that is soluble in non-polar solvents (e.g., hexane, tetrahydrofuran (THF) and / or chloroform) and substantially immiscible or insoluble in an aqueous solution. Some examples of lipophilic groups are hydrocarbon groups, eg, non-cyclic, preferably straight chain hydrocarbon groups (eg, straight chain hydrocarbon groups having at least 10 carbon atoms), such as the lipophilic chains of natural lipids.

[0444] As used herein, the term "perfluorinated" with respect to a compound, group or moiety means that in said compound, group or moiety, all CH moieties have been replaced by CF moieties. For example, the chemical formula of perfluorooctanoic acid is F 3 C(CF 2 ) 6 COOH.

[0445] The term "lipid" is broadly defined herein as a molecule comprising one or more hydrophobic parts or groups (such as one or more hydrophobic chains) and optionally also comprising one or more hydrophilic parts or groups. For example, the term "lipid" refers to a group of such organic compounds, characterized in that it is insoluble in water, but soluble in many organic solvents. Molecules comprising hydrophobic parts and hydrophilic parts are also often referred to as amphiphiles. Lipids are usually insoluble in water. In aqueous environments, amphiphilic properties allow molecules to self-assemble into organized structures and different phases. One of those phases is composed of lipid bilayers because they are present in vesicles, multilayer / unilayer liposomes or films in aqueous environments. Hydrophobicity can be imparted by comprising non-polar groups, which include but are not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, alicyclic or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, and include carbohydrates, phosphates, carboxyls, sulfates, amino groups, sulfhydryls, nitro groups, hydroxyls and other similar groups. Generally speaking, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from the condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fat, fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides and phospholipids), as well as metabolites containing sterols, such as cholesterol.

[0446] The term "amphiphilic" as used herein refers to a molecule having both a polar portion and a non-polar portion. An amphiphilic compound typically has a polar head connected to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have a formal positive charge or a formal negative charge. Alternatively, the polar portion may have both a formal positive charge and a negative charge, and may be a zwitterion or an inner salt. For the purposes of the present disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipidoid compounds.

[0447] The term "lipidoid substance", "lipidoid compound" or "lipidoid molecule" refers to a substance that is structurally and / or functionally related to a lipid but cannot be considered as a lipid in the strict sense. For example, the term includes compounds that can form an amphiphilic layer when it is present in a film in a vesicle, a multilamellar / unilamellar liposome or an aqueous environment, and includes a surfactant or a synthetic compound with both a hydrophilic and a hydrophobic part. In general, the term includes molecules that include hydrophilic and hydrophobic parts with different structural organizations that may or may not be similar to the structural organization of a lipid. Some examples of lipidoid compounds that can be spontaneously integrated into the cell membrane include functional lipid constructs, such as synthetic function-spacer-lipid constructs (synthetic function-spacer-lipid construct, FSL), synthetic function-spacer-sterol constructs (synthetic function-spacer-sterol construct, FSS) and artificial amphiphilic molecules. The lipid comprising two long alkyl chains and a polar head group is generally cylindrical. The area shared by the two alkyl chains is similar to the area shared by the polar head group. The lipid of this invention is low in solubility as monomer, and is easy to be aggregated into water-insoluble planar bilayer.The conventional surfactant monomer that only comprises a linear alkyl chain and hydrophilic head group is usually conical.The hydrophilic head group tends to occupy more molecular space than linear alkyl chain.In some embodiments, surfactant tends to be aggregated into water-soluble spherical or elliptical micelle.Although lipid also has the general structure identical with surfactant---polar hydrophilic head group and nonpolar hydrophobic tail---, lipid is different from surfactant in the shape of monomer, the type of aggregate formed in solution and in the concentration range required for aggregation.Unless otherwise noted herein or obviously contradictory with context, the term " lipid " used herein should be interpreted as including lipid and lipidoid material.

[0448] Some specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, amino lipids, and sphingolipids. In certain embodiments, the amphiphilic compound is a lipid.

[0449] Fatty acids or fatty acid residues are a class of different molecules consisting of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, which is usually 4 to 24 carbons long, can be saturated or unsaturated and can be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If the fatty acid contains a double bond, cis or trans geometric isomerism may occur, which significantly affects the molecular configuration. Cis double bonds cause the fatty acid chain to bend, which is an effect of compounding with more double bonds in the chain. The other major lipid species in the fatty acid class are fatty esters and fatty amides.

[0450] Glycerolipids consist of mono-, di- and tri-substituted glycerols, the most notable being tri-esters of glycerol fatty acids, known as triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, usually with a different fatty acid. Another subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues linked to glycerol by glycosidic linkages.

[0451] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) comprising a glycerol core connected to two fatty acid-derived "tails" by ester linkages and to a "head" group by phosphate linkages. Some examples of glycerophospholipids, commonly called phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (also known as PC, GPCho or phosphatidylcholine), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0452] Sphingolipids are a complex family of compounds that share a common structural feature - a sphingoid base skeleton. The main sphingoid base in mammals is commonly referred to as sphingosine. Ceramide (N-acyl-sphingoid base) is a major subclass of sphingoid base derivatives with amide-linked fatty acids. Fatty acids are typically saturated or monounsaturated with a chain length of 16 to 26 carbon atoms. The main sphingolipid of mammals is sphingomyelin (ceramide phosphorylcholine), while insects mainly contain ceramide phosphoethanolamine and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are different molecular families consisting of one or more sugar residues connected to sphingoid bases by glycosidic bonds. Some examples of these are simple and complex sphingolipids, such as cerebrosides and gangliosides.

[0453] Sterol lipids (such as cholesterol and its derivatives or tocopherol and its derivatives) are important components of membrane lipids together with glycerophospholipids and sphingomyelin.

[0454] Glycolipids describe compounds in which fatty acids are directly linked to a sugar backbone to form a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharide in Gram-negative bacteria. The typical lipid A molecule is a disaccharide of glucosamine that is derivatized with up to seven fatty acyl chains. The minimum lipopolysaccharide required for growth in Escherichia coli (E. coli) is Kdo2-lipid A, which is a glucosamine hexaacylated disaccharide glycosylated with two 3-deoxy-D-mannose-octulose acid (Kdo) residues.

[0455] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classical enzymes as well as iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources and have a large structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0456] According to the present disclosure, lipids and lipidoids can be cationic, anionic or neutral. Neutral lipids or lipidoids exist in the form of uncharged or neutral zwitterions at a selected pH. An example of a neutral zwitterionic lipid is a phospholipid.

[0457] As used herein, the term "functional moiety" refers to a group of atoms in a molecule having a unique chemical identity, 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 O, N and S. The functional moiety may be monovalent (e.g., hydroxyl, cyano, nitro or amide (e.g., -C(O)NHCH 3)) or divalent (e.g., amide (e.g., -C(O)NH-)), carbonyl (-C(O)-) or ester (e.g., -OC(O)-). In some embodiments, the functional moiety provides hydrophilicity to the group to which the functional moiety is attached, for example, by providing at least one hydrogen bond acceptor / donor and / or at least one charge (positive or negative) to the group to which the functional moiety is attached. In certain embodiments, the functional moiety comprises a hydrogen bond acceptor (e.g., a carbonyl moiety), a hydrogen bond donor (e.g., a hydroxyl moiety, -NH- (e.g., an amide moiety) or a thiol moiety) or both (e.g., an amide moiety), and / or is charged (e.g., a phosphate, an amino or an ammonium moiety). Some examples of monovalent functional moieties include hydroxyl, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, sulfamate / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester, imino (imine), thioimidate / ester, sulfinamido, carbonate / ester , thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties. Examples of divalent functional moieties include ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, sulfamate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioacid salt, imidate, imino (imine), thioimidate, sulfinamido, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties.

[0458]

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

[0459] The term "halogen" as used herein with respect to a functional moiety, particularly as a component of a linker, means fluorine, chlorine, bromine or iodine.

[0460] The term "cyano" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to the group -CN.

[0461] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "azido" refers to the group N 3 .

[0462] The term "nitro" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to the group -NO 2 .

[0463] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "amino" includes unsubstituted amino groups (i.e., -NH 2 The amino group may be monovalent (e.g., -NRR, wherein each R is independently H or an organic group, such as R as defined below). 72 or R 73 ) or a divalent (e.g. -NR-, wherein R is H or an organic group, such as R defined below 72 In some embodiments, the term "amino" refers to the group -N(R 72 )(R 73 ), where R 72 and R 73 is independently selected from -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl at each occurrence, or R 72 and R 73 can be linked together with the nitrogen atom to which they are attached to form the group -N=CR 75 R 76 , wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is optionally replaced by one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 Replacement; R 75 and R 76are independently selected from -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and -NH y R 80 2-y , or R 75 and R 76 The atoms to which they are attached may be linked together to form a ring, which is optionally substituted by one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the ring, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 substituted, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is optionally replaced by one or more (e.g., 1 to the maximum number of hydrogen atoms bound to alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 Substitution; y is an integer from 0 to 2; R 80 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is optionally replaced by one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 Replace; and R 70 is a first substituent, a second substituent, or a third substituent other than H, preferably as disclosed herein. 72 and R 73 Each of which is independently H or a hydrocarbon group, for example, selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl), wherein each of the hydrocarbon groups (e.g., C 1-6 Alkyl, aryl and aryl (C 1-6 Each of the alkyl) groups is optionally replaced by one or more (eg, 1 to 2) hydrocarbon groups (eg, C 1-6 Alkyl, aryl or aromatic (C 1-6 The maximum number of hydrogen atoms bound to the alkyl) group is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, for example, 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R 70 replace.

