Copolymers and lipid compositions for lipid nanoparticles

Abstract

Lipid composition comprising: (i) one or more cationic lipids, preferably cationically ionizable lipids, (ii) one or more phospholipids, and (iii) one or more statistical copolymers comprising repeating units of the following formula (I-a) and repeating units of formula (II-a) wherein R1 represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group; R2 represents a group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfone, sulfoxide, keto, ester, amide, carbamate and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; and an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds, and containing 1 to 5 divalent groups selected from ether, keto, ester, and carbonate groups and / or 1 to 5 fluorine atoms as substituents; and x and y independently represent 0 or 1.

Description

[0001] COPOLYMERS AND LIPID COMPOSITIONS FOR LIPID NANOPARTICLES

[0002] Field of the Invention

[0003] The present invention relates to a lipid composition comprising a specific statistical copolymer. The present invention also relates to a lipid nanoparticle (LNP) obtained or obtainable based on the lipid composition of the present invention. The present invention also relates to a method of producing LNPs, wherein the specific statistical copolymer is used. Furthermore, the present invention relates to the LNP or lipid composition according to the invention for use in the treatment or prevention of a disease or disorder in a subject. Also, the present invention relates to a method for delivering polynucleotides such as RNA to cells of a subject and a method for delivering a therapeutic peptide or protein to a subject, wherein a composition or LNP of the present invention is used. The present invention also relates to methods for treating or preventing a disease, wherein a composition or LNP according to the present invention is used. The present invention also relates to a method of producing the specific statistical copolymer of the present invention. The present invention further relates to a method of producing LNPs containing RNA or DNA encapsulated in said LNP. Finally, the present invention relates to a specific statistical copolymer.

[0004] Background of the Invention

[0005] LNPs are known. LNPs are useful in the field of drug delivery, particularly for delivering nucleic acid-based therapeutics like mRNA vaccines.

[0006] In general, LNPs are spherical structures having a diameter of less than 1000 nm, which are made up of lipid molecules forming a structure including a surface layer and an amorphous, isotropic core. LNPs, depending on their composition, have the potential to encapsulate and protect nucleic acids, such as mRNA or siRNA, within a hydrophobic core. Encapsulation of nucleic acids such as mRNA or siRNA is useful for the following reasons:

[0007] Nucleic acids are inherently unstable in the bloodstream and can be quickly degraded by enzymes. LNPs protect nucleic acids from enzymatic degradation.

[0008] LNPs facilitate the efficient delivery of nucleic acids into target cells. In fact, LNPs can be engineered to have certain properties, such as size, charge, and surface modifications, which can influence their distribution in the body and uptake by specific cell types. Once LNPs are taken up by cells through endocytosis, LNPs need to escape the endosome, a cellular compartment, to release the nucleic acid payload into the cytoplasm where it can be effective. LNPs must be designed to destabilize the endosomal membrane, aiding in the escape of the nucleic acid payload.

[0009] Lipids used in LNPs are generally biocompatible, which reduces the potential for adverse immune reactions. This makes LNPs a safer option for drug delivery applications.

[0010] By modifying the surface of LNPs, LNPs can be targeted to specific types of cells or tissues, enhancing the efficacy of the drug, and reducing side effects.

[0011] LNPs have gained significant attention recently due to their role in COVID-19 mRNA vaccines, wherein LNPs are used to encapsulate and deliver mRNA molecules that encode the spike protein of the SARS-CoV-2 virus. Once delivered into host cells, the mRNA instructs the cells to produce the spike protein, thereby triggering an immune response against the spike protein without using the live virus.

[0012] A typical RNA LNP is composed of, other than the RNA (such as mRNA or siRNA), a mixture of multiple different types of lipids, each serving a specific function. These components generally include:

[0013] 1. Ionizable lipids having a pKa allowing the molecules to be positively charged at acidic pH value, which aids in encapsulating the negatively charged RNA, and neutral at physiological pH value to ensure biocompatibility. These properties are crucial for encapsulating RNA, facilitating endosomal escape, and releasing the RNA into the cytoplasm of target cells.

[0014] 2. Phospholipids are the structural lipids that contribute to the formation of a lipid layer and provide structural integrity and stability to the nanoparticle. Commonly used phospholipids in LNPs include phosphatidylcholine or phosphatidylethanolamine.

[0015] 3. PEGylated lipids (PEG-lipids) which are polyethylene glycol (PEG) conjugated to lipids, are used to form a protective hydrophilic shell around the LNP. PEG-lipid is also used to control the particle size and act as a steric barrier to prevent aggregation during storage. 4. Cholesterol is generally included to modulate the fluidity and stability of the lipid layer and helps in stabilizing the nanoparticle structure while potentially affecting the release rate of the RNA payload.

[0016] The protective shell or layer formed by PEGylated lipids increases the circulation time of the nanoparticles in the bloodstream by reducing opsonization, i.e. the process by which particles are marked for clearance by the immune system. However, the amount of PEG and the type of PEG-lipid used must be carefully balanced to avoid reducing cellular uptake and endosomal escape. Specifically, PEGylated lipids give rise to accelerated blood clearance (ABC), sensitization, and the formation of undesired anti-PEG antibodies leading to immune reactions such as complement activation-related pseudo-allergy (CARPA).

[0017] Accordingly, there is a need for efficient RNA delivery methods avoiding disadvantages of PEGs. WO2023247064 A1 (NGP Polymers GmbH) relates to poly(oxazoline)- and poly(oxazine)-based lipids. These lipids are prepared for example by modifying the w-end group of poly(2-n-alkyl-2-oxazolines) or poly(2-n-2-alkyloxazines) with long-chain alkyl groups. The products have amphiphilic properties and are suitable as replacements of polyethylene glycol, in particular in active-ingredient formulations.

