Polyoxyalkylene-1, 2-dimyristoyl-glycerol compounds wherein polyoxyalkylene is poly (ethylene oxide) having c1 to c3-alkoxymethyl side chains

Abstract

The present invention relates to novel polyoxyalkylene-based compounds and to a process for the preparation thereof, as well as to compositions comprising at least one novel polyoxyalkylene-based compound and at least one active agent. Furthermore, the invention relates to the preparation of the compositions according to the invention and to their use for the treatment of mammalian or human diseases.

Description

Technical Field

[0001] The present invention relates to new polyoxyalkylene-based compounds and methods for their preparation, as well as compositions comprising at least one new polyoxyalkylene-based compound and at least one active agent. In particular, the compounds are suitable as novel lipids, which can be used in compositions suitable as lipid nanoparticles, which may optionally contain other lipid components. The compositions are suitable as delivery carriers for at least one active agent, in particular for facilitating intracellular delivery of therapeutic nucleic acids. Background Art

[0002] Polyethylene oxide (PEO)-based lipids are an important class of excipients. Early publications on such lipids include Frisch et al., Bioconjugate Chemistry (2004), 15(4), 754-764. They are used in lipid nanoparticle formulations, for example in vaccines against Covid-19. The main function of PEO is the so-called "stealth effect", which avoids recognition by the patient's reticuloendothelial system. The main problem with PEO is the immune response of the patient's body, which can lead to the formation of anti-PEO antibodies, as disclosed, for example, in J. Selva et al., ACS Nano (2022), 16(8), 11769-11780. Anti-PEO antibodies can lead to the loss of the desired "stealth effect", loss of drug function, and allergic reactions.

[0003] Therefore, there is a need for alternative compounds that have a "stealth effect" but exhibit less or no antibody formation, i.e., less or no immunogenic potential.

[0004] The inventors of the present invention have surprisingly found that the above object can be solved by polyoxyalkylene-based compounds according to the present invention, which have polyoxyalkylene units different from PEO. In addition, the obtained compounds surprisingly show better storage stability compared to similar common PEO lipids. Summary of the Invention

[0005] Accordingly, in a first aspect, the present invention relates to a compound having the following formula (I)

[0006]

[0007] wherein R 1 and R 2 are independently selected from saturated or unsaturated branched or straight-chain C3-C 20 hydrocarbyl groups having at most three -C═C- bonds or -H, provided that at least one of R 1 and R 2 is not -H;

[0008] and wherein R 3 is bonded to a carbon atom of the polyoxyalkylene group A, and said polyoxyalkylene group A is bonded through an oxygen atom to the remainder of the molecule opposite to R 3 ;

[0009] A is a polyoxyalkylene group comprising at least one of the following units

[0010] (a)

[0011] and at least one unit selected from the following:

[0012] (b)

[0013] (c)

[0014] (d) and

[0015] (e) and

[0016] R 3 is selected from -H; -OH; -SH; -NH2; -NHR 4 , -NR 4 R 5 , -OR 6 , -SR 6 or a straight-chain, branched-chain or cyclic alkyl group having up to 20 carbon atoms; wherein

[0017] R 4 to R 6 are independently selected from straight-chain, branched-chain or cyclic alkyl groups having up to 20 carbon atoms, where up to 5 carbon atoms may be replaced by an oxygen atom or a sulfur atom; and

[0018] wherein -A-R 3 has a molecular weight of from 1100 to 7500 g / mol.

[0019] In a second aspect, the present invention relates to a composition comprising at least one compound of formula (I) according to the present invention and at least one active agent.

[0020] In a third aspect, the present invention relates to a method for preparing a compound of formula (I) according to the present invention, which comprises the following steps or consists of the following steps:

[0021] (i) providing a precursor compound H-A-R 3 , wherein A and R 3 are as defined in formula (I),

[0022] (ii) Replace -H with a leaving group -X, where the leaving group is capable of undergoing a substitution reaction;

[0023] (iii) Subsequently, carry out the substitution reaction of X-A-R 3 with to obtain the compound Then

[0024] (iv) Protonate the compound (III) to obtain the compound and subsequently

[0025] (v) Carry out an esterification reaction with and , where Y is a leaving group capable of undergoing an esterification reaction with -H of the -OH group, and R 1 and R 2 are as defined in formula (I), to obtain the compound of formula (I).

[0026] In a fourth aspect, the present invention relates to a method for preparing a composition according to the present invention, which comprises the following steps: providing at least one compound of formula (I) according to the present invention, at least one active agent, and optionally other ingredients; and combining all the ingredients to obtain the composition according to the present invention.

[0027] In a fifth aspect, the present invention relates to a composition according to the present invention, which is used for treating human diseases.

[0028] In a sixth aspect, the present invention relates to a composition according to the present invention, which is used for treating mammalian diseases.

[0029] By studying the following detailed description and claims, these and other aspects, embodiments, features, and advantages of the present invention will become apparent to those skilled in the art. Any feature of one aspect of the present invention can be used in any other aspect of the present invention. In addition, those skilled in the art can easily understand that the examples included herein are intended to describe and illustrate the present invention, rather than limit the present invention, and specifically, the present invention is not limited to these examples. Description of the Drawings

[0030] Figure 1a : M: RNA Ladder

[0031] Figure 1b : 1: Agarose gel electrophoresis (AGE) of LNP formulated with different PEO / GME-lipids. 1: Free FLucmRNA, 2: PEO2k-DMG LNP, 3: g(9)

[0032] Figure 2 : Transfection efficiency of LNP formulated with different PEO / GME-lipids evaluated by luciferase assay in different cell lines.

[0033] Figure 3 : ELISA assay using different (1,2-dimyristoyl-glycerol) (DMG) lipids Detailed Description of the Invention

[0035] As used herein, unless otherwise specified, the following terms have the meanings ascribed to them.

[0036] Unless the context otherwise requires, throughout this specification and the claims, the word "comprising" and its variations, such as "comprises" and "comprising", shall be construed in an open, inclusive sense, i.e., construed to mean "including but not limited to".

[0037] 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. Unless the context clearly dictates otherwise, as used in the specification and claims, the singular forms "a", "an", and "the" include plural referents.

[0038] A numerical range indicated in the format "x to y" also includes the recited values. If several preferred numerical ranges are indicated in this format, it is clear that all ranges arising from the combination of the respective endpoints are also included.

[0039] As used herein, "one or more" refers to at least one and includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of the recited substances. Similarly, "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. As used herein, "at least one" with respect to any component refers to the number of chemically distinct molecules, i.e., the number of different types of the recited substances, but not the total number of molecules. For example, "at least one therapeutic agent" means at least one type of molecule that falls within the definition of a therapeutic agent, but there may also be two or more different types of therapeutic agents that fall within that definition, and does not mean that there is only one or more molecules of one type of therapeutic agent.

[0040] Unless otherwise expressly stated, all percentages given herein in relation to a composition relate to weight % relative to the total weight of the corresponding composition.

[0041] According to the present invention, with respect to a compound, "substantially free of" means that the compound can only be present in an amount that does not affect the properties of the composition, in particular, based on the total weight of the composition, the corresponding compound is present in less than 3% by weight, preferably less than 1% by weight, more preferably less than 0.01% by weight, or is absent altogether.

[0042] As used herein, the term "nucleic acid" refers to a compound containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form or triple-stranded form, and includes DNA, RNA, and their hybrid molecules. DNA can be in the form of an antisense molecule, plasmid DNA (pDNA), linear or circular DNA, a PCR product, or a vector. RNA can be self-amplifying RNA (saRNA) or small hairpin RNA (shRNA), small interfering RNA (siRNA), chemically modified or unmodified messenger RNA (mRNA), antisense RNA, circular RNA (circRNA) containing at least one coding sequence, microRNA (miRNA), micRNA, multivalent RNA, transfer RNA (tRNA), single-guide RNA (sgRNA), replicating RNA (repRNA), dicer substrate RNA, or viral RNA (vRNA), antisense oligonucleotide (ASO), double-stranded RNA (dsRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to those of a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless specifically restricted, this term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to those of a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence.

[0043] The term "lipid" refers to a class of organic compounds that includes, but is not limited to, esters of fatty acids, and is generally characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) "simple lipids", which include fats and oils and waxes; (2) "compound lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0044] A "cationic lipid" refers to a lipid capable of carrying a positive charge. Exemplary cationic lipids contain one or more amine groups that carry a positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions.

[0045] The term "neutral lipid" refers to any of a number of lipid substances that exist in an uncharged or neutral zwitterionic form at a selected pH value.

[0046] The term "ionizable lipid" refers to any of a number of lipid substances that exist in a positively or negatively charged form independent of pH within a useful physiological range, such as pH ~3 to pH ~9. Ionizable lipids can be synthetic or of natural origin.

[0047] An "effective amount" or "therapeutically effective amount" of an active agent such as a nucleic acid is an amount sufficient to produce a desired effect, e.g., an increase or inhibition of target sequence expression compared to the normal expression level detected in the absence of the nucleic acid. In the case where the expression product is absent in the absence of the nucleic acid, an increase in target sequence expression is achieved when any measurable level is detected. In the case where the expression product is present at a certain level prior to contact with the nucleic acid, an increase in expression is achieved when the fold increase of the value obtained with the nucleic acid, e.g., mRNA, relative to the control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of target gene or target sequence expression is achieved when the value obtained with a nucleic acid such as an antisense oligonucleotide is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the control. Suitable assays for determining the expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art, such as dot blot, RNA blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those skilled in the art.

[0048] The disclosure herein also encompasses all pharmaceutically acceptable compounds of the compounds of formula (I) as their pharmaceutically acceptable salts and / or being isotopically labeled by substituting one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine.

[0049] The embodiments disclosed herein also encompass the in vivo metabolites of the compounds of the invention. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, mainly due to enzymatic processes. Accordingly, the embodiments of the present disclosure include compounds produced by a method comprising administering to a mammal a compound of the present disclosure for a period of time sufficient to produce its metabolites.

[0050] The compounds of the present invention, including their pharmaceutically acceptable salts, may contain one or more stereogenic centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)- according to absolute stereochemistry, or as (D)- or (L)- for amino acids. The present invention is intended to embrace all such possible isomers, as well as their racemic and optically pure forms. The optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation / separation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other geometrically asymmetric center, the compounds are intended to include the E and Z geometric isomers unless otherwise specified. Also included are all tautomeric forms.

[0051] "Stereoisomers" refer to compounds that are composed of the same atoms, bonded by the same bonds, but have different three-dimensional structures and cannot be superimposed on each other. The present invention encompasses a variety of stereoisomers and mixtures thereof, and includes "enantiomers", which are two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0052] "Tautomers" refer to the migration of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes the tautomers of any of the said compounds.