[0464] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "ammonium" refers to the group -N+ (R 72 ) 2 (R 73 ), where R 72 and R 73 As defined for the term "amino".

[0465]

[00136] The term "thiol" or "sulfanyl" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to the group -SH.

[0466] The term "disulfide" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to the group -SSH.

[0467]

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

[0468] The term "amide" or "amido" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(O)NH- (including the structure -NHC(O)- of its isomeric arrangement, unless specified to the contrary). Preferably, each of the two ends of the amide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker) (if the two ends are linked to the same organic group, the amide moiety is also referred to as a lactam). The 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 the R shown above in the definition of the term "amino" 72 ) or a divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0469] The term "ester" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(O)O- (including the structure -OC(O)- of its isomeric arrangement, unless specified to the contrary). Preferably, each of the two ends of the ester structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker) (if both ends are linked to the same organic group, the ester moiety is also referred to as a lactone). The ester group may be monovalent (e.g., -C(O)OR or -OC(O)R, wherein R is independently an organic group, such as R shown above in the definition of the term "amino" 72 ) or a divalent organic group (e.g., -C(O)O- or -OC(O)-).

[0470] The term "ether" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -O-, wherein each of the two ends of the ether structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The ether group can be monovalent (e.g., -OR, wherein R is an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -O-).

[0471] The term "sulfide" or "thioether" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -S-, wherein each of the two ends of the thioether structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The thioether group can be monovalent (e.g., -SR, wherein R is an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -S-).

[0472] The term "disulfide" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SS-, wherein each of the two ends of the disulfide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The disulfide group can be monovalent (e.g., -SSR, wherein R is an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -SS-).

[0473] The term "diselenide" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SeSe-, wherein each of the two ends of the diselenide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The diselenide group can be monovalent (e.g., -SeSeR, wherein R is an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (e.g., -SeSe-).

[0474] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfoxide" refers to a group comprising a sulfinyl structure -S(O)-, wherein each of the two ends of the sulfoxide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The sulfoxide group may be monovalent (e.g., -S(O)R, wherein R is an organic group, such as R shown above in the definition of the term "amino"). 72 ) or divalent (such as, for example, -S(O)-).

[0475] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfone" refers to a sulfonyl structure -S(O) 2 -, wherein each of the two ends of the sulfone structure is covalently linked to the C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The sulfone group can be monovalent (e.g., -S(O) 2 R, wherein R is an organic group, such as R as shown above in the definition of the term "amino" 72 one of the organic groups specified in the definition of 2 -).

[0476] The term "sulfite" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OS(O)O-, wherein one of the two ends of the sulfite structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or to a C atom of the same or another organic group (e.g., an alkylene group as another component of the linker). The sulfite group may be monovalent (e.g., -OS(O)OR, wherein R is H or an organic group, such as R shown above in the definition of the term "amino" 72 ) or divalent (e.g., -OS(O)O-).

[0477] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfate" refers to a moiety comprising the structure -OS(O) 2 O-, wherein one of the two ends of the sulfate structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or a C atom of the same or another organic group (e.g., an alkylene group as another component of the linker). The sulfate group can be monovalent (e.g., -OS(O) 2 OR, wherein R is H or an organic group, such as R as shown above in the definition of the term "amino" 72 one of the organic groups specified in the definition of2 O-).

[0478] The term "phosphate" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -OP(O)(OR)O-, wherein one of the two ends of the phosphate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the other end is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino" 72 The phosphate group may be monovalent (e.g., -OP(O)(OR) 2 , wherein each R is independently H or an organic group, such as R shown above in the definition of the term "amino" 72 ) or a divalent (e.g., -OP(O)(OR)O-, wherein R is independently H or an organic group, such as R shown above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0479] The term "sulfenamide" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -S(O)N(R)-, wherein the S-terminus of the sulfenamide structure is covalently linked to a C-atom of an organic group (e.g., an alkylene group as another component of the linker) and the N-terminus is covalently linked to H or a C-atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino" 72 The sulfonamide group may be monovalent (e.g., -S(O)N(R) 2 , wherein each R is independently H or an organic group, such as R shown above in the definition of the term "amino" 72 ) or a divalent (e.g., -S(O)N(R)-, wherein R is H or an organic group such as R shown above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0480] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfonamide" refers to a sulfonamide comprising the structure -S(O) 2 N(R)-, wherein the S-terminus of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker) and the N-terminus is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, such as R shown above in the definition of the term "amino" 72The sulfonamide group may be monovalent (e.g., -S(O) 2 N(R) 2 , wherein each R is independently H or an organic group, such as R shown above in the definition of the term "amino" 72 one of the organic groups specified in the definition of 2 N(R)-, wherein R is H or an organic group, such as R shown above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0481] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfamate" refers to a sulfamate comprising the structure -OS(O) 2 N(R)-(including isomeric structures thereof unless otherwise specified -N(R)S(O) 2 O-) group, wherein one of the two ends of the aminosulfonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other 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 R as described above in the definition of the term "amino" 72 The aminosulfonate / ester group may be monovalent (e.g., -OS(O) 2 N(R) 2 or -N(R)S(O) 2 OR, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 one of the organic groups specified in the definition of 2 N(R)-or-N(R)S(O) 2 O-, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0482] The term "sulfite diamide" as used herein in relation to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)S(O)N(R)-, wherein one of the two ends of the sulfite diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other 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 the R described above in the definition of the term "amino" 72The sulfite 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 R described above in the definition of the term "amino" 72 ) or a divalent (e.g., -N(R)S(O)N(R)-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0483] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "sulfuric acid diamide" refers to a functional moiety comprising the structure -N(R)S(O) 2 N(R)-, wherein one of the two ends of the sulfuric acid diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other 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 R described in the definition of the term "amino" above) 72 The sulfuric acid diamide group may be monovalent (e.g., -N(R)S(O) 2 N(R) 2 , wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 is one of the organic groups specified in the definition of ), or is divalent (e.g., -N(R)S(O) 2 N(R)-, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0484] The term "urea" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)C(O)N(R)-, wherein one of the two ends of the urea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other 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 the R described above in the definition of the term "amino" 72 The urea group may be monovalent (e.g., -N(R)C(O)N(R) 2 , wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72or a divalent (e.g., -N(R)C(O)N(R)-, wherein each R is H or an organic group, such as R as described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0485] The term "thiourea" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -N(R)C(S)N(R)-, wherein one of the two ends of the thiourea structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other end is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino" 72 The thiourea group may be monovalent (e.g., -N(R)C(S)N(R) 2 , wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 or a divalent (e.g., -N(R)C(S)N(R)-, where each R is H or an organic group, such as R as described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0486] The term "carbonyl" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(O)-, wherein one of the two ends of the carbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other end is covalently linked to H or to a C atom of the same or another organic group (if both ends are linked to a C atom of an organic group, the carbonyl moiety is also referred to as a "keto" moiety). The carbonyl group can be monovalent (e.g., -C(O)R, wherein R is H or an organic group, such as R described above in the definition of the term "amino" 72 ) or divalent (e.g., -C(O)-).

[0487] The term "thiocarbonyl" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(S)-, wherein one of the two ends of the thiocarbonyl structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other end is covalently linked to H or to a C atom of the same or another organic group. The thiocarbonyl group can be monovalent (e.g., -C(S)R, wherein R is H or an organic group, such as R described in the definition of the term "amino" above). 72) or divalent (e.g., -C(S)-).

[0488] As used herein with respect to a functional moiety, particularly as a component of a linker, the term "orthoester" refers to a molecule comprising an alkoxy group (i.e., -OR, wherein R is an organic group (e.g., an alkylene group as a further component of the linker), such as R as described above in the definition of the term "amino" 72 The moiety of the C atom connected to one of the organic groups specified in the definition of . Exemplary formulas of orthoesters include the structure (-O) r C(OR) 3-r -, wherein each R is independently an organic group, for example independently selected from R described in the definition of the term "amino" above 72 wherein the orthoester comprises an organic group specified in the definition of ; r is 1 or 2; and each of the two ends of the orthoester structure is covalently linked to a C atom of another organic group or two additional separate organic groups. In some embodiments, the orthoester comprises the structure (—O) r C(OR 25 ) 3-r -, where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more first-stage substituents, second-stage substituents, or third-stage substituents as defined herein); r is 1 or 2; and each of the two termini of the orthoester structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The orthoester group can be monovalent (e.g., -C(OR) 3 Or-OC(OR) 2 R, wherein each R is an organic group, for example independently selected from R described above in the definition of the term "amino" 72 ) or a divalent (e.g., -(O) 2 C(OR)(R) or -OC(OR) 2 -, wherein each R is an organic group, for example independently selected from R described in the definition of the term "amino" above 72 (organic radicals as specified in the definition of ).

[0489] The term "thioate" or "thioester" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(O)- (including the structure -C(O)S-, isomeric arrangements thereof, unless otherwise indicated), wherein each of the two termini of the thioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The thioate group can be monovalent (e.g., -SC(O)R or -C(O)SR, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 ) or a divalent organic group (e.g., -SC(O)- or -C(O)S-).