[0018] WO2023166099 A1 (BIONTECH SE) discloses conjugates of polyoxazoline (POX) or polyoxazine (POZ) with hydrophobic chains at the polymer endgroups as alternative for PEGylated lipid components for RNA LNP assembly. POX and POZ are synthesized by living cationic ring-opening polymerization using unsubstituted or substituted 2-oxazoline and 2-oxazine compounds. Accordingly, LNPs are suggested which essentially comprise:

[0019] - RNA; a cationic or cationically ionizable lipid; and a conjugate of a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and one or more hydrophobic end groups.

[0020] The synthesis of the conjugates according to WO2023166099 A1 of (a) a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and (b) one or more hydrophobic chains includes polymerization of a monomer, such as 2-methyl-4,5-dihydro-1 ,3-oxazole, in dry acetonitrile in the presence of an initiator such as ethyl 3-bromopropionate at 80 °C for 18 h in a sealed tube. Polymerization termination is then performed by adding a methanolic solution of KOH. Subsequently, treatment with an amine containing one or more hydrophobic chains (such as myristyl amine), optionally in the presence of coupling additives (such as / V- hydroxysuccinimide and / or dicyclohexylcarbodiimide), results in a conjugate of the polymer and a lipid moiety. According to WO2023166099 A1 , multiple synthetic steps and the use of additional reagents increase the complexity of the preparation and thereby the challenges in process control and quality control, notably in a pharmaceutical context where high standards are applied. Moreover, a conjugate obtainable according to WO2023166099 A1 is problematic at least in view of safety, stability, and / or toxicity.

[0021] Specifically, a conjugate of a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and a hydrophobic chain according to WO2023166099 A1 is prone to shedding when forming part of an LNP. During shedding in a living organism, the conjugate is detached from the LNP and may continue circulation in the body independently from the LNP. Given that anchoring of the molecule on the LNP is driven by the hydrophobic effect of a hydrophobic end group and given that the major portion of the conjugate remains solvated in the aqueous medium, the anchoring of the conjugate on the LNP is of limited stability. A loss of the conjugate from the LNP weakens the protective hydrophilic layer around the LNP as well as the steric barrier to prevent aggregation of LNPs. Moreover, shedded conjugate molecules are potentially immunological hazards and may increase side-effects when used as medicines and administered to patients.

[0022] Similarly, CN114685784 A (BEIJING QINGKENG BIOTECHNOLOGY CO LTD) describes conjugates of poly(2-oxazoline) with terminal hydrophobic C12-C18 alkyl chains, and LNPs comprising them, for nucleic acid delivery. These LNPs have the corresponding disadvantages as described for WO2023166099 A1.

[0023] Therefore, it is an object of the present invention to provide a lipid composition, notably LNP, providing high or improved stability of the LNP, safety, to achieve low toxicity, high stability in the bloodstream, and thus efficient delivery to target cells. It is another object to provide LNP providing controlled cellular uptake, endosomal escape, and / or release of the payload, such as a peptide but preferably a nucleic acid (e.g. RNA), within the cell, and whereby shedding of components from the LNP is avoided.

[0024] Summary of the Invention

[0025] In order to solve these problems, the present invention provides:

[0026] 1) A lipid composition comprising:

[0027] (i) one or more cationic lipids, preferably cationically ionizable lipids,

[0028] (ii) one or more phospholipids, and (iii) one or more statistical copolymers obtainable by a method comprising cationic ring-opening polymerization of a mixture comprising:

[0029] (a) one or more monomers of the following formula (I), wherein

[0030] R1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group, x represents 0 or 1 ; and

[0031] (b) one or more monomers of the following formula (II) wherein

[0032] R2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds, and containing 1 to 5 divalent groups selected from ether, thioether, sufoxide, sulfone, keto, ester, amide, carbamate and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; y represents 0 or 1 ; in the presence of a polymerization initiator. ) The lipid composition according to item 1 , further comprising:

[0033] (iv) a sterol, preferably cholesterol. ) The lipid composition according to item 1 or 2, wherein the mole ratio of R1: R2in the statistical copolymer is [R1] : [R2]= 1 to 200 : 1. ) The lipid composition according to any one of the preceding items, wherein R2contains a sterol group, preferably a cholesterol group. ) The lipid composition according to any one of the preceding items, comprising:

[0034] (i) from 30 to 60 % by weight of the at least one cationic lipid,

[0035] (ii) from 3 to 20 % by weight of the at least one phospholipid,

[0036] (iii) from 0.2 to 20 % by weight of the at least one statistical copolymer, and

[0037] (iv) from 20 to 55 % by weight of a sterol, such as cholesterol, based on the total weight of all components (i) to (iv); or preferably:

[0038] (i) from 30 to 60 mol-% of the one or more cationic lipid,

[0039] (ii) from 3 to 20 mol-% of the one or more phospholipid,

[0040] (iii) from 0.2 to 20 mol-% of the one or more statistical copolymer, and

[0041] (iv) from 20 to 55 mol-% of a sterol, such as cholesterol, wherein the sum of all components (i) to (iv) is 100 mol-%; or, for lower sterol content:

[0042] (i) from 50 to 90 mol-% of said one or more cationic lipids,

[0043] (ii) from 8 to 30 mol-% of said one or more phospholipids,

[0044] (iii) from 0.5 to 25 mol-% of one or more statistical copolymers, and

[0045] (iv) from 0 to 20 mol-%, preferably from 0 to 10 mol-%, of a sterol, such as cholesterol, wherein the sum of all components (i) to (iv) is 100 mol-%. ) The lipid composition according to any one of the preceding items, wherein monomers of formulae (I) and monomers of formulae (II) represent at least 90 mol-%, preferably at least 95 mol-%, and most preferably at least 97 mol-% based of the total moles of all cationically polymerizable monomers present or added to said mixture. ) A lipid composition, in particular according to any one of the preceding items, comprising:

[0046] (i) one or more cationic lipids, preferably cationically ionizable lipids, (ii) one or more phospholipids, and