[0053] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

[0054] "Pharmaceutically acceptable acid addition salts" refers to those salts that retain the biological efficacy and properties of the free base, which are not biologically or otherwise undesirable, and which are formed from inorganic acids and organic acids, such as but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. for inorganic acids, and such as but not limited to acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylaminosulfonic acid (cyclamic acid), dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0055] The present invention particularly relates to compounds of the following formula (I)

[0056]

[0057] wherein R 1 and R 2 are each independently selected from branched or straight-chain C3-C hydrocarbon groups that are saturated or unsaturated and have at most three -C═C- bonds, or -H, provided that at least one of R 20 and R 1 is not -H, preferably R 2 and R 1 are independently selected from saturated straight-chain C 2 hydrocarbon groups, more preferably C 12-18 hydrocarbon groups, most preferably C 12-16 hydrocarbon groups; preferably R 12-14 and R 1 are the same; most preferably both are C 2 hydrocarbon groups; 14 hydrocarbon groups;

[0058] And wherein R 3 is attached to a carbon atom of the polyoxyalkylene group A, preferably a terminal carbon atom, and the polyoxyalkylene group A is attached to the remainder of the molecule opposite to R 3 through an oxygen atom, preferably a terminal oxygen atom;

[0059] A is a polyoxyalkylene group containing at least one of the following units

[0060]

[0061] and at least one unit selected from the following:

[0062] (b)

[0063] (c)

[0064] (d) and

[0065] (e) and

[0066] R 3 is selected from -H; -OH; -SH; -NH2; -NHR 4 、-NR 4 R 5 、-OR 6 、-SR 6 or a straight-chain, branched-chain or cyclic alkyl group having at most 20 carbon atoms;

[0067] wherein R 4 to R 6 are independently selected from straight-chain, branched-chain or cyclic alkyl groups having at most 20 carbon atoms, wherein at most 5 carbon atoms may be replaced by an oxygen atom or a sulfur atom;

[0068] Preferably, R 3 is -OR 6 wherein R 6 is selected from straight-chain, branched-chain or cyclic alkyl groups having at most 20 carbon atoms, wherein at most 5 carbon atoms may be replaced by an oxygen atom;

[0069] More preferably, R 3 is selected from methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, 2-ethylhexyloxy, dodecan-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propane-2-oxy, 1-octadecyloxy, 3-methylbutan-1-oxy, phenylmethoxy, 3-ethyl-butoxy and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy;

[0070] and

[0071] wherein the molecular weight of -A-R 3 is 1100 to 7500 g / mol, preferably 1500 to 3500 g / mol, more preferably 2000 to 3000 g / mol.

[0072] The molecular weight may be the theoretical molecular weight calculated in the field of chemistry -A-R 3 , for example, the unit has a theoretical molecular weight of 44 g / mol, and the unit has a theoretical molecular weight of 88 g / mol, where the value is rounded to an integer. Alternatively, the molecular weight may be the weight-average molecular weight determined by measuring the weight-average molecular weight of the precursor H-A-R 3 by size-exclusion chromatography and subtracting the theoretical molecular weight of -H (1 g / mol). Size-exclusion chromatography can preferably be carried out at 50 °C on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column using dimethylformamide (DMF, containing 1 g / L LiBr) as the mobile phase (flow rate 1 mL / min). The polymer concentration is 1 mg / mL. Calibration is carried out using poly(ethylene glycol) standards (from Polymer Standard Service, Mainz, Germany).

[0073] The compound of formula (I) is suitable as a lipid.

[0074] In one embodiment, R 1 and R 2 are independently selected from branched or straight-chain C4-C 20 hydrocarbyl groups having at most two -C=C- bonds or -H, which are saturated or unsaturated.

[0075] In one embodiment, R 1 and R 2 are independently selected from straight-chain C4-C 20 hydrocarbyl groups having at most two -C=C- bonds or -H, which are saturated or unsaturated.

[0076] In one embodiment, R 1 and R 2 are independently selected from branched or straight-chain C4-C 20 hydrocarbyl groups having one -C=C- bond or -H, which are saturated or unsaturated.

[0077] In one embodiment, R 1 and R 2 are independently selected from straight-chain C4-C 20 hydrocarbyl groups having one -C=C- bond or -H, which are saturated or unsaturated.

[0078] In one embodiment, R 1 and R 2 are independently selected from saturated branched or straight-chain C4-C 20 hydrocarbyl groups or -H.

[0079] In one embodiment, R 1 and R 2 are each independently selected from saturated straight-chain C4-C 20 hydrocarbyl groups or -H.

[0080] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated branched or straight-chain C8-C 18 hydrocarbyl groups having at most two -C═C- bonds or -H.

[0081] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated straight-chain C8-C 18 hydrocarbyl groups having at most two -C═C- bonds or -H.

[0082] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated branched or straight-chain C8-C 18 hydrocarbyl groups having one -C═C- bond or -H.

[0083] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated straight-chain C8-C 18 hydrocarbyl groups having one -C═C- bond or -H.

[0084] In one embodiment, R 1 and R 2 are each independently selected from saturated branched or straight-chain C8-C 18 hydrocarbyl groups or -H.

[0085] In one embodiment, R 1 and R 2 are each independently selected from saturated straight-chain C8-C 18 hydrocarbyl groups or -H.

[0086] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated branched or straight-chain C 12 -C 17 hydrocarbyl groups having at most two -C═C- bonds or -H.

[0087] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated straight-chain C 12-C 17 An alkyl group or -H.

[0088] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated branched or straight-chain C having one -C═C- bond 12 -C 17 alkyl group or -H.

[0089] In one embodiment, R 1 and R 2 are each independently selected from saturated or unsaturated straight-chain C having one -C═C- bond 12 -C 17 alkyl group or -H.

[0090] In one embodiment, R 1 and R 2 are each independently selected from saturated branched or straight-chain C 12 -C 17 alkyl group or -H.

[0091] In one embodiment, R 1 and R 2 are each independently selected from saturated straight-chain C 12 -C 17 alkyl group or -H.

[0092] In one embodiment, R 1 and R 2 are each independently selected from saturated straight-chain C 12 -C 14 alkyl group or -H.

[0093] In a preferred embodiment, R 1 and R 2 are the same.

[0094] The group -A-R 3 is obtained by providing the precursor H-A-R 3 wherein -H is bonded to the terminal oxygen atom of unit A. Subsequently, H-A-R 3 is further modified and then a substitution reaction is carried out to obtain the compound of formula (I).

[0095] H-A-R 3 is preferably obtained by anionic ring-opening copolymerization of the following substances:

[0096] Ethylene oxide

[0097] and

[0098] at least one comonomer selected from the following:

[0099] 2-(methoxymethyl)oxirane (glycidyl methyl ether), 1,2-epoxy-3-ethoxypropane, 1,2-epoxy-3-n-propoxypropane, 1,2-epoxy-3-isopropoxypropane, preferably said at least one comonomer is 2-(methoxymethyl)oxirane; and

[0100] an initiator which is suitable for forming -R as defined in the compound of formula (I) in the presence of a base 3 and preferably said initiator is 1-methoxy-3-(2-methoxyethoxy)propan-2-ol.

[0101] Preferably, said base is a base having a pKa of at least 16, preferably at least 19, and more preferably is potassium tert-butoxide.

[0102] In a preferred embodiment, a small amount of pure ethylene oxide is added after the copolymerization step such that there are 2 to 5 additional units derived from ethylene oxide at one or both ends of A.

[0103] The synthesis of HOCH2CH2-A-R via anionic ring-opening copolymerization 3 is described, for example, in PCT / EP2022 / 062896, in which HOCH2CH2-A-R 3 is referred to as a polymer, and this document is incorporated by reference.

[0104] Preferably, said anionic ring-opening copolymerization is carried out in a temperature range of -10°C to 90°C, more preferably -10°C to 70°C, and most preferably -10°C to 60°C.

[0105] After obtaining the precursor H-A-R 3 wherein A and R 3 are as defined in formula (I), to obtain the compound of formula (I):

[0106] replace -H with a leaving group -X, said leaving group such as a tosylate, tosyl or mesylate group, preferably tosylate or tosyl, which can undergo a substitution reaction;

[0107] subsequently carry out the substitution reaction of X-A-R 3 with to obtain the compound then

[0108] protonate the compound (III) to obtain the compound and subsequently carry out an esterification reaction with where Y is a leaving group capable of undergoing an esterification reaction with -H of the -OH group, such as -Cl, -F, -Br, -I, and R 1 and R 2As defined in formula (I), to obtain the compound of formula (I).

[0109] The introduction of the leaving group -X is well known to those skilled in the art.

[0110] The polyoxyalkylene group A comprises unit (a) and at least one of units (b) to (e). In one embodiment, the polyoxyalkylene group A consists essentially of or consists of unit (a) and at least one of units (b) to (e). In particular, the polyoxyalkylene group A is substantially free of residues or is free of residues.

[0111] In a preferred embodiment, the polyoxyalkylene group A comprises units (a) and (b) and optionally additional units selected from (c) to (e), or consists of units (a) and (b) and optionally additional units selected from (c) to (e). In a preferred embodiment, the polyoxyalkylene group A comprises units (a) and (b), or consists of units (a) and (b).

[0112] For the sake of clarity, the inclusion of unit (a) in a certain paragraph does not mean that there is only one unit (a), but at least one unit of (a) is present in the group, that is, several monomer units derived from ethylene oxide can be present. For example, 1 to 20 units of (a) can be present in group A. This also applies to paragraphs involving at least one of units (b) to (e). However, if not otherwise specifically defined, it can be that only one (quantity) unit of (a) or only one (quantity) unit of (b) to (e) is present in group A.

[0113] In a preferred embodiment, unit (a) constitutes 5% to 95% of group A, while the other units add up to 100%. In a further preferred embodiment, unit (b) is present in group A in an amount of up to 70%, more preferably, unit (b) is present in group A in an amount of 30% to 70%, and most preferably, the additional unit (a) is present in an amount of 30% to 70%, adding up to 100%.

[0114] In a preferred embodiment, the molar ratio of (a) to (b) to (e), preferably the molar ratio of (a) to (b), is from 1:9 to 9:1, preferably from 2:8 to 8:2, more preferably from 3:7 to 7:3.

[0115] In a preferred embodiment, -A-R 3The polydispersity index (PDI) is 1.15 or lower, more preferably 1.10 or lower, and most preferably 1.08 or lower, where the weight-average molecular weight and number-average molecular weight are preferably determined by size-exclusion chromatography as described above for Mw. Size-exclusion chromatography can preferably be carried out at 50 °C on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column using dimethylformamide (DMF, containing 1 g / L LiBr) as the mobile phase (flow rate 1 mL / min). The polymer concentration is 1 mg / mL. Calibration is carried out using poly(ethylene glycol) standards (from Polymer Standard Service, Mainz, Germany).

[0116] In one embodiment, -A-R 3 has a molecular weight, preferably a weight-average molecular weight, of from 1500 to 3500 g / mol, preferably from 2000 to 3000 g / mol. In one embodiment, -A-R 3 has a molecular weight, preferably a weight-average molecular weight, within a range having any lower limit of 1500 g / mol, 1550 g / mol, 1600 g / mol, 1650 g / mol, 1700 g / mol, 1750 g / mol, 1800 g / mol, 1850 g / mol, 1900 g / mol, 1950 g / mol or 2000 g / mol and any upper limit of 1550 g / mol, 1600 g / mol, 1650 g / mol, 1700 g / mol, 1750 g / mol, 1800 g / mol, 1850 g / mol, 1900 g / mol, 1950 g / mol, 2000 g / mol, 2050 g / mol, 2100 g / mol, 2150 g / mol, 2200 g / mol, 2250 g / mol, 2300 g / mol, 2350 g / mol, 2400 g / mol, 2450 g / mol, 2500 g / mol, 2550 g / mol, 2600 g / mol, 2650 g / mol, 2700 g / mol, 2750 g / mol, 2800 g / mol, 2850 g / mol, 2900 g / mol, 2950 g / mol, 3000 g / mol, 3050 g / mol, 3100 g / mol, 3150 g / mol, 3200 g / mol, 3250 g / mol, 3300 g / mol, 3350 g / mol, 3400 g / mol, 3450 g / mol or 3500 g / mol.