[0490] The term "dithioate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(S)- (including the structure -C(S)S-, unless otherwise specified, of isomeric arrangements thereof), wherein each of the two termini of the dithioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker). The dithioate group may be monovalent (e.g., -SC(S)R or -C(S)SR, wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 ) or divalent (e.g., -SC(S)- or -C(S)S-).

[0491] The term "imidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)- (including the structure -C(=NR)O-, unless otherwise specified, of isomeric arrangements thereof), wherein each of the two termini of the imidate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as a further component of the linker) (each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 The imidate / ester group may be monovalent (e.g., -OC(=NR)R' or -C(=NR)OR', wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72) or a divalent (e.g., -OC(=NR)- or -C(=NR)O-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0492] The term "imino" or "imine" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -C(=NR)-, wherein one of the two ends of the imino structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other 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 R as described above in the definition of the term "amino" 72 The moiety -C(=NR)H is also known as "aldimine", and the moiety -C(=NR)R' (wherein R' is an organic group, such as R as described in the definition of the term "amino" above) 72 The imino group may be monovalent (e.g., -C(=NR)R, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -C(=NR)-, wherein R is H or an organic group, such as R as described in the definition of the term "amino" above) 72 (one of the organic radicals specified in the definition of ).

[0493] The term "thioimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(=NR)S- (including the structure -SC(=NR)-, unless otherwise specified, of isomeric arrangements thereof), wherein one of the two termini of the thioimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as R as described above in the definition of the term "amino" 72 The thioimidate group may be monovalent (e.g., -C(=NR)SR or -SC(=NR)R, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72) or a divalent (e.g., -C(=NR)S- or -SC(=NR)-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0494] The term "sulfinamido" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(S)NR- (including, unless otherwise indicated, the structure -N(R)C(S)- of isomeric arrangements thereof), wherein one of the two termini of the sulfinamido structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other termini are covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino"). 72 The sulfinamido group can be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72 or a divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0495] The term "carbonate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(O)O-, wherein each of the two ends of the carbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonate group may be monovalent (e.g., -OC(O)OR', wherein R' is an organic group, such as R described above in the definition of the term "amino" 72 ) or divalent (e.g., -OC(O)O-).

[0496] The term "thiocarbonate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(S)O- or -OC(O)S- (including the structure -SC(O)O- of isomeric arrangements thereof unless otherwise indicated), wherein each of the two termini of the thiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The thiocarbonate group may be monovalent (e.g., -OC(S)OR' or -OC(O)SR' or -SC(O)OR', wherein each R' is independently an organic group, such as R' as described above in the definition of the term "amino" 72 ) or a divalent organic group (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).

[0497] The term "dithiocarbonate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(O)S- or -OC(S)S- (including the structure -SC(S)O- of isomeric arrangements thereof unless otherwise indicated), wherein each of the two termini of the dithiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The dithiocarbonate group may be monovalent (e.g., -SC(O)SR', -OC(S)SR' or -SC(S)OR', wherein each R' is independently an organic group, such as R' as described above in the definition of the term "amino" 72 ) or a divalent organic group (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).

[0498] The term "trithiocarbonate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(S)S-, wherein each of the two ends of the trithiocarbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The trithiocarbonate group may be monovalent (e.g., -SC(S)SR, wherein R is an organic group, such as R described above in the definition of the term "amino" 72 ) or a divalent organic group (e.g., -SC(S)S-).

[0499] The term "guanidinyl" or "imidamido" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a guanidinyl group comprising the structure -N(R)C(=NR)NR- (wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72The guanidinyl group may be a monovalent group (e.g., -N(R)C(=NR)NRR, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72 ) or a divalent (e.g., -N(R)C(=NR)NR-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0500] The term "carbamimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)NR- (including, unless otherwise indicated, the structure -N(R)C(=NR)O-, isomeric arrangements thereof), wherein the O terminus of the carbamimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other (N) terminus is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino"). 72 The 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 R as described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -OC(=NR)NR- or -N(R)C(=NR)O-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0501] The term "carbonimidate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)O-, wherein each end of the carbonimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72The carbonimidate group may be monovalent (e.g., -OC(=NR)OR', wherein R is H or an organic group, such as R as described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -OC(=NR)O-, wherein R is H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0502] The term "carbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(O)NR- (including, unless otherwise indicated, the structure -N(R)C(O)O-, which is an isomeric arrangement thereof), wherein the O terminus of the carbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino"). 72 The carbamate / ester group can be monovalent (e.g., -OC(O)NRR or -N(R)C(O)OR', wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0503] The term "dithiocarbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(S)NR- (including, unless otherwise indicated, the structure -N(R)C(S)S- of isomeric arrangements thereof), wherein the S terminus of the dithiocarbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino"). 72The dithiocarbamate group may be monovalent (e.g., -SC(S)NRR or -N(R)C(S)SR', wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -SC(S)NR- or -N(R)C(S)S-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0504] The term "carbonodithioimidate" as used herein with respect to a functional moiety, in particular as a component of a linker, relates to a group comprising the structure -SC(=NR)S-, wherein each end of the carbonodithioimidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 The carbonodithioimidate group may be monovalent (e.g., -SC(=NR)SR', wherein R is H or an organic group, such as R as described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -SC(=NR)S-, where R is H or an organic group, such as R as described in the definition of the term "amino" above) 72 (one of the organic radicals specified in the definition of ).

[0505] The term "carbamimidothioate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -SC(=NR)NR- (including, unless otherwise indicated, the structure -N(R)C(=NR)S- of isomeric arrangements thereof), wherein the S terminus of the carbamimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino" 72The 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 R as described above in the definition of the term "amino" 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -SC(=NR)NR- or -N(R)C(=NR)S-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0506] The term "thiocarbamate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including the structures -SC(O)NR- or -OC(S)NR-, isomeric arrangements thereof, unless otherwise indicated), wherein the O / S terminus of the thiocarbamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker), and the other terminus (the N terminus) is covalently linked to a C atom of the same or another organic group (each R independently being H or an organic group, such as the R described above in the definition of the term "amino"). 72 The thiocarbamate group may be monovalent (e.g., -N(R)C(O)SR' or -N(R)C(S)OR' or -SC(O)NRR or -OC(S)NRR, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino"). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -N(R)C(O)S- or -N(R)C(S)O- or -SC(O)NR- or -OC(S)NR-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino" 72 (one of the organic radicals specified in the definition of ).

[0507] The term "carbonimidothioate" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OC(=NR)S- (including, unless otherwise indicated, the structure -SC(=NR)O- of isomeric arrangements thereof), wherein the O / S terminus of the carbonimidothioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as another component of the linker), and the other terminus (the N terminus) is covalently linked to H or to a C atom of the same or another organic group (each R being independently H or an organic group, such as the R described above in the definition of the term "amino" 72 The 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 R as described in the definition of the term "amino" above). 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -OC(=NR)S- or -SC(=NR)O-, wherein each R is independently H or an organic group, such as R as described in the definition of the term "amino" above 72 (one of the organic radicals specified in the definition of ).

[0508] The term "acylhydrazone" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -C(R')(=NN(R)C(O)-) (including isomeric arrangements thereof, unless otherwise indicated (-C(O)(N(R)-N=)C(R')-) and / or =C(=NN(R)C(O)R'), wherein each R' is independently an organic group, such as R as described above in the definition of the term "amino". 72 one of the organic groups specified in the definition of "amino"; each R is H or an organic group, such as R described in the definition of the term "amino" above 72 The acylhydrazone structure is one of the organic groups specified in the definition of ; and each of the two ends of the acylhydrazone structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the acylhydrazone comprises the structure -C(R 25 )(=NN(R 26 )C(O)-)(Unless otherwise specified, structures including isomeric arrangements thereof (-C(O)(N(R 26 )-N=)C(R 25 )-) and / or =C(=NN(R26 )C(O)R 25 ), where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more 1st, 2nd, or 3rd substituents as defined herein); each R 26 are independently H or a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., substituted with one or more first-level substituents, second-level substituents, or third-level substituents as defined herein); and each of the two ends of the acylhydrazone structure is covalently linked to a C atom of another organic group or two additional separate organic groups. An exemplary chemical structure of an acylhydrazone is shown below:

[0509]

[0510] Each of these represents a bond by which the acylhydrazone is covalently linked to another organic group (e.g., an alkylene group as a further component of the linker). The acylhydrazone group may be monovalent (e.g., -C(R')(=NN(R)C(O)R') or -C(O)(N(R)-N=)C(R') 2 , wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 and each R' is independently an organic group, such as R described in the definition of the term "amino" above. 72 ) or a divalent organic group (e.g., -C(R')(=NN(R)C(O)-, -C(O)(N(R)-N=)C(R')-, or =C(=NN(R 26 )C(O)R 25 ), wherein each R' is independently an organic group, such as R described in the definition of the term "amino" above 72 and each R is H or an organic group, such as R described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0511] The term "hydrazine" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -N(R)N(R)-, wherein each R is independently H or an organic group, such as R as described above in the definition of the term "amino". 72One of the organic groups specified in the definition of ; and each of the two ends of the hydrazine structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the hydrazine comprises the structure -N(R 26 )N(R 26 )-, where each R 26 are independently H or a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more first-level substituents, second-level substituents, or third-level substituents as defined herein); and each of the two termini of the hydrazine structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The hydrazine group can be monovalent (e.g., -N(R)N(R) 2 , wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino" 72 ) or a divalent (e.g., -N(R)N(R)-, wherein each R' is independently an organic group, such as R as described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0512] The term "oxime" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure =C(=N(OH)), wherein each of the two ends of the oxime structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as an additional component of the linker). An exemplary chemical formula of an oxime is shown below:

[0513]

[0514] Each of these represents a bond through which the oxime is covalently attached to another organic group. The oxime group may be monovalent (e.g., -C(=N(OH))(R), wherein each R is independently H or an organic group, such as R described above in the definition of the term "amino". 72 ) or divalent (e.g., ═C(═N(OH))).

