RNA-Lipid Nanoparticles (LNPs) Containing Polyoxazoline and / or Polyoxazine Polymers

Abstract

The present invention relates to RNA nanoparticles (LNPs) for RNA delivery after target tissue administration, particularly after parenteral administration, such as intravenous, intramuscular, subcutaneous, intratumoral, intraarterial, intradermal, transdermal, intranasal, rectal or oral administration, and compositions comprising such RNA LNPs. In some embodiments, the RNA LNPs comprise single-stranded RNA, such as mRNA, that encodes a peptide or protein of interest, such as a pharma- ceutical active peptide or protein. The RNA is taken up by cells of the target tissue, and the RNA is translated into the encoded peptide or protein that can exert a physiological activity. Furthermore, the present invention relates to conjugates comprising (a) polyoxazoline (POX) and / or polyoxazine (POZ) polymers and (b) one or more hydrophobic chains, compositions comprising such conjugates, and uses of such conjugates and compositions.

Description

[Technical Field]

[0001] Technical Field The present invention relates to RNA nanoparticles (LNPs) and compositions comprising such RNA LNPs for delivery of RNA to target tissues after administration, particularly parenteral administration, such as intravenous, intramuscular, subcutaneous, intratumoral, intraarterial, intradermal, transdermal, intranasal, rectal, or oral administration. In some embodiments, the RNA LNPs comprise single-stranded RNA, such as mRNA, that encodes a peptide or protein of interest, such as a pharmaceutically active peptide or protein. The RNA is taken up by cells of the target tissue, and the RNA is translated into the encoded peptide or protein that can exert a physiological activity. Furthermore, the present invention relates to conjugates comprising (a) polyoxazoline (POX) and / or polyoxazine (POZ) polymers and (b) one or more hydrophobic chains, compositions comprising such conjugates, and uses of such conjugates and compositions. [Background technology]

[0002] background The use of RNA to deliver foreign genetic information to target cells is an attractive alternative to DNA. The advantages of using RNA include transient expression and non-transforming properties. RNA does not need to enter the nucleus to be expressed, and it cannot be integrated into the host genome, thereby eliminating the risk of carcinogenesis.

[0003] RNA can be delivered to a subject using a variety of delivery vehicles, most of which are based on cationic polymers or lipids that combine with the RNA to form nanoparticles. The nanoparticles are intended to protect the RNA from degradation, enable delivery of the RNA to the target site, and assist cellular uptake and processing by the target cells. In addition to molecular composition, parameters such as particle size, charge, and the implantation of molecular moieties such as polyethylene glycol (PEG) or ligands play a role in delivery efficacy. The implantation of PEG is thought to reduce serum interactions, increase serum stability, and extend circulation time, which may be useful for certain targeting approaches. Ligands that bind to receptors at the target site can help improve targeting efficacy. Furthermore, PEGylation can be used for particle engineering. For example, if LNPs are produced by mixing an aqueous phase of RNA with an organic phase of lipids, a specific fraction of PEG-conjugated lipids in the lipid mixture is required; otherwise, the particles will aggregate during the mixing process. It has been shown that particle size can be tuned by varying the molar fraction of PEG-lipids containing PEG at various molar masses. Similarly, particle size can be adjusted by varying the molar mass of the PEG moiety of the PEGylated lipid. Typical accessible sizes are between 30 and 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). Due to the PEG fraction, the particles thus formed have the added advantage of interacting less with serum components, which is desirable in many drug delivery approaches, and a longer circulation half-life. Without the PEG-lipid, particles of a specific size cannot be formed; they form large aggregates and precipitate.

[0004] Therefore, for techniques in which LNPs are formed from ethanolic and aqueous phases, one of the primary roles of PEG-lipids is to promote particle self-assembly by providing steric hindrance to the surface of nascent particles that form when nucleic acids are rapidly mixed to bind RNA in lipid-containing ethanolic solutions. PEG steric hindrance prevents interparticle fusion and promotes the formation of a uniform population of LNPs, with diameters of <100 nm being achievable.

[0005] PEG is the most widely used and gold-standard "stealth" polymer for drug delivery. PEG-lipids are typically incorporated into systems to produce uniform and colloidally stable nanoparticle populations due to their hydrophilic, steric hindrance properties (the PEG shell prevents electrostatic or van der Waals attractions that lead to aggregation). PEGylation attracts a water shell around the polymer, shielding the RNA complex from opsonization by serum proteins, prolonging serum half-life, reducing rapid renal clearance, and improving pharmacokinetic behavior. Variation in the lipid acyl chain length (C18, C16, or C14) modifies the stability of PEG-lipid incorporation into the particle, leading to modulation of pharmacokinetics. The use of PEG-lipids containing short (C14) acyl chains that dissociate from LNPs with half-lives of <30 minutes in vivo results in optimal hepatocyte gene silencing capacity (Chen et al., 2014, J. Control Release 196:106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163). Furthermore, tight control of particle size can be obtained by varying PEG-lipid parameters: the higher the PEG MW or the higher the PEG-lipid molar fraction in the particle, the smaller the particle.

[0006] Despite these advantages, PEGylation of nanoparticles can also have several adverse effects that are detrimental to their intended use in drug delivery. PEGylation of liposomes and LNPs is known to reduce cellular uptake and endosomal escape, ultimately lowering overall transfection efficiency. Indeed, the PEG shell provides steric hindrance for efficient particle binding to cells and also prevents endosomal release by preventing membrane fusion between liposomes and endosomal membranes. This is why the type and amount of PEG-lipid used must always be carefully regulated. On the one hand, it must provide sufficient stealth effect for in vivo and stabilization aspects, while on the other hand, it must not interfere with transfection. This phenomenon is known as the "PEG dilemma."

[0007] In addition to reducing transfection efficiency, PEGylation is associated with the accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and / or complement activation and storage diseases (Bendele A et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; SM Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes in rats significantly altered the pharmacokinetic behavior of a second dose when the second dose was administered several days apart (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 appears to correlate with the PEG content of liposomes. The presence of anti-PEG antibodies in the plasma induces high particle clearance by the monophagocyte system (MPS), ultimately reducing drug efficacy.

[0008] PEG has also been postulated to induce complement activation, which can lead to a hypersensitivity reaction also known as complement activation-associated pseudoallergy (CARPA). It is not yet clear from the literature whether complement activation is due to the presence of nanoparticles in general or PEG in particular.

[0009] The presence of PEG in other lipid particles may also induce specific immune responses. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotides or plasmid DNA induced a strong immune response in mice, resulting in rapid blood clearance of subsequent administration. The strength of this response was sufficient to induce significant morbidity and, in some cases, mortality. The rapid clearance of liposome-encapsulated oligodeoxynucleotides from the blood depended on the presence of PEG-lipids in the membrane, as the use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG-lipids negated this response. The production of anti-PEG antibodies and presumed complement activation may explain the rapid clearance of vesicles from the blood (Semple et al., 2005, J. Pharmacol. Exp. Ther. 312(3), 1020-6).

[0010] Because PEG can induce an immune response, multiple injections are necessary, which needs to be avoided in certain applications, such as treatments using mRNA for protein replacement therapy, where the risk is particularly high due to the potential intrinsic immunogenicity of RNA. Summary of the Invention [Problem to be solved by the invention]

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

[0012] The inventors have surprisingly discovered that the RNA LNP formulations described herein meet the above requirements. In particular, conjugates comprising hydrophobic chains and polyoxazoline (POX) and / or polyoxazine (POZ) polymers have been shown to be suitable components for the assembly of RNA LNPs. POX and POZ can be synthesized by activated cationic ring-opening polymerization using unsubstituted or substituted 2-oxazoline and 2-oxazine compounds. POX / POZ conjugates enable the production of RNA LNPs by various techniques, resulting in defined surface properties and a controlled size range. Production can be carried out using robust methods that meet the requirements of pharmaceutical manufacturing. Particles can be end-functionalized with various moieties for charge regulation or the introduction of specific molecular moieties such as ligands. [Means for solving the problem]

[0013] overview In a first aspect, the present invention relates to a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises: (i) RNA; (ii) cationic or cationic ionizable lipids in a molar fraction of 47.5:42.5-X:10; and (iii) a conjugate of (a) a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and (b) one or more hydrophobic chains.

[0014] In certain embodiments, the RNA LNPs are non-viral RNA particles.

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

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

[0017] In some embodiments, cationic or cationically ionizable lipids are combined with RNA together with (a) POX and / or POZ polymers and (b) one or more hydrophobic chain conjugates to form particles. In some embodiments, the cationically ionizable lipids are only positively charged at acidic pH and do not remain cationic at physiological pH. In some embodiments, LNPs comprise one or more additional lipids. In some embodiments, the POX and / or POZ polymers are conjugated to one or more hydrophobic chains via linkers. In some embodiments, the one or more hydrophobic chains are one or more hydrocarbyl groups, such as acyclic, preferably linear hydrocarbyl groups (e.g., linear hydrocarbyl groups having at least 10 carbon atoms), for example, the hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the linker comprises a functionalized moiety, such as a cleavable moiety (e.g., a moiety that can be cleaved under physiological conditions), that connects one or more hydrophobic chains to the POX and / or POZ polymer. In some embodiments, the functionalizing moiety is neutral at physiological conditions; in some embodiments, the complete linker is neutral at physiological conditions.

[0018] In some embodiments, the linker comprises at least one functionalized moiety. In some embodiments, the linker comprises an alkylene moiety substituted with at least one monovalent functionalized moiety and / or linked to a divalent functionalized moiety that terminating in the alkylene group being attached to one or more hydrophobic chains (e.g., C 1-6 Alkylene moieties, e.g., C 1-3 Alkylene moiety or C 2-3In certain embodiments, each monovalent functionalized moiety comprises a hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imide, or the like. and amide moieties. In certain embodiments, each divalent functionalized moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, and carboxylate. and independently selected from carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

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

[0020] In some embodiments, the linker further comprises an alkylene moiety (preferably C) connecting the functionalized moiety to the POX and / or POZ polymer. 1-6 alkylene moiety).

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

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

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

[0024] In some embodiments (especially when one or more hydrophobic chains are attached to the N-terminus (i.e., terminal N atom) of the POX and / or POZ polymer, e.g., as shown in formula (II) below), the linker is [*-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 1-3 -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 point of attachment of the linker to 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 is C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); R 26 is H, C 1-6 Alkyl, aryl and aryl(C 1-6 and r is an integer from 1 to 2. For example, the linker is [*-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(OR 25 ) 3-r ](C 1-3 -alkylene)-, [*-C(OR 25 )2O] 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 (C1-3 -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 point of attachment of the linker 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); R 25 is C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); R 26 is H, C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); and r is an integer from 1 to 2.

[0025] In some embodiments (especially when one or more hydrophobic chains are attached to the N-terminus (i.e., terminal N atom) of the POX and / or POZ polymer, e.g., as shown in formula (II) below), the linker is [*-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 )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)- or [*-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 )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)-, for example [*-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, [*-NHC(O)] p (C 1-6 -alkylene)- and [*-C(O)O] p (C 1-6 In some embodiments, the linker may be selected from the group consisting of [*-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)- and [*-S(O)2]p (C 1-6 -alkylene)-, preferably [*-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)-. In any of the above embodiments, C 1-6 -Alkylene is C such as methylene, ethylene or trimethylene 1-3 -alkylene is preferred.

[0026] In some embodiments, R 25 is C 1-3 Alkyl, phenyl and phenyl(C 1-3 alkyl), such as methyl, ethyl, phenyl, benzyl, and phenylethyl.

[0027] In some embodiments, R 26 is H, C 1-3 Alkyl, phenyl and phenyl(C 1-3 alkyl), such as H, methyl, ethyl, phenyl, benzyl, and phenylethyl.

[0028] In certain embodiments (particularly when one or more hydrophobic chains are attached to the N-terminus (i.e., terminal N atom) of the POX and / or POZ polymer, e.g., as shown in formula (II) below), the linker may be *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)-, *-C(O)NH-(CH2)2-, -(CH2)-CH(OC(O)-*)(CH2 The linker may be selected from the group consisting of -OC(O)-*), -(CH2)-CH(S-*), -(CH2)-CH(S-*)-CH2(S-*), *-S-(CH2)3-, *-S(O)2-(CH2)3- and *-OC(O)-(CH2)-, preferably the linker is *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-, where * represents the point of attachment of the linker to the hydrophobic chain.

[0029] In some embodiments (especially when one or more hydrophobic chains are attached to the N-terminus (i.e., terminal N atom) of the POX and / or POZ polymer, e.g., as shown in formula (II) below), the linker is [*-]NC(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 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 is [*-]2NC(O)(C 1-2 -alkylene)-, [*-OC(O)]2(C 2-4 -alkylene)-OP(O)2O(C 2-3 -alkylene)NHC(O)-(C1-2 -alkylene)- and *-C(O)(C 2-3 -C(O)-, where * represents the point of attachment of the linker to the hydrophobic chain.

[0030] In some embodiments (especially when one or more hydrophobic chains are attached to the (II') C-terminus (i.e., terminal C atom) of the POX and / or POZ polymer, e.g., as shown in the formula below), the linker comprises at least one bifunctional moiety via which the one or more hydrophobic chains are attached to the POX and / or POZ polymer. In some embodiments, the linker further comprises an alkylene moiety (e.g., C 1-6 Alkylene moieties, e.g., C 1-3 alkylene moiety), cycloalkylene moiety (preferably C 3-8 -cycloalkylene, e.g. C 3-6 -cycloalkylene moiety) or cycloalkenylene moiety (preferably C 3-8 -cycloalkenylene, e.g., C 3-6-cycloalkenylene moieties), each of which connects a difunctionalized moiety to the POX and / or POZ polymer (either directly to the end of the POX and / or POZ polymer or, preferably, via a further difunctionalized moiety). For example, one hydrophobic chain may be attached to the POX and / or POZ polymer via one difunctionalized moiety (either directly or via an alkylene, cycloalkylene, or cycloalkenylene moiety bearing an alkylene, cycloalkylene, or cycloalkenylene moiety or other difunctionalized moiety); two hydrophobic chains may be attached to the POX and / or POZ polymer via two difunctionalized moieties (which are then preferably attached to alkylene, cycloalkylene, or cycloalkenylene moieties bearing an alkylene, cycloalkylene, or cycloalkenylene moiety or other difunctionalized moiety); or two hydrophobic chains may be attached to the termini of the POX and / or POZ polymer via the same difunctionalized moiety (which then becomes a trifunctionalized moiety, which is attached to the termini of the POX and / or POZ polymer directly or via an alkylene, cycloalkylene, or cycloalkenylene moiety bearing an alkylene, cycloalkylene, or cycloalkenylene moiety or other difunctionalized moiety). In certain embodiments, each divalent functionalized moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, and carboxylate. and independently selected from carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.In certain preferred embodiments (especially when one or more hydrophobic chains are attached to the (II') C-terminus (i.e., terminal C atom) of the POX and / or POZ polymer, e.g., as shown in the formula below), the linker comprises at least one divalent functionalized moiety selected from the group consisting of amide, sulfide, sulfone, and amino moieties.

[0031] In some embodiments, the cycloalkylene moiety is C 3-8 -cycloalkylene, e.g. C 3-6 -cycloalkylene, e.g., cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, wherein the cycloalkylene moiety optionally contains one or more (e.g., 1, 2, 3, or 4) substituents (e.g., -OH, ═O, -SH, halogen, -CN, -N, and C 1-3 -alkyl).

[0032] In some embodiments, the cycloalkenylene moiety is C 3-8 -cycloalkenylene, e.g., C 3-6 -cycloalkenylene, e.g., cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, wherein the cycloalkenylene moiety optionally contains one or more (e.g., 1, 2, 3, or 4) substituents (e.g., -OH, ═O, -SH, halogen, -CN, -N, and C 1-3 -alkyl).

[0033] In some embodiments, the alkylene moiety is C 1-6 -Alkylene, e.g. C 1-3 - alkylene, for example methylene, ethylene or trimethylene or C 2-3 It is alkylene.

[0034] In certain embodiments (particularly when one or more hydrophobic chains are attached to the (II') C-terminus (i.e., terminal C atom) of the POX and / or POZ polymer, e.g., as shown in the formula below), the conjugate comprises one of the following structures (and can have the general formula (II')): (hydrophobic chain)-(difunctionalized moiety)-(POX and / or POZ polymer) [(hydrophobic chain)-(divalent functionalized moiety)] 1-2 -(alkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer) (hydrophobic chain)-(difunctionalized moiety)-(cycloalkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer) (hydrophobic chain)-(difunctionalized moiety)-(cycloalkenylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer) (hydrophobic chain)-(difunctionalized moiety)-(alkylene moiety)-(POX and / or POZ polymer) [(hydrophobic chain)2-(trifunctionalized moiety)]-(alkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer)

[0035] In certain embodiments (especially when one or more hydrophobic chains are attached to the (II') C-terminus (i.e., terminal C atom) of the POX and / or POZ polymer, e.g., as shown in the formula below), the conjugate has one of the following formulas (and can fall into general formula (II')): (hydrophobic chain)-(difunctionalized moiety)-(POX and / or POZ polymer)-(end group) [(hydrophobic chain)-(divalent functionalized moiety)] 1-2 -(alkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer)-(end group) (hydrophobic chain)-(difunctionalized moiety)-(cycloalkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer)-(end group) (hydrophobic chain)-(difunctionalized moiety)-(cycloalkenylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer)-(end group) (hydrophobic chain)-(difunctionalized moiety)-(alkylene moiety)-(POX and / or POZ polymer)-(end group) [(hydrophobic chain)2-(trifunctionalized moiety)]-(alkylene moiety)-(difunctionalized moiety)-(POX and / or POZ polymer)-(end group)

[0036] The POX and / or POZ polymers can contain neutral end groups (e.g., H, alkyl, alkoxy, ester, or amide end groups) or functionalized end groups (e.g., hydroxy, thiol, cyano, azido, or amino end groups). In some embodiments, the end groups are R, as defined herein. 3 (Especially formulas (II), (II'), (IIa), (IIa'), (IIb), (IIb'), (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi'), (II j'), (III), (III'), (IIIa), (IIIa'), (IIIb), (IIIb'), (VI), (VI'), (VIa), (VIa'), (VII), (VII'), (VIIa), with respect to any of formulas (VIIa'), (VIII), (VIII'), (VIIIa), (IX), (IX') and (IXa) or any of formulas (IIe), (IVc), (XV), (XV'), (XVI), (XVII), (XVII'), (XVIII), (XVIII'), (XIX), (XIX'), (XX'), (XXI'), (XXII') or (XXIII')) or R as defined herein 6 (particularly with respect to any of formulas (IV), (IV'), (IVa), (IVb), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (V) and (V') or formula (IVc)).

[0037] In some embodiments, particularly when the hydrophobic chain / linker is attached at the C-terminus, the linker is [*-Z] p (C 1-6 -Alkylene)-Z-, *-Z-(C 3-8 -cycloalkylene)-Z-, *-Z-(C 3-8 -cycloalkenylene)-Z-, (*=N)(C1-6 -Alkylene)-Z-, *-Z-(C 1-6 -Z-, where * 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 selected from -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, alkyl, aryl, aryl, arylsulfonyl ... 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 - independently selected from the group consisting of, where R 22 is selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl. For example, the linker can be [*-C(O)O] p (C 1-6 -Alkylene)-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 * represents the point of attachment to the hydrophobic chain; p is 1 or 2; C 1-6 - alkylene is divalent (if p is 1) or trivalent (if p is 2); C3-8 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, alkyl, aryl, aryl, arylsulfonyl ... 1-3 -alkylene)NH-, -NH(C 1-3 -alkylene)OP(O)2O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0038] In some embodiments, particularly when the hydrophobic chain / linker is attached at the C-terminus, the linker is [*-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 -Z-, where * 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 selected from -OH, =O, -SH, halogen, -CN, -N and C 1-3 -alkyl; and each Z is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of: -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-, -N(C 1-3 -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 * 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 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, alkyl, aryl, aryl, arylsulfonyl ... 1-2 -alkylene)NH-, -NH(C 1-2 -alkylene)OP(O)2O-, -OC(O)NH-, -NHC(O)O-, -O-, -S- and -NH-.

[0039] In some embodiments, particularly when the hydrophobic chain / linker is attached at the C-terminus, the linker is [*-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 -Z-, where * 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 3-6Each of the -cycloalkenylene groups is optionally selected from -OH, =O, -SH, halogen, -CN, -N and C 1-3 -alkyl; and each Z is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of: -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-, -N(C 1-3 -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 * 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 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of -OP(O)2O(CH2)2NH-, -NH(CH2)2OP(O)2O-, -OC(O)NH-, -NHC(O)O-, -O-, -S-, and -NH-.