[0047] (iii) one or more statistical copolymers comprising repeating units of the following formula (l-a) and repeating units of formula (ll-a): wherein

[0048] R1represents a hydrogen atom or a group selected from a C1.3 alkyl group, a cyclopropyl group, and a C3 alkenyl group;

[0049] R2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carboncarbon double or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; and an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double or triple bonds, and containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; x and y independently represent 0 or 1, preferably in an amount of at least 90 mol-% based of the total moles of repeating units present in the statistical copolymer. ) The lipid composition according to item 7, comprising monomer units of formula (ll-a) relative to the total content of all monomer units of formulae (l-a) and (ll-a) in an amount of: from 0.5 to 50 mol-%, preferably from 1 to 40 mol-%, more preferably from

[0050] 2 to 30 mol-%, still more preferably from 3 to 20 mol-%, and still more preferably from

[0051] 3 to 15 mol-%. ) The lipid composition according to item 7, 8 or 31 , comprising at least 90 mol-%, preferably at least 95 mol-%, and most preferably at least 97 mol-%, repeating units of formula (la) and formula (Ila) among all monomer units (repeating units) of said statistical copolymer. 0) The lipid composition according to any one of the preceding items, wherein the polymerization initiator introduces an end group into the statistical copolymer, which is preferably selected from a hydrogen atom, benzyl, Ci-3-alkyl or a Cs-alkenyl group, more preferably methyl or benzyl. 1) The lipid composition according to any one of the preceding items, wherein the statistical copolymer is end-capped, preferably with a halogen atom, such as fluorine, chlorine, bromine or iodine; an azide group -N3; a fluoro(Ci-6-alkyl)sulfonic acid ester group such as a nonaflate group -OSO2C4F9 or a trifluoromethane sulfonate group - OSO2CF3, a fluorosulfonate group -OSO2F; a Ci-s-aryl- or O-s-alkylsulfonic acid group such as a tosyl group CH3-C6H4-SO2- or the mesyl group CH3-SO2-; an unsubstituted, mono- or di-substituted amino group -NH2, -NHR’ or -NR’2, a hydroxyl group -OH, a thiol group -SH, an ester group -OCOR’, a thioester group -SCOR’; a phthalimide group or a cyano group -CN, or functional groups obtainable by modification of these end groups, wherein R’ is a monovalent organic radical of 1 to 6 carbon atoms. 2) A lipid composition, in particular according to any one of the preceding items, comprising:

[0052] (i) one or more cationic lipids, preferably cationically ionizable lipids,

[0053] (ii) one or more phospholipids, and

[0054] (iii) one or more statistical copolymers, wherein the one or more statistical copolymer has the following general formula (III):

[0055] T1— {[A]m, [B]n}staf- T2(III) wherein

[0056] T1is an end group derived from a polymerization initiator,

[0057] T2is an end group obtained by termination of the cationic polymerization using a nucleophile, or an end group obtainable by modification of an end group obtained by termination of the cationic polymerization using a nucleophile, A are repeating units of the formula (l-a) which may be the same or different, as defined in item 7;

[0058] B are repeating units of the formula (I l-a) which may be the same or different, as defined in item 7; m is an integer of at least 5 defining the number of repeating units A in the copolymer, n is an integer of at least 1 defining the number of repeating units B in the copolymer, stat indicates that repeating units A and B are statistically distributed in the copolymer backbone indicated by curly brackets { }, wherein n+m is preferably from 10 to 400, preferably from 20 to 200, more preferably from 30 to 150. ) The lipid composition wherein according to item 12, wherein T2is selected from a halogen atom, such as fluorine, chlorine, bromine or iodine; an azide group -N3; a fluoro(Ci-6-alkyl)sulfonic acid ester group such as a nonaflate group -OSO2C4F9 or a trifluoromethane sulfonate group -OSO2CF3, a fluorosulfonate group -OSO2F; a C1-8- aryl- or Ci-s-alkylsulfonic acid group such as a tosyl group CH3-C6H4-SO2- or the mesyl group CH3-SO2-; an unsubstituted, mono- or di-substituted amino group -NH2, - NHR’ or -NR’2, a hydroxyl group -OH, a thiol group -SH, an ester group -OCOR’, a thioester group -SCOR’; a phthalimide group or a cyano group -CN, or functional groups obtainable by modification of these end groups, wherein R’ is a monovalent organic radical of 1 to 6 carbon atoms. ) The lipid composition according to any one of the preceding items, wherein the statistical copolymer has a number average molecular weight Mnof from 1 .000 to 250.000, preferably from 3.000 to 50.000, more preferably from 3.000 to 15.000 Da. ) The lipid composition according to any one of the preceding items, wherein said composition does not contain PEG (polyethylene glycol) and none of said components (i) to (iii) and, if present (iv), is PEGylated. ) The lipid composition according to any one of the preceding items, wherein said cationic lipid is a cationically ionizable lipid and is preferably a compound of the following formula (IV): wherein

[0059] R3is R6R7N-(CmH2m)-, CH3-(CnH2n)-O-(CoH2o)-, HO-(CmH2m)-,

[0060] HO-CH2-CH(OH)-CH2-, CH3-(CH2)n-O-CO-(CmH2m)-, CH3-(CnH2n)-CO-O-(CmH2m)-, NC-(COH2O)-, HO-CH2-CH((COH2O)-CH3)-, CH3-(CoH2o)-CH(OH)-(CPH2p)-, CH3-(CnH2n)-CO-NH-(CoH2o)-, (HO-CH((CqH2q)-CH3)-CH((CoH2o)-OH)- or C6H10(OH)-,

[0061] R4and R5independently are linear or branched alkyl groups of 6 to 24 carbon atoms that may have an inserted ester group -CO-O- or -O-CO-, and / or are alkenyl groups of 6 to 24 carbon atoms and 1 , 2 or 3 non-conjugated double bonds,