[0515] The term "acetal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCH(R')O-, wherein R' is an organic group, such as R as described above in the definition of the term "amino". 72and each of the two O atoms of the acetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the acetal comprises the structure -OCH(R 25 )O-, where R 25 is a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more 1st, 2nd, or 3rd substituents as defined herein); and each of the two O atoms of the acetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The acetal group can be monovalent (e.g., -OCH(R')OR', wherein each R' is independently an organic group, such as independently selected from the R described in the definition of the term "amino" above). 72 ) or a divalent (e.g., -OCH(R')O-, wherein R' is an organic group, such as R as described in the definition of the term "amino" above) 72 (one of the organic radicals specified in the definition of ).

[0516] The term "hemiacetal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCH(OH)-, wherein each of the two ends of the hemiacetal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). The hemiacetal group may be monovalent (e.g., -OCH(OH)OR', wherein R' is an organic group, such as R described above in the definition of the term "amino" 72 ) or divalent (e.g., -OCH(OH)-).

[0517] The term "ketal" as used herein with respect to a functional moiety, particularly as a component of a linker, refers to a group comprising the structure -OC(R')(R')O-, wherein each R' is independently an organic group, for example independently selected from the R groups described above in the definition of the term "amino". 72 and each of the two O atoms of the ketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the ketal comprises the structure -OC(R 25 )(R 25 )O-, where each R 25 are independently hydrocarbon groups, such as C 1-6 Alkyl, aryl, and aryl (C1-6 alkyl), which is optionally substituted (e.g., with one or more first-level substituents, second-level substituents, or third-level substituents as defined herein); and each of the two O atoms of the ketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The ketal group can be monovalent (e.g., -OC(R')(R')OR', wherein each R' is independently an organic group, such as independently selected from the R described in the definition of the term "amino" above. 72 ) or a divalent (e.g., -OC(R')(R')O-, wherein each R' is independently an organic group, such as R as described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0518] The term "himeketal" as used herein with respect to a functional moiety, particularly as a component of a linker, relates to a group comprising the structure -OCR'(OH)-, wherein R' is an organic group, such as R as described above in the definition of the term "amino". 72 One of the organic groups specified in the definition of ; and each of the two ends of the hemiketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups (e.g., an alkylene group as an additional component of the linker). In some embodiments, the hemiketal comprises the structure -OCR 25 (OH)-, where R 25 is a hydrocarbon group, such as C 1-6 Alkyl, aryl, and aryl (C 1-6 alkyl), which is optionally substituted (e.g., with one or more primary 1, secondary, or tertiary substituents as defined herein); and each of the two termini of the hemiketal structure is covalently linked to a C atom of another organic group or two additional separate organic groups. The hemiketal group can be monovalent (e.g., -OC(R') 2 (OH), wherein each R' is independently an organic group, for example independently selected from the R described in the definition of the term "amino" above 72 ) or a divalent (e.g., -OCR'(OH)-, wherein R' is an organic group, such as R as described in the definition of the term "amino" above) 72 (one of the organic radicals specified in the definition of ).

[0519] The term "imide" as used herein with respect to a functional moiety, particularly as a component, refers to a group comprising the structure -C(O)N(R)C(O)-, wherein R is an organic group, such as R as described above in the definition of the term "amino". 72The imide group may be monovalent (e.g., -C(O)N(R)C(O)R', where R is independently H or an organic group, such as R described in the definition of the term "amino" above). 72 and R' is an organic group, such as R described in the definition of the term "amino" above. 72 or a divalent (e.g., -C(O)N(R)C(O)-, where R is H or an organic group, such as R as described in the definition of the term "amino" above. 72 (one of the organic radicals specified in the definition of ).

[0520] The term "hydrocarbyl" as used herein refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. Some typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Some specific examples of hydrocarbyl groups are C 1-6 Alkyl, aryl and aryl (C 1-6 In some embodiments, the hydrocarbyl group is optionally substituted (eg, with one or more 1st, 2nd, or 3rd substituents as defined herein).

[0521] The term "acyclic" as used herein in the context of an organic group relates to an open-chain organic group that does not contain a ring. "Open-chain" or "acyclic" organic groups can be linear (i.e. they contain only one unbranched chain without any side chains) or branched (i.e. the main chain contains one or more side chains).

[0522] An organic group "substituted with one or more substituents" means that one or more hydrogen atoms (e.g., 1 to the maximum number of hydrogen atoms bound to the organic group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the organic group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the one or more substituents may be selected from the first-level substituents, second-level substituents or third-level substituents described herein.

[0523] As used herein, the expression "hydrogen bond" or "H-bond" means a non-covalent bond (in some embodiments, primarily electrostatic attraction) between (i) a hydrogen atom covalently bonded to a more electronegative atom or group and (ii) a lone pair of electrons of another electronegative atom. In some embodiments, the more electronegative atom or group includes nitrogen atoms and oxygen atoms; thus, some examples of groups in which the hydrogen atom is covalently bonded to the more electronegative atom or group include amino groups with at least one covalently attached hydrogen atom, -NH- groups of amide groups, hydroxyl groups (as such (as in the corresponding alcohol) or as part of other functional groups (e.g., as part of carboxyl (-COOH))), and sulfanyl groups (as such (as in the corresponding thiol) or as part of other functional groups (e.g., as part of disulfanyl (-SSH) or thioester (-C(OSH) group))). In some embodiments, the lone pair of electrons of another electronegative atom is the lone pair of oxygen atoms present in a carbonyl group or the lone pair of nitrogen atoms present in a primary, secondary, or tertiary amino group.

[0524] The expression "hydrogen bond donor" as used herein means an atom, ion or molecular component that provides a hydrogen bond bridging (shared) hydrogen atoms. In some embodiments, hydrogen bond donors include amino groups with at least one covalently attached hydrogen atom, -NH- groups of amide groups, hydroxyl groups (either as such (as in the corresponding alcohols) or as part of other functional groups (e.g., as part of carboxyl groups (-COOH)), and sulfanyl groups (either as such (as in the corresponding thiols) or as part of other functional groups (e.g., as part of disulfanyl groups (-SSH) or thioesters (-C(OSH) groups))).

[0525] The expression "hydrogen bond acceptor" as used herein means an atom, ion or molecular component that does not provide a hydrogen bond bridging (shared) hydrogen atoms. In some embodiments, the hydrogen bond acceptor comprises at least one lone pair of electrons. Some examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary and tertiary amino groups.

[0526] The term "tocopherol" refers to a group of four compounds, namely, α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol, which have the following formula:

[0527]

[0528] Where R t1 and R t2 Each is independently hydrogen or methyl. In α-tocopherol, R t1 and R t2 Both are methyl groups; in β-tocopherol, R t1 is a methyl group, and R t2 is H; in γ-tocopherol, Rt1 is H, and R t2 is a methyl group; and in delta-tocopherol, R t1 and R t2 Both are H. The term "tocopherol moiety" or "tocopheryl moiety" means a monovalent radical of tocopherol, preferably a radical in which the hydrogen atom of the hydroxyl group has been removed.

[0529] The term "aqueous phase" used herein in connection with compositions / formulations containing particles, in particular LNPs, means a mobile or liquid phase, i.e. a continuous aqueous phase comprising all components dissolved therein but excluding (formally) particles. Thus, if particles (e.g. LNPs) are dispersed in an aqueous phase, and the aqueous phase is substantially free of compound X, then the aqueous phase is free of X in such a way that it is practically and practicable, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is possible that the particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

[0530] As used herein, the expression "substantially free of X" means that a mixture (e.g., a composition or formulation described herein or an aqueous phase of such a composition or formulation) is free of X in such a manner that it is practical and practicable to do so. For example, if the mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the mixture.

[0531] Thus, if the compositions described herein (e.g., RNALNP compositions) are substantially free of lipid or lipidoid substances comprising polyethylene glycol (PEG), then preferably the amount of lipid or lipidoid substances comprising PEG in the composition (e.g., RNALNP composition) is less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the composition.

[0532] If the aqueous phase of a composition described herein (e.g., an RNALNP composition) is substantially free of lipid or lipidoid materials comprising PEG, then preferably the amount of lipid or lipidoid materials comprising PEG in the aqueous phase of the composition (e.g., an RNALNP composition) is less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the aqueous phase.

[0533] If the compositions described herein (e.g., RNALNP compositions) are substantially free of PEG, then preferably the amount of PEG in the composition (e.g., RNALNP composition) is less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the composition.

[0534] If the aqueous phase of a composition described herein (e.g., an RNALNP composition) is substantially free of PEG, then preferably the amount of PEG in the aqueous phase of the composition (e.g., an RNALNP composition) is less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the aqueous phase.