[0040] In some embodiments, particularly when the hydrophobic chain / linker is attached at the C-terminus, the linker is [*-C(O)] p (C 1-6 -alkylene)-C(O)NH- and [*-]N-, where * represents the point of attachment 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 is -C(O)(C 2-3 -alkylene)-C(O)NH- or [*-]2N-, where * represents the point of attachment to the hydrophobic chain.

[0041] In some embodiments, the linker may be (*-C(O)O)(CH(OC(O)-*))(CH)-Z-, (*=N)(C 1-3 -alkylene)-NHC(O)-, (*-Z)(C 1-3 -Alkylene)-Z-, *-Z-(C 3-6 -cycloalkenylene)-Z- and *-Z-, where * represents the point of attachment to the hydrophobic chain; C 3-6 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -alkyl; and each Z is independently selected from the group consisting of -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-. For example, the linker can be (*-C(O)O)(CH(OC(O)-*))(CH2)-Z-, (*=N)(C 1-3 -alkylene)-NHC(O)-, (*-NH)(C 1-3 -alkylene)-NHC(O)-, *-C(O)NH-, *-NHC(O)-, *-Z-(C 3-6-cycloalkenylene)-Z-, -S-, and -S(O)2-, where * represents the point of attachment to the hydrophobic chain; C 3-6 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of -OP(O)2O(CH2)2NH-, -NH(CH2)2OP(O)2O-, -OC(O)NH-, -NHC(O)O-, -O-, -S-, and -NH-.

[0042] In one embodiment, the terminal group on the POX and / or POZ polymer side of the conjugate of (a) a POX and / or POZ polymer and (b) one or more hydrophobic chains is H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 , -SR 20 (e.g., SH), halogen, -CN, -N3, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C 1-6 The alkyl group may optionally be -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 20 is H, C 1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-3Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be joined to the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N, —C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl), sugars, amino acids, peptides, and members of a targeting pair.

[0043] 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-αvβ3 integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor. Thus, in some embodiments, members of the targeting pair include one of the following: antigen, antibody, avidin, streptavidin, folate, transferrin, aptamer; RGD peptide; NGR peptide; and galactose.

[0044] In one embodiment, the terminal group on the POX and / or POZ polymer side of the conjugate of (a) a POX and / or POZ polymer and (b) one or more hydrophobic chains is H, C 1-3 Alkyl, -OR 20 , -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 and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, SH, halogen, -CN, -N3, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R 20 is H, C 1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-3 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally -OH, SH, halogen, -CN, -N3, -COOH, -NR22 R 23 and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R 21 is C 1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally -OH, SH, halogen, -CN, -N3, -COOH, -NR 22 R 23 and one or more substituents independently selected from the group consisting of members of a targeting pair; and R 22 and R 23 Each of the groups independently represents H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl or R 22 and R 23 may join with the nitrogen atom to which they are attached to form a heterocyclyl group, where C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Each alkynyl group may optionally be -OH, SH, halogen, -CN, -N, -COOH, -NH, -NH(C 1-3 alkyl), -N(C 1-3 and one or more substituents independently selected from the group consisting of alkyl)2 and members of a targeting pair.

[0045] In some embodiments, this terminal group on the POX and / or POZ polymer side of the conjugate is H, C 1-3 Alkyl, -OR 20 , -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 and a member of a targeting pair, wherein C 1-3The alkyl group may optionally be -OH, -N3, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 and substituted with one or more substituents independently selected from the group consisting of members of a targeting pair; R 20 is H and C 1-3 alkyl; R 21 is optionally -OH, SH, halogen, -CN, -N3, -COOH, -NR 22 R 23 and C substituted with one or more substituents independently selected from the group consisting of members of a targeting pair. 1-3 alkyl; and R 22 and R 23 Each of the groups independently represents H, C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 alkynyl, wherein C 1-3 Alkyl, C 2-3 Alkenyl and C 2-3 Each alkynyl group may optionally be -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2 and one or more substituents independently selected from members of a targeting pair, or R 22 and R 23 can be taken together with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclyl group.

[0046] In some embodiments, this terminal group on the POX and / or POZ polymer side of the conjugate is H, C 1-3 Alkyl, -OH, -N3, C 2-6alkynyl, -COOH, -NH, -NHCH, -N(CH), -NH(CHCH), -NHC(O)(CH)COOH, -N(CHCH)C(O)(CH)COOH, -N(CHCH)C(O)CH, -C(O)NH, -C(O)NHCH, -OC(O)(CH)COOH and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, -N, C 2-6 Substituted with one or more (e.g., one or two) substituents independently selected from the group consisting of alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2 and members of a targeting pair.

[0047] In some embodiments, the POX and / or POZ polymer has the following general formula (I): [ka] [wherein a is an integer of 1 to 2; R 1 is alkyl, especially C 1-3 alkyl, e.g., methyl, ethyl, isopropyl, or n-propyl, selected independently for each repeat unit; and m is 2 to 200. In some embodiments, R 1 is the same in each instance (i.e., in each repeat unit) (e.g., R 1 (R can be methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is the R of another repeating unit 1 (e.g., R 1 is a particular alkyl (e.g., ethyl), and R 1 is a different specific alkyl (e.g., methyl).

[0048] In some embodiments, the POX and / or POZ polymer is a POX polymer and has the following general formula (Ia): [ka] [In the formula, R 1 is defined above for formula (I). It contains repeating units of

[0049] In some embodiments, the POX and / or POZ polymer is a POZ polymer and has the following general formula (Ib): [ka] [In the formula, R 1 is defined above for formula (I). It contains repeating units of

[0050] In any of the above embodiments of formula (I), (Ia) and (Ib), m (i.e., the number of repeat 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.

[0051] In some embodiments, the POX and / or POZ polymers have the following general formulas (Ia) and (Ib): [ka] [In the formula, R 1 is defined above for 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. It is a copolymer containing repeating units of

[0052] Preferred embodiments of formulae (I), (Ia) and (Ib) are shown below as "Conjugates of POX and / or POZ Polymers with One or More Hydrophobic Chains."

[0053] In certain embodiments, the conjugate has the following general formula (II) or (II'): [ka] [During the ceremony, a is an integer between 1 and 2; R 1 is alkyl, especially C 1-3 alkyl, e.g., methyl, ethyl, iso-propyl, or n-propyl, independently selected for each repeat unit; m is 2 to 200; R 2 is R 4 or -L 1 (R 4 ) p where each R 4 are independently hydrocarbyl groups; L 1 is a linker; and p is 1 or 2; and R 3 is H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 , -SR 20 , halogen, -CN, -N3, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C 1-6 The alkyl group may optionally be -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 20 is H, C1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-3 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be joined to the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N, —C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 and substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, sugars, amino acids, peptides, and members of targeting pairs. It has.

[0054] In some embodiments, R 1 is the same in each case (i.e., in each repeat unit) (e.g., R1 (R can be methyl in each repeat unit). In some embodiments, R in at least one repeat unit 1 is the R of another repeating unit 1 (e.g., R 1 is a particular alkyl (e.g., ethyl), and R 1 is a different specific alkyl (e.g., methyl).

[0055] In certain embodiments, a is 1, i.e., the conjugate has the following general formula (IIa) or (IIa′): [ka] It has.

[0056] In certain embodiments, a is 2, i.e., the conjugate has the following general formula (IIb) or (IIb'): [ka] It has.

[0057] In any of the above embodiments of formulas (IIa), (IIa'), (IIb) and (IIb'), R 1 , R 2 , R 3 and m is as defined for formula (II) or (II'). Preferred embodiments of formulas (II), (II'), (IIa), (IIa'), (IIb) and (IIb') (e.g., formulas (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi') and (IIj') and formula (IIe)) are set forth herein under "Conjugates of POX and / or POZ Polymers with One or More Hydrophobic Chains."

[0058] In certain embodiments, the conjugate has the following general formula (III) or (III'): [ka] [During the ceremony, a is an integer between 1 and 2; R 1 is methyl or ethyl, selected independently for each repeat unit; m is 10 to 100 (preferably 20 to 80, 30 to 70, or 40 to 50); R 2 is for formula (III) -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 ), wherein each R 4 are independently linear hydrocarbyl groups having at least 10 carbon atoms; and L 1 is selected from the group consisting of *-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 the group consisting of *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)- and *-C(O)NH-(CH2)2-, e.g., *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-), where * is R 4 or for formula (III'), R represents the point of attachment to 2 (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 C 3-6The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -alkyl; and each Z is independently selected from the group consisting of -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 is H, C 1-3 Alkyl, -OH, -N3, C 2-6 alkynyl, -COOH, -NH, -NHCH, -N(CH), -NH(CHCH), -NHC(O)(CH)COOH, -N(CHCH)C(O)(CH)COOH, -N(CHCH)C(O)CH, -C(O)NH, -C(O)NHCH, -OC(O)(CH)COOH and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, -N, C 2-6 and substituted with one or more (e.g., one or two) substituents independently selected from the group consisting of alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair. It has.

[0059] In certain embodiments, the conjugate has the general formula (III) or (III'), wherein: a is an integer between 1 and 2; R 1 is methyl or ethyl, selected independently for each repeat unit; m is 10 to 100; R 2 is for formula (III) (R 4 )2NC(O)(C 1-2 -alkylene)-, R 4C(O)OCH2CH(OC(O)R 4 )-CH2-OP(O)2O-(CH2)2-NHC(O)-CH2- and R 4 -C(O)(CH2)2-C(O)-, wherein each R 4 are independently a linear hydrocarbyl group having at least 10 carbon atoms; or for formula (III'), R 2 is R 4 -C(O)(CH2)2C(O)NH- or R 4 N-, where each R 4 are independently a linear hydrocarbyl group or a tocopherol moiety having at least 10 carbon atoms, 2 Part 1 R 4 is the tocopherol moiety; and R 3 is H, C 1-3 Alkyl, -OH, -N3, C 2-6 alkynyl, -COOH, -NH, -NHCH, -N(CH), -NH(CHCH), -NHC(O)(CH)COOH, -N(CHCH)C(O)(CH)COOH, -N(CHCH)C(O)CH, -C(O)NH, -C(O)NHCH, -OC(O)(CH)COOH and a member of a targeting pair, wherein C 1-3 The alkyl group may optionally be -OH, -N, C 2-6 Substituted with one or more (e.g., one or two) substituents independently selected from the group consisting of alkynyl, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2 and members of a targeting pair.

[0060] In certain embodiments, a is 1, i.e., the conjugate has the following general formula (IIIa) or (IIIa′): [ka] It has.

[0061] In certain embodiments, a is 2, i.e., the conjugate has the following general formula (IIIb) or (IIIb'): [ka] It has.

[0062] In any of the above embodiments of formulas (IIIa), (IIIa'), (IIIb) and (IIIb'), R 1 , R 2 , R 3 and m is as defined in formula (III) or (III').

[0063] Preferred embodiments of formulas (III), (III'), (IIIa), (IIIa'), (IIIb) and (IIIb') are shown below as "Conjugates of POX and / or POZ Polymers with One or More Hydrophobic Chains."

[0064] In certain embodiments, the conjugate has the following general formula (IV): R 5 -POXZ-R 6 [During the ceremony, R 5 is R 7 or -L 2 (R 7 ) q where each R 7 are independently hydrocarbyl groups; L 2 is a linker; and q is 1 or 2; POXZ has the following general formulas (Ia) and (Ib): [ka] where R 1 each independently represents alkyl, particularly C 1-3alkyl, e.g., methyl, ethyl, iso-propyl, or n-propyl, independently selected for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 199; the number of repeat units of formula (Ib) in the copolymer is 1 to 199; the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 2 to 200; and the repeat units of formula (Ia) and (Ib) are arranged in a random, periodic, alternating, or block unit manner; and R 6 is H, C 1-6 Alkyl, C 2-6 Alkynyl, -OR 20 , -SR 20 , halogen, -CN, -N3, -OC(O)R 21 , -C(O)R 21 , -NR 22 R 23 , -COOH, -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein C 1-6 The alkyl group may optionally be -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -COOCH3, -NR 22 R 23 , -C(O)NR 22 R 23 , -NR 22 C(O)R 21 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 20 is H, C 1-3 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-3 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23, substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of targeting pairs; R 21 is C 1-6 selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, wherein C 1-6 Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N, C 2-6 Alkynyl, -COOH, -NR 22 R 23 , substituted with one or more substituents independently selected from the group consisting of sugars, amino acids, peptides, and members of a targeting pair; and R 22 and R 23 each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 may be joined to the nitrogen atom to which they are attached to form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally —OH, —SH, halogen, —CN, —N, —C 2-6 Alkynyl, -COOH, -NH2, -NH(C 1-3 alkyl), -N(C 1-3 substituted with one or more substituents independently selected from the group consisting of alkyl, sugars, amino acids, peptides, and members of a targeting pair. It has.

[0065] In certain embodiments, the conjugate has the following general formula (V): R 5 -POXZ-R 6 [During the ceremony, R 5 When attached to the N-terminus of a POXZ copolymer, -L 2 R 7 , -(CH2)-CH(OC(O)R 7 )(CH2OC(O)R 7 ), -(CH2)-CH(SR7 )2 and -(CH2)-CH(SR 7 )-CH2(SR 7 ), wherein each R 7 are independently linear hydrocarbyl groups having at least 10 carbon atoms; and L 2 is selected from the group consisting of *-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 the group consisting of *-NHC(O)-(CH2)-, *-NHC(O)-(CH2)2-, *-C(O)NH-(CH2)- and *-C(O)NH-(CH2)2-, e.g., *-NHC(O)-(CH2)- or *-NHC(O)-(CH2)2-), where * is R 7 represents the point of attachment to R 5 When attached to the C-terminus of the POXZ copolymer, (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 C 3-6 The cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N and C 1-3 -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, aryl, methyl, methyl, methylamino ... 1is independently selected from the group consisting of -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 has the following general formulas (Ia) and (Ib): [ka] where R 1 are independently methyl or ethyl and are independently selected for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 99; the number of repeat units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 10 to 100 (preferably 20 to 80, 30 to 70, or 40 to 50); and the repeat units of formula (Ia) and (Ib) are arranged in a random, periodic, alternating, or block unit manner; and R 6 is H, C 1-3 is selected from the group consisting of alkyl, -OH, -N3, -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 a member of a targeting pair, wherein C 1-3 The alkyl group is optionally substituted with one or more (e.g., one or two) substituents independently selected from the group consisting of -OH, -N3, -COOH, -COOCH3, -NH2, -NHCH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)NH(CH2)2NH2, and members of a targeting pair. It has.

[0066] Preferred embodiments of formulas (IV) and (V) and conjugates of POX and / or POZ polymers with one or more hydrophobic chains (as well as further preferred embodiments of formulas (IV'), (V'), (VI), (VI'), (VIa), (VIa'), (VII), (VII'), (VIIa), (VIIa'), (VIII), (VIII'), (VIIIa), (IX), (IX') and (IXa) and formulas (IVc), (XV), (XV'), (XVI), (XVII), (XVII'), (XVIII), (XVIII'), (XIX), (XIX'), (XX'), (XXI'), (XXII') and (XXIII')) and specific examples thereof are shown below under the heading "Conjugates of POX and / or POZ polymers with one or more hydrophobic chains".

[0067] In certain embodiments, the conjugate of (a) a POX and / or POZ polymer and (b) one or more hydrophobic chains inhibits aggregation of LNPs.

[0068] In certain embodiments, 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.

[0069] In certain embodiments, the composition is substantially free of lipids comprising polyethylene glycol (PEG), preferably substantially free of PEG.

[0070] In certain embodiments, the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom that can be protonated under physiological conditions.

[0071] In some embodiments, the cationically ionizable lipid has the formula (X): [ka] [During the ceremony, L 10 and L 20One of the 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 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 a direct bond; G 1 and G 2 are each independently unsubstituted C-C 12 Alkylene or C 2-12 alkenylene; G 3 is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 is cycloalkenylene; R a is H or C 1-12 is alkyl; R 35 and R 36 are each independently C 6-24 Alkyl or C 6-24 is alkenyl; R 37 is H, OR 50 , CN, -C(=O)OR 40 , -OC(=O)R 40 or -NR 50 C(=O)R 40 and; R 40 is C1-12 is alkyl; R 50 is H or C 1-6 is alkyl; and x is 0, 1, or 2. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0072] In some embodiments, the cationically ionizable lipid is selected from structures X-1 through X-36 disclosed herein. In some embodiments, the cationically ionizable lipid is a lipid having structure X-3.

[0073] In some embodiments, the cationically ionizable lipid is selected from structures A through G disclosed herein.

[0074] In some embodiments, the cationically ionizable lipid has formula (XI): [ka] [During the ceremony, each of R1 and R2 is independently R5 or -G1-L1-R6, where at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 independently selected from the group consisting of cycloalkyl; each of R5 and R6 is independently R5 and R6; each of G1 and G2 is independently G1 and G2; Each of L1 and L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x independently selected from the group consisting of -, -SS-, -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 1-12is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2. It has the following structure.

[0075] In some embodiments, the cationically ionizable lipid has the structure (XIV-1), (XIV-2), or (XIV-3) disclosed herein.

[0076] In some embodiments, the cationic or cationically ionizable lipid is 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or mixtures thereof.

[0077] In certain embodiments, the cationic or cationically ionizable lipids comprise between about 20 mol % and about 80 mol % of the total lipids present in the LNP.

[0078] In certain embodiments, the LNPs further comprise one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof; more preferably, the LNPs comprise a cationically ionizable lipid, a conjugate, a phospholipid, and a steroid.

[0079] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphthanoyl-phosphatidylethanolamine (DPyPE). In some embodiments, the phospholipid is DOPE.

[0080] In certain embodiments, phospholipids comprise between about 1 mol % and about 30 mol % of the total lipids present in the LNP.

[0081] In some embodiments, the steroid comprises a sterol, such as cholesterol.

[0082] In certain embodiments, the steroid comprises between about 10 mol % and about 60 mol % of the total lipids present in the LNP.

[0083] In certain embodiments, cationic or cationically ionizable lipids comprise between about 20 mol% and about 70 mol% of the total lipids present in the LNP; (a) a POX and / or POZ polymer and (b) a conjugate of one or more hydrophobic chains comprise between about 0.5 mol% and about 15 mol% of the total lipids present in the LNP; phospholipids comprise between about 5 mol% and about 25 mol% of the total lipids present in the LNP; and steroids comprise between about 25 mol% and about 55 mol% of the total lipids present in the LNP.

[0084] In certain embodiments, the LNPs have a size of about 30 nm to about 500 nm.

[0085] In certain embodiments, the RNA is encapsulated or associated with the LNP.

[0086] In certain embodiments, the RNA is single-stranded RNA, such as mRNA.

[0087] In certain embodiments, the RNA comprises modified nucleosides in place of uridine, where the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0088] In certain embodiments, the RNA comprises at least one of, and preferably all of, the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

[0089] In certain embodiments, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

[0090] In certain embodiments, the 5' cap is a cap1 or cap2 structure.

[0091] In certain 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 against an antigen in a subject.

[0092] In a second aspect, the present invention relates to a method of delivering RNA to cells in a subject, comprising administering to the subject a composition of the first aspect.

[0093] In a third aspect, the present invention relates to a method of delivering a therapeutic peptide or protein to a subject, comprising administering to the subject a composition of the first aspect, wherein the RNA encodes the therapeutic peptide or protein.

[0094] In a fourth aspect, the present invention relates to a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of the first aspect, wherein delivery of RNA to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the present invention relates to the composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of RNA to cells of the subject is beneficial to treating or preventing the disease or disorder.

[0095] In a fifth aspect, the present invention relates to a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of the first aspect, wherein the RNA encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the present invention relates to the composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein the RNA encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder.

[0096] In certain embodiments of the second through fifth aspects, the subject is a mammal, such as a human.

[0097] In a sixth aspect, the present invention relates to a conjugate of (a) a POX or POZ polymer and (b) one or more hydrophobic chains, wherein the conjugate has the general formula (II) or (II') disclosed herein. In certain embodiments, the conjugate of the sixth aspect has the general formula (IIa), (IIa'), (IIb), (IIb'), (IIc), (IId), (IIe'), (IIf'), (IIg'), (IIh'), (IIi'), (IIj'), (III), (III'), (IIIa), (IIIa'), (IIIb), (IIIb'), (VI), (VI'), (VIa), (VIa'), (VII), (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), (II-2 2), (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) or any of formulas (IIe), (IVc), (XV), (XV'), (XVI), (XVII), (XVII'), (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).