[0062] R6and R7independently are hydrogen or alkyl groups of 1 to 5 carbon atoms, or R6and R7form, together with the nitrogen atom to which they are bound, a pyrrolidine or a piperidine group, z is O or l ; m is an integer of from 2 to 6, n is an integer of from 0 to 6, o and p independently are integers of from 1 to 6, q is an integer of from 2 to 16, and r is O or l . ) The lipid composition according to any one of the preceding items, further comprising an organic solvent. ) The lipid composition according to any one of the preceding items, further comprising a nucleic acid, such as RNA and / or DNA. ) A lipid nanoparticle (LNP) obtained or obtainable from the lipid composition as defined in any one of the preceding items and optionally comprising a peptide or a nucleic acid encapsulated in said LNP. ) An LNP composition comprising said LNP of item 19 and an aqueous solvent, preferably for use in medicine. ) The LNP composition according to item 20, said composition not comprising PEG or PEGylated components, preferably for use in medicine. ) A method of producing LNPs, comprising mixing (i) at least one cationic lipid, (ii) at least one phospholipid, (iii) at least one statistical copolymer as defined in any one of items 1 to 14, and (iv) optionally a sterol. ) A method of producing LNPs containing a nucleic acid encapsulated in said LNPs, comprising mixing (i) at least one cationic lipid, (ii) at least one phospholipid, (iii) said at least one statistical copolymer as defined in any one of items 1 to 14, (iv) optionally a sterol, and (v) a nucleic acid to produce said LNP. ) The LNP composition or said LNP according to any one of items 18 to 21 for use in the treatment or prevention of a disease or disorder in a subject. ) A method for delivering a nucleic acid into cells of a subject, the method comprising administering to the subject the LNP composition or LNP according to any one of items 18 to 21. ) A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject the LNP composition or LNP according to any one of items 18 to 21 , wherein said LNP contains a nucleic acid encoding the therapeutic peptide or protein. ) A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject the LNP composition or LNP according to any one of items 18 to 21 , wherein delivering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder. ) A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject the LNP composition or LNP according to any one of items 18 to 21 , wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. ) A statistical polymer obtainable by a method comprising cationic ring-opening polymerization of a mixture comprising: (a) one or more monomers of the following formula (I), wherein

[0063] R1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group, x represents 0 or 1 ; and

[0064] (b) one or more monomers of the following formula (II) wherein

[0065] R2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds (preferably double bonds); a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds (preferably double bonds), and containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; y represents 0 or 1 ; in the presence of a polymerization initiator; or a statistical copolymer comprising repeating units of the following formula (l-a) and repeating units of formula (ll-a): wherein x, y, R1and R2are as defined above.

[0066] 30) The statistical copolymer according to item 29, wherein one or more groups R2is / are a group of the following formula (V): wherein

[0067] R6are independently hydrogen atoms or methyl groups,

[0068] R7is a linear or branched C1-10 alkyl group, or a linear or branched C2-10 alkenyl group,

[0069] L is a linear or branched C1-5 alkylene group or a linear or branched C2-5 alkenylene group,

[0070] M is a carbonyl group or single bond linking the adjacent groups, and the dashed line next to the bond means that the bond is either a single bond or a double bond.

[0071] 31) The lipid composition according to item 7, comprising monomer units of formula (I l-a) relative to the total content of all monomer units of formulae (l-a) and (I l-a) in an amount of: from 0.5 to 40 mol-%, preferably from 1 to 30 mol-%, more preferably from 1 to 20 mol-%, still more preferably from 1 to 15 mol-%, and still more preferably from 1 to 10 mol-%. The present invention provides a lipid composition comprising a specific statistical copolymer which may be prepared by a simple and straightforward one-pot polymerization method and which may be used for LNPs having improved safety and efficacy by ensuring low toxicity, high stability in the bloodstream, efficient delivery to the target cells, controlled cellular uptake, endosomal escape, and release of the RNA payload within the cell, whereby shedding of the polymer from the LNP can be reduced or avoided.

[0072] The present invention is based on the recognition that LNPs comprising a statistical copolymer of the present invention offer improved safety and stability compared to PEGylated LNPs or conjugates of a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and one or more hydrophobic chains of WO2023166099A1. The polymer topology of the statistical copolymer of the present invention gives rise to a fundamentally different structure of the protective layer compared to the structure available by using PEGylated lipids or conjugates of a polyoxazoline (POX) and / or polyoxazine (POZ) polymer and one or more hydrophobic chains according to WO2023166099A1. Specifically, the conjugates used in the prior art are oriented radially outward on the LNPs while being anchored by a terminal hydrophobic chain. On the other hand, the statistical copolymer of the present invention attaches with multiple hydrophobic side chains along the backbone of the statistical polymer to the LNP, whereby an orientation of the polymer chains results which is essentially orthogonal to what is known from the prior art. Accordingly, the hydrophobic side chains in the statistical copolymer of the present invention enhance the interaction with the LNP to optimize the balance of stability during circulation and local de-shedding to improve mRNA delivery in the cytosol.

[0073] Moreover, the present invention is based on the recognition that the statistical copolymer of the present invention presents a lower risk of inducing undesired immune responses, notably against PEG, whereby LNPs based on the statistical copolymer of the present invention are more suitable for applications requiring multiple administrations.

[0074] Moreover, the present invention is based on the recognition that the molecular structure of the statistical copolymers of the present invention influence the size and particle size distribution of LNPs which affect the biodistribution, cellular uptake, and overall efficacy of the LNPs.

[0075] Moreover, the present invention is based on the recognition that the statistical copolymers of the present invention provide different surface characteristics compared to PEG or the conjugates of WO2023166099 A1, which is advantageous for certain applications. Finally, the statistical copolymer of the present invention provides release kinetics of the RNA payload from LNPs which differ from those of PEGylated LNPs, leading to improved release profiles.