[0117] Group R 3 can be modified by selecting a suitable initiator and / or by chemically modifying the end groups, which are in the precursor H-A-R 3is initially formed during anionic ring-opening copolymerization in the preparation thereof. Such reactions are well known in the art. R 3 can be, for example, a functional group selected from the following: acetal (dialkoxy), aldehyde (formyl), amide (formamido), azide, carbonate ((alkoxycarbonyl)oxy), carboxyl (carboxyl), carboxylic anhydride, ester (alkoxycarbonyl), ether, halogen, halocarbonyl (carbonohaloridoyl), hemiacetal (alkoxy alcohol), hemiketal (alkoxy alcohol), hydroxyl, imide (imido), imine (imino), ketal (dialkoxy), ketone (acyl), orthoester (trialkoxy), primary amino, secondary amino, tertiary amino, primary alkoxy, secondary alkoxy and tertiary alkoxy, mercapto (thioalkyl, H-S-), thioether and combinations thereof. In a preferred embodiment, the end group is selected from alkyl, hydrogen, hydroxyl, alkoxy, thioalkyl, phthalimide, amide, amine and combinations thereof. The end group can be a primary alkoxy group selected from the formula R-(CH2) n -O-, where R is a straight-chain, branched-chain or cyclic alkyl or phenyl group, and n is equal to 1 to 20.

[0118] In a preferred embodiment, R 3 is -OR 6 , where R 6 is selected from straight-chain, branched-chain or cyclic alkyl groups having up to 20 carbon atoms, where up to 5 carbon atoms can be replaced by oxygen atoms; more preferably R 3 is selected from methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, 2-ethylhexyloxy, dodecan-1-oxy, 1-methoxy-3-(2-methoxyethoxy)propane-2-oxy, 1-octadecyloxy, 3-methylbutan-1-oxy, phenylmethoxy, 3-ethyl-butoxy and 2,3-dialkoxypropoxy, 1-methoxy-3-(2-methoxyethoxy)propoxy. The end group can be introduced by a suitable initiator, which can be an alkyl anion and a hydride anion, such as a metal alkyl or metal hydride compound of the above end group -R 3 . However, preferably the alkoxy anion and the thioalkoxy anion are not tertiary alkoxy anions. The imide anion is preferably a phthalimide anion. The metal counterion is preferably Na + , K + or Cs + .

[0119] In one embodiment, the initiator is MeOCH2CH2O - , MeO(CH2CH2O)2 -, benzyl OCH2CH2O - , BzO(CH2CH2O)2 - , (Bz)2N-CH2CH2O - , (Bz)2N(CH2CH2O)2 - , phthalimide-CH2CH2O - , phthalimide-(CH2CH2O)2 - salts, where Me is methyl and Bz is benzyl. Most preferred is MeO(CH2CH2O)2 - , BzOCH2CH2O - and (Bz)2N-CH2CH2O - . The counterion is preferably Na + , K + or Cs + .

[0120] The initiator can be provided in an inert solvent. The solvent is preferably an aprotic solvent, and most preferably dimethyl sulfoxide (DMSO) or toluene. In addition, the copolymerization reaction is preferably carried out in the same solvent.

[0121] The end-group fidelity of the group -A-R of the present invention 3 can be determined by known methods by MALDI TOF or by a combination of MALDI TOF and 1 H NMR on the corresponding precursor H-A-R 3 . The group -A-R of the present invention 3 preferably has an end-group fidelity of at least 95%, more preferably at least 98%.

[0122] The polyoxyalkylene group A of the present invention can be a random copolymer. Such groups provide the lowest immunogenicity because they do not provide a blueprint for antibodies to the immune system. They inherently resist immune responses and are therefore a preferred embodiment of the present invention.

[0123] In another embodiment, the polyoxyalkylene group A of the present invention can have a block-like structure or a tapered or layered structure. Methods for preparing such polymers are known to those skilled in the art of polyoxyalkylene. In such an embodiment, preferably no more than 5% of the group A contains blocks having more than 15 ethylene oxide-derived repeating units, and more preferably no more than 5% of the macromolecules of the polymer contain blocks having more than 8 ethylene oxide-derived repeating units.

[0124] Crystallization generally produces solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of a compound of the present invention and one or more solvent molecules. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present invention can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as the corresponding solvated forms. The solvates of the compounds of the present invention can be true solvates, while in other cases, the compounds of the present invention can retain only adventitious water or a mixture of water plus some adventitious solvent.

[0125] The present invention also relates to a composition that includes at least one compound of formula (I) of the present invention and at least one active agent. The at least one active agent is preferably included in an effective amount.

[0126] As used herein, an active agent includes any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or individual. Such an effect can be, for example, biological, physiological, or cosmetic. An active agent can be any type of molecule or compound, including, for example, nucleic acids, nucleic acid analogs, peptides, and polypeptides, including, for example, antibodies, such as polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized TM antibodies, cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.

[0127] In one embodiment, the active agent is a therapeutic agent or a salt or derivative thereof. Therapeutic agent derivatives can themselves be therapeutically active, or they can be prodrugs that become active upon further modification.

[0128] In one embodiment, the therapeutic agent includes any therapeutically effective medicament or drug, such as an anti-inflammatory compound, an antidepressant, a stimulant, an analgesic, an antibiotic, a birth control drug, an antipyretic, a vasodilator, an anti-angiogenic agent, a cytovascular agent, a signal transduction inhibitor, a cardiovascular drug, such as an antiarrhythmic agent, a vasoconstrictor, a hormone, and a steroid.

[0129] In one embodiment, the therapeutic agent is an oncology drug, which may also be referred to as an anti-tumor drug, anti-cancer drug, tumor drug, anti-tumor agent, etc. Examples of oncology drugs that can be used according to the present invention include, but are not limited to, doxorubicin, melphalan (alkeran), allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous injection, busulfan oral preparation, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporine A, cytarabine, cytarabine, daunorubicin, cyclophosphamide (Cytoxan), daunorubicin, dexamethasone, dexrazoxane, docetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab ozogamicin, goserelin acetate, hydroxyurea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megestrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituximab, streptozocin, STI-571, tamoxifen, taxotere, temozolomide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP16 and vinorelbine. Other examples of oncology drugs that can be used according to the present invention are ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecins.

[0130] In a preferred embodiment, the at least one active agent is selected from proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogs, organic molecules having a molecular weight of up to 1000 g / mol, and combinations thereof.

[0131] Any known protein is generally suitable. Exemplary proteins include glycoproteins and apolipoproteins. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins known to those of skill in the art, their variants and fragments, as well as apolipoprotein agonists, analogs or fragments thereof, and chimeric constructs of apolipoproteins. Apolipoproteins for use in the present invention also include recombinant, synthetic, semi-synthetic or purified apolipoproteins.

[0132] Any known peptide is generally suitable. The term peptide of the present invention includes peptidomimetics. The peptide or peptidomimetic can be about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or antimicrobial peptide PI (Ceropin PI)), peptides containing disulfide bonds (e.g., α-defensins, β-defensins or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also include nuclear localization signals (NLS). For example, the cell-penetrating peptide can be a bipartite amphiphilic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T.

[0133] In one embodiment, the targeting peptide linked to the iRNA reagent and / or the vector oligomer can be an amphiphilic α-helical peptide.

[0134] Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, such as D-peptides or L-peptides; α-peptides, β-peptides or γ-peptides; N-methyl peptides; azapeptides; peptides having one or more amides, i.e., peptides, with linkages replaced by one or more ureas, thioureas, carbamates or sulfonylureas; or cyclic peptides.

[0135] Any known carbohydrate is generally suitable. Exemplary carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid.

[0136] As described herein, the compositions of the present invention can be particularly used for delivering nucleic acids or nucleic acid analogs, including, for example, siRNA molecules, mRNA molecules, plasmids, microRNAs, antagomirs, nucleic acid aptamers and ribozymes. Thus, the compositions of the present invention can be used to regulate the expression of target genes and proteins both in vitro and in vivo by contacting cells with the compositions of the present invention associated with nucleic acids (e.g., siRNA or microRNA) that reduce the expression of the target gene or nucleic acids (e.g., mRNA or plasmid encoding the desired protein) that can be used to increase the expression of the desired protein.

[0137] Any known nucleic acid and nucleic acid analog or plasmid are generally suitable. Methods for their preparation include, but are not limited to, chemical synthesis and enzymatic cleavage, chemical cleavage, or in vitro transcription of longer precursors. Methods for synthesizing DNA and RNA nucleotides are widely used and well-known in the art.

[0138] Nucleic acids and nucleic acid analogs include polymers containing at least two deoxyribonucleotides or ribonucleotides in single-stranded, double-stranded, or triple-stranded forms, and include DNA, RNA, and their hybrids. DNA can be in the form of linear DNA, circular DNA, plasmid DNA (pDNA), antisense molecules, PCR products, or vectors. RNA can be in the form of chemically modified or unmodified messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA) containing at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single-guide RNA (sgRNA), or viral RNA (vRNA) and combinations thereof. The nucleic acids can include one or more oligonucleotide modifications.

[0139] The nucleic acids of the present invention can have various lengths, typically depending on the specific form of the nucleic acid. For example, in certain embodiments, the length of a plasmid or gene can be about 1,000 to 100,000 nucleotide residues. In certain embodiments, the length of an oligonucleotide can be about 10 to 100 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can be in the range of about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, about 20 to about 30 nucleotides.

[0140] The term "circular DNA" includes any DNA that forms a closed loop and has no ends. Examples of circular DNA are plasmid DNA, minicircle DNA, and doggybone DNA (db DNA TM )

[0141] For plasmid DNA, the preparations used in conjunction with the embodiments of the present invention generally utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the target plasmid. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the target plasmid allows those bacteria containing the target plasmid to grow selectively in a medium containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in the art. Plasmid isolation can be carried out using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0142] In a preferred embodiment, the present invention specifically relates to compositions for delivering mRNA or siRNA molecules.

[0143] For mRNA, the main method of preparation is, but not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for generating long sequence-specific mRNA. In vitro transcription describes a template-directed method for synthesizing RNA molecules from an engineered DNA template, which is composed of an upstream phage promoter sequence (e.g., including but not limited to T7, T3, and SP6 Escherichia coli) and a downstream sequence encoding the gene of interest linked thereto. The template DNA can be prepared from a variety of sources using suitable techniques well-known in the art for in vitro transcription, and the suitable techniques include but are not limited to plasmid DNA and polymerase chain reaction amplification.

[0144] Transcription of RNA is carried out in vitro using a linearized DNA template, in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), under conditions that support polymerase activity while minimizing the potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits, including but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), and can also be carried out using commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well-known in the art.

[0145] The desired in vitro transcribed mRNA is then purified from the unwanted components of the transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc. Techniques for isolating mRNA transcripts are well-known in the art. Well-known methods include phenol / chloroform extraction or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0146] Additional non-limiting examples of purification methods that can be used include size exclusion chromatography, silica-based affinity chromatography, and polyacrylamide gel electrophoresis. Purification can be carried out using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and the In Vitro Transcription Purification and Concentration Kit (Norgen Biotek).

[0147] In addition, while reverse transcription can generate large amounts of mRNA, the product can contain many aberrant RNA impurities associated with unwanted polymerase activities, which may need to be removed from the full-length mRNA preparation. These include short RNAs generated by transcriptional initiation aborting, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from an RNA template, and the extension of self-complementary 3'. It has been demonstrated that these contaminants with dsRNA structures can cause unwanted immune-stimulatory activities by interacting with multiple innate immune sensors in eukaryotic cells, which are used to recognize specific nucleic acid structures and induce an effective immune response. This, in turn, can significantly reduce mRNA translation because protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to scalable HPLC purification. It has been reported that HPLC-purified mRNA translates at higher levels, particularly within primary cells and in vivo.

[0148] A variety of modifications for altering the specific properties of in vitro transcribed mRNA and improving its utility have been described in the art. These include but are not limited to modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5'-end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is responsible for enhancing mRNA stability and the efficiency of mRNA translation in cells. Therefore, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-terminal nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Other modifications include methylation of the most terminal and the penultimate 5'-nucleotide at the 2'-hydroxyl.