[0098] In a seventh aspect, the present invention provides a conjugate of (a) a POX and POZ polymer and (b) one or more hydrophobic chains, wherein the POX and POZ polymers have the following general formulas (Ia) and (Ib): [ka] [In the formula, R 1 each independently represents alkyl, particularly C 1-3 alkyl, such as methyl, ethyl, isopropyl, or n-propyl, selected independently 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 of Formulas (IV), (IV'), (IVa), (IVb), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (V) and (V') disclosed herein, or Formula (IVc) disclosed herein.

[0099] In an eighth aspect, the present invention relates to a composition comprising: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the sixth or seventh aspect.

[0100] In certain embodiments of the eighth aspect, the conjugate comprises from about 0.25 mol % to about 50 mol % of the total lipid present in the composition.

[0101] In certain embodiments of the eighth aspect, the composition is substantially free of lipids comprising polyethylene glycol (PEG), preferably substantially free of PEG.

[0102] In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid comprises a head group comprising at least one nitrogen atom capable of being protonated under physiological conditions. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid has a structure of formula (X) shown herein. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid has a structure of formula (X) disclosed herein. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid is selected from structures X-1 through X-36 disclosed herein. In certain embodiments of the eighth aspect, the cationically ionizable lipid is a lipid having structure X-3. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid is selected from structures A through G disclosed herein. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid has a structure of formula (XI) disclosed herein. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid has the structure (XIV-1), (XIV-2), or (XIV-3) disclosed herein. In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid is 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ... The compound may comprise 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 certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol % of the total lipid present in the composition.

[0103] In certain embodiments of the eighth aspect, the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably the composition comprises a cationically ionizable lipid, a conjugate, a phospholipid, and a steroid. In certain embodiments of the eighth aspect, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether The phospholipid is selected from the group consisting of 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphthanoyl-phosphatidylethanolamine (DPyPE). In some embodiments, the phospholipid is DOPE. In some embodiments of the eighth aspect, the phospholipid comprises about 1 mol% to about 30 mol% of the total lipids present in the composition. In some embodiments of the eighth aspect, the steroid comprises a sterol, such as cholesterol.In certain embodiments of the eighth aspect, the steroid comprises about 10 mol % to about 60 mol % of the total lipids present in the composition.

[0104] In certain embodiments of the eighth aspect, the cationic or cationically ionizable lipid comprises about 20 mol% to about 70 mol% of the total lipid present in the composition; the conjugate comprises about 0.5 mol% to about 15 mol% of the total lipid present in the composition; the phospholipid comprises about 5 mol% to about 25 mol% of the total lipid present in the composition; and the steroid comprises about 25 mol% to about 55 mol% of the total lipid present in the composition.

[0105] 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 or cationically ionizable lipid 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 (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In some embodiments of the eighth aspect, the particles comprise or are LNPs. In some embodiments of the eighth aspect, the particles comprise or are liposomes. In some embodiments of the eighth aspect, the particles comprise or are LPXs. In some embodiments of the eighth aspect, the particles comprise or are a mixture of LNPs and liposomes. In some embodiments of the eighth aspect, the particles comprise or are a mixture of LNPs and LPXs. In some embodiments of the eighth aspect, the particles comprise or are a mixture of liposomes and LPXs. In some embodiments of the eighth aspect, the particles comprise or are a mixture of LNPs, liposomes, and LPXs. In certain embodiments of the eighth aspect, the particles have a size of about 30 nm to about 500 nm. In certain embodiments of the eighth aspect, the nucleic acid is encapsulated within or associated with the particles.

[0106] In some embodiments of the eighth aspect, the nucleic acid is DNA or RNA, particularly RNA, such as single-stranded RNA, particularly mRNA. In some embodiments of the eighth aspect, the RNA contains 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, preferably all of, the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In some embodiments of the eighth aspect, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably an interrupted sequence of A nucleotides. In some embodiments of the eighth aspect, the 5' cap is a Cap1 or Cap2 structure.

[0107] In certain 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 against an antigen in a subject.

[0108] The composition of the eighth aspect may be used in (i) a method of delivering a nucleic acid (e.g., DNA or RNA) to cells of a subject, the method comprising administering to the subject a composition of the eighth aspect; (ii) a method of delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid (e.g., DNA or RNA) encodes a therapeutic peptide or protein; (iii) a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein delivery of the nucleic acid (e.g., DNA or RNA) to cells of the subject is beneficial in treating or preventing the disease or disorder; and / or (iv) a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid (e.g., DNA or RNA) encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

[0109] It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth and seventh aspects is also applicable to any embodiment of the eighth aspect (including the use of any embodiment of the eighth aspect).

[0110] In a ninth aspect, the present invention provides a method for transfecting cells, comprising adding a composition of the first or eighth aspect to cells and incubating the mixture of the composition and cells for a sufficient period of time. In some embodiments, particularly when the nucleic acid (e.g., DNA or RNA) encodes a pharmaceutically active protein, the mixture of the composition and cells is incubated for a sufficient period of time to allow expression of the pharmaceutically active protein. In some embodiments, the sufficient period of time is at least 1 hour (e.g., at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or up to about 48 hours (e.g., up to about 36 hours or up to about 24 hours). In some embodiments of the ninth aspect, the method is performed in vivo (i.e., the cells form part of a subject's organ, tissue, and / or organism). In some embodiments of the ninth aspect, the method is performed in vitro (i.e., the cells do not form part of a subject's organ, tissue, and / or organism, e.g., the cells are in ex vivo cell culture).

[0111] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect can also be applied to any embodiment of the ninth aspect.

[0112] In a tenth aspect, the present invention provides a use of the composition of the first or eighth aspect to transfect cells. In some embodiments of the tenth aspect, the use is an in vivo use (i.e., the cells form part of a subject's organ, tissue, and / or organism). In some embodiments of the tenth aspect, the use is an in vitro use (i.e., the cells do not form part of a subject's organ, tissue, and / or organism, e.g., the cells are in ex vivo cell culture).

[0113] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect can also be applied to any embodiment of the tenth aspect.

[0114] In a further aspect, the present invention provides a kit comprising a composition of the first or eighth aspect, a conjugate of the sixth or seventh aspect, or a pharmaceutical composition described herein. In some embodiments, the kit is for use in therapy, such as inducing an immune response. In some embodiments, the kit is for use in inducing an immune response against a pathogen, such as in treating or preventing an infectious disease. [Brief explanation of the drawings]

[0115] [Figure 1] Simplified scheme for POX synthesis via activated cationic ring-opening polymerization using unsubstituted (R=H) or substituted (R≠H) 2-oxazoline compounds.

[0116] [Figure 2] Examples of RNA LNP composition production and storage.

[0117] [Figure 3] Evaluation of the % grafting of poly(2-oxazoline)-grafted lipids into LNPs. Four different poly(2-oxazoline)-grafted lipids (hereafter POX-lipids) were used to form lipid nanoparticles: C14-PMeOx (C14-PMeOx45-50-OH; a tetradecyl alkyl chain followed by 45-50 units of poly-2-methyl-2-oxazoline); C14-PEtOx (C14-PEtOx45-50-OH; a tetradecyl alkyl chain followed by 45-50 units of poly-2-ethyl-2-oxazoline); C14-NHCOPMeOx (C14-NHCOPMeOx45-50-N3; a tetradecyl alkyl chain linked via an amide bond to a block of 45-50 units of poly-2-methyl-2-oxazoline); and bisC14-COO-PMeOX (bisC14-COO-PMeOx45-50-N3; two tetradecyl alkyl chains each linked via an ester bond to a single block of 45-50 units of poly-2-methyl-2-oxazoline). LNPs were fabricated (using various amounts of POX-lipid) and tested for the following parameters: (A) size; (B) accessible mRNA; and (C) in vitro luciferase expression.

[0118] [Figure 4] The effect of introducing a polar linker between the hydrophilic and hydrophobic blocks of POX-lipids. Three different stealth lipids were used to form LNPs: POX-lipid C14-PMeOx (C14-PMeOx45-50-OH; a tetradecyl alkyl chain followed by 45-50 units of poly-2-methyl-2-oxazoline); POX-lipid C14-NHCOPMeOx (C14-NHCOPMeOx45-50-N3; a tetradecyl alkyl chain linked via an amide bond to a 45-50 unit block of poly-2-methyl-2-oxazoline); and the reference stealth lipid C14-PSar (a tetradecyl alkyl chain followed by 23 units of poly(N-methyl-glycine)). LNP formulations were evaluated for size, RNA accessibility, zeta potential, and in vitro luciferase expression.

[0119] [Figure 5] Determining the effect of the end group on POX-lipids. Four different POX-lipids were used to form lipid nanoparticles: C14-NHCOPMeOx-N3 (a tetradecyl alkyl chain terminated with an azide group and linked via an amide bond to a 45–50-unit block of poly-2-methyl-2-oxazoline); C14-NHCOPMeOx-NH (a tetradecyl alkyl chain terminated with a primary amino group and linked via an amide bond to a 45–50-unit block of poly-2-methyl-2-oxazoline); C14-NHCOPMeOx-COOH (a tetradecyl alkyl chain terminated with a carboxyl group and linked via an amide bond to a 45–50-unit block of poly-2-methyl-2-oxazoline); and C14-NHCOPMeOx-COOH / NH (a physical mixture of C14-NHCOPMeOx-COOH and C14-NHCOPMeOx-NH in a 1:1 ratio). P formulations were evaluated for size, RNA accessibility, zeta potential and in vitro luciferase expression.

[0120] [Figure 6]Particle size and PDI of LNP containing C14-PMeOX25-NH2 using HY501 as the cationic lipid. Particle size and PDI of BM_2 LNP formulation are also included.

[0121] [Figure 7] Zeta potential of LNP containing C14-PMeOX25-NH2 using HY501 as the cationic lipid. Zeta potential of BM_2 LNP formulation is also included.

[0122] [Figure 8] Accessible RNA of LNP-containing C14-PMeOX25-NH2 using HY501 as a cationic lipid measured via Ribogreen assay.

[0123] [Figure 9] Agarose gel electrophoretic image of LNP containing C14-PMeOX25-NH2 using HY501 as the cationic lipid.

[0124] [Figure 10] Terminal complement complex (SC5b-9) formation after incubation of human serum with C14-PMeOX25-NH2-containing LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0125] [Figure 11] Hemolysis analysis after incubation (neutral pH conditions) of C14-PMeOX25-NH2-containing LNPs with human whole blood and HY501 as the ionizable lipid.

[0126] [Figure 12]In vitro expression (Figure 12A-C) and viability (Figure 12D-F) of BM_2 and C14-PMeOX25-NH2-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 12A, D), a liver cancer cell line (HepG2) (Figure 12B, E), and a mouse macrophage cell line (viable cells) (Figure 12C, F). Firefly luciferase expression after 24 h of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0127] [Figure 13] Particle size and PDI of C14-PMeOX25-COCH3-containing LNPs using BM_2 and HY501 as an ionizable lipid.

[0128] [Figure 14] Zeta potential of C14-PMeOX25-COCH3-containing LNPs using BM_2 and HY501 as the ionizable lipid.

[0129] [Figure 15] Accessible RNA of BM_2 and C14-PMeOX25-COCH3-containing LNPs using HY501 as the ionizable lipid, as measured by Ribogreen assay.

[0130] [Figure 16] Agarose gel electrophoretic image of C14-PMeOX25-COCH3-containing LNPs using HY501 as the ionizable lipid.

[0131] [Figure 17] Terminal complement complex (SC5b-9) formation after incubation of human serum with C14-PMeOX25-COCH3-containing LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0132] [Figure 18]Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with BM_2 and C14-PMeOX25-COCH3-containing LNPs using HY501 as an ionizable lipid.

[0133] [Figure 19] In vitro mRNA expression (Figure 19A-C) and cell viability (Figure 19D-F) of BM_2 and C14-PMeOX25-COCH3-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 19A, D), a liver cancer cell line (HepG2) (Figure 19B, E), and a mouse macrophage cell line (live cells) (Figure 19C, E). Firefly luciferase expression after 24 h of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0134] [Figure 20] Particle size and PDI of C14-PMeOX25-COOH-containing LNPs using BM_2 and HY501 as an ionizable lipid.

[0135] [Figure 21] Zeta potential of C14-PMeOX25-COOH-containing LNPs using BM_2 and HY501 as the ionizable lipid.

[0136] [Figure 22] Accessible RNA of C14-PMeOX25-COOH-containing LNPs using HY501 as a cationic lipid measured via Ribogreen assay.

[0137] [Figure 23] Agarose gel electrophoretic image of C14-PMeOX25-COOH-containing LNPs using BM_2 and HY501 as the ionizable lipid.

[0138] [Figure 24]Terminal complement complex (SC5b-9) formation after incubation of human serum with C14-PMeOX25-COOH-containing LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0139] [Figure 25] Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with BM_2 and C14-PMeOX25-COOH-containing LNPs using HY501 as an ionizable lipid.

[0140] [Figure 26] In vitro expression (Figures 26A-C) and viability (Figures 26D-F) of BM_2 and C14-PMeOX25-COOH-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 26A,D), a liver cancer cell line (HepG2) (Figure 26B,E), and a mouse macrophage cell line (live cells) (Figure 26C,F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0141] [Figure 27] Particle size and PDI of C14-PMeOX25-OH-containing LNPs using BM_2 and HY501 as an ionizable lipid.

[0142] [Figure 28] Zeta potential of C14-PMeOX25-OH-containing LNPs using BM_2 and HY501 as the ionizable lipid.

[0143] [Figure 29] Accessible RNA of BM_2 and C14-PMeOX25-OH-containing LNPs.

[0144] [Figure 30] Agarose gel electrophoretic images of BM_2 and C14-PMeOX25-OH-containing LNPs.

[0145] [Figure 31] Terminal complement complex (SC5b-9) formation after incubation of human serum with C14-PMeOX25-OH-containing LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

[0146] [Figure 32] Hemolysis analysis after incubation (neutral pH condition) of human whole blood with BM_2 and C14-PMeOX25-OH-containing LNPs.

[0147] [Figure 33] In vitro expression (Figures 33A-C) and viability (Figures 33D-F) of BM_2 and C14-PMeOX25-OH-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 33A,D), a liver cancer cell line (HepG2) (Figure 33B,E), and a mouse macrophage cell line (live cells) (Figure 33C,F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0148] [Figure 34] (A) Accessible RNA measured by Ribogreen assay and (B) agarose gel electrophoresis image of POX-grafted lipid-containing LNPs for in vivo experiments.

[0149] [Figure 35] In vitro expression (Figure 35A-C) and viability (Figure 35D-F) of BM_3 and POX-transplanted lipid-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 35A, D), a liver cancer cell line (HepG2) (Figure 35B, E), and a mouse macrophage cell line (live cells) (Figure 35C, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0150] [Figure 36]In vivo whole-body bioluminescence imaging (BLI) of animals (n=3) treated with test formulation iv at 6 hours (top row), 24 hours (middle row) and ex vivo BLI at 6 hours (bottom row).

[0151] [Figure 37] (A) In vivo luciferase expression 6 and 24 h after i.v. injection of LNPs (from in vivo imaging). (B) Ex vivo luciferase expression 6 h after i.v. injection of LNPs (from organ imaging).

[0152] [Figure 38] Particle size and PDI of BM_2 and C14-PMeOX45-NH2-containing LNPs.

[0153] [Figure 39] Zeta potential of BM_2 and C14-PMeOX45-NH2-containing LNPs.

[0154] [Figure 40] Accessible RNA of C14-PMeOX45-NH2-containing LNPs using HY501 as a cationic lipid measured via Ribogreen assay.

[0155] [Figure 41] Agarose gel electrophoretic images of BM_2 and C14-PMeOX45-NH2-containing LNPs.

[0156] [Figure 42] In vitro expression (Figures 42A-C) and viability (Figures 42D-F) of BM_2 and C14-PMeOX45-NH2-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 42A,D), a liver cancer cell line (HepG2) (Figure 42B,E), and a mouse macrophage cell line (live cells) (Figure 42C,F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0157] [Figure 43] Particle size and PDI of BM_2 and C14-PMeOX45-COOH-containing LNPs.

[0158] [Figure 44] Zeta potential of BM_2 and C14-PMeOX45-COOH-containing LNPs.

[0159] [Figure 45] Accessible RNA of C14-PMeOX45-COOH-containing LNPs using HY501 as an ionizable lipid measured via Ribogreen assay.

[0160] [Figure 46] Agarose gel electrophoretic image of C14-PMeOX45-COOH-containing LNPs using HY501 as the ionizable lipid.

[0161] [Figure 47] In vitro expression (Figures 47A-C) and viability (Figures 47D-F) of BM_2 and C14-PMeOX45-COOH-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 47A,D), a liver cancer cell line (HepG2) (Figure 47B,E), and a mouse macrophage cell line (live cells) (Figure 47C,F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0162] [Figure 48] Particle size and PDI of BM_2 and C14-PMeOX45-OH-containing LNPs.

[0163] [Figure 49] Zeta potential of C14-PMeOX45-OH-containing LNPs using BM_2 and HY501 as cationic lipid.

[0164] [Figure 50]Accessible RNA of C14-PMeOX45-OH-containing LNPs using HY501 as a cationic lipid measured via Ribogreen assay.

[0165] [Figure 51] Agarose gel electrophoretic image of C14-PMeOX25-OH-containing LNPs using HY501 as the ionizable lipid.

[0166] [Figure 52] In vitro expression (Figures 52A-C) and viability (Figures 52D-F) of BM_2 and C14-PMeOX45-OH-containing LNPs in a skeletal muscle cell line (C2C12) (Figure 52A, D), a liver cancer cell line (HepG2) (Figure 52B, E), and a mouse macrophage cell line (live cells) (Figure 52C, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng (black bars), 25 ng (gray bars), and 50 ng (white bars) of mRNA-loaded LNPs per well.

[0167] [Figure 53] Particle size and PDI of BM_2 and C14-PMeOX45-NH-AC-containing LNPs.

[0168] [Figure 54] Zeta potential of C14-PMeOX45-NH-AC-containing LNPs using BM_2 and HY501 as cationic lipid.

[0169] [Figure 55] Accessible RNA of C14-PMeOX45-NH-AC-containing LNPs using HY501 as a cationic lipid measured via Ribogreen assay.

[0170] [Figure 56] Agarose gel electrophoretic image of C14-PMeOX45-NH-AC-containing LNPs using HY501 as the ionizable lipid.

[0171] [Figure 57] Particle size and PDI of BM_2 and VE-PMeOX50-CH3-containing LNPs.

[0172] [Figure 58] Zeta potential of VE-PMeOX50-CH3-containing LNPs using BM_2 and HY501 as cationic lipid.

[0173] [Figure 59] Accessible RNA of VE-PMeOX50-CH3-containing LNPs using HY501 as a cationic lipid measured via Ribogreen assay.

[0174] [Figure 60] Agarose gel electrographic image of VE-PMeOX50-CH3-containing LNPs using HY501 as the ionizable lipid.

[0175] Sequence description The following table provides a list of certain sequences referred to herein. [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0176] Detailed Description Although the present invention is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, as these may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0177] The elements of the present invention are described in further detail below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention, but merely as explicitly describing embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of all elements described in the present invention should be construed as disclosed by the description of the present invention, unless otherwise indicated by the context. For example, if in one embodiment the composition comprises a conjugate of formula (III) and in another embodiment the cationically ionizable lipid has one of structures XIV-1, XIV-2, and XIV-3, in a further embodiment the composition comprises a conjugate of formula (III) and a cationically ionizable lipid having one of structures XIV-1, XIV-2, and XIV-3.

[0178] Preferably, the terms used herein are those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0179] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, as explained in the literature of the field (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. (See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0180] All methods described herein can be used in any suitable order unless otherwise specified herein or clearly indicated from the context. The use of examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0181] The recitation of ranges of values ​​herein is intended to serve merely as a shorthand method of referring to each individual value falling within the range, and unless otherwise stated herein, each individual value is encompassed herein to the same extent as if it were individually set forth herein.

[0182] Several documents are cited herein. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such documents.

[0183] definition The following definitions are provided that apply to all aspects of the present invention. The following terms have the following meanings unless otherwise specified: Any undefined term has its art-recognized meaning.

[0184] In this specification and the claims that follow, unless the context requires otherwise, the terms "comprise" and variations thereof, such as "comprises" and "comprising," should be understood to imply the inclusion of the stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" intends the exclusion of other members, integers, or steps of any essential significance. The term "comprising" includes the term "consisting essentially of," which in turn includes the term "consisting of." Thus, in each instance herein, the term "comprising" can be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each instance herein, the term "consisting essentially of" can be replaced with the term "consisting of."

[0185] As used in the context of the description of the invention (particularly in the context of the claims), the singular forms "a," "the," and "the" are to be construed as including both the singular and the plural unless otherwise specified herein or clearly contrary to the context.