[0076] Brief description of the figures

[0077] Fig. 1 Schematic comparison of components in prior art LNPs and the invention. A depicts PEGylated neutral lipids used in prior art LNPs. B shows a schematic polymer, e.g. a poly(2- oxazoline) (POX), having a hydrophilic backbone and multiple lipophilic side chains. The multiple lipophilic side chains can interact with other lipids of LNPs, whereby the polymer is considered to arrange in “loops” on the surface of the LNP. C. Schematic representation of a POX having lipophilic side chains derived from soy lipids (left) or from nonyl side chains (right).

[0078] Fig. 2 Cationic ring-opening polymerization to obtain statistical copolymers comprising units from the hydrophilic monomers MeOx / EtOx and hydrophobic monomers NonOx / SoyOx. The SoyOx monomers are typically used in a mixture, e.g. in the molar ratio indicated. Mixtures of all monomers shown can be cationically polymerized using methyl tosylate (MeTos) as initiator in acetonitrile under conditions as indicated. The polymerization may be stopped by addition of water. The product is a statistical poly(2-oxazoline) copolymer with side chains R1and R2.

[0079] Fig. 3 Cationic ring-opening polymerization to obtain statistical copolymers comprising the hydrophilic monomers MeOx / EtOx and the hydrophobic monomer EtPentOx.

[0080] Fig. 4 SEC elugrams (CHCh, RID) of the statistical copolymers containing SoyOx / EtOx (left) and NonOx / EtOx (right).

[0081] Fig. 5 SEC elugrams (DMAc, RID) of the statistical copolymers containing SoyOx / MeOx (left) and NonOx / MeOx (right).

[0082] Fig. 6 SEC elugrams of the statistical copolymers comprising EtPentOx. Left: SEC (CHCI3, RID) for EtOx-containing copolymers. Right: SEC (DMAc, RID) for MeOx-containing copolymers.

[0083] Fig. 7 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising EtOx / SoyOx units and assignment of the signals used to estimate the copolymer composition.

[0084] Fig. 8 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising EtOx / NonOx units and assignment of the signals used to estimate the copolymer composition. Fig. 9 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx / SoyOx units and assignment of the signals used to estimate the copolymer composition.

[0085] Fig. 10 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx / NonOx units and assignment of the signals used to estimate the copolymer composition.

[0086] Fig. 11 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising EtOx / EtPentOx units and assignment of the signals used to estimate the copolymer composition.

[0087] Fig. 12 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx / EtPentOx and assignment of the signals used to estimate the copolymer composition.

[0088] Fig. 13 Schematic representation of the synthesis route yielding CholOx.

[0089] Fig. 14 Schematic representation of the cationic ring-opening polymerization (CROP) to obtain statistical copolymers comprising the hydrophilic monomers MeOx / EtOx and the hydrophobic monomer CholOx. Polymerization times were 33 min for the CROP of CholOx / MeOx and 43 min for the CROP of CholOx / EtOx, respectively.

[0090] Fig. 15 SEC elugrams of the statistical copolymers containing CholOx. Left: SEC (DM Ac, RID). Right: SEC (CHCI3, RID).

[0091] Fig. 161H NMR spectrum (CDCI3, 300 MHz) of the statistical copolymer comprising EtOx / CholOx and assignment of the signals used to estimate the copolymer composition. Fig. 171H NMR spectrum (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx / CholOx and assignment of the signals used to estimate the copolymer composition. Fig. 18 Characterization of POX-LNP, see Example 4.

[0092] Fig. 19 Characterization of POX-LNP2, see Example 4.

[0093] Fig. 20 Characterization of JC8OI-BNT-D0E. (A) Z-average diameter and polydispersity index. (B) Encapsulation efficacy.

[0094] Fig. 21 Heat maps JC8OI-BNT-D0E. (A) Z-average diameter. (B) Polydispersity index.

[0095] Fig. 22: Characterization of POX-BNT-mRNA. (A) Z-average diameter and polydispersity index. (B) Encapsulation efficacy.

[0096] Fig. 23 Images of transfection with HEK293 cells of Biontech benchmark (A1) 100ng mRNA applied, (A2) 200ng mRNA applied. Transfection of POX793-BNT-mRNA (B1) 100ng mRNA applied, (B2) 200ng mRNA applied.

[0097] Fig. 24 GFP expression in HEK293 cells after LNP treatment (LNP labelled with arrows are benchmark LNP without PEG replacement polymers in pre-tested or commercial formulation). Fig. 25 GFP expression in Jurkat cells after LNP treatment (LNP labelled with arrows are benchmark LNP without PEG replacement polymers in pre-tested or commercially used formulation, connecting lines indicate LNP with the same lipid composition, but prepared on different days or using different mRNA; these formulations were used for further testing together with the additional formulations SP187, SP188, SP193 and SP194).

[0098] Fig. 26 Images (phase and green fluorescence) of HEK293 cells after 24 h of treatment with different LNP.

[0099] Fig. 27 Images (phase and green fluorescence) of Jurkat cells after 24 h of treatment with different LNP.

[0100] Fig. 28 GFP expression in HEK cells after LNP treatment (n = 2 for S134-138, n = 1 for S146-150).

[0101] Fig. 29 GFP expression in Jurkat cells after LNP treatment (n = 4 for S134-138, n = 1 for S146-150).

[0102] Fig. 30 Cytotoxicity of selected LNP on HEK293 (top) and Jurkat (bottom) cells compared to a benchmark LNP (Comir) (n = 1).

[0103] Fig. 31 Microfluidics set up 0. Numeral 1 indicates lipids in EtOH, numeral 2 indicates Poly-A in acetate buffer.

[0104] Fig. 32 Microfluidics set up 1. Numerals 1 and 2 are as in Fig. 31.

[0105] Fig. 33 Microfluidics set up 2. Numerals 1 and 2 are as in Fig. 31 , numeral 3 indicates EtOH to rinse the system.