[0149] Multiple different cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of the synthetic mRNA can be carried out co-transcriptionally (i.e., capping during in vitro transcription) using chemical cap analogs. For example, the anti-reverse cap analog (ARC A) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcriptional process, up to 20% of the transcripts remain uncapped, and the synthetic cap analogs are different from the 5'-cap structures of authentic cellular mRNAs, which potentially reduces translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more authentic 5'-cap structures that structurally or functionally more closely mimic the endogenous 5'-cap, which have enhanced binding to cap-binding proteins, increased half-life, and reduced sensitivity to 5'-endonucleases and / or reduced 5'-decapping. Many synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.

[0150] Typically during RNA processing, a long chain of adenine nucleotides (poly-A tail) is added to the mRNA molecule at the 3'-end. After transcription, the 3'-end of the transcript is immediately cleaved to release a 3'-hydroxyl group, and poly-A polymerase adds a chain of adenine nucleotides to this RNA at the hydroxyl group in a process called polyadenylation. It has been widely shown that the poly-A tail enhances the translation efficiency and stability of mRNA.

[0151] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including but not limited to cloning a poly(T) tract into the DNA template or by post-transcriptional addition using poly(A) polymerase. The first case allows for the in vitro transcription of mRNA with a defined length of poly(A) tail, which depends on the size of the poly(T) tract, but requires additional manipulation of the template. The latter case involves using poly(A) polymerase to enzymatically add a poly(A) tail to in vitro transcribed mRNA, which catalyzes the incorporation of adenine residues onto the 3'-end of the RNA, does not require additional manipulation of the DNA template, but results in mRNA with a wide variety of lengths of poly(A) tail. 5'-capping and 3'-poly(A) tailing can be carried out using a variety of commercially available kits, including but not limited to the poly(A) polymerase tailing kit (Epicenter), the mMESSAGE mMACHINE T7 Ultra kit, and the poly(A) tailing kit (Life Technologies), as well as commercially available reagents, a variety of ARCA caps, poly(A) polymerase, etc.

[0152] In addition to the 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger an effective innate immune response. The ability to distinguish pathogenic and self DNA and RNA has been shown to be based at least in part on structure and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications and is thus immunostimulatory, which in turn can inhibit efficient mRNA translation as described above. Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby alleviating this undesired immunostimulatory activity and enhancing translational capacity. The modified nucleosides and nucleotides for the synthesis of modified RNA can be prepared, monitored, and utilized using general methods and procedures known in the art. A variety of nucleoside modifications are available, which can be incorporated individually or in combination with other modified nucleosides to a certain extent into in vitro transcribed mRNA, as disclosed in, for example, US2012 / 0251618. The in vitro synthesis of nucleoside-modified mRNA has been reported to have a reduced ability to activate immune sensors and an accompanying enhanced translational capacity.

[0153] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular RNA or viral RNA), either both or independently, has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA.

[0154] In one embodiment, the RNA is self-amplifying RNA. When delivered to a vertebrate cell, even in the absence of any protein, a self-amplifying RNA molecule (replicase) can result in the production of multiple progeny RNAs by self-transcription (via an antisense copy generated by itself). Thus, a self-amplifying RNA molecule is, in certain embodiments, a (+) strand molecule that can be directly translated after delivery to the cell, and this translation provides an RNA-dependent RNA polymerase, which then produces antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple progeny RNAs. These progeny RNAs, as well as collinear subgenomic transcripts, can be translated themselves to provide in situ expression of the encoded protein, or can be transcribed to provide further transcripts that are synonymous with the delivered RNA, which are translated to provide in situ expression of the protein. The overall result of this transcriptional sequence is the amplification of the number of introduced self-amplifying RNAs, and thus the encoded protein becomes the major polypeptide product of the host cell.

[0155] In one embodiment, the RNA is circular RNA (circRNA), a single-stranded RNA that, unlike linear RNA, forms a covalently closed continuous loop by joining the 3' and 5' ends that are normally present in an RNA molecule. Like mRNA, circRNA can be designed to encode and express proteins. In certain embodiments, the oligonucleotide (or strand thereof) of the invention specifically hybridizes or is complementary to a target polynucleotide.

[0156] In one embodiment, the RNA is a hairpin siRNA having a duplex region equal to or at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length. The length of the duplex region can be equal to or less than 200, 100, or 50. In certain embodiments, the length of the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs. The hairpin can have a single-stranded overhang or a terminal unpaired region. In certain embodiments, the overhang length is 2 to 3 nucleotides. In some embodiments, the overhang is located on the sense side of the hairpin, and in some embodiments, it is located on the antisense side of the hairpin.

[0157] In one embodiment, the RNA is siRNA. siRNA is an RNA duplex that is typically 16 to 30 nucleotides in length and can bind to a cytoplasmic multi-protein complex called the RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts, and thus siRNA can be designed to knockdown protein expression with high specificity. Unlike other antisense technologies, siRNA acts through a natural mechanism that has evolved to control gene expression through non-coding RNA.

[0158] As used herein, a "single-stranded siRNA compound" is an siRNA compound that consists of a single molecule. It can contain a duplex region formed by intrastrand pairing; for example, it can be or contain a hairpin or a pan-handle structure. A single-stranded siRNA compound can be antisense with respect to a target molecule.

[0159] A single-stranded siRNA compound can be long enough such that it can enter RISC and participate in RISC-mediated cleavage of the target mRNA. The length of the single-stranded siRNA compound is at least 14, and in other embodiments at least 15, 20, 25, 30, 35, 40, or 50 nucleotides. In certain embodiments, its length is less than 200, 100, or 60 nucleotides.

[0160] As used herein, a "double-stranded siRNA compound" is an siRNA compound that comprises more than one, and in some cases more than two strands, wherein regions of intermolecular hybridization can form duplex structures.

[0161] The length of the antisense strand of the double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides. Its length can be equal to or less than 200, 100, or 50 nucleotides. Ranges can be lengths of 17 to 25, 19 to 23, and 19 to 21 nucleotides. As used herein, the term "antisense strand" refers to the strand of an siRNA compound that is sufficiently complementary to a target molecule (e.g., a target RNA).

[0162] The length of the sense strand of the double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides. Its length can be equal to or less than 200, 100, or 50 nucleotides. Ranges can be lengths of 17 to 25, 19 to 23, and 19 to 21 nucleotides.

[0163] The length of the double-stranded portion of the double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs. Its length can be equal to or less than 200, 100, or 50 nucleotide pairs.

[0164] Ranges can be lengths of 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0165] In many embodiments, the siRNA compound is large enough such that it can be cleaved by endogenous molecules such as Dicer to produce smaller siRNA compounds, e.g., siRNA reagents.

[0166] The sense and antisense strands can be selected such that the double-stranded siRNA compound includes single-stranded or unpaired regions at one or both ends of the molecule. Thus, the double-stranded siRNA compound can contain a sense strand and an antisense strand that pair to contain overhangs, e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides. The overhangs can be the result of one strand being longer than the other, or the result of the two strands of the same length being staggered. Some embodiments will have at least one 3' overhang. In one embodiment, both ends of the siRNA molecule will have 3' overhangs. In some embodiments, the overhangs are 2 nucleotides.

[0167] In certain embodiments, the length of the duplex region is 15 to 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, such as within the scope of the siRNA compounds described above. The length and structure of the siRNA compounds can be similar to the products of natural Dicer processing of long dsRNA. Also included are embodiments where the two strands of the siRNA compound are linked, such as covalently linked. Hairpins or other single-stranded structures that provide the desired duplex region and 3' overhangs are also within the scope of the present invention.

[0168] The siRNA compounds described herein, including double-stranded siRNA compounds and single-stranded siRNA compounds, can mediate the silencing of target RNAs such as mRNAs, such as transcripts of genes encoding proteins. For convenience, such mRNAs are also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNAs can also be targeted, such as tRNAs and viral RNAs.

[0169] As used herein, the phrase "mediating RNAi" refers to the ability to silence a target RNA in a sequence-specific manner. While not wishing to be bound by theory, it is believed that silencing uses the RNAi mechanism or method and a guide RNA, such as a 21- to 23-nucleotide siRNA compound.

[0170] In one embodiment, the siRNA compound is "sufficiently complementary" to the target RNA, such as the target mRNA, such that the siRNA compound silences the production of the protein encoded by the target mRNA. In another embodiment, the siRNA compound is "perfectly complementary" to the target RNA, such as annealing of the target RNA and the siRNA compound, for example, to form a hybrid made specifically of Watson-Crick base pairs in the perfectly complementary region. A "sufficiently complementary" target RNA can include an internal region (e.g., at least 10 nucleotides) that is perfectly complementary to the target RNA. In addition, in certain embodiments, the siRNA compound specifically discriminates single nucleotide differences. In such cases, if perfect complementarity is found in the region of the single nucleotide difference (e.g., within 7 nucleotides), the siRNA compound only mediates RNAi.

[0171] In addition to conventional siRNAs, dicer substrate siRNAs can also be used as alternatives with lower immunogenicity. DsiRNAs are 25 to 30 nucleotides in length and are further cleaved and processed by Dicer enzymes after cellular uptake, converting them into an active form, which then binds to RISC.

[0172] Antisense RNA against a target polynucleotide. The term "antisense RNA" or simply "antisense" is intended to include RNA that is complementary to a targeted polynucleotide sequence. Antisense RNA is a single-stranded RNA that is complementary to a selected sequence such as the target gene mRNA. Antisense RNA is thought to inhibit gene expression by binding to the complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression, which is achieved by preventing translation of the complementary mRNA strand by binding to the complementary mRNA strand, or by causing degradation of the target mRNA. In certain embodiments, the antisense RNA contains from about 10 to about 50 nucleotides, more preferably from about 15 to about 30 nucleotides. The term also encompasses antisense RNAs that may not be precisely complementary to the desired target gene.

[0173] MicroRNA (miRNA) is a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but not translated into proteins. The processed miRNA is a single-stranded RNA molecule of 17 to 25 nucleotides (nt) that is incorporated into the RNA-induced silencing complex (RISC) and has been identified as a key regulator of development, cell proliferation, apoptosis, and differentiation.

[0174] In one embodiment, the RNA is transfer RNA (tRNA). Transfer RNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between mRNA and the amino acid sequence of a protein. Transfer RNA accomplishes this by carrying amino acids to the protein synthesis machinery of the cell called the ribosome. The anticodon of the tRNA, which is a trinucleotide, is complementary to the codon, which is also a trinucleotide, in messenger RNA (mRNA), resulting in protein synthesis based on the mRNA coding. Thus, tRNA is an essential component of translation, biosynthesizing new proteins according to the genetic code.

[0175] In one embodiment, the nucleic acid is a single-stranded guide RNA applied to direct CRISPR / Cas9-mediated gene editing. The single-stranded guide RNA hybridizes to a target sequence in the cell genome and complexes with the Cas9 protein at the target site, thereby inducing a single-stranded or double-stranded break.

[0176] In one embodiment, the at least one active agent is selected from antagomir, aptamer, ribozyme, immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimetic, antimir or miRNA inhibitor, and UI adaptor.

[0177] Antagomirs are RNA-like oligonucleotides that have multiple modifications and pharmacological properties protected by RNase, such as enhanced tissue and cell uptake. They differ from normal RNA, for example, by complete 2'-O-methylation of the sugar, a phosphorothioate backbone, and a cholesterol moiety at the 3'-end, for example.

[0178] An aptamer is a nucleic acid or peptide molecule that binds to a specific molecule of interest with high affinity and specificity. DNA or RNA aptamers that bind many different entities, from large proteins to small organic molecules, have been successfully produced. Aptamers can be RNA- or DNA-based and can contain riboswitches. A riboswitch is part of an mRNA molecule that can directly bind a small target molecule, and whose binding to the target affects gene activity. The aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. Additionally, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers" that contain a consensus sequence derived by comparing two or more known aptamers to a given target.