[0186] As used herein, "and / or" is to be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" is to be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0187] In the context of the present invention, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to be certain of the technical effect of the nature in question. This term typically indicates deviations of ±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% from the indicated numerical value. As will be apparent to a person skilled in the art, the specific deviation of the numerical value of a certain technical effect depends on the nature of the technical effect. For example, natural or biological technical effects generally have larger deviations than artificial or engineered technical effects.

[0188] As used herein, terms such as "reduction" or "inhibition" refer to the ability to cause a total decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibition" or similar terms includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0189] Terms such as "increase" or "enhancement" refer in certain embodiments 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%.

[0190] As used herein, "physiological pH" refers to a pH of about 7.4. In certain embodiments, "physiological pH" as used herein refers to a neutral pH, i.e., a pH of about 7.0.

[0191] "Physiological conditions" as used herein refers to the conditions (especially pH and temperature) in a living subject, particularly a human. Preferably, physiological conditions refer to physiological pH and / or a temperature of about 37°C.

[0192] As used herein, "% w / v" refers to weight / volume percent, which is a unit of concentration that measures the amount of solute (in grams (g)) as a percentage of the total volume of a solution (in milliliters (mL)).

[0193] As used herein, "weight %" or "%(w / w)" (or "% w / w") refers to weight percent, which is a unit of concentration that measures the amount of a substance (in grams (g)) as a percentage of the total weight (in grams (g)) of the total composition.

[0194] As used herein, "mol %" is defined as the ratio of the number of moles of a component to the number of moles of all components x 100.

[0195] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species and their respective charges in a particular solution. Thus, ionic strength, I, is given by the formula

number

[0196] According to this specification, the term "ionic strength" refers to the presence of monovalent ions in some embodiments. With respect to the presence of divalent ions, particularly divalent cations, the concentration or effective concentration (presence of free ions) due to the presence of a chelating agent is low enough to prevent RNA degradation in some embodiments. In some embodiments, the concentration or effective concentration of divalent ions is lower than the catalytic level for the hydrolysis of phosphodiester bonds between RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, there is no or essentially no free divalent ions.

[0197] A "monovalent" compound refers to a compound that has only one functional group of interest. For example, a monovalent anion refers to a compound that has only one negatively charged group, preferably under physiological conditions.

[0198] A "divalent" or "dibasic" compound refers to a compound that has two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is Ca. 2+ is.

[0199] A "polyhydric" or "polybasic" compound refers to a compound having three or more functional groups of interest. For example, a polybasic organic acid has three or more acid groups.

[0200] A "monovalent moiety" refers to a monoradical, i.e., a moiety having a valence of 1. Typical monovalent moieties include alkyl, alkenyl, aryl, and the like.

[0201] A "divalent moiety" or "divalent moiety" refers to a diradical, i.e., a moiety having a valence of two. Exemplary divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, and the like. A further example of a divalent moiety is [*-S] when p is 1. p (C 1-6 -alkylene)- in C 1-6 -Alkylene moiety (group *-S(C 1-6 -alkylene)-, for example *-S-(CH2)6- or *-S-CH2-, where * is R 4 or R 7 represents the attachment point to

[0202] A "polyvalent moiety" refers to a polyradical, i.e., a moiety having a valence of at least 3. For example, a "trivalent moiety" refers to a triradical, i.e., a moiety having a valence of 3. For example, by further removing an H atom from an alkylene group, the resulting alkylene is trivalent. A further example of a trivalent moiety is the group [*-S] if p is 2. p (C 1-6-alkylene)- in C 1-6 -Alkylene moiety (group [*-S]2(C 1-6 -alkylene)-, for example *-S-CH(S-*)(CH2)5- or *-S-CH(S-*)(CH2)-, where * is R 4 or R 7 Another example of a trivalent moiety is the group (*=N)(C 1-3 -alkylene)-Z- (where two hydrophobic chains are attached to the N atom, which are then C 1-3 -bonded to the alkylene moiety).

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

[0204] The term "freezing" refers to a phase transition from a liquid to a solid state, usually caused by a reduction in the temperature of a system below a critical temperature, accompanied by a characteristic change in the enthalpy of the system.

[0205] The term "lyophilize" or "lyophilization" refers to freeze-drying a substance by freezing, followed by reducing the surrounding pressure to allow the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0206] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with a fluid that is being atomized (atomized) in a vessel (spray dryer) where the solvent evaporates from the previously formed droplets, resulting in a solid powder.

[0207] The term "cryoprotectant" is a substance added to a formulation to protect the active ingredient during the freezing step.

[0208] The term "lyoprotectant" is a substance added to a formulation to protect the active ingredient during the drying step.

[0209] The term "reconstitution" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

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

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

[0212] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably 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 include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0213] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon monoradical. Preferably, an alkyl group has 1 to 14 (e.g., 1 to 12 or 1 to 10) carbon atoms, i.e., C 1-14 Alkyl is a group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., C 1-10alkyl (abbreviated as alkyl) contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, iso-propyl (also known as 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. "Substituted alkyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms attached to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkylene group have been replaced with non-hydrogen substituents (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents specified herein. Examples of substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

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

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

[0216] The term "alkenylene" refers to an unsaturated straight-chain or branched hydrocarbon diradical having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the number of carbon atoms in the alkenylene group divided by 2, and if the number of carbon atoms in the alkenylene group is odd, the result of the division is rounded down to the next lower integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group 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, e.g. 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in certain embodiments, an alkenylene group contains 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in the cis (Z) or trans (E) configuration. Examples of alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, and the like.If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkenylene group have been replaced with non-hydrogen substituents (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0217] The term "alkynyl" refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the number of carbon atoms in the alkynyl group divided by 2, and if the number of carbon atoms in the alkynyl group is odd, the result of the division is rounded down to the next lower 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, an alkynyl group contains 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5 (preferably 1, 2, or 3)) carbon-carbon triple bonds, more preferably 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.Examples of 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-octyn ... Alkynyl groups include octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonylyl, 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 alpha to the nitrogen atom. "Substituted alkynyl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkynyl group have been replaced with non-hydrogen substituents (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

[0218] The term "cycloalkyl" or "cycloaliphatic" refers to a cyclic, non-aromatic version of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 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 certain embodiments, the cycloalkyl group has one, two, or more (preferably one or two) double bonds. Examples of cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. The term "cycloalkyl" also includes its bicyclic and tricyclic versions. If a bicyclic ring is formed, it is preferred that each ring is joined to each other at two adjacent carbon atoms, or alternatively the two rings are formed through the same carbon atom, i.e., forming a spirocyclic or "bridged" ring system. Preferred examples of cycloalkyl include C3-C8-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]decanyl, 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]decanyl (i.e. decalinyl)."Substituted cycloalkyl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of a cycloalkyl group have been replaced with non-hydrogen substituents (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

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

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

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

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

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

[0224] As used herein, the phrase "partially hydrogenated form" of an unsaturated compound or group means that a portion of the unsaturation has been removed by the formal addition of hydrogen to an originally unsaturated compound or group, without removing all of the unsaturation. The phrase "fully hydrogenated form" of an unsaturated compound or group is used interchangeably with the term "perhydro" and means that all unsaturation has been removed by the formal addition of hydrogen to an originally unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl group (containing two double bonds in the ring, e.g., furan) includes a dihydro form of the 5-membered heteroaryl group (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while a tetrahydro form of the 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a fully hydrogenated (or perhydro) form of the 5-membered heteroaryl group. Similarly, for a 6-membered heteroaryl group having three double bonds in the ring (e.g., pyridyl), the partially hydrogenated forms include the di- and tetrahydro forms (e.g., di- and tetrahydropyridyl), while the hexahydro forms (e.g., piperidinyl in the case of heteroarylpyridyl) are fully hydrogenated (or perhydro) derivatives of the 6-membered heteroaryl group. Consequently, hexahydro forms of an aryl or heteroaryl are only considered as partially hydrogenated forms according to the present invention if the aryl or heteroaryl contains at least four unsaturated moieties consisting of double and triple bonds between ring atoms.

[0225] The term "aromatic," as used in the context of hydrocarbons, means that the entire molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this invention. Similarly, if a bicyclic or polycyclic aryl (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this invention (even if one ring is still aromatic, such as in 1,2-dihydronaphthyl). A similar distinction is made herein between heteroaryl and heterocyclyl. For example, indolinyl, i.e., the dihydro variant of indolyl, is classified as a heterocyclyl for purposes of this invention because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0226] The term "optionally substituted" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 1-5, 1-4 or 1-3 or 1 or 2) hydrogen atoms are substituted with a group different from hydrogen (i.e., first level substituents), such as alkyl (preferably C 1-6 alkyl), alkenyl (preferably C 2-6 alkenyl), alkynyl (preferably C 2-6 alkynyl), aryl (preferably 6- to 14-membered aryl), heteroaryl (preferably 3- to 14-membered heteroaryl), cycloalkyl (preferably 3- to 14-membered cycloalkyl), heterocyclyl (preferably 3- to 14-membered heterocyclyl), halogen, -CN, azide, -NO2, -OR 71 , -N(R 72 )(R 73 ), -S(O) 0-2 R 71 , -S(O) 1-2 OR 71 , -OS(O) 1-2 R 71 , -OS(O) 1-2 OR 71 , -S(O) 1-2 N(R72 )(R 73 ), -OS(O) 1-2 N(R 72 )(R 73 ), -N(R 71 )S(O) 1-2 R 71 , -NR 71 S(O) 1-2 OR 71 , -NR 71 S(O) 1-2 N(R 72 )(R 73 ), -OP(O)(OR 71 )2, -C(=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 attached to the same carbon atom of a cycloalkyl or heterocyclyl group together represent =X 1 wherein each of the first-level substituent alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups may itself form C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl, halogen, -CF3, -CN, azide, -NO2, -OR 81 , -N(R 82 )(R 83 ), -S(O) 0-2 R 81 , -S(O) 1-2 OR 81 , -OS(O) 1-2 R 81 , -OS(O) 1-2 OR 81 , -S(O) 1-2 N(R 82 )(R 83), -OS(O) 1-2 N(R 82 )(R 83 ), -N(R 81 )S(O) 1-2 R 81 , -NR 81 S(O) 1-2 OR 81 , -NR 81 S(O) 1-2 N(R 82 )(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 attached to the same carbon atom of a cycloalkyl or heterocyclyl group that is a first-level substituent may be substituted together with ═X. 2 wherein the C of the second level substituent may form 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, and 3- to 14-membered heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3alkyl), -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 is independently methyl, ethyl, propyl, or isopropyl, and / or any two third-level substituents attached to the same carbon atom of a 3- to 14-membered cycloalkyl or heterocyclyl group that is a second-level substituent are together =O, =S, =NH, or =N(C 1-3 alkyl); where: R 71 , R 72 and R 73 Each of the groups independently represents H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each of the alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 3- to 7-membered heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, =O, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH2-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; R 81 , R 82 and R 83 Each of the groups independently represents 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 groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -OCF3, =O, -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)(C1-3 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 X 1 and X 2 each independently represents O, S, and N(R 84 ), where R 84 is H or C 1-3 It is alkyl.

[0227] Typical first level substituents are preferably C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 6- to 14-membered (e.g., 6- to 10-membered) aryl, 3- to 14-membered (e.g., 5- or 6-membered) heteroaryl, 3- to 14-membered (e.g., 3- to 7-membered) cycloalkyl, 3- to 14-membered (e.g., 3- to 7-membered) heterocyclyl, halogen, -CN, azide, -NO2, -OR 71 , -N(R 72 )(R 73 ), -S(O) 0-2 R 71 , -S(O) 1-2 OR 71 , -OS(O) 1-2 R 71 , -OS(O) 1-2 OR 71 , -S(O) 1-2 N(R 72 )(R 73 ), -OS(O) 1-2 N(R72 )(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 , e.g. C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7-membered cycloalkyl, 3- to 7-membered (e.g., 5- or 6-membered) heterocyclyl, halogen, —CF3, —CN, azide, —NO2, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z wherein each z is independently 0, 1, or 2; and each C 1-3 Alkyl is independently methyl, ethyl, propyl, or isopropyl; where X1 are independently selected from O, S, NH, and N(CH); and R 71 , R 72 and R 73 are as defined above or preferably independently H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 and selected from the group consisting of alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z wherein each z is independently 0, 1, or 2; and each C 1-3 Alkyl is independently methyl, ethyl, propyl, or isopropyl. In some embodiments, the first level substituent is C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z, —C(═O)OH and —C(═O)OCH3, where z is 0, 1 or 2; and C 1-3 Alkyl is methyl, ethyl, propyl, or isopropyl. In some embodiments, the first level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br) and -CF3.

[0228] Typical second level substituents are preferably C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF3, —CN, azido, —NO2, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z wherein each z is independently 0, 1, or 2; and each C 1-3 Alkyl is independently methyl, ethyl, propyl, or isopropyl. Specific examples of second-level substituents are C 1-3Alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF3, —CN, —OH, —O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z wherein each z is independently 0, 1, or 2; and each C 1-3 Alkyl is independently methyl, ethyl, propyl, or isopropyl. Particularly preferred second-level substituents are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, ═O, and ═S.

[0229] Typical third level substituents are preferably C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , —C(═O)OH and —C(═O)OCH3, where z is 0, 1 or 2; and C 1-3 Alkyl is methyl, ethyl, propyl, or isopropyl. Particularly preferred third level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and -CF, such as halogen (e.g., F, Cl, or Br) and -CF.

[0230] According to the present invention, the term "peptide" includes oligo- and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide," "polypeptide," and "protein" are generally used synonymously herein.

[0231] "Therapeutic peptide or protein" refers to a peptide or protein that can be used for treatment in an individual in whom expression of the peptide or protein would be beneficial, e.g., to alleviate disease symptoms. In certain embodiments, a therapeutic peptide or protein, when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "therapeutic peptide or protein" includes whole peptides or proteins, as well as therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, vaccination antigens and immunostimulants such as cytokines.

[0232] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may designate a contiguous or discontinuous fraction of the structure.

[0233] The terms "portion" and "fragment" are used interchangeably and refer to a continuous sequence. For example, a portion of a structure such as an amino acid sequence or a protein refers to a continuous sequence of the structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0234] A "fragment" with respect to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0235] According to the present invention, a portion or fragment of a peptide or protein preferably possesses 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 possesses at least one of the pharmacological activities of the peptide or protein from which it is derived. A portion or fragment of a peptide or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

[0236] As used herein, "variant" in reference to an amino acid sequence (peptide or protein) refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid or a modification of the same amino acid). The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence or can be a modified version of the wild-type amino acid sequence. In certain embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences, and preferably 1 to about 10 or 1 to about 5 amino acid differences, compared to the parent.

[0237] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found 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.

[0238] For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes total mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0239] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific positions in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the modifications are at amino acid sequence positions that are not conserved between homologous proteins or peptides and / or replace amino acids with others with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change is the substitution of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four groups: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions are within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0240] Preferably, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Similarity or identity is preferably expressed over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, similarity or identity is preferably shown for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, similarity or identity is shown for the leading edge of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix:Blosum62, Gap Open 10.0, Gap Ext end 0.5.

[0241] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0242] The terms "% identical" and "% identity" or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical between the compared sequences in optimal alignment. The percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed across the length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "comparison window" to identify local regions of corresponding sequences after optimal alignment. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the local homology 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 Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In one embodiment, the algorithm parameters used in the BLASTN algorithm on the NCBI website include: (i) an expectation threshold setting of 10; (ii) a word size setting of 28; (iii) a maximum match in the query range setting of 0; (iv) a match / mismatch score setting of 1, -2; (v) a gap cost setting of linear; and (vi) a filter for low complexity regions. In one embodiment, the algorithm parameters used in the BLASTP algorithm on the NCBI website include: (i) an expectation threshold setting of 10; (ii) a word size setting of 3; (iii) a maximum match in the query range setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap costs settings of presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0243] Percentage identity is determined by determining the number of identical positions where the compared sequences correspond, dividing this number by the total number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0244] In some embodiments, similarity or identity is shown for the region of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference sequence.For example, when the reference nucleic acid sequence is composed of 200 nucleotides, identity is shown for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, similarity or identity is shown for the full length of the reference sequence.

[0245] Homologous amino acid sequences according to the present disclosure exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0246] The amino acid sequence variants described herein can be readily produced by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, such as, for example, solid-phase synthesis and similar methods.

[0247] 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 refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. For antigens or antigenic sequences, a particular function is one or more of the immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered, particularly compared to the amino acid sequence of the parent molecule or sequence, but still retains one or more functions of the parent molecule or sequence, such as inducing an immune response. In some embodiments, modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0248] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein can be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0249] In certain embodiments, "isolated" refers to alteration or removal (e.g., purification) from a natural state or an artificial composition, e.g., a composition resulting from a manufacturing process. For example, a nucleic acid (e.g., RNA), peptide, or polypeptide naturally occurring in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide partially or completely separated from coexisting materials in its natural state is. An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell. In certain embodiments, the nucleic acid (e.g., DNA or RNA, particularly mRNA) used in the present invention is in substantially purified form. In certain embodiments, a solution (preferably an aqueous solution) of nucleic acid (e.g., DNA or RNA, particularly mRNA) in substantially purified form comprises a first buffer system.

[0250] The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid.

[0251] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids described herein can be present in vitro or in vivo; for example, the cells may form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is expressed only transiently. RNA can be transfected into cells for the transient expression of its encoded protein. Because the nucleic acid introduced during the transfection process is not usually integrated into the nuclear genome, the foreign nucleic acid is diluted out via mitosis or degradation. Cells that allow episomal amplification of nucleic acids have a significantly reduced dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must be performed. Such stable transfection can be achieved using a viral or transposon-based system for transfection. Generally, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells for transient expression of the encoded protein.

[0252] The present invention includes analogs of peptides, polypeptides, or proteins. According to the present invention, a peptide, polypeptide, or protein analog is a modified form of the peptide, polypeptide, or protein from which it is derived, and retains at least one functional property of the peptide, polypeptide, or protein. For example, a pharmacologically active peptide, polypeptide, or protein analog retains at least one pharmacological activity of the peptide, polypeptide, or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule attached to the peptide, polypeptide, or protein, such as carbohydrates, lipids, and / or proteins or peptides. In certain embodiments, a "peptide, polypeptide, or protein" "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or other cellular ligand. The term "analog" also extends to fully functional chemical equivalents of the peptide, polypeptide, or protein.

[0253] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of two or more sequences or components or domains.

[0254] According to the present invention, a cell, which may be in vitro or in a subject, preferably results in expression of a peptide, polypeptide, or protein once a nucleic acid, such as DNA or RNA (e.g., mRNA), encoding the peptide, polypeptide, or protein has been taken up or introduced into the cell, i.e., transfected or transduced. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or nucleus), and may secrete or express the encoded peptide, polypeptide, or protein on its surface.

[0255] According to the present invention, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms (e.g., "encoding RNA") are used interchangeably in reference to a particular peptide, polypeptide, or protein and mean that the nucleic acid, when present in the appropriate environment, preferably within a cell, can be expressed to produce the peptide, polypeptide, or protein.

[0256] As used herein, " activation " or " stimulation " refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term " activated immune effector cells " refers, in particular, to immune effector cells undergoing cell division.

[0257] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first comes into contact with a specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0258] The term "expansion" refers to the process by which a specific entity proliferates. In the context of the present invention, the term is preferably used in the context of an immunological response, in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize the antigen are expanded. In some embodiments, expansion leads to differentiation of immune effector cells.

[0259] An "antigen" according to the present invention encompasses any substance that induces an immune response and / or any substance against which an immune response or immune mechanism, e.g., a cellular response, is directed. This also includes situations in which an antigen is processed into antigenic peptides, particularly when presented in the context of MHC molecules, and an immune response or immune mechanism is directed against one or more antigenic peptides. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that contains at least one epitope, such as a T cell epitope. In certain embodiments, an antigen, in the context of the present invention, is a molecule that, after processing, optionally induces an immune response that may be specific to the antigen (including cells expressing the antigen). In certain embodiments, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0260] In some embodiments, antigens are presented or present on the surface of cells of the immune system, such as antigen-presenting cells, such as dendritic cells or macrophages. In some embodiments, antigens or their processed products, such as T cell epitopes, are bound by antigen receptors. Thus, antigens or their processed products can specifically react with immune effector cells, such as T lymphocytes (T cells).

[0261] According to the present invention, any suitable antigen can be used that is a candidate for an immune response, where the immune response can be either a humoral or cellular immune response. In the context of one embodiment of the present invention, the antigen is preferably presented by a cell, preferably an antigen-presenting cell, in the context of an MHC molecule, which results in an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can be a tumor antigen. According to the present invention, the antigen corresponds to a naturally occurring product, for example, a viral protein or a portion thereof.