[0106] Figs. 34-40 show the total green object integrated intensity over time of each cell type after the LNP transfection. Fig. 34 Astrocytes. Fig. 35 Cardiomyocytes. Fig. 36 HEK. Fig. 37 Macrophages. Fig. 38 Neurons. Fig. 39 T cells. Fig. 40 Jurkat.

[0107] Figs. 41-47 Viability / Toxicity of selected LNP (S134 - S138) by the CellTiter-GLO Luminescent Cell Viability Assay (Promega).

[0108] Fig. 48 Correlation of PEG substitute percentage with LNP size (scale left vertical axis) and PDI (scale right vertical axis).

[0109] Fig. 49 GFP expression in terms of total green integrated intensity after 24 h of Jurkat and HEK293 cells after treatment with LNPs containing different percentages of PEG substitute. Fig. 50 GFP expression in terms of total green integrated intensity of Jurkat cells after treatment with LNPs SP460 - SP502 (benchmark LNPs are marked with arrows).

[0110] Fig. 51 GFP expression in terms of total integrated green intensity of HEK and Jurkat cells after treatment with LNPs S262 - S271 (benchmark LNPs are marked with arrows).

[0111] Fig. 52 Schematic representation of the cationic ring-opening polymerization (CROP) used to obtain statistical copolymers containing the hydrophilic monomers MeOx / EtOx (left, top) and the hydrophobic monomers HeptOx / MyrOx (top, middle and rights). Fig. 53 SEC elugrams of the statistical copolymers containing EtOx (CHCI3, RID). Left: MyrOx-containing copolymers. Right: HeptOx-containing copolymers.

[0112] Fig. 54 SEC elugrams of the statistical copolymers containing MeOx (DMAc, RID). Left: MyrOx-containing copolymers. Right: HeptOx-containing copolymers.

[0113] Fig. 55 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising EtOx and MyrOx including assignment of the signals used to estimate the copolymer composition.

[0114] Fig. 56 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising EtOx and HeptOx including assignment of the signals used to estimate the copolymer composition.

[0115] Fig. 57 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx and MyrOx including assignment of the signals used to estimate the copolymer composition.

[0116] Fig. 58 Overlay of the1H NMR spectra (CDCI3, 300 MHz) of the statistical copolymers comprising MeOx and HeptOx including assignment of the signals used to estimate the copolymer composition.

[0117] Fig. 59 Schematic representation of the cationic ring-opening polymerization (CROP) used to obtain statistical copolymers containing MeOx and NonOx with a DP value of 120.

[0118] Fig. 601H NMR-spectra (CDCI3, 300 MHz) of the statistical copolymers of MeOx and NonOx with DP = 120 including assignment of the signals used to estimate the copolymer composition.

[0119] Fig. 61 SEC elugrams of the statistical copolymers of MeOx and NonOx with DP = 120 (DMAc, RID).

[0120] Fig. 62 Characterization of JC793-BNT-DoE. (A) Z-average diameter and polydispersity index. (B) Encapsulation efficacy. (C) Z-average diameter and polydispersity index of samples stored at 5°C for 1 week. (D) Encapsulation efficacy of samples stored at 5°C for 1 week.

[0121] Fig. 63 Heat maps JC793-BNT-DoE. (A) Z-average diameter. (B) Polydispersity index. (C) Encapsulation Efficiency.

[0122] Detailed Description of the Invention

[0123] The lipid composition

[0124] The present invention provides inter alia a lipid composition. In general, the term "lipid” as used here refers to molecules comprising one or more hydrophobic moieties and also one or more hydrophilic moieties or groups. Typically, lipids are essentially insoluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature of lipids allows the molecules to self-assemble into organized structures and different phases. Hydrophobicity can be conferred by the inclusion of nonpolar groups that include long-chain saturated and unsaturated aliphatic hydrocarbon groups. The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, hydroxyl, and other like groups.

[0125] The lipid composition according to the present invention is a composition containing functional lipids from at least three different classes of functional lipids:

[0126] (i) cationic lipids, preferably cationically ionizable lipids,

[0127] (ii) phospholipids, and

[0128] (iii) specific statistical copolymers comprising a first type of repeating units having a short sidechain for interacting with a hydrophilic medium, and a second type of repeating units having a hydrophobic sidechain for interaction with a hydrophobic medium such as lipids of an LNP.

[0129] Optionally, a sterol such as cholesterol may also be present in the mixture.

[0130] In a composition of the present invention, one or more different elements of each of the classes (i) to (iv) may be present.

[0131] The cationic lipids (i)

[0132] A "cationic lipid" refers to a lipid that may have a net positive charge. Cationic lipids in LNPs bind and stabilize negatively charged nucleic acid by electrostatic interaction and by having a hydrophobic (in some embodiments lipophilic) moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carrying the positive charge. It is believed that the cationic or cationically ionizable lipid may combine together with a nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

[0133] A cationic lipid may be either of the type having a constant cationic group, whereby the lipid may be referred to as constant cationic lipid. Alternatively, and preferably, it is of the type having a group that may assume a positive charge depending on the environment (such as depending on pH or presence of charged compounds), whereby the lipid is referred to as cationically ionizable lipid. A constant cationic group may be a quaternary ammonium group. A cationically ionizable lipid may comprise a basic, ionizable functional group (e.g. a primary, secondary or tertiary amine, or a nitrogen-containing heteroaryl) which is present in neutral or charged form. For example, a basic, ionizable functional group can be a nitrogen functional group (e.g., -NH2, guanidine, amidine, a mono- or dialkylamine, 5- to 6-membered heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl) that can be converted to a charged group by protonation or to a non-charged group by deprotonation.

[0134] Preferably, a cationically ionizable lipid has a net positive charge depending on the pH, so that a positive charge exists at lower pH, whereas it has preferably no net positive charge at a different, preferably higher pH, such as physiological pH. Accordingly, efficacy may be enhanced through helping with endosomal escape and reducing toxicity. Thus, depending e.g. on the pH of the composition or medium in which the cationically ionizable lipid is present, it is either positively charged or neutral.