[0535] Nucleic Acids

[0536] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded and linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to the present disclosure, the term "isolated nucleic acid" means such nucleic acids, which (i) are amplified in vitro for DNA, such as by polymerase chain reaction (PCR), or are transcribed in vitro for RNA (using, for example, RNA polymerase), (ii) are produced by cloning recombination, (iii) are purified, such as by cleavage and separation by gel electrophoresis, or (iv) are synthesized, such as by chemical synthesis.

[0537] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide consists of a nucleoside and one or more phosphate groups. Some examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0538] The five standard nucleosides that usually constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. Five nucleosides are usually abbreviated as their single-letter codes, respectively U, A, T, C and G. However, thymidine is more usually written as "dT" ("d" stands for "deoxy") because it contains 2'-deoxyfuranose moieties, rather than the furanose ring present in uridine. This is because thymidine is present in deoxyribonucleic acid (DNA) rather than ribonucleic acid (RNA). On the contrary, uridine is present in RNA rather than DNA. The remaining three nucleosides can be present in both RNA and DNA. In RNA, it will be represented as A, C and G, and in DNA, it will be represented as dA, dC and dG.

[0539] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably replaced by one or more alkyl groups, more preferably one or more C 1-4 Some specific examples of modified purine or pyrimidine base moieties include N 7 -alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C1-4 Alkyl-uracil and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0540] DNA

[0541] In some embodiments of the disclosure, the nucleic acid is DNA.

[0542] The term "DNA" herein refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In certain embodiments, DNA comprises all or most of the deoxyribonucleotide residues. "Deoxyribonucleotides" used herein refer to nucleotides lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA encompasses but is not limited to double-stranded DNA, single-stranded DNA, isolated DNA (e.g., partially purified DNA), substantially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that is different from naturally occurring DNA by adding, deleting, replacing and / or changing one or more nucleotides. Such changes may refer to adding non-nucleotide substances to internal DNA nucleotides or to DNA ends. It is also contemplated herein that the nucleotides in the DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered to be analogs of naturally occurring DNA. A molecule contains "mostly deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0543] The DNA may be recombinant DNA and may be obtained by cloning nucleic acids (particularly cDNA). cDNA may be obtained by reverse transcription of RNA.

[0544] RNA

[0545] In some embodiments of the disclosure, the nucleic acid is RNA.

[0546] According to the present disclosure, the term "RNA" means a nucleic acid molecule comprising ribonucleotide residues. In certain embodiments, RNA comprises all or most of the ribonucleotide residues. "Ribonucleotide" used herein refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes but is not limited to double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides. Such a change may refer to adding a non-nucleotide substance to an internal RNA nucleotide or to an RNA end. It is also contemplated herein that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these changed / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA (i.e., changed / modified RNA) comprising such changed / modified nucleotides may be referred to as analogs of naturally occurring RNA. In some embodiments, the RNA used according to the present disclosure comprises a population of different RNA molecules, such as a mixture of different RNA molecules that optionally encode different peptides and / or proteins. Therefore, according to the present disclosure, the term "RNA" may comprise a mixture of RNA molecules.

[0547] A molecule contains "mostly ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0548] "RNA" includes mRNA, tRNA, ribosomal RNA (ribosomal RNA, rRNA), small nuclear RNA (small nuclear RNA, snRNA), self-amplifying RNA (self-amplifying RNA, saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (small interfering RNA, siRNA) or microRNA (microRNA, miRNA)), activating RNA (e.g., small activating RNA) and immunostimulatory RNA (immunostimulatory RNA, isRNA). In certain embodiments, the RNA used in accordance with the present disclosure is mRNA.

[0549] In certain embodiments, RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. The RNA can express the encoded peptide, polypeptide or protein. For example, the RNA can be an RNA that encodes and expresses a pharmaceutically active peptide or protein. In some embodiments, RNA can interact with the cell translation machinery to allow translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), can secrete the encoded peptide or protein, or can make it produce on the surface. Alternatively, RNA can be a non-coding RNA, such as an ...

Claims

1. A composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises: (i) RNA; (ii) cationic lipids or lipids that can ionize into cations; and (iii) (a) Gathering Oxazoline (POX) and / or poly Conjugates of polyoxazine (POZ) polymers and (b) one or more hydrophobic chains.

2. The composition of claim 1, wherein the total number of POX and / or POZ repeating units in the polymer is from 2 to 200.

3. The composition of claim 1 or 2, wherein the POX and / or POZ polymer comprises the following general formula (I): wherein a is an integer from 1 to 2; R 1 is an alkyl group, especially C 1-3 Alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; and m is 2 to 200.

4. The composition of any one of claims 1 to 3, wherein the conjugate has the following general formula (II) or (II'): in: a is an integer from 1 to 2; R 1 is an alkyl group, especially C 1-3 Alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; m is 2 to 200; R 2 YesR 4 or -L 1 (R 4 ) p , where each R 4 are independently a hydrocarbon group; L 1 is a linker; and p is 1 or 2; and R 3 Selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 、-SR 20 、Halogen、-CN、-N3、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and members of targeting pairs.

5. The composition of claim 3 or 4, wherein R 1 It is methyl or ethyl, preferably methyl.

6. The composition of any one of claims 3 to 5, wherein m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120, or 10 to 100.

7. The composition of any one of claims 4 to 6, wherein L 1 Contains at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or between the alkylene and R 4 An alkylene moiety connected to a divalent functional moiety at the terminal end of the connection, wherein preferably each monovalent functional moiety is independently selected from hydroxyl, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, aminosulfonate / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester ester, imino, thioimidate, sulfinamide, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone , hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties; and / or each divalent functional moiety is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, aminosulfonate / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester, imino, thioimidate / ester, sulfinamido, carbonate / ester, thiocarbonic acid [0043] In some embodiments, the present invention relates to salts, dithiocarbonates, trithiocarbonates, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties.

8. The composition of any one of claims 4 to 7, wherein L 1 Selected from [*-NHC(O)] p (C 1-6 Alkylene)-, [*-C(O)NH] p (C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-,[*-OC(O)] p (C 1-6 Alkylene)-, [*-S] p (C 1-6 Alkylene)-, [*-SS] p (C 1-6 Alkylene)-,[*-S(O)2] p (C 1-6 Alkylene)-, [(*-O) r C(OR 25 ) 3-r ](C 1-6 Alkylene)-, [*-C(OR 25 )2O] p (C 1-6 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] p (C 1-6 Alkylene)-,[*-C(O)(N(R 26 )-N=)C(R 25 )-] p (C 1-6 Alkylene)-[*=C(=NN(R 26 )C(O)(R 25 ))] p (C 1-6 Alkylene)-, [*-N(R 26 )N(R 26 )] p (C 1-6 alkylene), [*=C(=N(OH))] p (C 1-6 Alkylene)-, and [*-OC(R 25 )(R 26 )O] p (C 1-6 Alkylene)-, Where * indicates the same as R 4 The connection point of 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); and r is an integer from 1 to 2.

9. The composition of any one of claims 4 to 8, wherein L 1 Selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, -(CH2)-CH(OC(O)-*)(CH2OC (O)-*), -(CH2)-CH(S-*)2, -(CH2)-CH(S-*)-CH2(S-*), *-S-(CH2)3-, *-S(O)2-(CH2)3-, and *-OC(O)-(CH2)-, Where * indicates the same as R 4 connection point.

10. The composition of any one of claims 4 to 9, wherein R 2 Selected from R 4 -L 1 R 4 ,-(CH2)-CH(OC(O)R 4 )(CH2OC(O)R 4 ), -(CH2)-CH(SR 4 )2, and -(CH2)-CH(SR 4 )-CH2(SR 4 ); and L 1 Selected from *-NHC(O)-(CH2)-, *-NHC(O)- (CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, *-S-(CH2)3-, *S(O)2-(CH2)3-, and *-OC(O)-(CH2)- (preferably, L 1 Selected from *-NHC(O)-(CH2)-, *-NHC(O)- (CH2)2-, *-C(O)NH-(CH2)-, and *-C(O)NH-(CH2)2-, for example *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2 - ), where * indicates the same as R 4 connection point.

11. The composition of any one of claims 4 to 7, wherein L 1 Selected from [*-]2NC(O)(C 1-6 Alkylene)-, [*-C(O)O] p (C 1-6 Alkylene)-OP(O)2O(C 1-6 Alkylene)NHC(O)-(C 1-6 Alkylene)-,[*-OC(O)] p (C 1-6 Alkylene)-OP(O)2O(C 1-6 Alkylene)NHC(O)-(C 1-6 alkylene)- and [*-C(O)] p (C 1-6 Alkylene)-C(O)-, where * represents 4 connection point; p is 1 or 2; and C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2).

12. The composition of any one of claims 4 to 7 and 11, wherein L 1 Selected from [*-]2NC(O)(C 1-2 Alkylene)-,[*-OC(O)]2(C 2-4 Alkylene)-OP(O)2O(C 2-3 Alkylene)NHC(O)-(C 1-2 alkylene)-, and *-C(O)(C 2-3 Alkylene)-C(O)-, Where * indicates the same as R 4 connection point.

13. The composition of any one of claims 4 to 7, 11 and 12, wherein R 2 Selected from (R 4 )2NC(O)(C 1-2 Alkylene)-, R 4 C(O)OCH2CH(OC(O)R 4 )-CH2-OP(O)2O-(CH2)2-NHC(O)-CH2-, and R 4 -C(O)(CH2)2-C(O)-.