[0262] 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 containing an epitope that stimulates the host's immune system to mount a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by a microorganism, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, such as tumor antigens, typically tumors.

[0263] In some embodiments, antigen is tumor antigen, i.e., it is a part of tumor cell, particularly mainly occurs as intracellular or surface antigen of tumor cell.In some embodiments, antigen is pathogen-associated antigen, i.e., antigen derived from pathogen, for example, virus, bacteria, unicellular organism or parasite, for example, viral antigen, such as viral ribonucleoprotein or coat protein.In particular, antigen must be presented by MHC molecule, which can be expressed by immune system cells, preferably CD4, especially through regulating the activity of T cell receptor. + and CD8 + It results in the regulation of lymphocyte activation.

[0264] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to an antigen produced as an intracellular or surface antigen of a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, fetal sulfur glycoprotein, α2-H-ferroprotein, and γ-fetoprotein, as well as various viral tumor antigens. According to the present invention, tumor antigens preferably include any antigen that is characteristic of a tumor or cancer and of a tumor or cancer cell in terms of type and / or expression level.

[0265] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0266] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0267] The term "epitope" refers to an antigenic determinant of a molecule such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. Protein epitopes preferably include continuous or discontinuous portions of the protein and are preferably about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 0, and most preferably about 10 to about 25 amino acids in length; for example, epitopes may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that epitopes in the context of the present invention are T cell epitopes.

[0268] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably and are preferably incomplete representations of an antigen, which are preferably capable of inducing an immune response against the antigen or a cell that expresses, contains, and preferably presents 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 when present 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 molecule, such as a portion or fragment of an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0269] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invasive microorganisms) to T cells. For class I MHC / peptide complexes, binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. For class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may be effective.

[0270] Peptide and protein antigens can be 2 to 100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0271] A peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibody and T cell responses against the peptide or protein.

[0272] In certain embodiments, the vaccine antigen, i.e., the antigen whose inoculation into a subject induces an immune response, is recognized by immune effector cells. Preferably, once recognized by immune effector cells, the vaccine antigen can be induced in the presence of appropriate costimulatory signals, stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of this embodiment of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell.

[0273] In certain embodiments, the antigen is expressed on a diseased cell (e.g., a tumor cell or an infected cell).

[0274] In some embodiments, the antigen is presented by diseased cells (for example, tumor cells or infected cells). In some embodiments, the antigen receptor is a TCR that binds to the epitope of the antigen presented in the context of MHC. In some embodiments, when TCR is expressed by T cell and / or present on T cell, binding to the antigen presented by cells such as antigen-presenting cells leads to the stimulation, priming and / or expansion of the T cell. In some embodiments, when TCR is expressed by T cell and / or present on T cell, binding to the antigen presented by diseased cells leads to the cytolysis and / or apoptosis of diseased cells, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzyme.

[0275] In some embodiments, the antigen is expressed on the surface of diseased cells (e.g., tumor cells or infected cells). In some embodiments, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In some embodiments, the CAR binds to the natural epitope of the antigen present on the surface of living cells. In some embodiments, when the CAR is expressed by and / or present on T cells, binding to the antigen presented by cells such as antigen-presenting cells leads to the stimulation, priming and / or expansion of the T cells. In some embodiments, when the CAR is expressed by and / or present on T cells, binding to the antigen presented by diseased cells leads to the cytolysis and / or apoptosis of diseased cells, wherein the T cells preferably release cytotoxic factors, such as perforin and granzyme.

[0276] In certain embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope of the antigen. In certain embodiments, the antibody or B cell receptor binds to a natural epitope of the antigen.

[0277] The terms "expressed on the cell surface" or "bound to the cell surface" mean that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, for example, by an antibody located on the outside of the cell. In this context, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The binding can be direct or indirect. For example, the binding can be via one or more transmembrane domains, one or more lipid anchors, or via interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule bound to the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that binds to the surface of a cell by interacting with another protein that is a transmembrane protein.

[0278] "Cell surface" or "surface of a cell" is used according to its usual meaning in the art, and thus includes the outside of a cell that is accessible for binding by proteins and other molecules.If an antigen is located on the cell surface, it is expressed on the cell surface and is accessible for binding by, for example, an antigen-specific antibody added to the cell.In some embodiments, the antigen expressed on the cell surface is an integral membrane protein that has an extracellular portion that can be recognized by CAR.

[0279] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell by a binding molecule, such as an antibody, located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or a domain or fragment thereof.

[0280] The terms "T cell" and "T lymphocyte" are used interchangeably and refer to T helper cells (CD4 + T cells) and cytotoxic T cells, including cytolytic T cells (CTL, CD8 + The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen that the T cell targets, particularly when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, in the context of an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if the cell kills a target cell that expresses the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the invention, a nucleic acid (e.g., DNA or RNA, particularly mRNA) encodes at least one epitope.

[0281] The term "target" refers to a factor, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses the antigen, preferably presenting the antigen in conjunction with class I MHC.

[0282] "Antigen processing" refers to the breakdown of an antigen into processing products that are fragments of the antigen (e.g., breakdown of a protein into peptides) and the association (e.g., via binding) of one or more of these fragments with an MHC molecule for delivery to a specific T cell by a cell, preferably an antigen-presenting cell. Antigen-presenting cells can be divided into professional and non-professional antigen-presenting cells.

[0283] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When a T cell binds to the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0284] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that express MHC class II molecules not constitutively but upon stimulation with certain cytokines, such as interferon-gamma. By way of example, non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0285] The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with a presentable antigen, they begin to activate into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade proteins into small fragments, and upon maturation, present these fragments on the cell surface using MHC molecules. At the same time, they upregulate cell surface receptors, such as CD80, CD86, and CD40, which act as coreceptors in T cell activation, greatly enhancing their ability to activate T cells. They also upregulate the chemotactic receptor CCR7, which guides dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. There, they act as antigen-presenting cells, and activate helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Therefore, dendritic cells can actively induce T cell or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0286] The term "macrophage" refers to a subgroup of phagocytes produced by the differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products nonspecifically phagocytose and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, causing the pathogens to degrade. Peptides of degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of immune responses. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0287] "Antigen-responsive CTL" refers to CD8 CTLs that are reactive to antigens presented together with class I MHC on the surface of antigen-presenting cells or peptides derived from such antigens. + Refers to T cells.

[0288] According to the present invention, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0289] The terms "immune response" and "immune reaction" are used interchangeably herein with their conventional meaning and refer to the integrated body response to an antigen, preferably a cellular immune response, a humoral immune response, or both. According to the present invention, the term "immune response to" or "immune response against" an agent such as an antigen, cell, or tissue relates to an immune response, such as a cellular response, to said agent. An immune response is the development of antibodies against one or more antigens and antigen-specific T lymphocytes, preferably CD4 + and CD8 + T lymphocytes, more preferably CD8 + The present invention may include one or more responses selected from the group consisting of expansion of T lymphocytes, which may be detected in vitro by various proliferation or cytokine production tests.

[0290] The terms "induction of an immune response" and "elicitation of an immune response" and similar terms, in the context of the present invention, 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 can be protective / preventive / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably 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, "induction" can mean not only that there was no immune response against a particular antigen or pathogen before induction, but also that there was a certain level of immune response against a particular antigen or pathogen before induction, and that the immune response is enhanced after induction. Thus, in this context, "induction of an immune response" also includes "enhancement of an immune response." Preferably, after induction of an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or a disease state that is alleviated by the induction of an immune response.

[0291] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include cellular responses directed against cells characterized by expression of antigens and / or presentation of antigens with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (CD4 + T cells (also called killer cells) play a more central role in regulating the immune response and are responsible for the production of immune cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, such as diseased cells.

[0292] The term "humoral immune response" refers to the process in living organisms by which antibodies are produced in response to factors and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide and produce memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope as that recognized by the antigen receptor. Memory B lymphocytes remain quiescent until subsequently activated by a specific antigen. These lymphocytes provide the cellular basis for memory and the resulting increased antibody response upon re-exposure to the specific antigen.

[0293] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an epitope of an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interrupted by more conserved 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 carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxy terminus): CH1, CH2, CH3. The constant region of an antibody may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0294] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are structural domains with a characteristic immunoglobulin (Ig) fold, i.e., immunoglobulin domains. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins and are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains, linked via disulfide bonds. These chains are mainly V L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domains and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H The immunoglobulin chain consists of immunoglobulin domains such as α, δ, ε, γ, and μ. There are five types of mammalian immunoglobulin heavy chains, namely, α, δ, ε, γ, and μ, which are responsible for 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 the carboxy terminus. In mammals, there are two types of light chains, namely, lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among different immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0295] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, as described herein, particularly in the form of DNA or RNA (particularly mRNA) encoding same, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

[0296] "Cells characterized by the presentation of an antigen" or "cells presenting antigen" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions refer to cells, such as diseased cells, particularly tumor or infected cells, or antigen-presenting cells that present antigens or antigenic peptides, either 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.

[0297] In the context of the present invention, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA (especially mRNA), which can subsequently be translated into peptides, polypeptides or proteins.

[0298] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0299] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide, polypeptide, or protein.

[0300] In the context of the present invention, the term "RNA encodes" refers to the process by which RNA, if present in the appropriate environment, such as within the cells of a target tissue, can direct the assembly of amino acids to produce the encoded peptide or protein during the process of translation.

[0301] As used herein, the term "optionally" or "optionally" means that the subsequently described event, event, or condition may or may not occur, and the description includes instances where the event, event, or condition occurs and instances where it does not occur.

[0302] The medical preparations described herein, particularly kits, may include instructions or directions. As used herein, "instructions" or "instructions" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructions for the kits of the present invention can be, for example, attached to a container containing the compositions / formulations of the present invention or shipped together with the container containing the compositions / formulations. Alternatively, the instructions can be shipped separately from the container with the intention that the instructions and the composition will be used cooperatively by the recipient.

[0303] A prodrug of a specific compound described herein is a compound that undergoes chemical conversion under physiological conditions upon administration to an individual to provide the specific compound. Furthermore, prodrugs can be converted to the specific compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the specific compound when placed in a transdermal patch reservoir with, for example, a suitable enzyme or chemical reagent. Examples of prodrugs are in vivo hydrolyzable esters (using an alcohol or carboxy group contained in the specific compound) or amides (using an amino or carboxy group contained in the specific compound). Specifically, any amino group contained in the specific compound that bears at least one hydrogen atom can be converted to a prodrug form. Exemplary N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0304] In this specification, the structural formula of a compound may represent a certain isomer of the compound. However, it is understood that the present invention includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, structurally occurring stereoisomers, tautomers, and isomeric mixtures, and is not limited to the description of the formula. Furthermore, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it is understood that the present invention includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0305] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) position of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even in pure form, spontaneously and reversibly interconvert into each other by the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is a keto-enol-tautomeric isomer. "Conformers" are stereoisomers that can formally be interconverted only by rotation of a single bond, and include—in particular—those that lead to different three-dimensional forms of (hetero)cyclic rings, such as the chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0306] As used herein, the term "solvate" refers to an addition complex of a substance dissolved in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), wherein the addition complex exists in crystalline or mixed crystalline form. The amount of solvent contained in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solvent is water.

[0307] In an isotopically labeled compound, one or more atoms are replaced with a corresponding atom having the same number of protons but a different number of neutrons. For example, a hydrogen atom is replaced with a deuterium or tritium atom. Examples of isotopes that can be used in the present invention are deuterium, tritium, 11 C. 13 C. 14 C. 15 N, 18 F, 32 P, 32 S, 35 S, 36 Cl and 125 Includes I.

[0308] The term "average diameter" refers to the diameter measured by dynamic light scattering (DLS), resulting in the so-called Z 平均 The "average diameter", "diameter" or "size" of a particle is defined as the average hydrodynamic diameter of the particle analyzed using the so-called cumulant algorithm, which provides the particle diameter Z, together with the dimension of length and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). 平均 Used synonymously with this value of

[0309] In one embodiment, the "polydispersity index" is calculated based on dynamic light scattering measurements by so-called cumulant analysis as described in the definition of "average diameter." Subject to certain requirements, it can be interpreted as an indicator of the size distribution of an ensemble of nanoparticles.

[0310] The "cross-sectional radius of gyration" of the particle about the axis of rotation (here R g ) is the radial distance of a point from the axis of rotation, at which the total mass of the particle is assumed to be concentrated, and its moment of inertia about that axis is the same as the actual mass distribution. Mathematically, R g is the root mean square distance of a particle component from the center of mass or some axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at i For a polymer consisting of n mass sequences (i=1, 2, 3, . . . , n), R g is the s over the entire mass array i 2 is the mass-averaged square root of and can be calculated as follows:

number

[0311] The cross-sectional radius of gyration can be determined experimentally or calculated, for example, using light scattering techniques.

number

number

[0312] The "D10 value," particularly with respect to quantitative particle size distribution, is the diameter below which 10% of the particles have a diameter. The D10 value is a means of describing the proportion of smallest particles within a particle population (e.g., within a particle peak obtained by field-flow fractionation).

[0313] The "D50 value," particularly with respect to a quantitative particle size distribution, is the diameter below which 50% of the particles have a diameter. The D50 value is a means of describing the average particle size of a population of particles (e.g., within a particle peak obtained by field-flow fractionation).

[0314] The "D90 value," particularly with respect to the quantitative size distribution of particles, is the diameter below which 90% of the particles have a diameter. The "D95," "D99," and "D100" values ​​have corresponding meanings. The D90, D95, D99, and D100 values ​​are a means of describing the proportion of large particles within a particle population (e.g., within a particle peak obtained by field-flow fractionation).

[0315] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle. The hydrodynamic radius relates to the movement of the particle, taking into account not only size but also solvent effects. For example, a small, highly hydrated charged particle may have a larger hydrodynamic radius than a large, weakly hydrated charged particle. This is because the small particle drags a greater number of water molecules with it as it moves through the solution. While the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is defined by the Stokes-Einstein equation:

number

[0316] As used herein, the term "aggregation" refers to a conglomeration of particles, where the particles are identical or very similar and are attached to one another in a non-covalent manner (e.g., via ionic interactions, H-bridge interactions, dipole interactions and / or van der Waals interactions).

[0317] As used herein, the expression "light scattering" refers to the physical process by which light is deflected from a straight-line trajectory by one or more paths due to localized non-uniformities in the medium through which it travels.

[0318] The term "UV" means ultraviolet and designates the band of the electromagnetic spectrum between 10 nm and 400 nm, i.e., wavelengths shorter than visible light but longer than X-rays.

[0319] As used herein, the expression "multi-angle light scattering" or "MALS" refers to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at a variety of different angles, e.g., measured with a single detector moving over a range that includes a selected specific angle, or with an array of detectors fixed at specific angular positions. In one embodiment, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the cross-sectional radius of gyration (R) can be calculated using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot). g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is depicted graphically using the following formula (or its reciprocal):

number

number

number

[0320] As used herein, the phrase "dynamic light scattering" or "DLS" refers to a technique used to determine particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is directed through a polarizer and into the sample. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution impinge on the light, causing it to diffract in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated at short intervals, and the resulting set of spot patterns is analyzed with an autocorrelator, which compares the light intensity of each spot over time.

[0321] As used herein, the phrase "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's cross-sectional radius of gyration and / or particle molar mass. A high-intensity monochromatic light, usually a laser, is projected into a solution containing the particles. One or many detectors are used to measure the scattered intensity at one or many angles. The angular dependence is necessary for accurate determination of both the molar mass and size of the entire radial macromolecule. Therefore, simultaneous measurements at several angles relative to the incident light direction, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0322] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is the relatively nonspecific and immediate component of the immune system. Along with the adaptive immune system, it is one of the two main components of the vertebrate immune system.

[0323] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0324] As used herein, the term "exogenous" refers to any substance introduced from or produced outside an organism, cell, tissue or system.

[0325] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of two or more sequences, components, or domains.

[0326] As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, ranging in size from 1 to 1000 nanometers in diameter. The mixture is called a colloid or colloidal suspension. The term "colloid" can sometimes refer only to the particles in the mixture, rather than the entire suspension.

[0327] In the context of the present invention, the term "nucleic acid particle" refers to a particle comprising nucleic acid (e.g. DNA or RNA). Thus, the term "RNA particle" relates to a particle comprising RNA.

[0328] As used herein, "polymer" has its conventional meaning, i.e., a molecular structure comprising one or more repeating units (monomers) covalently linked. The repeating units may all be identical, 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 such as a protein. In some cases, additional moieties, such as targeting moieties, such as those described herein, may also be present in the polymer.

[0329] The term "repeating unit" refers to a basic unit that is repeated periodically along the polymer chain of a polymer and is derived from one monomer. The structures of a repeating unit and its corresponding monomer often match but can differ from each other.

[0330] If more than one type of repeat unit is present in a polymer, the polymer is referred to as a "copolymer." In any embodiment using a polymer, it is understood that the polymer used may, in some cases, be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeat units may be arranged in a random manner, a periodic manner, an alternating manner, or a block unit manner. In this regard, the term "randomly arranged" means that the arrangement of the repeat units of the copolymer follows statistical laws (such copolymers are referred to as statistical copolymers). The term "periodically arranged" means that the repeat units occur in a repeating pattern in the copolymer (e.g., a periodic copolymer consisting of repeat units A and B would have the formula (-ABAAABBB-) n The term "alternatingly arranged" means that the corresponding copolymer may have, for example, the formula: -ABABABABAB- or -(-AB-) n- means having regularly alternating repeating units such as in (A). The term "arranged in a block unit manner" means that the corresponding copolymer contains at least two homopolymer subunits (i.e., blocks) covalently linked. A block copolymer can have two (diblock copolymer), three (triblock copolymer) or more different blocks. An example of a triblock copolymer arrangement consisting of A and B repeating units is as follows:-(A) a -(B) b -(A) c -where a, b and c represent the number of repeat units in each block).

[0331] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in place of somatic histones in the sperm cells of various animals (e.g., fish), particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not aggregate on heating, and hydrolyzes to yield primarily arginine. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin. According to the present invention, the term "protamine" as used herein is meant to include any protamine amino acid sequence (including fragments thereof and multimers of said amino acid sequence or fragments) obtained or derived from natural or biological sources, as well as artificial and specifically designed polypeptides for specific purposes that cannot be isolated from natural or biological sources (synthetic).

[0332] The term "hydrophobic" as used herein with respect to a compound, group, or moiety means that the compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, aggregates and excludes water molecules. In certain embodiments, the term "hydrophobic" refers to any compound, group, or moiety that is substantially immiscible or insoluble in an aqueous solution. In certain embodiments, the hydrophobic compound, group, or moiety is substantially non-polar. Examples of hydrophobic groups are hydrocarbyl groups and fluorinated (e.g., perfluorinated) hydrocarbyl groups. In certain embodiments, the hydrophobic compound, group, or moiety is lipophilic. In this regard, the term "lipophilic" as used herein with respect to a compound, group, or moiety means that the compound, group, or moiety is soluble in a non-polar solvent (e.g., hexane, tetrahydrofuran (THF) and / or chloroform). In certain embodiments, the term "lipophilic" refers to any compound, group, or moiety that is soluble in a non-polar solvent (e.g., hexane, tetrahydrofuran (THF) and / or chloroform) and substantially immiscible or insoluble in an aqueous solution. Examples of lipophilic groups are hydrocarbyl groups, such as acyclic, preferably straight-chain, hydrocarbyl groups (eg straight-chain hydrocarbyl groups having at least 10 carbon atoms), such as the lipophilic chains of natural lipids.

[0333] The term "perfluorinated," as used herein with respect to a compound, group, or moiety, means that in the compound, group, or moiety, all CH moieties have been replaced with CF moieties. For example, perfluorinated n-octanoic acid has the formula F3C(CF2)6COOH.

[0334] The term "lipid" is broadly defined herein as a molecule containing one or more hydrophobic moieties or groups (e.g., one or more hydrophobic chains) and, optionally, one or more hydrophilic moieties or groups. For example, the term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphilic. Lipids are usually poorly soluble in water. In an aqueous environment, amphiphilicity causes the molecules to self-assemble into organized structures and various phases. One of the phases is a lipid bilayer, as long as it exists in a vesicle, a multilamellar / unilamellar liposome, or a membrane in an aqueous environment. Hydrophobicity can be conferred by nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups can contain polar and / or charged groups, including carbohydrates, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Lipids are generally classified 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). The term "lipid" is sometimes used interchangeably with fat, which is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids) and sterol-containing metabolites such as cholesterol.

[0335] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion may have a formal positive or negative charge. Alternatively, the polar portion may have both formal positive and negative charges and may be a zwitterion or inner salt. For purposes of the present invention, an amphiphilic compound may be, but is not limited to, one or more of natural or non-natural lipids and lipid-like compounds.