[0179] A ribozyme is an RNA molecular complex with a specific catalytic domain that has endonuclease activity. For example, a large number of ribozymes promote phosphotransfer reactions with high specificity, typically cleaving only one of several phosphates in an oligonucleotide substrate. This specificity has been attributed to the need for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme through specific base-pairing interactions prior to the chemical reaction.

[0180] The nucleic acid associated with the lipid particles of the present invention can be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that are capable of inducing an immune response when administered to an individual, which can be a mammal or other patient.

[0181] Because transcription factors recognize their relatively short binding sequences, short oligonucleotides carrying the consensus binding sequence of a specific transcription factor can be used as tools to manipulate gene expression in living cells even in the absence of surrounding genomic DNA. This strategy involves intracellular delivery of such "decoy oligonucleotides," which are then recognized and bound by the target factor. Occupation of the DNA-binding site of the transcription factor by the decoy prevents the transcription factor from subsequently binding to the promoter region of the target gene.

[0182] A supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which has a nucleotide sequence that is substantially the same as and antisense to a miRNA with respect to its target. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, and which contain at least one non-naturally occurring moiety that functions similarly. Due to desired properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases, the modified or substituted oligonucleotides are superior to the native form.

[0183] MiRNA mimics represent a class of molecules that can be used to mimic the gene-silencing ability of one or more miRNAs. Thus, the term "microRNA mimic" refers to a synthetic non-coding RNA that is capable of entering the RNAi pathway and regulating gene expression (i.e., the miRNA is not obtained by purification from an endogenous miRNA source). MiRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimicked precursors (e.g., primary-miRNA (pri-mRNA) or pre-miRNA (pre-mRNA)).

[0184] The terms "antimir", "microRNA inhibitor", "miR inhibitor", or "inhibitor" are synonymous and refer to an oligonucleotide or modified oligonucleotide that interferes with the ability of a specific miRNA. Generally, the inhibitor is a nucleic acid or modified nucleic acid in nature, including oligonucleotides containing RNA, modified RNA, DNA, modified DNA, locked nucleic acid (LNA), or any combination of the above. Modifications include 2'-modifications and internucleotide modifications (e.g., phosphorothioate modifications), which can affect delivery, stability, specificity, intracellular compartmentalization, or potency. In addition, miRNA inhibitors can contain conjugates, which can affect delivery, intracellular compartmentalization, stability, and / or potency. Inhibitors can adopt various configurations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally speaking, microRNA inhibitors include one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the miRNA to be targeted. In addition, the miRNA inhibitor can also contain additional sequences located 5' and 3' of the sequence that is reverse complementary to the mature miRNA. The additional sequence can be the reverse complementary sequence of the sequence adjacent to the mature miRNA in the primary-miRNA from which the mature miRNA is derived, or the additional sequence can be any sequence (with a mixture of A, G, C, or U).

[0185] The U1 adaptor suppresses polyA sites and is a bifunctional oligonucleotide having a target domain complementary to a site in the terminal exon of a target gene and a 'U1 domain' that binds to the U1 small nuclear RNA component of U1 snRNP. U1 snRNP is a ribonucleoprotein complex that is primarily used to direct early steps in spliceosome formation by binding to pre-mRNA exon-intron boundaries. Nucleotides 2-11 at the 5' end of the U1 snRNA base pairs bind to the 5'ss of the pre-mRNA. In one embodiment, the oligonucleotide of the invention is a U1 adaptor.

[0186] In a preferred embodiment, the at least one active agent is selected from: linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (sRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA (circRNA) comprising at least one coding sequence, short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single-stranded guide RNA (sgRNA), or viral RNA (vRNA), and combinations thereof.

[0187] In one embodiment, the at least one active agent is an organic molecule having a molecular weight of up to 1000 g / mol, also known as a small molecule in the pharmaceutical field, and preferably the organic molecule is selected from paclitaxel, doxorubicin, irinotecan, vincristine, and oxaliplatin.

[0188] The composition according to the invention may further comprise a compound selected from lipids, which are different from the compound of formula (I), such as ionizable lipids, cationic lipids, neutral lipids or structural lipids, sterols or sterol derivatives; buffers, pharmaceutically acceptable salts, cryoprotectants, or any combination thereof.

[0189] Suitable lipids according to the invention that may further be present and which are different from the compounds of formula (I) are, for example, diacyl phosphatidylcholines, diacyl phosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins and cerebrosides or mixtures thereof. Lipids with various acyl chain groups of different chain lengths and degrees of saturation are available or can be isolated or synthesized by well-known techniques. In one embodiment, lipids comprising saturated fatty acids with a carbon chain length in the range of C10 to C20 are preferred. In one embodiment, lipids with a mono-unsaturated or di-unsaturated fatty acid with a carbon chain length in the range of C10 to C20 are used. Additionally, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Preferred lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoyl phosphatidylcholine (DPPC) or any related phosphatidylcholine.

[0190] Other suitable lipids consist of sphingomyelins, dihydrosphingomyelins, or phospholipids with other head groups such as serine and inositol, and sterols, in particular cholesterol and phytosterols.

[0191] In one embodiment, the other lipids are ionizable lipids, preferably selected from 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-dioleyloxy-3-dimethylaminopropane, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate, 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine, [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0192] In one embodiment, the other lipids are cationic lipids, preferably salts selected from: 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium-propane, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylformamido)ethyl]-3,4-di[oleoyloxy]-benzamide, N 4-cholesteryl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol, O,O'-di(tetradecanoyl)-N-(α-trimethylammonioacetyl) diethanolamine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, dimethyldi(octadecyl)ammonium, 1,2-dimyristoyl-3-trimethylammonio-propane, 1,2-dipalmitoyl-3-trimethylammonio-propane, 1,2-stearoyl-3-trimethylammonio-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) prop-1-ammonium and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol. The salt can be any pharmaceutically acceptable salt and is preferably a fluoride or chloride salt.

[0193] Other lipids suitable for the compositions of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutaroylphosphatidylethanolamine, lysylphosphatidylglycerol and other anionic modifying groups in combination with neutral lipids.

[0194] In one embodiment, the other lipids are selected from phospholipids, aminolipids and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds can also be used, such as sphingolipids, the glycosphingolipid family, diacylglycerol and β-acyl oxy acids. Additionally, the amphiphilic lipids can be readily mixed with other lipids such as triglycerides and sterols.

[0195] In one embodiment, the other lipids are selected from polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (polyoxyethylene(40) stearate) and Brij TMS10 (polyoxyethylene (10) stearyl ether) or combinations thereof. These lipids are known in the art as stabilizers and, in addition to the compound (I) of the present invention and any other lipids described herein, they may also be present in the compositions of the present invention.

[0196] Cryoprotectants are agents that protect the composition from experiencing adverse effects upon freezing and thawing. For example, in the present invention, cryoprotective agents such as polyols and / or carbohydrates etc. may be added to prevent a large amount of particle aggregation.

[0197] Buffers may also be included. Suitable buffers are, for example, phosphates, acetates, citrates, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, amino acids and other organic compounds; antioxidants, including ascorbic acid and methionine.

[0198] In addition, at least one of the following additives may further be present in the composition: preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates, including monosaccharides, disaccharides and other sugar compounds, such as glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; counterions for salting, such as sodium; metal complexes (such as Zn-protein complexes), solvents, binders, disintegrants, immunoadjuvants, such as cell-penetrating peptides, such as human lactoferrin or fragments thereof, Tat, Ant, Rev, FHV, HSV-1 protein VP22, C6, C6M1, PF20, NAP, POD, polyarginine, polylysine, PTD-5, Transportan, MAP, TP10, Pep-7, azurin p18, azurin p28, hCT18-32, Bac 7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, melittin, penetratin, EB1, MPG, CADY, Pep4, preferably human lactoferrin or fragments thereof, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colorants, sweeteners, suspending / dispersing agents, film-forming / coating agents, flavoring agents, printing inks.

[0199] In one embodiment, the composition, preferably the lipid nanoparticle, comprises at least one active agent and a compound of formula (I) in a weight-to-weight ratio of from 1:0.01 to 1:100.

[0200] In one embodiment, the composition, preferably the lipid nanoparticle, comprises the following compounds: at least one other compound selected from one or more lipids different from the compound of formula (I); a buffering agent; a pharmaceutically acceptable salt different from the buffering agent; a cryoprotectant, or any combination thereof. In a preferred embodiment, the composition, preferably the lipid nanoparticle, further comprises one or more lipids different from the compound of formula (I), more preferably further comprises one, two or three other lipids different from the compound of formula (I). In a preferred embodiment, the composition, preferably the lipid nanoparticle, consists of a compound of formula (I), at least one active agent and one or more lipids different from the compound of formula (I), more preferably one, two or three other lipids different from the compound of formula (I).

[0201] In one embodiment of the composition, preferably the lipid nanoparticle, the compound of formula (I) is present in a ratio of from about 0.1 mol% to about 10 mol% based on the total lipid content. In one embodiment, the compound of formula (I) is present in a ratio greater than 10 mol% based on the total lipid content. In one embodiment, the compound of formula (I) is present in a ratio of from 0.5 mol% to 5 mol% based on the total lipid content. In some embodiments, the compound of formula (I) is present in a ratio of 1.5 mol%.

[0202] In one embodiment of the composition, preferably the lipid nanoparticle, which contains other lipids different from the compound of formula (I) and which are cationic lipids, the cationic lipids are preferably present in a ratio of from about 10 mol% to about 80 mol% based on the total lipid content. In one embodiment, the cationic lipid is present in a ratio of about 50 mol% based on the total lipid content.

[0203] In one embodiment of the composition, preferably the lipid nanoparticle, which contains other lipids different from the compound of formula (I) and which are ionizable lipids, the ionizable lipids are preferably present in a ratio of from about 10 mol% to about 80 mol% based on the total lipid content. In one embodiment, the ionizable lipid is present in a ratio of about 50 mol% based on the total lipid content.

[0204] In one embodiment of the composition, preferably a lipid nanoparticle, which contains other lipids different from the compound of formula (I) and which are structural lipids, also known as "helper lipids", having a neutral or negative net charge, the structural lipids are preferably present in a ratio of from about 10 mol% to about 40 mol% based on the total lipid content. In one embodiment, the structural lipids are present in a ratio of about 10 mol% based on the total lipid content.

[0205] In one embodiment of the composition, preferably a lipid nanoparticle, which contains other lipids different from the compound of formula (I) and which are sterols, such as cholesterol or phytosterols or derivatives thereof, the sterols are preferably present in a ratio of from about 10 mol% to about 60 mol% based on the total lipid content. In one embodiment, the sterols are present in a ratio of from about 35 mol% to about 41 mol% based on the total lipid content. In one embodiment, the sterols are present in a ratio of about 38.5 mol% based on the total lipid content.

[0206] In one embodiment of the composition, preferably a lipid nanoparticle, which contains other lipids different from the compound of formula (I) and which are stabilizers, the stabilizers are preferably present in a ratio of from about 0 mol% to about 10 mol% based on the total lipid content.

[0207] In one embodiment of the composition, preferably a lipid nanoparticle, in which there is at least one buffering agent present, the at least one buffering agent being present at a molar concentration of from 0.1 mM to 1000 mM relative to the total volume of the phase in which the composition is dispersed.

[0208] In one embodiment of the composition, preferably a lipid nanoparticle, in which there is at least one cryoprotectant present, the at least one cryoprotectant being present at a mass concentration of from 0.1 wt% to 50 wt% relative to the total volume of the phase in which the composition is dispersed.

[0209] The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, intracisternal injection or infusion techniques.

[0210] The compositions of the present invention, preferably the pharmaceutical compositions of the present invention, are formulated such that the active ingredient contained therein is bioavailable after administration of the composition to a patient. In some embodiments, the composition administered to an individual or patient takes the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of a compound of formula (I) of the present invention in aerosol form can hold multiple dosage units. The actual methods of preparing such dosage forms are known or obvious to those skilled in the art. In some embodiments, in any case, the composition to be administered will contain a therapeutically effective amount of a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof for treating a disease or condition of interest according to the teachings of the present disclosure.