14. The composition of any one of claims 4 to 7, wherein the conjugate has the general formula (II'), and L 1 Selected from [*-Z] p (C 1-6 Alkylene)-Z-, *-Z-(C 3-8 Cycloalkylene)-Z-, *-Z-(C 3-8 Cycloalkenylene)-Z-, (*=N)(C 1-6 Alkylene)-Z-, *-Z-(C 1-6 Alkylene)- and *-Z-, wherein * represents 4 The connection point of 1-6 The alkylene group is divalent (if p is 1) or trivalent (if p is 2); the C 3-8 Cycloalkylene and C 3-8 Each of the cycloalkenylene groups is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from: -OH, =O, -SH, halogen, -CN, -N3, and C 1-3 Alkyl; and each Z is independently selected from -OP(O)2O(C 1-3 Alkylene)NH-,-NH(C 1-3 alkylene)OP(O)2O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O), -S-, -S(O2)-, and -NR 22 -.

15. The composition of any one of claims 4 to 7, wherein the conjugate has the general formula (II'), and L 1 Yes[*-C(O)] p (C 1-6 Alkylene)-C(O)NH- or [*-]2N-, where * represents 4 connection point; p is 1 or 2; and C 1-6 Alkylene is divalent (if p is 1) or trivalent (if p is 2).

16. The composition of any one of claims 4 to 7 and 15, wherein the conjugate has the general formula (II'), and L 1 is *-C(O)(C 2-3 Alkylene)-C(O)NH- or [*-]2N-, where * represents 4 connection point.

17. The composition of any one of claims 4 to 7, 15 and 16, wherein the conjugate has the general formula (II'), and R 2 YesR 4 -C(O)(CH2)2C(O)NH- or R 4 N-.

18. The composition of any one of claims 4 to 17, wherein each R 4 are independently acyclic hydrocarbon groups, preferably straight-chain hydrocarbon groups.

19. The composition of any one of claims 4 to 18, wherein each R 4 is independently a hydrocarbyl group having at least 8 carbon atoms, for example a hydrocarbyl group having at least 10 carbon atoms.

20. The composition of any one of claims 4 to 17 and 19, wherein one R 4 It is the tocopherol part.

21. The composition of any one of claims 4 to 20, wherein R 3 Selected from H, C 1-3 Alkyl, -OR 20 、-N3、C 2-6 Alkynyl, -OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 and members of a targeting pair, wherein the C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 and members of the targeting pair; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and members of the targeting pair; R 21 Selected from C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a member of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl, or R 22 and R 23 may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein the C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and a member of a targeting pair.

22. The composition of any one of claims 4 to 21, wherein R 3 Selected from H,C 1-3 Alkyl, -OR 20 ,-N3,C 2-6 Alkynyl, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 ,-NR 22 C(O)R 21 , and members of the targeted pair, Wherein C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and members of the targeting pair; R 20 Selected from H and C 1-3 Alkyl; R 21 is C optionally substituted by one or more substituents independently selected from the following 1-3 Alkyl: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a member of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Alkynyl, wherein the C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and a member of a targeting pair, or R 22 and R 23 They may be linked together with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclic group.

23. The composition of any one of claims 4 to 22, wherein R 3 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(O)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of a targeted pair, Wherein C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair.

24. The composition of any one of claims 1 to 23, wherein the conjugate has the following general formula (III) or (III'): in: a is an integer from 1 to 2; R 1 is methyl or ethyl and is independently selected for each repeat unit; m is 10 to 100; For formula (III), R 2 Selected from -L 1 R 4 , -(CH2)- CH(OC(O)R 4 )(CH2OC(O)R 4 ), -(CH2)-CHCR 4 )2, and -(CH2)-CH(SR 4 )-CH2(SR 4 ), where each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; and L 1 Selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, *-S-(CH2)3-, *-S(O)2-(CH2)3-, and *-OC(O)-(CH2)- (preferably, L 1 is selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, and *-C(O)NH-(CH2)2-, such as *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-), wherein * represents a 4 or for formula (III'), R 2 Selected from (R 4 C(O)O)(CH(OC(O)R 4 ))(CH2)-Z-, (R 4 )2N(C 1-3 Alkylene)-Z-, R 4 Z(C 1-3 Alkylene)-Z-, R 4 Z-(C 3-6 Cycloalkenylene)-Z- and R 4 Z-, wherein the C 3-6 The cycloalkenylene group is optionally substituted with one or more (eg, 1, 2, 3, or 4) substituents independently selected from: -OH, =O, -SH, halogen, -CN, -N3, and C 1-3 Alkyl; and each Z is independently selected from -OP(O)2O(CH2)2NH-, -NH(CH2)2OP(O)2O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O)2-, and -NH-; and R 3 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(O)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of the targeted pair, wherein the C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -CoOCH3, -NH2, -NHCH3, -N(CH3)2, C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeted pair.

25. The composition of any one of claims 1 to 23, wherein the conjugate has the general formula (III) or (III') as shown in claim 24, wherein: a is an integer from 1 to 2; R 1 is methyl or ethyl and is independently selected for each repeat unit; m is 10 to 100; For formula (III), R 2 Selected from (R 4 )2NC(O)(C 1-2 -alkylene)-R 4 C(O)OCH2CH(OC(O)R 4 )-CH2-OP(O)2O-(CH2)2-NHC(O)-CH2-, and R 4 -C(O)(CH2)2-C(O)-, Each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; or for formula (III'), R 2 YesR 4 -C(O)(CH2)2C(O)NH- or R 4 N-, where each R 4 are independently a straight chain hydrocarbon group having at least 10 carbon atoms, or a tocopherol moiety, wherein the same R 2 An R in part 4 is a tocopherol moiety; and R 3 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(O)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of the targeted pair, wherein the C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2-C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair.

26. The composition of any one of claims 3 to 25, wherein a is 1.

27. The composition of any one of claims 3 to 25, wherein a is 2.

28. The composition of claim 1 or 2, wherein the POX and / or POZ polymer is a copolymer comprising repeating units of the following general formulae (Ia) and (Ib): Where R 1 Each of which is independently an alkyl group, especially C 1-3 an alkyl group, such as a methyl group, an ethyl group, an isopropyl group or an n-propyl group, and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 199; the number of repeating units of formula (Ib) in the copolymer is 1 to 199; and the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 200.

29. The composition of any one of claims 1, 2 and 28, wherein the conjugate has the following general formula (IV): R 5 -POXZ-R 6 in: R 5 YesR 7 or -L 2 (R 7 ) q , where each R 7 are independently a hydrocarbon group; L 2 is a linker; and q is 1 or 2; POXZ is a copolymer comprising repeating units of the following general formulae (Ia) and (Ib): Where R 1 Each of which is independently an alkyl group, especially C 1-3 alkyl, such as methyl, ethyl, isopropyl or n-propyl, and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 199; the number of repeating units of formula (Ib) in the copolymer is 1 to 199; the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 200; and the repeating units of formula (Ia) and (Ib) are arranged randomly, periodically, alternately or in blocks; and R 6 Selected from H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 、-SR 20 、Halogen、-CN、-N3、-OC(O)R 21 、-C(O)R 21 、-NR 22 R 23 、-COOH、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs, wherein the C 1-6 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 、-C(O)NR 22 R 23 、-NR 22 C(O)R 21 , sugars, amino acids, peptides, and members of targeting pairs; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; R 21 Selected from C 1-6 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , sugars, amino acids, peptides, and members of targeting pairs; and R 22 and R 23 Each of R is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R 22 and R 23 The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2, sugars, amino acids, peptides, and members of targeting pairs.

30. The composition of claim 28 or 29, wherein R 1 Each of is independently methyl or ethyl, preferably methyl, and is independently selected for each repeating unit.

31. The composition of any one of claims 28 to 30, wherein the number of repeating units of formula (Ia) in the copolymer is 1 to 179, for example, 1 to 159, 1 to 139, 1 to 119, or 1 to 99; the number of repeating units of formula (Ib) in the copolymer is 1 to 179, for example, 1 to 159, 1 to 139, 1 to 119, or 1 to 99; and the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 2 to 180, for example, 4 to 160, 6 to 140, 8 to 120, or 10 to 100.

32. The composition of any one of claims 29 to 31, wherein L 2 Contains at least one functional moiety, such as an alkylene moiety substituted with at least one monovalent functional moiety and / or between the alkylene and R 7 An alkylene moiety connected to a divalent functional moiety at the terminal end of the connection, wherein preferably each monovalent functional moiety is independently selected from hydroxyl, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, thioether, disulfide, sulfoxide, sulfone, sulfite / ester, sulfate / ester, phosphate / ester, sulfenamide, sulfonamide, aminosulfonate / ester, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt / ester, dithioacid salt / ester, imidate / ester ester, imino, thioimidate, sulfinamide, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acyl The divalent functional groups are selected from the group consisting of hydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties; and / or each divalent functional group is independently selected from ether, amino, ester, thioether, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfenamide, sulfonamide, aminosulfonate, diselenide, sulfodiamide, sulfuric acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioacid salt, dithioacid salt, imidate, imino, thioimidate, sulfinamido, carbonate, thio Carbonate, dithiocarbonate, trithiocarbonate, imidamido, carbamimidate, carbonimidate, carbamate, dithiocarbamate, carbonodithioimidate, carbamimidothioate, thiocarbamate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide and amide moieties.