[0336] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, the term includes compounds that can form amphiphilic layers when present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, including surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules containing hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble lipids. Examples of lipid-like compounds that can spontaneously incorporate into cell membranes include functional lipid constructs, such as synthetic functional group-spacer-lipid constructs (FSLs), synthetic functional group-spacer-sterol constructs (FSSs), and artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are generally cylindrical. The region occupied by the two alkyl chains resembles the region occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Classical surfactant monomers, which contain only one linear alkyl chain and a hydrophilic head group, are generally conical in shape. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble. Lipids have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—but lipids differ from surfactants in the shape of the monomer, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" is intended to include both lipids and lipid-like substances, unless otherwise specified herein or clearly contradicted by the context.

[0337] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids. In some embodiments, the amphiphilic compound is a lipid.

[0338] Fatty acids or fatty acid residues are a diverse group of 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, typically 4 to 24 carbons long, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains double bonds, there is the possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis double bonds cause bending of the fatty acid chain, and the more double bonds in the chain, the greater the effect. Other major lipid classes in the fatty acid category are fatty esters and fatty amides.

[0339] Glycerolipids consist of mono-, di-, and tri-substituted glycerols, with the most well-known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically each esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by one or more sugar residues attached to glycerol via glycosidic bonds.

[0340] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by ester bonds to two fatty acid-derived "tails" and a phosphate ester bond to one "head" group. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0341] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The major sphingoid base in mammals is commonly called sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The major phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi contain phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues linked via glycosidic bonds to a sphingoid base. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0342] Sterol lipids, such as cholesterol and its derivatives or tocopherol and its derivatives, are important components of membrane lipids together with glycerophospholipids and sphingomyelins.

[0343] Glycolipids are compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with membrane bilayers. 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 the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexa-acylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0344] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes as well as repetitive multimodular enzymes that share mechanistic properties with fatty acid synthases. They encompass a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and represent a large amount of structural diversity. Many polyketides are cyclic molecules whose main chains are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0345] According to the present invention, lipids and lipid-like substances are cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH. An example of a neutral zwitterionic lipid is a phospholipid.

[0346] As used herein, the term "functionalized moiety" refers to a group of atoms in a molecule having a characteristic chemical property, where the atoms of the functionalized moiety are covalently linked to each other and to the rest of the molecule. Preferably, the atoms of the functionalized moiety include at least one atom selected from the group consisting of O, N, and S. The functionalizing moiety can be monovalent (e.g., hydroxy, cyano, nitro, or amido (e.g., -C(O)NHCH3)) or divalent (e.g., amide (e.g., -C(O)NH-), carbonyl (-C(O)-), or ester (e.g., -OC(O)-). In certain embodiments, the functionalizing moiety confers hydrophilicity on the group to which it is attached, for example, by providing at least one hydrogen bond acceptor / donor and / or at least one charge (positive or negative) on the group to which it is attached. In certain embodiments, the functionalizing moiety comprises a hydrogen bond acceptor (e.g., a carbonyl moiety), a hydrogen bond donor (e.g., a hydroxyl moiety, -NH- (e.g., in an amide moiety) or a thiol moiety), or both (e.g., in an amide moiety) and / or is charged (e.g., a phosphate, amino, or ammonium moiety). Examples of monovalent functionalizing moieties include hydroxy, ether, halogen, cyano, azido, nitro, azido, hydroxyl, ether ... Amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionyl Includes amide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.Examples of divalent functionalized moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfuric diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino(imine), imidothioate, thionylamide, carbonate, The term "carbonimidothioate" includes thioates, carbonothioates, carbonodithioates, carbonotrithioates, guanidino(imidamide), carbamimidates, carbonimidates, carbamates, carbamodithioates, carbonodithioimidates, carbamimidothioates, carbamothioates, carbonimidothioates, acylhydrazones, hydrazines, oximes, acetals, hemiacetals, ketals, hemiketals, imide and amide moieties.

[0347] The term "hydroxyl" or "hydroxy" as used herein with respect to a functionalized moiety as a component of a linker refers to the group --OH.

[0348] The term "halogen" as used herein as a functionalizing moiety, particularly as a component of a linker, means fluoro, chloro, bromo, or iodo.

[0349] The term "cyano" as used herein, particularly as a functionalizing moiety as a component of a linker, refers to the group --CN.

[0350] The term "azide" as used herein as a functionalizing moiety, particularly as a component of a linker, refers to the group N3.

[0351] The term "nitro" as used herein, particularly as a functionalizing moiety as a component of a linker, refers to the group -NO2.

[0352] The term "amino," as used herein, particularly as a functionalized moiety as a component of a linker, includes unsubstituted amino (i.e., the group -NH) and substituted amino (i.e., mono- or di-substituted amino in which one or two of the hydrogen atoms are replaced with a group other than hydrogen). Amino groups are monovalent (e.g., -NRR, where each R is independently an R group as defined below). 72 or R 73 or a divalent (e.g., —NR—, where R is R as defined below) 72 In some embodiments, the term "amino" refers to the group -N(R 72 )(R 73 ), where R 72 and R 73 is, in each occurrence, independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 72 and R 73 together with the nitrogen atom to which they are attached form the group -N=CR 75 R 76 wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups optionally has one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected R 70 is substituted with;R 75 and R 76 is -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and -NH y R 80 2-y or R 75 and R 76may be joined together with the atoms to which they are attached to form a ring, which may optionally contain one or more (e.g., from 1 to the maximum number of hydrogen atoms on the carbon atoms attached to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected R 70 wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups optionally has one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected R 70 y is an integer from 0 to 2; R 80 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups optionally has one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected R 70 and R 70 is other than H, preferably a first level substituent, second level substituent, or third level substituent disclosed herein. In certain embodiments, R 72 and R 73 Each of the groups independently represents H, C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl), wherein each of the hydrocarbyl groups (e.g., C 1-6 Alkyl, aryl and aryl(C 1-6Each of the alkyl groups may optionally contain one or more (e.g., from 1 to the maximum number of hydrogen atoms (e.g., C 1-6 Alkyl, aryl or aryl(C 1-6 alkyl) groups), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2, independently selected R 70 is replaced by .

[0353] The term "ammonium" as used herein as a functionalized moiety, particularly as a component of a linker, refers to the group -N + (R 72 )2(R 73 ) (where R 72 and R 73 is as defined for the term "amino".

[0354] The term "thiol" or "sulfanyl" as used herein, particularly as a functionalizing moiety as a component of a linker, refers to the group --SH.

[0355] The term "disulfanyl" as used herein as a functionalizing moiety, particularly as a component of a linker, refers to the group -SSH.

[0356] The term "carboxyl" or "carboxy" as used herein as a functionalizing moiety, particularly as a component of a linker, refers to the group --COOH.

[0357] The term "amide" or "amido" as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)NH- (including its isomeric arrangements of the structure -NHC(O)-, unless otherwise specified). Preferably, each end of the amide structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker) (if both ends are linked to the same organic group, the amide moiety is also referred to as a lactam). The amide group is monovalent (e.g., -C(O)NRR or -NRC(O)R), where each R is H or an organic group, e.g., R as set forth above in the definition of the term "amino." 72 or divalent (e.g., —C(O)NR— or —NRC(O)—, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 (It is one of the organic groups specified in the definition of

[0358] The term "ester," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)O- (including its isomeric arrangements of the structure -OC(O)-, unless otherwise specified). Preferably, each end of the ester structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). (If both ends are linked to the same organic group, the ester moiety is also referred to as a lactone.) The ester group is monovalent (e.g., -C(O)OR or -OC(O)R), where R is independently an organic group, e.g., R as defined above in the definition of the term "amino." 72 (wherein the aryl group is one of the organic groups specified in the definition) or divalent (for example, —C(O)O— or —OC(O)—).

[0359] The term "ether," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -O-, where each end of the ether structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). An ether group is monovalent (e.g., -OR, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (eg, -O-).

[0360] The term "sulfide" or "thioether" as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group containing the structure -S-, where the sulfide structure is covalently linked to the C atoms of each of the two ends of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfide group is monovalent (e.g., -SR, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (e.g., -S-).

[0361] The term "disulfide," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -SS-, where the disulfide structure is covalently linked to the C atoms of each of the two ends of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). Disulfide groups are monovalent (e.g., -SSR, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (eg, -SS-).

[0362] The term "diselenide," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group containing the structure -SeSe-, where the diselenide structure is covalently linked to the C atoms of each of the two ends of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The diselenide group is monovalent (e.g., -SeSeR, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition) or divalent (e.g., -SeSe-).

[0363] The term "sulfoxide," as used herein, particularly as a functionalizing moiety as a component of a linker, refers to a group containing the sulfinyl structure -S(O)-, where the sulfoxide structure is covalently linked to the C atoms of each of the two ends of the same organic group or to two separate organic groups (e.g., alkylene groups as further components of a linker). The sulfoxide group is monovalent (e.g., -S(O)R, where R is an organic group, e.g., R as shown above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (e.g., -S(O)-).

[0364] The term "sulfone," as used herein, particularly as a functionalizing moiety as a component of a linker, refers to a group containing the sulfonyl structure -S(O)-, where the sulfone structure is covalently linked to a C atom at each end of the same organic group or to two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfone group is monovalent (e.g., -S(O)R, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (e.g., —S(O)2—), or divalent (e.g., —S(O)2—).

[0365] The term "sulfite," as used herein, particularly as a functionalizing moiety as a component of a linker, refers to a group containing the structure -OS(O)O-, where one of the two ends of the sulfite structure is covalently linked to a C atom of an organic group and the other end is covalently linked to H or a C atom of the same or another organic group (e.g., an alkylene group as a further component of the linker). The sulfite group is monovalent (e.g., -OS(O)OR, where R is H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, -OS(O)O-).

[0366] The term "sulfate," as used herein, particularly as a functionalizing moiety as a component of a linker, refers to a group containing the structure -OS(O)2O-, where 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 a further component of the linker). The sulfate group is monovalent (e.g., -OS(O)2OR, where R is H or an organic group, such as R as set forth above in the definition of the term "amino"). 72 (i.e., one of the organic groups specified in the definition of —OS(O)2O—) or divalent (e.g., —OS(O)2O—).

[0367] The term "phosphate" as used herein as a functionalized moiety, particularly as a component of a linker, refers to a phosphate having the structure -OP(O)(OR)O-, where 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 a further 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, e.g., R as shown above in the definition of the term "amino"). 72 The phosphate group is monovalent (e.g., —OP(O)(OR)2, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72or divalent (e.g., —OP(O)(OR)O—, where R is independently H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0368] The term "sulfinamide" as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -S(O)N(R)-, where the S terminus of the sulfinamide 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 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, e.g., R as set forth above in the definition of the term "amino"). 72 A sulfinamide group is a monovalent (e.g., —S(O)N(R)2, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —S(O)N(R)—, where R is H or an organic group, such as R as shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0369] The term "sulfonamide," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -S(O)N(R)-, where the S terminus of the sulfonamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the 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, e.g., R as set forth above in the definition of the term "amino"). 72 A sulfonamide group is a monovalent (e.g., —S(O)N(R) where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72(e.g., —S(O)N(R)—, where R is H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0370] The term "sulfamate", as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -OS(O)N(R)- (including its isomeric arrangements -N(R)S(O)O-, unless otherwise specified), in which one of the ends of the sulfamate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently linked to H or a C atom of the same or another organic group (R is H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 The sulfamate group is a monovalent group (e.g., —OS(O)N(R) or —N(R)S(O)OR, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 or divalent (e.g., —OS(O)N(R)— or —N(R)S(O)O—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0371] The term "sulfur-containing diamide," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -N(R)S(O)N(R)-, where one end of the sulfur-containing diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72The sulfur-containing diamide group is monovalent (e.g., —N(R)S(O)N(R)2, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)S(O)N(R)—, where each R is independently H or an organic group, such as the R groups set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0372] The term "sulfuric acid diamide," as used herein, particularly as a functionalized moiety as a component of a linker, has the structure -N(R)S(O)N(R)-, where one end of the sulfuric acid diamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 The sulfonamide group is monovalent (e.g., —N(R)S(O)N(R) where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)S(O)N(R)—, where each R is independently H or an organic group, such as the R groups set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0373] The term "urea," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -N(R)C(O)N(R)-, where one of the 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 a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72The urea group is a monovalent (e.g., —N(R)C(O)N(R)2, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)C(O)N(R)—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0374] The term "thiourea," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -N(R)C(S)N(R)-, where one of the 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 a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 The thiourea group is monovalent (e.g., —N(R)C(S)N(R)2, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)C(S)N(R)—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0375] The term "carbonyl," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)-, where one of the ends of the carbonyl 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 a C atom of the same or another organic group (if both ends are linked to C atoms of organic groups, the carbonyl moiety is also referred to as a "keto" moiety). The carbonyl group is monovalent (e.g., -C(O)R, where R is H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (e.g., —C(O)—).

[0376] The term "thiocarbonyl," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group containing the structure -C(S)-, where one of the ends of the thiocarbonyl 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 a C atom of the same or another organic group. The thiocarbonyl group is monovalent (e.g., -C(S)R, where R is H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (e.g., -C(S)-).

[0377] The term "orthoester," as used herein, particularly as a functionalized moiety as a component of a linker, refers to an orthoester that is an ester of three alkoxy groups (i.e., -OR, where R is an organic group (e.g., an alkylene group as a further component of a linker), such as the R groups shown above in the definition of the term "amino." 72 The moiety contains a C atom to which is attached an organic group (which is one of the organic groups specified in the definition of (-O)). An example of an orthoester formula is the structure (-O) r C(OR) 3-r -, where each R is independently an organic group, such as R as set forth above in the definition of the term "amino." 72wherein R is one of the organic groups specified in the definition of (—O) r C(OR 25 ) 3-r where each R 25 are independently optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein) hydrocarbyl groups, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and R is 1 or 2; and each end of the orthoester structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. The orthoester group is monovalent (e.g., —C(OR) or —OC(OR)R, where each R is an organic group, such as the R groups shown above in the definition of the term “amino.” 72 (R) or -OC(OR)-, where each R is an organic group, such as an R shown above in the definition of the term "amino." 72 (independently selected from the organic groups specified in the definition of

[0378] The term "thioate" or "thioester," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(O)- (including its isomeric arrangement, -C(O)S-, unless otherwise specified), where each end of the thioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). A thioate group is monovalent (e.g., -SC(O)R or -C(O)SR, where each R is independently H or an organic group, such as the R groups set forth above in the definition of the term "amino." 72(one of the organic groups specified in the definition of —S—C(O)—) or divalent (e.g., —S—C(O)— or —C(O)S—).

[0379] The term "dithioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)- (including its isomeric arrangement, -C(S)S-, unless otherwise specified), where each end of the dithioate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). Dithioate groups are monovalent (e.g., -SC(S)R or -C(S)SR, where each R is independently H or an organic group, such as the R groups set forth above in the definition of the term "amino." 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, -SC(S)- or -C(S)S-).

[0380] The term "imidate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -OC(=NR)- (including its isomeric arrangements, unless otherwise specified), where each end of the imidate structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker), where each R is independently H or an organic group, such as the R group set forth above in the definition of the term "amino." 72 An imidate group is a monovalent (e.g., —OC(═NR)R′ or —C(═NR)OR′, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 and each R' is independently one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(═NR)— or —C(═NR)O—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72(the group is one of the organic groups specified in the definition of

[0381] The term "imino" or "imine," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -C(=NR)-, where one end of the imino 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 a C atom of the same or another organic group (each R is independently H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 The moiety -C(=NR)H is also known as an "aldimine" and the moiety -C(=NR)R' (where R' is an organic group, such as one of the R groups specified above in the definition of the term "amino"). 72 An imino group is one of the organic groups specified in the definition of the term "amino." An imino group is a monovalent (e.g., -C(=NR)R, where each R is independently H or an organic group, such as the R shown above in the definition of the term "amino." 72 and each R' is independently one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —C(═NR)—, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0382] The term "imidothioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -C(=NR)S- (including its isomeric arrangement, -SC(=NR)-, unless otherwise specified), in which one end of the imidothioate 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 a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72The imidothioate group is a monovalent (e.g., —C(═NR)SR or —SC(═NR)R, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 or divalent (e.g., —C(═NR)S— or —SC(═NR)—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0383] The term "thionylamino," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -C(S)NR- (including its isomeric arrangements, unless otherwise specified), in which one of the ends of the thionylamino 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 a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 A thionylamino group is a monovalent (e.g., —C(S)NRR or —N(R)C(S)R, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 or divalent (e.g., —C(S)NR— or —N(R)C(S)—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0384] The term "carbonate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(O)O-, where each end of the carbonate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonate group is monovalent (e.g., -OC(O)OR', where R' is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, -OC(O)O-).

[0385] The term "carbonothioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group containing the structure -OC(S)O- or -OC(O)S- (including its isomeric arrangements, -SC(O)O-, unless otherwise specified), where each end of the carbonothioate 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 carbonothioate group is monovalent (e.g., -OC(S)OR' or -OC(O)SR' or -SC(O)OR', where each R' is independently an organic group, e.g., an R as set forth above in the definition of the term "amino." 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, —OC(S)O— or —OC(O)S— or —SC(O)O—).

[0386] The term "carbonodithioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group containing the structure -SC(O)S- or -OC(S)S- (including its isomeric arrangements of the structure -SC(S)O-, unless otherwise specified), where each end of the carbonodithioate 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 carbonodithioate group is monovalent (e.g., -SC(O)SR'-OC(S)SR' or -SC(S)OR', where each R' is independently an organic group, e.g., an R as set forth above in the definition of the term "amino." 72(wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, -SC(O)S- or -OC(S)S- or -SC(S)O-).

[0387] The term "carbonotrithioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)S-, where each end of the carbonotrithioate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonotrithioate group is monovalent (e.g., -SC(S)SR, where R is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (e.g., -SC(S)S-).