[0135] Any cationic lipids commonly used for the purpose may be employed in a lipid composition according to the present invention. In the invention, cationically ionizable lipids are preferred. According to a preferred embodiment, a cationically ionizable lipid is a compound of the following formula (IV): wherein

[0136] R3is R6R7N-(CmH2m)-, CH3-(CnH2n)-O-(CoH2o)-, HO-(CmH2m)-,

[0137] HO-CH2-CH(OH)-CH2-, CH3-(CH2)n-O-CO-(CmH2m)-, CH3-(CnH2n)-CO-O-(CmH2m)-,

[0138] NC-(COH2O)-, HO-CH2-CH((COH2O)-CH3)-, CH3-(CoH2o)-CH(OH)-(CpH2p)-,

[0139] CH3-(CnH2n)-CO-NH-(CoH2o)-, (HO-CH((CqH2q)-CH3)-CH((CoH2o)-OH)- or C6Hw(OH)-,

[0140] R4and R5which may be the same or different, independently represent alkyl groups of 6 to 24 carbon atoms that may have an inserted ester group -CO-O- or — O-CO-, and / or are alkenyl groups of 6 to 24 carbon atoms and 1 , 2 or 3 nonconjugated double bonds,

[0141] R6and R7, which may be the same or different, independently represent a hydrogen atom or alkyl groups of 1 to 5 carbon atoms, or R6and R7form, together with the nitrogen atom to which they are bound, a pyrrolidine or a piperidine group, m is an integer of from 2 to 6, n is an integer of from 0 to 6, o and p independently are integers of from 1 to 6, q is an integer of from 2 to 16, r is 0 or 1 and z is 0 or 1 , preferably it is 0.

[0142] Examples of cationically ionizable lipids are e.g. those referred to as X-1 to X-36 in WO 2023 / 166099. Preferred examples are ALC-0315 and SM-102 used in the Covid-19 vaccines of Biontech / Pfizer and Moderna, respectively, of the following formulae.

[0143] ALC-0315 SM-102

[0144] The lipid composition may comprise from 30 to 90 % by weight, preferably from 40 to 80 % by weight of said one or more cationic lipid based on the total weight of all components (i) to (iv) in the lipid composition. In these embodiments, the cationic lipid preferably contains a cationically ionizable lipid, more preferably the cationic lipid is entirely a cationically ionizable lipid.

[0145] The lipid composition according to the present invention may contain, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 50 to 90 % by weight, preferably from 60 to 80 % by weight, of said one or more cationic lipid based on the total weight of all components (i) to (iv) in the composition. In an embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 30 to 60 % by weight, preferably 40 to 55 % by weight, of said one or more cationic lipid based on the total weight of all components (i) to (iv) in the composition. Also in these embodiments, the cationic lipid preferably contains a cationically ionizable lipid, more preferably the cationic lipid is entirely a cationically ionizable lipid.

[0146] Alternatively and preferably, the lipid composition according to the present invention may contain, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 50 to 90 mol-%, preferably from 60 to 80 mol-%, of said one or more cationic lipid, wherein all components (i) to (iv) in the composition are 100 mol-%. In an embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 30 to 60 mol-%, preferably 40 to 55 mol-%, of said one or more cationic lipid based on the all components (i) to (iv) in the composition (100 mol-%). Also in these embodiments, the cationic lipid preferably contains a cationically ionizable lipid, more preferably the cationic lipid is entirely a cationically ionizable lipid.

[0147] The phospholipids (ii)

[0148] For the purpose of the present invention, any phospholipids commonly used for the purpose may be employed in a lipid composition according to the present invention. Specific phospholipids may be selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipahnitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains. Phospholipids with saturated hydrophobic chains are preferred. A particularly preferred phospholipid is DSPC.

[0149] The lipid composition may comprise from 3 to 30 % by weight, more preferably 5 to 20 % by weight of said one or more phospholipids based on the total weight all components (i) to (iv) in the composition.

[0150] Preferably, the lipid composition comprises, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 8 to 30 % by weight, preferably from 12 to 20 % by weight, of said one or more phospholipid based on the total weight of all components (i) to (iv) in the composition. In another embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 3 to 20 % by weight, preferably from 5 to 15 % by weight, of said one or more phospholipid based on the total weight of all components (i) to (iv) in the composition.

[0151] Alternatively and preferably, the lipid composition comprises, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 8 to 30 mol-%, preferably from 12 to 20 mol-%, of said one or more phospholipid based on the total of all components (i) to (iv) in the composition that represent 100 mol-%. In another embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 3 to 20 mol-%, preferably from 5 to 15 mol-%, of said one or more phospholipid based on the total of all components (i) to (iv) in the composition (that represent 100 mol-%).

[0152] The statistical copolymer (Hi)

[0153] The statistical copolymers are obtainable by a method comprising cationic ring-opening polymerization (CROP) of a monomer mixture. The mixture contains specific substituted 2- oxazoline and / or 2-oxazine compounds. Optionally, unsubstituted 2-oxazoline and 2-oxazine may also be present in the mixture. CROP is generally known in the art, see e.g. the references cited in the Examples.

[0154] CROP of substituted or unsubstituted 2-oxazolines and 2-oxazines may be used to synthesize the statistical copolymers of the present invention. In a first step, a cationic species is formed, which acts as an initiator. Examples of initiators are electrophiles, such as salts or esters of aromatic sulfonic acids, or salts or esters of aliphatic sulfonic acids, or aromatic halogen compounds or aliphatic halogen compounds. Examples of preferred initiators are esters of arylsulfonic acids, such as methyl tosylate, esters of alkanesulfonic acids, such as of trifluoromethanesulfonic acid, or mono- or dibromomethylbenzene, or mono- or diiodomethylbenzene or oxazolinium salts. In the Examples, methyl tosylate was used, which is a preferred initiator. The polymerization is usually carried out in a polar aprotic solvent, for example in acetonitrile or benzonitrile, e.g. depending on the solubility of the produced copolymer (see Examples).