[0211] The composition, preferably the pharmaceutical composition of the present invention, can be in solid or liquid form. In one aspect, the carrier is particulate such that the composition is in the form of, for example, a tablet or powder. The carrier can be liquid, where the composition is, for example, an oral syrup, an injectable liquid or an aerosol, which can be used for, for example, inhalation administration.

[0212] When used for oral administration, the compositions of the present invention, preferably pharmaceutical compositions, are preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included in the forms considered herein to be solid or liquid.

[0213] As a solid composition for oral administration, the composition, preferably the pharmaceutical composition, can be formulated as a powder, granule, compressed tablet, pill, capsule, chewing gum or wafer. Such solid compositions generally contain one or more inert diluents or edible carriers. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavor; and coloring agents. When the pharmaceutical composition of some embodiments is in capsule form, such as a gelatin capsule, it can also contain a liquid carrier such as polyethylene glycol or an oil in addition to materials of the above types.

[0214] The composition of the present invention, preferably the pharmaceutical composition of the present invention, can be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. As two examples, the liquid can be used for oral administration or for delivery by injection. When used for oral administration, in addition to the compound of formula (I), the preferred composition further comprises one or more of a sweetening agent, a preservative, a dye / colorant and a flavor enhancer. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent can be included.

[0215] The liquid compositions of the present invention, preferably liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, can contain one or more of the following excipients: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic glycerol monoesters or glycerol diesters which can be used as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose; agents used as cryoprotectants such as sucrose or trehalose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred excipient. Injectable pharmaceutical compositions are preferably sterile.

[0216] The composition of the present invention, preferably the pharmaceutical composition, can be used for topical administration, in which case the carrier can suitably comprise a matrix of a solution, an emulsion, an ointment or a gel. For example, the matrix can comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifying and stabilizing agents. Thickeners can be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoresis device.

[0217] The composition of the present invention, preferably the pharmaceutical composition, can be used for rectal administration, for example in the form of a suppository which will melt in the rectum and release the drug. The composition for rectal administration can contain an oily matrix as a suitable non-irritating excipient. Such matrices include but are not limited to lanolin, cocoa butter and polyethylene glycol.

[0218] The composition of the present invention, preferably the pharmaceutical composition, can contain a variety of materials which alter the physical form of a solid or liquid dosage unit. For example, the composition can include materials which form a coating shell around the active ingredient. The materials forming the coating shell are generally inert and can be selected from, for example, sugars, shellac and other enteric coating agents.

[0219] The compositions of the present invention, preferably pharmaceutical compositions, may consist of dosage units suitable for administration as an aerosol. The term "aerosol" is used to denote a variety of systems ranging from those preparations of colloidal nature to systems consisting of pressurized packages. Delivery can be effected by liquefied or compressed gases or by a suitable pump system for dispensing the active ingredient. The aerosol of the compound of formula (I) of the present invention can be delivered in a single-phase, two-phase or three-phase system to deliver the active ingredient. The delivery of the aerosol includes the necessary container, activator, valve, sub-container, etc., which together can form a kit.

[0220] In a preferred embodiment, the composition is a lipid nanoparticle. In certain embodiments, the active agent is encapsulated within the aqueous interior of the lipid nanoparticle. In other embodiments, the active agent is present within one or more lipid layers of the lipid nanoparticle. In other embodiments, the active agent is bound to the outer or inner lipid surface of the lipid nanoparticle. Lipid nanoparticles include, but are not limited to, liposomes. As used herein, a liposome is a structure having a lipid-containing membrane encapsulating an aqueous interior. Liposomes can have one or more lipid membranes. Liposomes can be unilamellar, referred to as unilamellar, or multilamellar, referred to as multilamellar. When forming a complex with nucleic acids, the lipid particle can also be a lipoplex, which consists of a cationic lipid bilayer sandwiched between layers of DNA.

[0221] The lipid nanoparticles of the present invention can be formulated into a pharmaceutical composition, for example, which further comprises a pharmaceutically acceptable diluent, excipient or carrier selected according to the route of administration and standard pharmaceutical practice, such as physiological saline or phosphate buffer.

[0222] In a specific embodiment, the lipid nanoparticles of the present invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Generally, physiological saline will be used as the pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.9% saline, 0.3% glycine, etc., including glycoproteins for enhancing stability, such as albumin, lipoprotein, globulin, etc. In a composition containing saline or other saline-containing carriers, it is preferred to add the carrier after the formation of the lipid particles. Thus, after the formation of the lipid nanoparticles, the composition can be diluted into a pharmaceutically acceptable carrier such as physiological saline.

[0223] The obtained pharmaceutical preparation can be sterilized by conventional and well-known sterilization techniques. Then the aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition can contain pharmaceutically acceptable auxiliary substances required for near-physiological conditions, such as pH regulators and buffers, tonicity regulators, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. In addition, the lipid suspension can include lipid protectants that protect the lipids from free radical and lipid peroxidation damage during storage. Lipophilic radical quenchers such as α-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

[0224] The term "lipid nanoparticle" refers to a particle having at least one nanoscale (e.g., 1 - 1,000 nm) dimension and comprising one or more compounds of formula (I). In some embodiments, lipid nanoparticles containing at least one compound of formula (I) are included in formulations that can be used to deliver therapeutic agents such as nucleic acids (e.g., mRNA) to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles contain a compound of formula (I) and a nucleic acid. In some embodiments, the therapeutic agent such as a nucleic acid can be encapsulated in the lipid portion of the lipid nanoparticles or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticles, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, such as adverse immune responses.

[0225] In various embodiments, the average diameter of the lipid nanoparticles is about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm, preferably determined by dynamic light scattering according to ISO 22412:2017, where the sample is diluted 1:10 in RNase-free water, corresponding to an RNA concentration of 5 ng / μL. Preferably, the measurement can be performed with a Malvern Zetasizer NanoZS.

[0226] Some administration techniques can result in systemic delivery of certain active agents but not others. Systemic delivery means that a useful, preferably therapeutically effective amount of the active agent is exposed to a large part of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0227] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within a living organism. For example, an agent can be locally delivered by direct injection into a diseased site such as a tumor, other target sites such as an inflammatory site, or a target organ such as the liver, heart, pancreas, kidney, etc. Local delivery can also include topical application or local injection techniques, such as intramuscular injection, subcutaneous injection, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0228] The compositions of the present invention can also be administered concomitantly with, before, or after the administration of one or more other active agents. Such combination therapies include formulations of the compositions of the present invention and one or more additional active agents in a single pharmaceutical dosage form, as well as the compositions of the present invention and each active agent administered in their respective separate pharmaceutical dosage forms. For example, the compositions of the present invention and the other active agents can be administered to a patient together in a single oral dosage composition such as a tablet or capsule, or each agent can be administered in separate oral dosage formulations. In the case of using separate dosage formulations, the compound of formula (I) of the present invention and one or more additional active agents can be administered at substantially the same time, i.e., concomitantly, or at separate staggered times, i.e., sequentially; combination therapy is understood to include all such regimens.

[0229] The compositions of the present invention, preferably pharmaceutical compositions, can be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a dispersion. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the compounds of the present disclosure in order to facilitate the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0230] The compositions of the present invention are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent employed; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; drug combinations; the severity of the particular disorder or condition; and the individual undergoing treatment.

[0231] In a preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating human diseases. In a further preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating mammalian diseases. Examples

[0232] Synthesis of Polyoxyalkylene Copolymer

[0233] Reagents and chemicals

[0234] Unless otherwise stated, reagents and chemicals were purchased from TCI (Tokio, Japan), Thermo Fisher Scientific (Waltham, MA, USA), Carl Roth GmbH (Karlsruhe, Germany) and Merck KGaA (Darmstadt, Germany). Ethylene oxide (EO) was obtained from Air Liquide (Paris, France). Tetrahydrofuran (THF) was passed rapidly through basic alumina before use. Glycidyl methyl ether (GME) was dried over calcium hydride (CaH2) and transferred at low temperature before polymerization.

[0235] Example 1: Synthesis of glycidyl methyl ether (GME)

[0236]

[0237] 1-Chloro-3-methoxy-propan-2-ol (50.0 g, 401 mmol) was added to a flask equipped with a magnetic stirrer and cooled with an ice bath. Finely ground sodium hydroxide (NaOH, 20.9 g, 522 mmol) was added in portions with stirring. After complete reaction (TLC control), the crude product was transferred at low temperature from the reaction flask and dried over CaH2 while cooling with an ice bath. After additional low-temperature transfer and filtration steps, GME (30.1 g, 85%) was obtained as a colorless liquid.

[0238] Example 2: Synthesis of 1-methoxy-3-(2-methoxyethoxy)propan-2-ol

[0239]

[0240] Ethylene glycol monomethyl ether (8.64 g, 9.00 mL, 113 mmol) was added to a flask equipped with a reflux condenser. NaOH solution (19 M, 3 mL) was added with stirring, and the resulting solution was heated to 55 °C. GME (5.00 g, 5.10 mL, 56.7 mmol) was added and the solution was stirred overnight. The solution was cooled to room temperature and extracted three times with dichloromethane (DCM, 50 mL). The combined organic phases were dried over magnesium sulfate (MgSO4). After filtration, the solvent was evaporated under reduced pressure. After fractional distillation of the residue, 1-methoxy-3-(2-methoxyethoxy)propan-2-ol, obtained as a colorless liquid (3.74 g, 40%).

[0241] Example 3: Synthesis of mP(EO 15 -co-GME 15 )(Entry b)

[0242]

[0243] Potassium tert-butoxide (KOtBu, 1.01 g, 8.95 mmol) was dissolved in THF without stabilizer and a small amount of Millipore water and transferred to a flame-dried and argon-flushed flask equipped with a Teflon stopcock valve and septum. 1-Methoxy-3-(2-methoxyethoxy)propan-2-ol (1.50 g, 9.14 mmol) of Example 2 was dissolved in benzene and transferred to the flask. High vacuum was applied to the flask and the solvent was removed under high vacuum. The resulting initiator salt was dried overnight at 55 °C under high vacuum. The residue was dissolved in anhydrous dimethyl sulfoxide (DMSO, 86 mL). After freezing the resulting solution at -80 °C, GME (11.3 g, 11.5 mL, 128 mmol) of Example 1 was added to the flask via syringe. EO (5.63 g, 5.81 mL, 128 mmol) was added to the flask from a graduated ampoule by cryotransfer. The cooling bath was removed and the reaction mixture was stirred for 2 days with the stopcock valve closed under high vacuum at 30 °C. The solvent was evaporated at 50 °C under high vacuum. Millipore water (100 mL) and acidic ion exchange resin (DOWEX) (1 g) were added to the residue. The resulting suspension was stirred overnight. The suspension was filtered and the resulting solution was lyophilized. The residue was dissolved in diethyl ether (400 mL). The resulting suspension was filtered and the organic phase was dried over MgSO4. After the filtration step, the solvent was evaporated to yield the statistical copolymer mP(EO 15 -co-GME 15 (15.2 g, 83%), as a viscous liquid.

[0244] In addition, mP(EO-co-GME) with different stoichiometries was prepared according to the same procedure (Entries a, c, and d in Tables 1 and 2).