33. The composition of any one of claims 29 to 32, wherein L 2 Selected from [*-NHC(O)] q (C 1-6 Alkylene)-, [*-C(O)NH] q (C 1-6 Alkylene)-, [*-C(O)O] q (C 1-6 Alkylene)-,[*-OC(O)] q (C 1-6 Alkylene)-, [*-S] q (C 1-6 Alkylene)-, [*-SS] q (C 1-6 Alkylene)-,[*-S(O)2] p (C 1-6 Alkylene)-, [(*-O) s C(OR 25 ) 3-s ](C 1-6 Alkylene)-, [*-C(OR 25 )2O] q (C 1-6 Alkylene)-, [*-C(R 25 )(=NN(R 26 )C(O)-)] q -(C 1-6 Alkylene)-,[*-C(O)(N(R 26 )-N=)C(R 25 )-] q (C 1-6 Alkylene)-, [*=C(=NN(R 26 )C(O)(R 25 ))] q (C 1-6 Alkylene)-, [*-N(R 26 )N(R 26 )] q (C 1-6 alkylene)-, [*=C(=N(OH))] q (C 1-6 Alkylene)-, and [*-OC(R 25 )(R 26 )O] q (C 1-6 Alkylene)-, Where * indicates the same as R 7 The connection point of ; q is 1 or 2; C 1-6 Alkylene is divalent (if q is 1) or trivalent (if q is 2); R 25 Selected from C 1-6 Alkyl, aryl and aryl (C 1-6 Alkyl); R 26 Selected from H, C 1-6 Alkyl, aryl and aryl (C 1-6 alkyl); and s is an integer from 1 to 2.

34. The composition of any one of claims 29 to 33, wherein L 2 Selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, -(CH2)-CH(OC(O)-*)(CH2OC (O)-*), -(CH2)-CH(S-*)2, -(CH2)-CH(S-*)-CH2(S-*), *-S-(CH2)3-, *-S(O)2-(CH2)3-, and *-OC(O)-(CH2)-, Where * indicates the same as R 7 connection point.

35. The composition of any one of claims 29 to 34, wherein R 5 Selected from R 7 -L 2 R 7 ,-(CH2)-CH(OC(O)R 7 )(CH2OC(O)R 7 ), -(CH2)-CH(SR 7 )2, and -(CH2)-CH(SR 7 )-CH2(SR 7 ); and L 2 Selected from *-NHC(O)-(CH2), *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, *-S-(CH2)3-, *-S(O)2-(CH2)3-, and *-OC(O)-(CH2)- (preferably L 2 Selected from *-NHC(O)-(CH2), *-NHC(O)- (CH2)2-, *-C(O)NH-(CH2)-, and *-C(O)NH-(CH2)2-, such as *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-), wherein * represents the 7 connection point.

36. The composition of any one of claims 29 to 32, wherein L 2 Selected from [*-]2NC(O)(C 1-6 Alkylene)-, [*-C(O)O] q (C 1-6 Alkylene)-OP(O)2O(C 1-6 Alkylene)NHC(O)-(C 1-6 Alkylene)-,[*-OC(O)] q (C 1-6 Alkylene)-OP(O)2O(C 1-6 Alkylene)NHC(O)-(C 1-6 alkylene)-, and [*-C(O)] q (C 1-6 Alkylene)-C(O)-, where * represents 7 The connection point of q is 1 or 2; and C 1-6 Alkylene is divalent (if q is 1) or trivalent (if q is 2).

37. The composition of any one of claims 29 to 32 and 36, wherein L 2 Selected from [*-]2NC(O)(C 1-2 Alkylene)-,[*-OC(O)]2(C 2-4 Alkylene)-OP(O)2O(C 2-3 Alkylene)NHC(O)-(C 1-2 alkylene)-, and *-C(O)(C 2-3 Alkylene)-C(O)-, Where * indicates the same as R 7 connection point.

38. The composition of any one of claims 29 to 32, 36 and 37, wherein R 5 Selected from (R 7 )2NC(O)(C 1-2 Alkylene)-, R 7 C(O)OCH2CH(OC(O)R 7 )-CH2-OP(O)2O-(CH2)2-NHC(O)-CH2-, and R 7 -C(O)(CH2)2-C(O)-.

39. The composition of any one of claims 29 to 32, wherein R 6 connected to the N-terminus of the POXZ copolymer; and L 2 Connected to the C-terminus of the POXZ copolymer and selected from [*-Z 1 ] q (C 1-6 Alkylene)-Z 1 -, *-Z 1 -(C 3-8 Cycloalkylene)-Z 1 -, *-Z 1 -(C 3-8 Cycloalkenylene)-Z 1 -、(*=N)(C 1-6 Alkylene)-Z 1 -, *-Z 1 (C 1-6 alkylene)- and *-Z 1 -, where * indicates the same as R 7 The connection point of ; q is 1 or 2; C 1-6 The alkylene group is divalent (if q is 1) or trivalent (if q is 2); the C 3-8 Cycloalkylene and C 3-8 Each of the cycloalkenylene groups is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from: -OH, =O, -SH, halogen, -CN, -N3, and C 1-3 alkyl; and each Z 1 Independently selected from -OP(O)2O(C 1-3 Alkylene)NH-,-NH(C 1-3 alkylene)OP(O)2O-,-C(O)NH-,-NHC(O)-,-OC(O)NH-,-NHC(O)O-,-O-,-C(O)O-,-OC(O)-,-S-,-S(O)2-, and -NR 22 -.

40. The composition of any one of claims 29 to 32, wherein R 6 connected to the N-terminus of the POXZ copolymer; and L 2 Connected to the C-terminus of the POXZ copolymer and selected from [*-C(O)] q (C 1-6 Alkylene)-C(O)NH- and [*-]2N-, where * represents the 7 The connection point of q is 1 or 2; and C 1-6 Alkylene is divalent (if q is 1) or trivalent (if q is 2).

41. The composition of any one of claims 29 to 32 and 40, wherein R 6 connected to the N-terminus of the POXZ copolymer; and L 2 Connected to the C-terminus of the POXZ copolymer and is *-C(O)(C 2-3 Alkylene)-C(O)NH- or [*-]2N-, where * represents 7 connection point.

42. The composition of any one of claims 29 to 32, 40 and 41, wherein R 6 connected to the N-terminus of the POXZ copolymer; and R 5 Connected to the C-terminus of the POXZ copolymer and is R 7 -C(O)(CH2)2C(O)NH- or R 7 N-.

43. The composition of any one of claims 29 to 42, wherein each R 7 are independently acyclic hydrocarbon groups, preferably straight-chain hydrocarbon groups.

44. The composition of any one of claims 29 to 43, wherein each R 7 is independently a hydrocarbyl group having at least 8 carbon atoms, for example a hydrocarbyl group having at least 10 carbon atoms.

45. The composition of any one of claims 29 to 42 and 44, wherein one R 7 It is the tocopherol part.

46. ​​The composition of any one of claims 29 to 45, wherein R 6 Selected from H,C 1-3 Alkyl, -OR 20 ,-N3,C 2-6 Alkynyl, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 ,-COOH,-C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and members of the targeting pair, Wherein C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and members of the targeting pair; R 20 Selected from H, C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-3 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and members of the targeting pair; R 21 Selected from C 1-3 alkyl and a 3 to 6-membered heterocyclic group, wherein the C 1-6 Each of the alkyl and 3 to 6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a member of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl, or R 22 and R 23 may be linked together with the nitrogen atom to which they are attached to form a heterocyclic group, wherein the C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and a member of a targeting pair.

47. The composition of any one of claims 29 to 46, wherein R 6 Selected from H,C 1-3 Alkyl, -OR 20 ,-N3,C 2-6 Alkynyl-OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and members of the targeting pair, Wherein C 1-3 The alkyl group is optionally substituted with one or more substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , and members of the targeting pair; R 20 Selected from H and C 1-3 Alkyl; R 21 is C optionally substituted with one or more substituents independently selected from the following 1-3 Alkyl: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NR 22 R 23 and a member of a targeting pair; and R 22 and R 23 Each of which is independently selected from H, C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Alkynyl, wherein the C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Each of the alkynyl groups is optionally substituted with one or more substituents independently selected from: -OH, SH, halogen, -CN, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 alkyl)2 and a member of a targeting pair, or R 22 and R 23 They may be linked together with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclic group.

48. The composition of any one of claims 29 to 47, wherein R 6 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(0)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of a targeted pair, Wherein C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair.