[0388] The term "guanidino" or "imidamide" as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -N(R)C(=NR)NR-, where each R is independently H or an organic group, such as the R groups set forth above in the definition of the term "amino." 72 The guanidino group relates to a group comprising a guanidino structure, one of whose ends is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of a linker) and the other end is covalently linked to H or a C atom of the same or another organic group. The guanidino group is monovalent (e.g., —N(R)C(═NR)NR R—R, where each R is independently H or an organic group, e.g., R as set forth above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)C(═NR)NR—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0389] The term "carbamidate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -OC(=NR)NR- (including its isomeric arrangements, unless otherwise specified to the contrary, of the structure -N(R)C(=NR)O-), where the O terminus of the carbamidate structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other (N) terminus is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R group as set forth above in the definition of the term "amino"). 72 A carbamimidate group is a monovalent (e.g., —OC(═NR)NRR or —N(R)C(═NR)OR′, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(═NR)NR— or —N(R)C(═NR)O—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0390] The term "carbonimidate," as used herein, particularly as a functionalized moiety as a component of a linker, is the structure -OC(=NR)O-, where each of the termini 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, e.g., an R group as set forth above in the definition of the term "amino"). 72 A carbon imidate group is a monovalent (e.g., —OC(═NR)OR′, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72or divalent (e.g., —OC(═NR)O—, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0391] The term "carbamate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -OC(O)NR- (including its isomeric arrangements, unless otherwise specified), in which 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 (N-terminus) is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 A carbamate group is a monovalent (e.g., —OC(O)NRR or —N(R)C(O)OR′, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(O)NR— or —N(R)C(O)O—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0392] The term "carbamodithioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -SC(S)NR- (including its isomeric arrangements -N(R)C(S)S-, unless otherwise specified to the contrary), in which the S terminus of the carbamodithioate 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 (N terminus) is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 A carbamodithioate group is a monovalent (e.g., —SC(S)NRR or —N(R)C(S)SR′, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —SC(S)NR— or —N(R)C(S)S—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0393] The term "carbonodithioimidate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a carbonodithioimidate having the structure -SC(=NR)S-, where each of the termini 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, e.g., R as set forth above in the definition of the term "amino"). 72 The carbonodithioimidate group is a monovalent (e.g., —SC(═NR)SR′, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72or divalent (e.g., —SC(═NR)S—, where R is H or an organic group, such as R as set forth above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0394] The term "carbamimidothioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -SC(=NR)NR- (including its isomeric arrangements, -N(R)C(=NR)S-, unless otherwise specified to the contrary), in which the S terminus of the carbamimidothioate 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 (N terminus) is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 A carbamimidothioate group is a monovalent (e.g., —SC(═NR)NRR or —N(R)C(═NR)SR′, where each R is independently H or an organic group, such as the R group shown above in the definition of the term “amino.” 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —SC(═NR)NR— or —N(R)C(═NR)S—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0395] The term "carbamothioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -N(R)C(O)S- or -N(R)C(S)O- (including its isomeric arrangements -SC(O)NR- or -OC(S)NR-, unless otherwise specified), where the O / S terminus of the carbamothioate 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 (N-terminus) is covalently linked to a C atom of the same or another organic group (each R is independently H or an organic group, e.g., an R as set forth above in the definition of the term "amino"). 72 A carbamothioate group is a monovalent (e.g., —N(R)C(O)SR′ or —N(R)C(S)OR′ or —SC(O)NRR or —OC(S)NRR, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 and each R' is independently one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —N(R)C(O)S— or —N(R)C(S)O— or —SC(O)NR— or —OC(S)NR—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0396] The term "carbonimidothioate," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -OC(=NR)S- (including its isomeric arrangement, -SC(=NR)O-, unless otherwise specified to the contrary), in which 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 a further component of the linker) and the other terminus (N terminus) is covalently linked to H or a C atom of the same or another organic group (each R is independently H or an organic group, e.g., R as set forth above in the definition of the term "amino"). 72The carbonimidothioate group is monovalent (e.g., —OC(═NR)SR′ or —SC(═NR)OR′, where each R is independently H or an organic group, such as R shown above in the definition of the term “amino.” 72 and each R' is independently one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —OC(═NR)S— or —SC(═NR)O—, where each R is independently H or an organic group, such as the R shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0397] The term "acylhydrazone," as used herein, particularly as a functionalized moiety as a component of a linker, refers to the structure -C(R')(=NN(R)C(O)-) (including its isomeric arrangements, -C(O)(N(R)-N=)C(R')-, unless otherwise specified to the contrary) and / or =C(=NN(R)C(O)R'), where each R' is independently an organic group, such as the R groups set forth above in the definition of the term "amino." 72 each R is H or an organic group, such as the R shown above in the definition of the term "amino" 72 and each of the ends of the acylhydrazone structure is linked by a group containing one additional organic group or two additional separate organic groups (e.g., alkylene groups as additional components of a linker) covalently linked to a C atom. In some embodiments, the acylhydrazone has the structure -C(R 25 )(=NN(R 26 )C(O)-) (unless otherwise specified, the isomerically arranged structure (-C(O)(N(R 26 )-N=)C(R 25 )-) and / or =C(=NN(R 26 )C(O)R 25 ), where each R 25are independently optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein) hydrocarbyl groups, such as C 1-6 Alkyl, aryl and aryl(C 1-6 alkyl); each R 26 are independently H or an optionally substituted (e.g., with one or more of the first level substituents, second level substituents, or third level substituents defined herein) hydrocarbyl group, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and each end of the acylhydrazone structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. An example of the chemical structure of an acylhydrazone is shown below: [ka] [During the ceremony, each [ka] represents a bond covalently linking the acylhydrazone to an additional organic group (e.g., an alkylene group as an additional component of a linker). The acylhydrazone group is monovalent (e.g., —C(R′)(═NN(R)C(O)R′) or —C(O)(N(R)—N═)C(R′)2, where each R is independently H or an organic group, such as the R groups listed above in the definition of the term “amino.” 72 and each R' is independently one of the organic groups specified in the definition of the term "amino"; 72 ) or divalent (e.g., -C(R')(=NN(R)C(O)-, -C(O)(N(R)-N=)C(R')- or =C(=NN(R 26 )C(O)R 25 ), where each R′ is independently an organic group, such as the R shown above in the definition of the term “amino.” 72 and each R is H or an organic group, such as the R shown above in the definition of the term "amino." 72(the group is one of the organic groups specified in the definition of

[0398] The term "hydrazine," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a hydrazine having the structure -N(R)N(R)-, where each R is independently H or an organic group, such as the R groups shown above in the definition of the term "amino." 72 and each of the ends of the hydrazine structure is linked to a group containing one additional organic group or two additional separate organic groups (e.g., alkylene groups as additional components of a linker) covalently linked to a C atom. In some embodiments, the hydrazine has the structure -N(R 26 )N(R 26 )-, where each R 26 are independently H or an optionally substituted (e.g., with one or more of the first level substituents, second level substituents, or third level substituents defined herein) hydrocarbyl group, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and each end of the hydrazine structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. The hydrazine group is monovalent (e.g., —N(R)N(R)2, where each R is independently H or an organic group, such as the R groups listed above in the definition of the term “amino.” 72 or divalent (e.g., —N(R)N(R)—, where each R′ is independently an organic group, e.g., one of the organic groups specified in the definition of the term “amino”). 72 (the group is one of the organic groups specified in the definition of

[0399] The term "oxime," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure =C(=N(OH)), where each end of the oxime structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). An example of the chemical formula of an oxime is shown below: [ka] [During the ceremony, each [ka] represents a bond covalently connecting the oxime to an additional organic group.] The oxime group is monovalent (e.g., —C(═N(OH))(R), where each R is independently H or an organic group, such as the R groups shown above in the definition of the term “amino.” 72 (wherein the aryl group is one of the organic groups specified in the definition of ═C(═N(OH))) or divalent (e.g., ═C(═N(OH))).

[0400] The term "acetal," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -OCH(R')O-, where R' is an organic group, such as the R group shown above in the definition of the term "amino." 72 and each of the O atoms of the acetal structure is linked by a covalent bond to a C atom of one additional organic group or two additional separate organic groups (e.g., an alkylene group as a further component of a linker). In some embodiments, the acetal has the structure -OCH(R 25 )O—, where R 25 is an optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein) hydrocarbyl group, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the O atoms of the acetal structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. The acetal group is monovalent (e.g., —OCH(R′)OR′, where each R′ is independently an organic group, such as the R′ shown above in the definition of the term “amino.” 72 (independently selected from the organic groups specified in the definition of the term "amino") or divalent (e.g., -OCH(R')O-, where R' is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (the group is one of the organic groups specified in the definition of

[0401] The term "hemiacetal," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group comprising the structure -OCH(OH)-, where each end of the hemiacetal structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups (e.g., alkylene groups as additional components of the linker). A hemiacetal group is monovalent (e.g., -OCH(OH)OR', where R' is an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (wherein the aryl group is one of the organic groups specified in the definition of aryl) or divalent (for example, —OCH(OH)—).

[0402] The term "ketal," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -OC(R')(R')O-, where each R' is independently an organic group, such as the R's shown above in the definition of the term "amino." 72 and each of the O atoms of the ketal structure is linked to a group containing one additional organic group or two additional separate organic groups (e.g., alkylene groups as additional components of a linker) covalently linked to the C atoms of the O ... 25 )(R 25 )O—, where each R 25 are independently optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein) hydrocarbyl groups, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and each of the O atoms of the ketal structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. The ketal group is monovalent (e.g., —OC(R′)(R′)OR′, where each R′ is independently an organic group, such as the R′ group shown above in the definition of the term “amino.” 72(independently selected from the organic groups specified in the definition of the term "amino") or divalent (e.g., -OC(R')(R')O-, where each R' is independently an organic group, e.g., R as set forth above in the definition of the term "amino"). 72 (the group is one of the organic groups specified in the definition of

[0403] The term "hemiketal," as used herein, particularly as a functionalized moiety as a component of a linker, has the structure -OCR'(OH)-, where R' is an organic group, such as the R group shown above in the definition of the term "amino." 72 and each of the ends of the hemiketal structure is linked by a group containing one additional organic group or two additional separate organic groups (e.g., alkylene groups as additional components of a linker) covalently linked to a C atom. In some embodiments, the hemiketal has the structure -OCR 25 (OH)—, where R 25 is an optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein) hydrocarbyl group, such as C 1-6 Alkyl, aryl and aryl(C 1-6 and each end of the hemiketal structure is covalently linked to a C atom of one additional organic group or two additional separate organic groups. The hemiketal group is monovalent (e.g., —OC(R′)2(OH), where each R′ is independently an organic group, such as the R′ group shown above in the definition of the term “amino.” 72 (independently selected from the organic groups specified in the definition of the term "amino") or divalent (e.g., -OCR'(OH)-, where R' is an organic group, e.g., R shown above in the definition of the term "amino"). 72 (the group is one of the organic groups specified in the definition of

[0404] The term "imide," as used herein, particularly as a functionalized moiety as a component of a linker, refers to a group having the structure -C(O)N(R)C(O)-, where R is an organic group, such as the R group shown above in the definition of the term "amino." 72and each end of the imide structure contains a covalent bond to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker). The imide group is monovalent (e.g., —C(O)N(R)C(O)R′, where R is independently H or an organic group, such as R as set forth above in the definition of the term “amino”). 72 and R' is one of the organic groups specified in the definition of the term "amino"; 72 or divalent (e.g., —C(O)N(R)C(O)—, where R is H or an organic group, such as R as shown above in the definition of the term “amino.” 72 (the group is one of the organic groups specified in the definition of

[0405] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (e.g., arylalkyl (aralkyl) and the like). Specific examples of hydrocarbyl groups include C 1-6 Alkyl, aryl and aryl(C 1-6 In certain embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more first-level substituents, second-level substituents, or third-level substituents, as defined herein).

[0406] The term "acyclic," as used herein in the context of organic groups, refers to an open-chain organic group that does not contain a ring. An "open-chain" or "acyclic" organic group can be straight-chained (i.e., containing only one unbranched chain with no side chains) or branched (i.e., the main chain contains one or more side chains).

[0407] An organic group "substituted with one or more substituents" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bonded to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the organic group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the one or more substituents can be selected from the first-level substituents, second-level substituents, or third-level substituents described herein.

[0408] As used herein, the phrase "hydrogen bond" or "H-bond" refers to a non-covalent bond (primarily electrostatic attraction in some embodiments) between (i) a hydrogen atom covalently bonded to a more electronegative atom or group and (ii) a lone pair of electrons on another electronegative atom. In some embodiments, the more electronegative atom or group includes a nitrogen atom and an oxygen atom; thus, examples of groups in which a hydrogen atom is covalently bonded to a more electronegative atom or group include an amino group, an -NH- group of an amide group, a hydroxyl group (per se (e.g., in an alcohol) or part of another functional group (e.g., a carboxyl (-COOH) group)), and a sulfanyl group (per se (e.g., in a thiol) or part of another functional group (e.g., a disulfanyl (-SSH) or thioester (-C(OSH) group))) bearing at least one covalently bonded hydrogen atom. In some embodiments, the lone pair of electrons on the other electronegative atom is the lone pair of an oxygen atom present in a carbonyl group or the lone pair of a nitrogen atom present in a primary, secondary, or tertiary amino group.

[0409] As used herein, the phrase "hydrogen bond donor" refers to an atomic, ionic, or molecular component of a hydrogen bond that provides a bridging (covalent) hydrogen atom. In certain embodiments, hydrogen bond donors include amino groups, -NH- groups of amide groups, hydroxyl groups (by themselves (such as in alcohols) or as part of other functional groups (e.g., carboxyl (-COOH) groups)), and sulfanyl groups (by themselves (such as in thiols) or as part of other functional groups (e.g., disulfanyl (-SSH) or thioester (-C(OSH) groups))) that bear at least one covalently bonded hydrogen atom.

[0410] As used herein, the phrase "hydrogen bond acceptor" refers to an atomic, ionic, or molecular component of a hydrogen bond that does not contribute a bridging (covalent) hydrogen atom. In some embodiments, the hydrogen bond acceptor contains at least one lone pair of electrons. Examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary, or tertiary amino groups.

[0411] The term "tocopherol" refers to a group of four compounds having the following formula: α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol: [ka] [In the formula, R t1 and R t2 Each of R is independently H or methyl. t1 and R t2 are both methyl; in β-tocopherol, R t1 is methyl and R t2 is H; in γ-tocopherol, R t1 is H and R t2 is methyl; and in δ-tocopherol, R t1 and R t2 are both H. The term "tocopherol moiety" or "tocopheryl moiety" refers to a monovalent radical of tocopherol, preferably one in which the hydrogen atom of the hydroxy group has been removed.

[0412] The term "aqueous phase," as used herein in connection with compositions / formulations comprising particles, particularly LNPs, refers to the mobile or liquid phase, i.e., the continuous aqueous phase, which (formally) contains all dissolved components other than the particles. Thus, if particles such as LNPs are dispersed in an aqueous phase, and the aqueous phase is substantially free of compound X, the aqueous phase is virtually and practically free of X in a manner that is feasible, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, it is also possible that particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

[0413] As used herein, the phrase "substantially free of X" means that a mixture (e.g., a composition or formulation described herein or the aqueous phase of such a composition or formulation) is, in a manner that is virtually and practically practicable, free of X. For example, if a mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, less than 0.001 wt. %) based on the total weight of the mixture.

[0414] Thus, if a composition (e.g., an RNA LNP composition) described herein is substantially free of lipids or lipid-like substances comprising polyethylene glycol (PEG), the amount of lipids or lipid-like substances comprising PEG in the composition (e.g., an RNA LNP composition) is preferably less than 1% by weight (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, or less than 0.001% by weight) based on the total weight of the composition.

[0415] If the aqueous phase of a composition (e.g., an RNA LNP composition) described herein is substantially free of lipids or lipid-like substances, including PEG, then the amount of lipids or lipid-like substances, including PEG, in the aqueous phase of the composition (e.g., an RNA LNP composition) is preferably less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the aqueous phase.

[0416] If the compositions (e.g., RNA LNP compositions) described herein are substantially free of PEG, then the amount of PEG in the composition (e.g., RNA LNP compositions) is preferably less than 1% by weight (e.g., less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.001% by weight), based on the total weight of the composition.

[0417] If the aqueous phase of a composition (e.g., an RNA LNP composition) described herein is substantially free of PEG, then the amount of PEG in the aqueous phase of the composition (e.g., an RNA LNP composition) is preferably less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the aqueous phase.

[0418] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded molecules and as linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present invention, means that a nucleic acid is (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) for DNA or in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0419] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0420] The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by the single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they are represented as A, C, and G, while in DNA, they are represented as dA, dC, and dG.

[0421] The modified purine (A or G) or pyrimidine (C, T or U) base moiety preferably contains one or more alkyl groups, more preferably one or more C 1-4 and more preferably modified with one or more alkyl groups, and even more preferably modified with one or more methyl groups. 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0422] DNA In certain embodiments of the invention, the nucleic acid is DNA.

[0423] As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In some embodiments, DNA contains all or most deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA, e.g., partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the ends of the DNA. It is also contemplated herein that the nucleotides of DNA may be non-standard nucleotides, e.g., chemically synthesized nucleotides or ribonucleotides. For purposes of the present invention, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the deoxyribonucleotide residue content in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0424] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0425] RNA In certain embodiments of the invention, the nucleic acid is RNA.

[0426] According to the present invention, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In some embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to internal RNA nucleotides or the addition of non-nucleotide material to the ends of the RNA. It is also contemplated herein that the nucleotides of the RNA may be non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For purposes of the present invention, these altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. In certain embodiments, the RNA used in accordance with the present invention comprises a population of different RNA molecules, e.g., a mixture of different RNA molecules that optionally encode different peptides and / or proteins. Thus, according to the present invention, the term "RNA" includes a mixture of RNA molecules.

[0427] A molecule contains a "majority of ribonucleotide residues" if the ribonucleotide residue content in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0428] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA) or microRNA (miRNA)), activating RNA (e.g., small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, the RNA used by the present invention is mRNA.

[0429] In some embodiments, RNA comprises an open reading frame (ORF) that encodes a peptide, polypeptide, or protein. The RNA can express the peptide, polypeptide, or protein that it encodes. 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 cellular translation machinery to enable the translation of peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), or can secrete or produce the encoded peptide or protein on its surface. Alternatively, RNA can be non-coding RNA, such as antisense RNA, microRNA (miRNA), or siRNA.

[0430] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT uses transcription machinery extracted from cells (e.g., a cell lysate or isolated components thereof containing an RNA polymerase, preferably T7, T3, or SP6 polymerase) rather than live / cultured cells.

[0431] According to the present invention, the term "mRNA" means "messenger RNA" and includes "transcripts" that may be produced by using a DNA template. Generally, mRNA encodes a peptide or protein.

[0432] Although mRNA is single stranded, it may contain self-complementary sequences that allow the mRNA to fold back on itself and pair to form a double helix.

[0433] According to the present invention, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0434] In certain embodiments of the present invention, mRNA relates to an RNA transcript that encodes a peptide or protein.

[0435] In one embodiment, preferably the RNA encoding the peptide or protein has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0436] In some embodiments, the RNA (e.g., mRNA) comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. In some embodiments, the RNA (e.g., mRNA) is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2004. ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Additionally, various in vitro transcription kits are available, for example, from Thermo Fisher Scientific (e.g., TranscriptAid TM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (e.g., HiScribe TM T7 Kit, HiScribe TM T7 ARCA mRNA kit), Promega (e.g., RiboMAX TM , HeLaScribe®, Riboprobe® systems), Jena Bioscience (e.g., SP6 or T7 transcription kits) and Epicentre (e.g., AmpliScribe TM ) To provide modified RNA (e.g., mRNA), correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be made and / or added to the mRNA after transcription.

[0437] In some embodiments, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for transcription control can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and can be introduced into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0438] In certain embodiments of the present invention, the RNA (e.g., mRNA) is a "replicon RNA" (e.g., a "replicon mRNA") or simply a "replicon," particularly a "self-replicating RNA" (e.g., a "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (e.g., a self-replicating mRNA) is derived from or contains sequences derived from ssRNA viruses, particularly positive-stranded ssRNA viruses such as alphaviruses. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically includes a 5' cap and a 3' poly(A) tail. The genome of an alphavirus contains nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (virion formation). The genome typically contains two open reading frames (ORFs). Four nonstructural...

Claims

1. A composition comprising a lipid nanoparticle (LNP), wherein the LNP is (i) RNA; (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of (a) a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and (b) one or more hydrophobic chains. A composition comprising:

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

3. The POX and / or POZ polymers have the following general formula (I): 【Chemistry 1】 [wherein a is an integer of 1 to 2; R １ is alkyl, especially C １－３ alkyl, e.g., methyl, ethyl, iso-propyl, or n-propyl, selected independently for each repeat unit; and m is from 2 to 200. The composition of claim 1 or 2, comprising:

4. The conjugate has the following general formula (II) or (II'): 【Chemistry 2】 [During the ceremony, a is an integer from 1 to 2; R １ is alkyl, especially C １－３ alkyl, e.g., methyl, ethyl, iso-propyl, or n-propyl, independently selected for each repeat unit; m is 2 to 200; R ２ R ４ Or -L １ (R ４ ) ｐ where each R ４ are independently hydrocarbyl groups; L １ is a linker; and p is 1 or 2; and R ３ H, C １－６ Alkyl, C ２－６ Alkynyl, -OR ２０ , -SR ２０ , halogen, -CN, -N ３ , -OC(O)R ２１ , -C(O)R ２１ , -NR ２２ R ２３ , -COOH, -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, -COOH, -COOCH ３ , -NR ２２ R ２３ , -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; R ２０ H, C １－３ is selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, １－３ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally selected from -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; R ２１ C １－６ is selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally selected from -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may join to the nitrogen atom to which they are attached to form a heterocyclyl group, where each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NH ２ , -NH(C １－３ alkyl), -N(C １－３ Alkyl) ２ , saccharides, amino acids, peptides, and members of a targeting pair. The composition according to any one of claims 1 to 3, comprising:

5. R １ A composition according to claim 3 or 4, wherein is methyl or ethyl, preferably methyl.

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

7. L １ is substituted with at least one monovalent functionalized moiety and / or an alkylene moiety linked to a divalent functionalized moiety (the divalent functionalized moiety ultimately converts the alkylene group into R ４ (which is bonded to the aryl group), wherein preferably each monovalent functional moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfate diamide, urea, thiourea, carbonyl, thiocarbon ... ester, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketate and / or each divalent functionalized moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfate diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, 7. The composition of claim 4, wherein the aryl group is independently selected from the group consisting of aryl ester, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

8. L １ is [*-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 )(=N-N(R ２６ )C(O)-)] ｐ (C １－６ -alkylene)-, [*-C(O)(N(R ２６ )-N=)C(R ２５ )-] ｐ (C １－６ -alkylene)-, [*=C(=N-N(R ２６ )C(O)(R ２５ )) ｐ (C １－６ -alkylene)-, [*-N(R ２６ )N(R ２６ )] ｐ (C １－６ -alkylene)-, [*=C(=N(OH))] ｐ (C １－６ -alkylene)- and [*-OC(R ２５ )(R ２６ )O] ｐ (C １－６ -alkylene)-, where * is selected from the group consisting of R ４ represents a point of attachment to １－６ - alkylene is divalent (if p is 1) or trivalent (if p is 2); R ２５ C １－６ Alkyl, aryl and aryl (C １－６ alkyl); R ２６ H, C １－６ Alkyl, aryl and aryl (C １－６ 8. The composition of claim 4, wherein the aryl group is selected from the group consisting of aryl, arylalkyl, arylsulfates ...