[0245] Example 4: Synthesis of mP(EO 15 -co-GME 15 )-b-PEO2 (entry e)

[0246]

[0247] Dissolve KOtBu (489 mg, 4.36 mmol) in stabilizer-free THF and a small amount of Millipore water and transfer to a flame-dried and argon-flushed flask equipped with a Teflon stopcock valve and a septum. Dissolve 1-methoxy-3-(2-methoxyethoxy)propan-2-ol (730 mg, 4.45 mmol) from Example 2 in benzene and transfer to the flask. Apply a high vacuum to the flask and remove the solvent under high vacuum. The resulting initiator salt is dried overnight at 55 °C under high vacuum. Dissolve the residue in anhydrous DMSO (42 mL). After freezing the resulting solution at -80 °C, add GME (5.48 g, 5.60 mL, 62.2 mmol) from Example 1 to the flask via syringe. Add EO (2.74 g, 2.83 mL, 62.2 mmol) from a graduated ampoule to the flask by cryogenic transfer. Remove the cooling bath and stir the reaction mixture at 30 °C under high vacuum for 1 day. Cool the solution to -80 °C and add EO (392 mg, 404 μL, 8.89 mmol) from a graduated ampoule to the flask by cryogenic transfer. Remove the cooling bath and stir the reaction mixture at 30 °C under high vacuum for 1 day. Evaporate the solvent at 50 °C under high vacuum. Add Millipore water (100 mL) and acidic ion-exchange resin (DOWEX) (500 mg) to the residue. Stir the resulting suspension overnight. Filter the suspension and lyophilize the resulting solution. Dissolve the residue in diethyl ether (400 mL). Filter the resulting suspension and dry the organic phase with MgSO4. After the filtration step, evaporate the solvent to obtain mP(EO 15 -co-GME 15 )-b-PEO2 (7.19 g, 77%), as a viscous liquid.

[0248] In addition, mP(EO with different stoichiometries is prepared according to the same procedure 21 -co-GME 22 )-b-PEO2 (entries f in Tables 1 and 2).

[0249] Example 5: Quantification of the ratio of primary alcohol end groups and secondary alcohol end groups in polymer samples

[0250] Dissolve 20 mg of polymer b(3), e(4) or f(4) in deuterated acetonitrile (CD3CN) (1 mL). Add trifluoroacetic anhydride (50 μL), and shake the resulting solution for 10 minutes. Transfer 0.6 mL of the solution to an NMR tube and measure 1 the 1H NMR spectrum. The ratio of primary hydroxyl end groups to secondary hydroxyl end groups in the polymer sample was determined by the quantitative esterification of hydroxyl end groups with excess trifluoroacetic anhydride in CD3CN. The 1 1H NMR spectrum of the polymer after the esterification reaction showed two distinct signals at 5.35 (CHOC(O)) and 4.45 ppm (CH2OC(O)). The ratio of the signal integrations was directly related to the percentages of primary and secondary hydroxyl end groups in the polymer sample. The amounts of primary hydroxyl end groups in samples of polymers b(3), e(4) and f(4) were 47%, 87% and 71%, respectively. These results confirmed that primary hydroxyl groups were effectively accumulated at the chain ends through the EO capping step of polymers e(4) and f(4).

[0251] Example 6: Synthesis of mPEO 46 (Entry g)

[0252]

[0253] Dissolve KOtBu (289 mg, 2.57 mmol) in THF without stabilizer and a small amount of Millipore water, and transfer it to a flame-dried and argon-flushed flask equipped with a Teflon stopcock valve and a septum. Dissolve triethylene glycol monomethyl ether (431 mg, 2.62 mmol) in benzene and transfer it to the flask. Apply a high vacuum to the flask and remove the solvent under high vacuum. The resulting initiator salt was dried overnight at 55 °C under high vacuum. Dissolve the residue in anhydrous DMSO (24 mL). After freezing the resulting solution at -80 °C, add EO (4.86 g, 5.01 mL, 110 mmol) from a graduated ampoule to the flask by cryotransfer. Remove the cooling bath and stir the reaction mixture at 30 °C under high vacuum for 1 day. Evaporate the solvent at 50 °C under high vacuum. Add Millipore water (75 mL) and acidic ion exchange resin (DOWEX) (260 mg) to the residue. Stir the resulting suspension overnight. Filter the suspension and lyophilize the resulting solution. Dissolve the residue in chloroform (350 mL). Filter the resulting suspension and dry the organic phase with MgSO4. After the filtration step, evaporate the solvent to obtain mPEO 46 (4.04 g, 76%) as a solid. Determine the degree of polymerization (DP) of 46 by 1 1H NMR spectroscopy.

[0254] Table 1: Composition of the prepared polymers

[0255]

[0256] DP = Degree of Polymerization

[0257] Table 2: Characterization Data of the Prepared Polymers

[0258]

[0259] Method for Characterizing Polyoxyalkylene Copolymers

[0260] 1 The 1H NMR spectra were recorded on a Bruker Avance III HD 300 spectrometer with 300 MHz, and the residual proton signals of the internal reference deuterated solvent were used.

[0261] The M w , M n and dispersity (M w / M n = PDI) were determined from the corresponding size exclusion chromatograms (refractive index (RI) detector, DMF, calibrated with PEO standards). Size exclusion chromatography (SEC) was carried out at 50 °C using dimethylformamide (DMF, containing 1 g / L lithium bromide (LiBr)) as the mobile phase (flow rate 1 mL / min) on a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column. The polymer concentration was 1 mg / mL. Calibration was carried out using PEO standards (from Polymer Standard Service, Mainz, Germany).

[0262] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) measurements were performed on a Bruker autoflex maX MALDI-TOF / TOF. Potassium salt of trifluoroacetic acid and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) were used as the ionization salt and matrix, respectively.

[0263] Synthesis of mP(EO-copoly-GME)-CH2CH2-DMG (1,2-Dimyristoyl-glycerol)

[0264] Reagents and Chemicals

[0265] DCM, triethylamine (NEt3), sodium sulfate (Na2SO4), 1 M hydrochloric acid (HCl) solution, Py, anhydrous tetrahydrofuran (THF, up to 0.05% H2O), myristoyl chloride (MyCl, 97%), 4-dimethylaminopyridine (DMAP), 1,2-isopropylidene-rac-glycerol (IPG) (97%), KOtBu, and acetonitrile (ACN) were obtained from Merck KgaA. p-Toluenesulfonyl chloride (TsCl) was purchased from TCI Chemicals (Tokio, Japan). Deionized water was used for all experiments.

[0266] Example 7: Synthesis of mP(EO 27 -co-GME 16 )-CH2CH2-OTs (p-toluenesulfonyl)

[0267]

[0268] At room temperature, mP(EO 28 -co-GME 16 )(2.46 g, 910 μmol, compound c from Example 3) and DCM (2.46 mL) were charged into a 50 mL round-bottom flask. The solution was cooled to 0 - 5 °C with stirring. Subsequently, DMAP (11.2 mg, 91.7 μmol), NEt3 (166 mg, 1.64 mmol), and p-toluenesulfonyl chloride TsCl (263 mg, 1.37 mmol) were added sequentially. The reaction mixture was stirred at 0 - 5 °C for an additional 1 hour, then heated to 20 - 25 °C and stirred for 72 hours. The conversion was quantified by HPLC. Due to incomplete conversion of the starting material, NEt3 (55.3 mg, 546 μmol) and TsCl (87.7 mg, 460 μmol) were added at 20 - 25 °C, and stirring was continued for 18 hours. Then, DCM (22.1 mL) and water (14.8 mL) were added, and the two-phase mixture was stirred vigorously for 5 minutes. The phases were separated, and the organic layer was mixed with water (4.92 mL) and 1 N HCl solution (0.54 mL). After stirring for 10 minutes, the phases were separated and the organic layer was washed with water (4.92 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated on a rotary evaporator at a water bath temperature of 60 °C and a pressure of 700 - 5 mbar. mP(EO 27 -co-GME 16 )-CH2CH2-OTs was isolated as an orange oil (2.14 g, 749 μmol, 82.3% yield). The purity was determined by HPLC (88.7% a).

[0269] Example 8: mP(EO 27 -co-GME 16)-Synthesis of -CH2CH2-DHG (Dihydroxyglycerol)

[0270]

[0271] Charge mP(EO 27 -co-GME 16 )-CH2CH2-OTs (1.00 g; 350 μmol; 88.7% a purity), anhydrous THF (4 mL) into a 50 mL round-bottom flask and heat to 30 °C. In a separate glass container, mix IPG (92.6 mg, 700 μmol) and KOtBu (78.6 mg, 700 μmol) in anhydrous THF (3 mL). Add the orange suspension in portions to the mP(EO 27 -co-GME 16 )-CH2CH2-OTs solution and continue stirring at 30 °C for 6 hours. Add water (4 mL) and remove THF on a rotary evaporator with a water bath temperature of 60 °C and a pressure of 400 - 150 mbar. Extract the remaining aqueous solution with DCM (8 mL) and concentrate the organic layer on a rotary evaporator with a water bath temperature of 60 °C and a pressure of 700 - 20 mbar. Isolate the intermediate mP(EO 27 -co-GME 16 )-CH2CH2-IPG (0.63 g), as an orange oil.

[0272] Subsequently, dissolve the mP(EO 27 -co-GME 16 )-CH2CH2-IPG intermediate in water (6.30 mL) and adjust the pH to 1.5 - 1.6 using 0.1 N HCl solution (227 μL). Heat the yellow solution to 40 °C, hold for 75 minutes, cool to room temperature, and extract with DCM (2 × 12.6 mL). Combine the organic phases and concentrate on a rotary evaporator with a water bath temperature of 60 °C and a pressure of 700 - 5 mbar. Isolate mP(EO 27 -co-GME 16 )-CH2CH2-DHG (430 mg; 160 μmol), as an orange oil. Analyze the product by HPLC.

[0273] Example 9: Synthesis of mP(EO 27 -co-GME 16 )-CH2CH2-DMG

[0274]

[0275] Charge mP(EO 27-Copolymerization - GME 16 ) - CH2CH2 - DHG (430 mg; 160 μmol) and DCM (2.2 mL). The solution was stirred for 5 minutes under ambient conditions. Py (61.6 mg; 780 μmol) and MyCl (173 mg; 700 μmol) were added in portions, and the reaction mixture was stirred at room temperature for 40 hours. DCM (3.2 mL) and water (1.7 mL) were added to the crude product mixture, and the pH was adjusted to 1.5 using 1 M HCl solution (0.15 mL). The DCM phase was separated, washed with water (3.9 mL), and the organic solvent was removed on a rotary evaporator at a water bath temperature of 60 °C and a pressure of 700 - 5 mbar to obtain crude mP(EO 27 -Copolymerization - GME 16 ) - CH2CH2 - DMG (359 mg), an oily substance. For purification, the crude product was dissolved in ACN (3.85 mL), heated to 50 °C and filtered. Further purification was carried out by flash column chromatography on a Buchi (Essen, Germany) Pure C - 850 FlashPrep system using a 4 g Macherey - Nagel (Düren, Germany) CHROMABOND Flash RS4SiOH cartridge (40 - 63 μm), an ACN to iPrOH gradient, and a flow rate of 5 mL / min. The fractions containing the product were combined and concentrated. Subsequently, the substance was purified using a 12 g Buchi FlashPure EcoFlex C18 cartridge (40 - 60 μm), an ACN / water (1:1) to iPrOH gradient, and a flow rate of 20 mL / min. Compound h, namely mP(EO 27 -Copolymerization - GME 16 ) - CH2CH2 - DMG (45.6 mg, 14.3 μmol; 8.9%), an orange oil. The product was characterized by 1 1H - NMR spectroscopy and HPLC (purity 67% a).