49. The composition of any one of claims 1, 2, and 28 to 48, wherein the conjugate has the following general formula (V): R 5 -POXZ-R 6 in: R 5 When attached to the N-terminus of the POXZ copolymer, it is selected from -L 2 R 7 ,-(CH2)-CH(OC(O)R 7 )(CH2OC(O)R 7 ), -(CH2)-CH(SR 7 )2, and -(CH2)-CH(SR 7 )-CH2(SR 7 ), where each R 7 are independently a straight chain hydrocarbon group having at least 10 carbon atoms; and L 2 Selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, *-S-(CH2)3-, *-S(O)2-(CH2)3-, and *-OC(O)-(CH2)- (preferably L 2 is selected from *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, and *-C(O)NH-(CH2)2-, such as *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-), wherein * represents a 7 or R 5 When connected to the C-terminus of the POXZ copolymer, it is selected from (R 7 C(O)O)(CH(OC(O)R 7 ))(CH2)-Z 1 -、(R 7 )2N(C 1-3 Alkylene)-Z 1 -、R 7 Z 1 (C 1-3 Alkylene)-Z 1 -、R 7 Z 1 -(C 3-6 Cycloalkenylene)-Z 1 - and R 7 Z 1 -, wherein said C 3-6 The cycloalkenylene group is optionally substituted with one or more (eg, 1, 2, 3, or 4) substituents independently selected from: -OH, =O, -SH, halogen, -CN, -N3, and C 1-3 alkyl; and each Z 1 independently selected from -OP(O)2O(CH2)2NH-, -NH(CH2)2OP(O)2O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -O-, -C(O)O-, -OC(O)-, -S-, -S(O)2-, and -NH-; POXZ is a copolymer comprising repeating units of the following general formulae (Ia) and (Ib): Where R 1 Each of the above is independently a methyl group or an ethyl group, and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 99; the number of repeating units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 10 to 100; and the repeating units of formula (Ia) and (Ib) are arranged randomly, periodically, alternately or in blocks; and R 6 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(O)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of the targeted pair, wherein the C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair.

50. The composition of any one of claims 1, 2, and 28 to 48, wherein the conjugate has the general formula (V) as shown in claim 49, wherein: R 5 When attached to the N-terminus of the POXZ copolymer, (R 7 )2NC(O)(C 1-2 Alkylene), R 7 C(O)OCH2CH(OC(O)R 7 )-CH2-OP(O)2O-(CH2)2-NHC(O)-CH2-, and R 7 -C(O)(CH2)2-C(O)-, Each R 7 is independently a straight chain hydrocarbon group having at least 10 carbon atoms; or R 5 When connected to the C-terminus of the POXZ copolymer, it is R 7 -C(O)(CH2)2C(O)NH- or R 7 N-, where each R 7 are independently a straight chain hydrocarbon group having at least 10 carbon atoms, or a tocopherol moiety, wherein the same R 5 An R in part 7 It is the tocopherol part; POXZ is a copolymer comprising repeating units of the general formula (Ia) and (Ib) as shown in claim 49, wherein R 1 Each of the above is independently a methyl group or an ethyl group and is independently selected for each repeating unit; the number of repeating units of formula (Ia) in the copolymer is 1 to 99; the number of repeating units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeating units of formula (Ia) and the number of repeating units of formula (Ib) in the copolymer is 10 to 100; and the repeating units of formula (Ia) and (Ib) are arranged randomly, periodically, alternately or in blocks; and R 6 Selected from H,C 1-3 Alkyl, -OH, -N3, C 2-6 Alkynyl, -COOH, -NH2, -NHCH3, -N(CH3)2, -NH(CH2CH3), -NHC(O)(CH2)2COOH, -N(CH2CH3)C(O)(CH2)2COOH, -N(CH2CH3)C(O)CH3, -C(O)NH2, -C(O)NHCH3, -OC(O)(CH2)2COOH, and members of the targeting pair, wherein the C 1-3 The alkyl group is optionally substituted with one or more (eg, one or two) substituents independently selected from: -OH,-N3,C 2-6 Alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair.

51. The composition of any one of claims 1 to 50, wherein the conjugate of (a) a POX and / or POZ polymer and (b) one or more hydrophobic chains inhibits the aggregation of the LNP.

52. The composition of any one of claims 1 to 51, wherein the conjugate of (a) a POX and / or POZ polymer and (b) one or more hydrophobic chains comprises about 0.25 mol% to about 50 mol% of the total lipid present in the LNP.

53. The composition of any one of claims 1 to 52, wherein the composition comprises substantially no lipid or lipid-like material containing polyethylene glycol (PEG), preferably substantially no PEG.

54. The composition of any one of claims 1 to 53, wherein the LNP is a non-viral particle.

55. The composition of any one of claims 1 to 54, wherein the lipid that can ionize into a cation comprises a head group comprising at least one nitrogen atom that can be protonated under physiological conditions.

56. The composition of any one of claims 1 to 55, wherein the lipid that can ionize into a cation has a structure of formula (X): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L 10 and L 20 One of them is -O(C=O)-,-(C=O)O-,-C(=O)-,-O-,-S(O) x -,-SS-,-C(=O)S-,SC(=O)-,-NR a C(=O)-,-C(=O)NR a -, NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O-, and L 10 and L 20 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond; G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene or C 2-12 alkenylene; G 3 It is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 Cycloalkenylene; R a Is H or C 1-12 alkyl; R 35 and R 36 Each independently is C 6-24 Alkyl or C 6-24 alkenyl; R 37 is H, OR 50 , CN, -C(=O)OR 40 , -OC(=O)R 40 or -NR 50 C(=O)R 40 ; R 40 It is C 1-12 alkyl; R 50 Is H or C 1-6 Alkyl; and x is 0, 1, or 2.

57. The composition of any one of claims 1 to 55, wherein: (α) The lipid that can ionize into cations is selected from the following structures X-1 to X-36: (β) The lipid that can ionize into cations is selected from the following structures A to G: or (γ) The lipid that can ionize to release cations is a lipid having structure X-3.

58. The composition of any one of claims 1 to 55, wherein the cationic lipid or the lipid that can ionize out a cation has a structure of formula (XI): in Each of R1 and R2 is independently R5 or -G1-L1-R6, wherein at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 Cycloalkyl; Each of R5 and R6 is independently a non-cyclic hydrocarbon group having at least 10 carbon atoms; Each of G1 and G2 is independently an unsubstituted C 1-12 Alkylene or C 2-12 alkenylene; Each of L1 and L2 is independently selected from -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S--SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-; Ra is H or C 1-12 alkyl; m is 0, 1, 2, 3 or 4; and x is 0, 1, or 2.

59. The composition of any one of claims 1 to 55 and 58, wherein the lipid that can ionize into a cation is selected from the following structures (XIV-1), (XIV-2) and (XIV-3):

60. The composition of any one of claims 1 to 54, wherein the cationic lipid or cationic lipid that can be ionized to give off cations comprises 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ... propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

61. The composition of any one of claims 1 to 60, wherein the cationic lipid or lipid that can ionize into a cation comprises about 20 mol% to about 80 mol% of the total lipid present in the LNP.

62. The composition of any one of claims 1 to 61, wherein the LNP further comprises one or more additional lipids, preferably selected from phospholipids, steroids, and combinations thereof, more preferably the LNP comprises the lipid that can ionize out cations, the conjugate, a phospholipid and a steroid.

63. The composition of claim 62, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DMPC), dioctanoylphosphatidylcholine (DP ... The following are the phosphatidylcholine compounds: dioleoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), ditricosanoylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE).

64. The composition of claim 62 or 63, wherein the phospholipids comprise about 5 mol% to about 30 mol% of the total lipids present in the LNP.

65. The composition of any one of claims 62 to 64, wherein the steroid comprises a sterol, such as cholesterol.

66. The composition of any one of claims 62 to 65, wherein the steroid comprises about 10 mol% to about 60 mol% of the total lipids present in the LNP.

67. compositions described in any one in claim 62 to 66, wherein said cationic lipid or the lipid that can ionize out cation accounts for about 20mol% to about 70mol% of the total lipid present in said LNP; (a) the conjugate of POX and / or POZ polymer and (b) one or more hydrophobic chains accounts for about 0.5mol% to about 15mol% of the total lipid present in said LNP; said phospholipid accounts for about 5mol% to about 25mol% of the total lipid present in said LNP; and said steroid accounts for about 25mol% to about 55mol% of the total lipid present in said LNP.

68. The composition of any one of claims 1 to 67, wherein the LNP has a size of about 30 nm to about 500 nm.

69. The composition of any one of claims 1 to 68, wherein the RNA is encapsulated within or associated with the LNP.

70. The composition of any one of claims 1 to 69, wherein the RNA is mRNA.

71. The composition of any one of claims 1 to 70, wherein the RNA comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

72. The composition of any one of claims 1 to 71, 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.

73. The composition of claim 72, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted sequence of A nucleotides.

74. The composition of claim 72 or 73, wherein the 5' cap is a cap 1 or cap 2 structure.

75. The composition of any one of claims 1 to 74, wherein the RNA encodes one or more peptides or proteins, wherein preferably the one or more peptides or proteins are therapeutic peptides or proteins and / or comprise an epitope for inducing an immune response to an antigen in a subject.

76. A method for delivering RNA to cells of a subject, the method comprising administering to the subject the composition of any one of claims 1 to 75.

77. A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject the composition of any one of claims 1 to 75, wherein the RNA encodes the therapeutic peptide or protein.

78. A method for treating or preventing a disease or condition in a subject, the method comprising administering to a subject the composition of any one of claims 1 to 75, wherein delivery of the RNA to cells of the subject is beneficial for treating or preventing the disease or condition.

79. A method for treating or preventing a disease or condition in a subject, the method comprising administering to a subject a composition as described in any one of claims 1 to 75, wherein the RNA encodes a therapeutic peptide or protein, and wherein delivering the therapeutic peptide or protein to the subject is beneficial for treating or preventing the disease or condition.

80. The method of any one of claims 76-79, wherein the subject is a mammal.

81. The method of claim 80, wherein the mammal is a human.

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