9. L １ is *-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 )-, where * is R ４ The composition of any one of claims 4 to 8, which represents a point of attachment to

10. R ２ R ４ , -L １ R ４ , -(CH ２ )-CH(OC(O)R ４ )(CH ２ O.C.O.R. ４ ), -(CH ２ )-CH(SR ４ ) ２ and -(CH ２ )-CH(SR ４ )-CH ２ (S.R. ４ ) selected from the group consisting of; and L １ is *-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 is *-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 * is R ４ The composition of any one of claims 4 to 9, which represents a point of attachment to

11. L １ but[*-] ２ N.C.O. 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)-, where * is R ４ represents a point of attachment to １－６ A composition according to any one of claims 4 to 7, in which alkylene is divalent (if p is 1) or trivalent (if p is 2).

12. L １ but[*-] ２ N.C.O. 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)-, where * is R ４ The composition of any of claims 4 to 7 and 11, which represents a point of attachment to:

13. R ２ (R ４ ) ２ N.C.O. 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 The composition according to any one of claims 4 to 7, 11 and 12, wherein the alkyl group is selected from the group consisting of -C(O)-.

14. The conjugate has the general formula (II'), １ is [*-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-, where * is R ４ represents a point of attachment to １－６ - 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 -OH, ═O, -SH, halogen, -CN, -N ３ and C １－３ -alkyl; and each Z is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of -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 The composition of any one of claims 4 to 7, wherein each of said compounds is independently selected from the group consisting of:

15. The conjugate has the general formula (II'), １ is [*-C(O)] ｐ (C １－６ -alkylene)-C(O)NH- or [*-] ２ N-, where * is R ４ represents a point of attachment to １－６ A composition according to any one of claims 4 to 7, in which alkylene is divalent (if p is 1) or trivalent (if p is 2).

16. The conjugate has the general formula (II'), １ *-C(O)(C ２－３ -alkylene)-C(O)NH- or [*-] ２ N-, where * is R ４ The composition of any of claims 4 to 7 and 15, which represents a point of attachment to:

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

18. Each R ４ A composition according to any one of claims 4 to 17, wherein are independently acyclic, preferably linear hydrocarbyl groups.

19. Each R ４ A composition according to any one of claims 4 to 18, wherein are independently hydrocarbyl groups having at least 8 carbon atoms, such as at least 10 carbon atoms.

20. One R ４ The composition of any of claims 4 to 17 and 19, wherein is a tocopherol moiety.

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

22. R ３ H, C １－３ Alkyl, -OR ２０ , -N ３ , 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 a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —N ３ , C ２－６ Alkynyl, -COOH, -COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and one or more substituents independently selected from the group consisting of members of a targeting pair; R ２０ H and C １－３ alkyl; R ２１ optionally represents -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ and C substituted with one or more substituents independently selected from the group consisting of members of a targeting pair. １－３ alkyl; and R ２２ and R ２３ each independently represents H, C １－３ Alkyl, C ２－３ Alkenyl and C ２－３ alkynyl, wherein C １－３ Alkyl, C ２－３ Alkenyl and C ２－３ Each alkynyl group is optionally -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NH ２ , -NH(C １－３ alkyl), -N(C １－３ Alkyl) ２ and substituted with one or more substituents independently selected from members of the targeting pair, or R ２２ and R ２３ may be combined with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclyl group.

23. R ３ 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ 23. The composition of any of claims 4 to 22, substituted with one or more (e.g. 1 or 2) substituents independently selected from the group consisting of:

24. The conjugate has the following general formula (III) or (III'): 【Chemistry 3】 [During the ceremony, a is an integer from 1 to 2; R １ is methyl or ethyl, selected independently for each repeat unit; m is 10 to 100; R ２ With respect to formula (III), -L １ R ４ , -(CH ２ )-CH(OC(O)R ４ )(CH ２ O.C.O.R. ４ ), -(CH ２ )-CH(SR ４ ) ２ and -(CH ２ )-CH(SR ４ )-CH ２ (S.R. ４ ), wherein each R ４ are independently linear hydrocarbyl groups having at least 10 carbon atoms; and L １ is *-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 is *-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 * is R ４ or for formula (III'), R ２ (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-, wherein C ３－６ -Cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N ３ and C １－３ -alkyl; and each Z is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of -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 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ and is substituted with one or more (e.g., 1 or 2) substituents independently selected from the group consisting of members of a targeting pair. The composition according to any one of claims 1 to 23, having the formula:

25. 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, selected independently for each repeat unit; m is 10 to 100; R ２ With respect to formula (III) (R ４ ) ２ N.C.O. 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 linear hydrocarbyl group having at least 10 carbon atoms; or for formula (III'), R ２ R ４ -C(O)(CH ２ ) ２ C(O)NH- or R ４ N-, where each R ４ are independently linear hydrocarbyl groups or tocopherol moieties having at least 10 carbon atoms, where the same R ２ One R in the part ４ is a tocopherol moiety; and R ３ 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ 24. The composition of any of claims 1 to 23, substituted with one or more (e.g. 1 or 2) substituents independently selected from the group consisting of:

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

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

28. The POX and / or POZ polymers have the following general formulas (Ia) and (Ib): 【Chemistry 4】 [In the formula, R １ Each of these is independently alkyl, particularly C １－３ alkyl, such as methyl, ethyl, iso-propyl or n-propyl, selected independently for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 199; the number of repeat units of formula (Ib) in the copolymer is 1 to 199; and the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 2 to 200.

3. The composition of claim 1, which is a copolymer comprising repeat units of:

29. The conjugate has the following general formula (IV): R ５ -POXZ-R ６ [During the ceremony, R ５ R ７ Or -L ２ (R ７ ) ｑ where each R ７ are independently hydrocarbyl groups; L ２ is a linker; and q is 1 or 2; POXZ is represented by the following general formulas (Ia) and (Ib): 【Chemistry 5】 where R １ Each of these is independently alkyl, particularly C １－３ alkyl, such as methyl, ethyl, iso-propyl or n-propyl, selected independently for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 199; the number of repeat units of formula (Ib) in the copolymer is 1 to 199; the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 2 to 200; and the repeat units of formula (Ia) and (Ib) are arranged in a random, periodic, alternating or block-wise manner; and R ６ H, C １－６ Alkyl, C ２－６ Alkynyl, -OR ２０ , -SR ２０ , halogen, -CN, -N ３ , -OC(O)R ２１ , -C(O)R ２１ , -NR ２２ R ２３ , -COOH, -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein C １－６ The alkyl group may optionally be —OH, SH, halogen, —CN, —N ３ , C ２－６ Alkynyl, -COOH, -COOCH ３ , -NR ２２ R ２３ , -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; R ２０ H, C １－３ is selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, １－３ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally selected from -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; R ２１ C １－６ is selected from the group consisting of alkyl and 3- to 6-membered heterocyclyl, １－６ Each of the alkyl and 3- to 6-membered heterocyclyl groups is optionally selected from -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ , substituted with one or more substituents independently selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ２２ and R ２３ each is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ２２ and R ２３ may join to the nitrogen atom to which they are attached to form a heterocyclyl group, where each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NH ２ , -NH(C １－３ alkyl), -N(C １－３ Alkyl) ２ , saccharides, amino acids, peptides, and members of a targeting pair.

29. The composition of any of claims 1, 2 and 28, having the following structure:

30. R １ is each independently methyl or ethyl, preferably methyl, and is independently selected for each repeat unit.

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

32. L ２ comprises at least one functionalized moiety, such as at least one functionalized moiety, wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfate diamide, urea, thiourea, carbonyl , thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal and / or each divalent functionalized moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfur-containing diamide, sulfate diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, 32. The composition of any of claims 29-31, wherein the moiety is independently selected from carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

33. L ２ is [*-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 ) ２ O] ｑ (C １－６ -alkylene)-, [*-C(R ２５ )(=N-N(R ２６ )C(O)-)] ｑ -(C １－６ -alkylene)-, [*-C(O)(N(R ２６ )-N=)C(R ２５ )-] ｑ (C １－６ -alkylene)-, [*=C(=N-N(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 * is selected from the group consisting of R ７ represents a point of attachment to １－６ - alkylene is divalent (if q is 1) or trivalent (if q is 2); R ２５ C １－６ Alkyl, aryl and aryl (C １－６ alkyl); R ２６ H, C １－６ Alkyl, aryl and aryl (C １－６ 33. The composition of any of claims 29-32, wherein s is selected from the group consisting of: alkyl; and s is an integer from 1 to 2.

34. L ２ is *-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 )-, where * is R ７ The composition of any of claims 29 to 33, which represents a point of attachment to:

35. R ５ R ７ , -L ２ R ７ , -(CH ２ )-CH(OC(O)R ７ )(CH ２ O.C.O.R. ７ ), -(CH ２ )-CH(SR ７ ) ２ and -(CH ２ )-CH(SR ７ )-CH ２ (S.R. ７ ) selected from the group consisting of; and L ２ is *-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 is *-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 * is R ７ The composition of any of claims 29 to 34, which represents a point of attachment to:

36. L ２ but[*-] ２ N.C.O. １－６ -alkylene)-, [*-C(O)O] ｑ (C １－６ -alkylene)-OP(O) ２ O(C １－６ -alkylene)NHC(O)-(C １－６ -alkylene)-, [*-OC(O)] ｑ (C １－６ -alkylene)-OP(O) ２ O(C １－６ -alkylene)NHC(O)-(C １－６ -alkylene)- and [*-C(O)] ｑ (C １－６ -alkylene)-C(O)-, where * is R ７ represents a point of attachment to １－６ A composition according to any one of claims 29 to 32, wherein alkylene is divalent (if q is 1) or trivalent (if q is 2).

37. L ２ but[*-] ２ N.C.O. 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)-, where * is R ７ The composition of any of claims 29 to 32 and 36, which represents a point of attachment to:

38. R ５ (R ７ ) ２ N.C.O. １－２ -alkylene)-, R ７ C(O)OCH ２ CH(OC(O)R ７ )-CH ２ -OP(O) ２ O-(CH ２ ) ２ -NHC(O)-CH ２ - and R ７ -C(O)(CH ２ ) ２ The composition of any one of claims 29 to 32, 36 and 37, wherein the moiety is selected from the group consisting of -C(O)-.

39. R ６ is attached to the N-terminus of the POXZ copolymer; and L ２ is bound to the C-terminus of the POXZ copolymer, 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 * is R ７ represents a point of attachment to １－６ - alkylene is divalent (if q is 1) or trivalent (if q is 2); C ３－８ -Cycloalkylene and C ３－８ Each of the -cycloalkenylene groups is optionally selected from -OH, ═O, -SH, halogen, -CN, -N ３ and C １－３ -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, aryl ... １ GA-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 The composition of any of claims 29 to 32, independently selected from the group consisting of:

40. R ６ is attached to the N-terminus of the POXZ copolymer; and L ２ is bound to the C-terminus of the POXZ copolymer, and [*-C(O)] ｑ (C １－６ -alkylene)-C(O)NH- and [*-] ２ N-, where * is R ７ represents a point of attachment to １－６ A composition according to any one of claims 29 to 32, wherein alkylene is divalent (if q is 1) or trivalent (if q is 2).

41. R ６ is attached to the N-terminus of the POXZ copolymer; and L ２ is attached to the C-terminus of the POXZ copolymer, and *-C(O)(C ２－３ -alkylene)-C(O)NH- or [*-] ２ N-, where * is R ７ The composition of any of claims 29 to 32 and 40, which represents a point of attachment to:

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

43. Each R ７ 43. The composition of any of claims 29 to 42, wherein are independently acyclic, preferably linear, hydrocarbyl groups.

44. Each R ７ A composition according to any one of claims 29 to 43, wherein are independently hydrocarbyl groups having at least 8 carbon atoms, such as at least 10 carbon atoms.

45. One R ７ The composition of any of claims 29 to 42 and 44, wherein is a tocopherol moiety.

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

47. R ６ H, C １－３ Alkyl, -OR ２０ , -N ３ , 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 a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —OH, —N ３ , C ２－６ Alkynyl, -COOH, -COOCH ３ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , -C(O)NR ２２ R ２３ , -NR ２２ C(O)R ２１ and one or more substituents independently selected from the group consisting of members of a targeting pair; R ２０ H and C １－３ alkyl; R ２１ optionally represents -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NR ２２ R ２３ and C substituted with one or more substituents independently selected from the group consisting of members of a targeting pair. １－３ alkyl; and R ２２ and R ２３ each independently represents H, C １－３ Alkyl, C ２－３ Alkenyl and C ２－３ alkynyl, wherein C １－３ Alkyl, C ２－３ Alkenyl and C ２－３ Each alkynyl group is optionally -OH, SH, halogen, -CN, -N ３ , C ２－６ Alkynyl, -COOH, -NH ２ , -NH(C １－３ alkyl), -N(C １－３ Alkyl) ２ and substituted with one or more substituents independently selected from members of the targeting pair, or R ２２ and R ２３ can be combined with the nitrogen atom to which they are attached to form a 5- or 6-membered heterocyclyl group.

48. R ６ 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ 48. The composition of any of claims 29 to 47, substituted with one or more (e.g. 1 or 2) substituents independently selected from the group consisting of:

49. The conjugate has the following general formula (V): R ５ -POXZ-R ６ [During the ceremony, R ５ is attached to the N-terminus of the POXZ copolymer, -L ２ R ７ , -(CH ２ )-CH(OC(O)R ７ )(CH ２ O.C.O.R. ７ ), -(CH ２ )-CH(SR ７ ) ２ and -(CH ２ )-CH(SR ７ )-CH ２ (S.R. ７ ), wherein each R ７ are independently linear hydrocarbyl groups having at least 10 carbon atoms; and L ２ is *-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 is *-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 * is R ７ or R ５ When attached to the C-terminus of the POXZ copolymer, (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 -, wherein C ３－６ -Cycloalkenylene group optionally includes -OH, =O, -SH, halogen, -CN, -N ３ and C １－３ -substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, aryl ... １ is independent -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-; POXZ is represented by the following general formulas (Ia) and (Ib): 【Chemistry 6】 where R １ are each independently methyl or ethyl and are independently selected for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 99; the number of repeat units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 10 to 100; and the repeat units of formula (Ia) and (Ib) are arranged in a random, periodic, alternating or block wise manner; and R ６ 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ and is substituted with one or more (e.g., 1 or 2) substituents independently selected from the group consisting of members of a targeting pair. The composition of any one of claims 1, 2 and 28 to 48, having the formula:

50. The conjugate has the general formula (V) as shown in claim 49, wherein: R ５ When is attached to the N-terminus of the POXZ copolymer, (R ７ ) ２ N.C.O. １－２ -alkylene)-, R ７ C(O)OCH ２ CH(OC(O)R ７ )-CH ２ -OP(O) ２ O-(CH ２ ) ２ -NHC(O)-CH ２ - and R ７ -C(O)(CH ２ ) ２ -C(O)-, where each R ７ are independently linear hydrocarbyl groups having at least 10 carbon atoms, or R ５ When attached to the C-terminus of the POXZ copolymer, R ７ -C(O)(CH ２ ) ２ C(O)NH- or R ７ N-, where each R ７ are independently linear hydrocarbyl groups having at least 10 carbon atoms or tocopherol moieties, where the same R ５ One R in the part ７ is a tocopherol moiety; POXZ is a copolymer comprising repeating units of the general formula (Ia) and (Ib) as shown in claim 49, wherein R １ are each independently methyl or ethyl and are independently selected for each repeat unit; the number of repeat units of formula (Ia) in the copolymer is 1 to 99; the number of repeat units of formula (Ib) in the copolymer is 1 to 99; the sum of the number of repeat units of formula (Ia) and the number of repeat units of formula (Ib) in the copolymer is 10 to 100; and the repeat units of formula (Ia) and (Ib) are arranged in a random, periodic, alternating or block wise manner; and R ６ 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 and a member of a targeting pair, wherein C １－３ The alkyl group may optionally be —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 N.H. ２ and members of a targeting pair.

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

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

53. The composition according to any one of claims 1 to 52, which is substantially free of lipids or lipid-like substances comprising polyethylene glycol (PEG), preferably being substantially free of PEG.

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

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

56. The cationically ionizable lipid has the formula (X): 【Chemistry 7】 [During the ceremony, L １０ and L ２０ One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, N.R. ａ C(=O)NR ａ --OC(=O)NR ａ -or-NR ａ C(=O)O-, and L １０ and L ２０ The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, N.R. ａ C(=O)NR ａ --OC(=O)NR ａ -or-NR ａ C(═O)O— or a direct bond; G １ and G ２ are each independently unsubstituted C １ -C １２ Alkylene or C ２－１２ alkenylene; G ３ C １－２４ Alkylene, C ２－２４ Alkenylene, C ３－８ Cycloalkylene or C ３－８ is cycloalkenylene; R ａ is H or C １－１２ is alkyl; R ３５ and R ３６ Each independently represents C ６－２４ Alkyl or C ６－２４ alkenyl; R ３７ H, OR ５０ , CN, -C(=O)OR ４０ , -OC(=O)R ４０ Or -NR ５０ C(=O)R ４０ and R ４０ C １－１２ is alkyl; R ５０ is H or C １－６ is alkyl; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

57. 56. The composition of any one of claims 1 to 55, which is: (α) The cationically ionizable lipid is selected from the following structures X-1 to X-36: 【Table 1】 【Table 2】 【Table 3】 (β) The cationically ionizable lipid is selected from the following structures A through G: 【Table 4】 or (γ) The cationically ionizable lipid is a lipid having the structure X-3.

58. The cationic or cationically ionizable lipid has the formula (XI): 【Chemistry 8】 [During the ceremony, R １ and R ２ Each of the groups is independently R ５ Or -G １ -L １ -R ６ where at least one of R1 and R2 is -G １ -L １ -R ６ and R ３ and R ４ Each of the C １－６ Alkyl, C ２－６ Alkenyl, aryl and C ３－１０ cycloalkyl; R ５ and R ６ each independently is an acyclic hydrocarbyl group having at least 10 carbon atoms; G １ and G ２ Each of １－１２ Alkylene or C ２－１２ alkenylene; L １ and L ２ Each of the above is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ independently selected from the group consisting of -, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-; Ra is H or C １－１２ is alkyl; m is 0, 1, 2, 3 or 4; and x is 0, 1 or 2.

56. The composition of any one of claims 1 to 55, having the structure:

59. Cationic ionizable lipids having the following structures (XIV-1), (XIV-2) and (XIV-3): 【Chemistry 9】 The composition according to any one of claims 1 to 55 and 58, selected from:

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

55. The composition of any of claims 1 to 54, comprising dimethyl ether chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

61. 61. The composition of any of claims 1 to 60, wherein the cationic or cationically ionizable lipid comprises from about 20 mol% to about 80 mol% of the total lipid present in the LNP.

62. 62. The composition of any of claims 1 to 61, wherein the LNP further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids and combinations thereof, more preferably the LNP comprises a cationically ionizable lipid, a conjugate, a phospholipid and a steroid.

63. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 63. The composition of claim 62, wherein the phosphatidylethanolamine is selected from the group consisting of dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), dilauroyl phosphatidylethanolamine (DLPE) and diphthanoyl phosphatidylethanolamine (DPyPE).

64. The composition of claim 62 or 63, wherein the phospholipid comprises from about 5 mol% to about 30 mol% of the total lipid present in the LNP.

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

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

67. The composition of any of claims 62-66, wherein the cationic or cationically ionizable lipids comprise about 20 mol% to about 70 mol% of the total lipids present in the LNP; the conjugates of (a) POX and / or POZ polymers and (b) one or more hydrophobic chains comprise about 0.5 mol% to about 15 mol% of the total lipids present in the LNP; the phospholipids comprise about 5 mol% to about 25 mol% of the total lipids present in the LNP; and the steroids comprise about 25 mol% to about 55 mol% of the total lipids present in the LNP.

68. 68. The composition of any of claims 1 to 67, wherein the LNPs have a size of about 30 nm to about 500 nm.

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

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

71. 71. The composition of any 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. 72. The composition of any of claims 1 to 71, wherein the RNA comprises at least one, and preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

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

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

75. 75. The composition of any 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 epitopes for inducing an immune response against an antigen in a subject.

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

77. 76. A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition according to 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 disorder in a subject, the method comprising administering to the subject a composition described in any of claims 1 to 75, wherein delivery of RNA to cells of the subject is beneficial in treating or preventing the disease or disorder.

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

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

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

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