[0276] Lipid Nanoparticle (LNP) Preparation and Biological Evaluation

[0277] Materials for LNP Preparation and Biological Assays

[0278] Fluc mRNA was obtained from TriLink BioTechnologies (San Diego, CA, USA). D-Lin-MC3-DMA was obtained from MedChemExpress (Monmouth Junction, NJ, USA), cholesterol and PEO2k-DMG were obtained from Merck KGaA, and DSPC was obtained from NOF (White Plains, NY, USA). All cell lines were provided by the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

[0279] Example 10: Preparation of LNP

[0280] An aqueous phase containing 0.133 g / L FLuc mRNA and 11 mM acetic acid was mixed with an ethanol phase containing 9.43 mM total lipid (50 mol% DLin-MC3-DMA, 38.5 mol% cholesterol, 10 mol% DSPC, 1.5 mol% PEO lipid or mP(EO-co-GME) lipid) at a volume ratio of 3:1. The crude LNP colloidal dispersion was dialyzed against phosphate buffered saline (PBS) for 3 hours (3x buffer change). The purified LNP was stored at 4 °C until further use.

[0281] Example 11: Determination of the average diameter (z-average) and ζ potential (z-potential) of LNP

[0282] The measurements were performed using a Zetasizer NanoZS from Malvern Instruments GmbH (Herrenberg, Germany). A DTS1070 transparent disposable folding capillary cell from Malvern Panalytical GmbH (Kassel, Germany) was used. For particle size determination, the sample was diluted 1:10 in RNase-free water, corresponding to an RNA concentration of 5 ng / μL. For z-potential determination, the colloidal LNP dispersion from Example 10 was diluted 1:30 in RNase-free water, corresponding to an RNA concentration of 1.67 ng / μL. The z-average, the width of the fitted Gaussian distribution shown as the polydispersity index (PDI), and the average z-potential were calculated based on data from at least 10 runs.

[0283] Table 3: Size and ζ potential of LNP

[0284]

[0285] Example 12: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

[0286] MTS assay was performed using HeLa cells. One day before transfection, 10,000 cells / well were seeded into the corresponding medium (containing 10% FBS and 30 μg / mL gentamicin) with a volume of 100 μL in a 96-well plate and cultured at 37 °C and 5% CO2 for 24 hours. On the second day, the old medium was removed and 90 μL of fresh medium was added to the cells. For polymer testing, the compounds from Example 3 and Example 6 were dissolved in sterile water to meet the final concentrations shown in Table 4, and the samples were added in a volume of 10 μL. For LNP testing, the colloidal LNP dispersion from Example 10 was diluted using ribonuclease-free water to adjust the mRNA concentration to 5 - 20 ng / μL. 10 μL of the corresponding diluted sample was added to the cells, equivalent to an amount of 50 - 200 ng mRNA per well, with a total volume of 100 μL. The cells were further incubated at 37 °C and 5% CO2 for 24 hours. On the third day, according to the manufacturer's protocol (Promega GmbH), CellTiter AQueous non-radioactive cell proliferation assay (MTS) was used to determine cell viability. The absorbance signal (at 400 nm) was quantified using a multiplate reader ( 200PRO, Tecan, Switzerland).

[0287] Table 4: Results of MTS assay for mP(EO - co - GME) compounds

[0288]

[0289] Table 5: Results of MTS assay for LNP

[0290]

[0291] Example 13: Characterization of LNP by agarose gel electrophoresis (AGE)

[0292] The colloidal LNP dispersion from Example 10 was characterized by agarose gel electrophoresis. The assay was performed using the E - Gel TM power snap electrophoresis system from Thermo Fisher Scientific. To evaluate the samples, a 1% agarose gel with a volume capacity of 20 μL / well was used. The results are summarized in Figure 1a and 1b . Agarose gel electrophoresis indicated complete encapsulation of mRNA for all tested LNP compositions (within the detection range of SYBR safe staining). Thus, the LNP prepared with mP(EO - co - GME) lipids exhibited a similar mRNA encapsulation rate to the reference prepared with conventional PEO lipids.

[0293] Example 14: Characterization of LNP by RiboGreen assay

[0294] Characterize the colloidal LNP dispersion from Example 10 by RiboGreen assay. Use Thermo Fischer Quant-iT TM RiboGreen TM RNA Assay Kit. Perform the method according to the manufacturer's protocol with slight adjustments. Dilute the samples to a theoretical RNA concentration of 0.4 μg / mL using TE buffer or Triton buffer and add them to a 96-well plate in a volume of 100 μL. To dissolve the LNP in the presence of Triton-buffer, place the plate in an incubator at 37 °C and 5% CO2 for 10 minutes. Add 100 μL of the dye solution to each well and then mix thoroughly with a pipette. Measure the fluorescence signal with a 200 PRO microplate reader at an excitation / emission value of 480 / 520 nm. All samples and standards are assayed in duplicate.

[0295] Table 6: Ribogreen assay results of LNP

[0296]

[0297] Example 15: Determination of LNP transfection efficiency by luciferase assay

[0298] Perform a luciferase assay using HeLa cells. The cell line is grown according to standard cell culture conditions. One day before transfection, seed 10,000 cells / well into the corresponding medium (containing 10% FBS and 30 μg / mL gentamicin) in a volume of 100 μL in a 96-well plate and culture at 37 °C and 5% CO2 for 24 hours. On the second day, remove the old medium and add 90 μL of fresh medium (without FBS and antibiotics) to the cells. Dilute with ribonuclease-free water and adjust the LNP nanodispersion prepared according to Example 13 to an mRNA concentration of 10 ng / μL. Add 10 μL of the corresponding diluted sample to the cells, equal to an amount of 100 ng mRNA per well, with a total volume of 100 μL. After 4 hours, remove the old medium containing the residual sample and replace it with 100 μL of fresh medium (containing 10% FBS and 30 μg / mL gentamicin). Incubate the cells at 37 °C and 5% CO2 for a further 20 hours. On the third day, use a Dual- Luciferase Reporter Assay System (Promega) to determine the transfection efficiency. Use a The 200PRO multiplate microplate reader quantifies the luminescence signal. For all transfection experiments, jetMessenger was used as a positive control. Reagents were prepared according to the manufacturer's protocol and administered at an RNA dose / well equal to that of the test samples. The results are summarized in Figure 2 below.

Claims

1. A compound having the formula (I) wherein R 1 and R 2 are each independently selected from branched or straight-chain C3-C 20 hydrocarbyl groups having at most three -C=C- bonds, which are saturated or unsaturated, or -H, provided that at least one of R 1 and R 2 is not -H; and wherein R 3 is bonded to a carbon atom of the polyoxyalkylene group A, and said polyoxyalkylene group A is bonded through an oxygen atom to the remainder of the molecule opposite to R 3 ; A is a polyoxyalkylene group comprising at least one of the following units (a) and at least one unit selected from the following: (b) (c) (d) and (e) and R 3 selected from -H; -OH; -SH; -NH2; -NHR 4 , -NR 4 R 5 , -OR 6 , -SR 6 or a straight-chain, branched-chain or cyclic alkyl group having at most 20 carbon atoms; wherein R 4 to R 6 are independently selected from straight-chain, branched-chain or cyclic alkyl groups having up to 20 carbon atoms, where up to 5 carbon atoms may be replaced by an oxygen atom or a sulfur atom; and wherein -A-R 3 has a molecular weight of from 1100 to 7500 g / mol.

2. The compound according to claim 1, wherein R 1 and R 2 are each independently selected from i) a branched or straight-chain C4-C hydrocarbon group that is saturated or unsaturated and has at most two -C=C- bonds, or -H; or 20 -H; or ii) a straight-chain C4-C hydrocarbon group having at most two -C=C- bonds, which is saturated or unsaturated, or -H; or 20 -H; or iii) a branched or straight-chain C4-C hydrocarbon group having a -C=C- bond, which is saturated or unsaturated, or -H; or 20 -H; or iv) a straight-chain C4-C hydrocarbon group having a -C=C- bond, which is saturated or unsaturated, or -H; or 20 -H; or v) a saturated branched or straight-chain C4-C 20 hydrocarbyl group or -H; or vi) saturated straight-chain C4-C 20 hydrocarbyl or -H; or vii) a branched or straight-chain C8-C hydrocarbon radical having at most two -C=C- bonds, which is saturated or unsaturated, or -H; or 18 or viii) a straight-chain C8-C hydrocarbon group that is saturated or unsaturated and has at most two -C=C- bonds, or -H; or 18 -H; or ix) A branched or straight-chain C8-C hydrocarbon radical having one -C=C- bond, which is saturated or unsaturated, or -H; or 18 -H; or x) a straight-chain C8-C hydrocarbon group having one -C=C- bond, which is saturated or unsaturated, or -H; or 18 or xi) a saturated branched or straight-chain C8-C 18 hydrocarbyl group or -H; or xii) saturated straight-chain C8-C 18 hydrocarbyl or -H; or xiii) a branched or straight-chain C having at most two -C=C- bonds, which is saturated or unsaturated 12 -C 17 hydrocarbyl or -H; or xiv) A straight-chain C having at most two -C=C- bonds, which is saturated or unsaturated, or -H; or 12 -C 17 hydrocarbyl or -H; or xv) A branched or straight-chain C having one -C=C- bond, which is saturated or unsaturated 12 -C 17 hydrocarbyl or -H; or xvi) A straight-chain C having a -C=C- bond, saturated or unsaturated 12 -C 17 -hydrocarbyl or -H; or xvii) saturated branched or straight-chain C 12 -C 17 hydrocarbyl or -H; or xviii) saturated straight-chain C 12 -C 18 hydrocarbyl or -H; or xix) saturated straight-chain C 12 -C 17 hydrocarbyl or -H; or xx) saturated straight-chain C 12 -C 16 hydrocarbyl or -H; or xx) saturated straight-chain C 12 -C 14 hydrocarbyl or -H; or xxi) saturated straight-chain C 14 hydrocarbyl or -H.

3. The compound according to claim 1 or 2, wherein R 1 and R 2 are the same.

4. A compound according to any one of the preceding claims, wherein -A-R 3 has a dispersibility of 1.15 or lower.

5. A compound according to any one of the preceding claims, wherein R 3 is -OR 6 , wherein R 6 is selected from straight-chain, branched-chain or cyclic alkyl groups having up to 20 carbon atoms, wherein up to 5 carbon atoms may be replaced by oxygen atoms.

6. A compound according to any one of the preceding claims, wherein -A-R 3 has a molecular weight of from 1500 to 3500 g / mol.

7. A composition comprising at least one compound of formula (I) according to any one of claims 1 to 6 and at least one active agent.

8. The composition according to claim 7, wherein the at least one active agent is selected from proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogs, organic molecules having a molecular weight of up to 1000 g / mol, and combinations thereof.

9. The composition according to claim 8, wherein the at least one active agent is selected from: linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA (circRNA) comprising at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single-stranded guide RNA (sgRNA), or viral RNA (vRNA); and combinations thereof.

10. The composition according to any one of claims 7 to 9, further comprising: a compound selected from lipids other than the compound of formula (I); a buffer; a pharmaceutically acceptable salt different from the buffer; a cryoprotectant, or any combination thereof.

11. The composition according to any one of claims 7 to 10, which is a lipid nanoparticle.

12. A method for preparing a compound of formula (I) according to any one of claims 1 to 6, comprising the following steps or consisting of the following steps: i) Provide the precursor compound H-A-R 3 , wherein A and R 3 are defined as in formula (I), ii) replacing -H with a leaving group -X, which is capable of undergoing a substitution reaction; (iii) Subsequently, perform X-A-R 3 with for a substitution reaction to obtain the compound Then (iv) Protonate the compound (III) to obtain the compound and then (v) React with to carry out an esterification reaction, where Y is a leaving group capable of esterifying -H of the -OH group, and R 1 and R 2 are as defined in formula (I) to obtain the compound of formula (I).

13. A method for preparing the composition according to any one of claims 7 to 11, comprising the following steps: Providing at least one compound of formula (I) according to any one of claims 1 to 6, at least one active agent, and optionally other ingredients; and combining all the ingredients to obtain a composition according to any one of claims 7 to 11.

14. The composition according to any one of claims 7 to 11, for the treatment of human diseases.

15. The composition according to any one of claims 7 to 11, for the treatment of diseases in mammals.

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