[0155] The initiator reacts with substituted or unsubstituted 2-oxazoline or 2-oxazine monomer to form a cationic species of the monomer and a generally anionic counter ion. The electrophilic moiety of the initiator forms end group T1described below (methyl in the case of a methyl sulfonic acid ester). The ring of the first cationic monomer opens upon reaction with a second monomer molecule, generating a new cationic species at the end of the growing polymer chain. In subsequent propagation steps in CROP, the cationic end of the polymer chain reacts with another monomer molecule. The ring of the cationic monomer unit at the end opens, and the chain extends again by one repeating (monomer) unit. This step repeats continuously, adding more monomers to the growing chain. The polymerization reaction can be terminated by various methods, such as the addition of a nucleophilic terminating agent (such as water) that reacts with the cationic end group, or by proton transfer to neutralize the cationic species. The terminating agent may lead to terminal group T2described below.

[0156] Preferably, CROP is controlled in a manner akin to living polymerization, where the chain growth occurs without significant chain termination or chain transfer reactions, whereby copolymers with narrow molecular weight distribution can be obtained. The statistical copolymer of the invention covers copolymers where the repeating units may be distributed along the polymer chain in a random, alternating, periodic, or gradient manner.

[0157] The monomer mixture for the CROP comprises one or more monomers of the following formula (I),

[0158] In formula (I), R1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group. Preferably, R1is a hydrogen atom, a methyl group or an ethyl group. Even more preferably, R1is a methyl or ethyl group, and most preferably a methyl group.

[0159] In formula (I), x represents 0 or 1 , whereby 0 is preferred. Thus, the monomer is preferably an oxazoline.

[0160] The mixture further comprises one or more monomers (b) of the following formula (II): wherein y represents 0 or 1 , whereby 0 is preferred, i.e. the monomer is preferably an oxazoline.

[0161] In formula (II), R2represents preferably a non-aromatic group, i.e. it does not contain an aromatic moiety. Possible groups as R2are described in the following.

[0162] Saturated hydrocarbyl group as R2

[0163] R2may be a saturated C4-40 hydrocarbyl group. The saturated C4-40 hydrocarbyl group may be straight-chain, branched chain hydrocarbyl group or may be or comprise a cyclic moiety. The saturated C4-40 hydrocarbyl group is preferably a saturated C5-36 hydrocarbyl group, more preferably a saturated C5-34 hydrocarbyl group, even more preferably a saturated C5-32 hydrocarbyl group, and even more preferably a saturated C5-30 hydrocarbyl group. In a further preferred embodiment, it is a C5-22 hydrocarbyl group. The saturated hydrocarbyl group may, for example, have 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16, 18, 20 or 22 carbon atoms.

[0164] In another embodiment, the saturated hydrocarbyl group is a C18-40 (preferably Ci 8-35) hydrocarbyl group.

[0165] The C4-40 hydrocarbyl group may be an alkyl group, a cycloalkyl group, a cycloalkyl alkyl group, an alkylcycloalkyl group, or an alkylcycloalkylalkyl group, whereby the preferred carbon atom numbers given in the preceding paragraph also apply to these embodiments. The alkyl moieties of these groups may be linear or branched.

[0166] Examples of the alkyl group include n-butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, secpentyl, neo-pentyl, 1 ,2-dimethyl- propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n- decyl, n-undecyl, n-dodecyl, and the like. According to a preferred embodiment, R2is a group derived from naturally occurring fatty acids and is selected from 2-ethylpentyl, n-butyl, n-pentyl, n-heptyl, n-nonyl, n-undecyl, n-tridecyl, n- pentadecyl, n-heptadecyl, and n-nonadecyl.

[0167] Examples of cycloalkyl group are cyclopentyl, cyclohexyl and cycloheptyl groups. Such a group may be a moiety of a cycloalkylalkyl group, an alkylcycloalkyl group, or an alkylcycloalkylalkyl group as the hydrocarbyl group.

[0168] Cycloalkylalkyl groups are groups wherein a cycloalkyl group is linked to an alkylene group. Examples are cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, and cyclohexylethyl. Alkylcycloalkyl groups are groups wherein an alkyl group is linked to a cycloalkylene group. Examples of alkylcycloalkyl groups are methylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, butylcyclopentyl, pentylcyclopentyl, hexylcylopentyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, butylcyclohexyl, pentylcyclohexyl and hexylcylohexyl.

[0169] Unsaturated hydrocarbyl group as R2

[0170] Alternatively, R2may be a, preferably non-aromatic, unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds. The number of double and / or triple bonds (i.e. the sum of double and triple bonds) may be from 1 to 4, preferably from 1 to 3, more preferably 1 or 2. Double bonds are preferred. Thus, the unsaturated C4-40 hydrocarbyl group may have one or more carbon-carbon double bonds, such as from 1 to 4, preferably from 1 to 3, more preferably 1 or 2 double bonds. If the unsaturated C4-40 hydrocarbyl group has more than one double or triple bond, these double or triple bonds are preferably non-conjugated double bonds, e.g. are separated by at least one methylene moiety -CH2-.

[0171] The unsaturated C4-40 hydrocarbyl group is preferably an unsaturated C5-36 hydrocarbyl group, more preferably an unsaturated Ce-34 hydrocarbyl group, even more preferably an unsaturated C7-32 hydrocarbyl group. In a further embodiment, it is an unsaturated C8-22 hydrocarbyl group. The hydrocarbyl group may, for example, have 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16, 18, 20 or 22 carbon atoms. In another embodiment, the unsaturated hydrocarbyl group is a C18-40 (preferably C18-35) hydrocarbyl group.

[0172] The unsaturated C4-40 hydrocarbyl group may be or comprise an alkenyl group, a cycloalkenyl group, a cycloalkylalkenyl group, a cycloalkenylalkyl group, a cycloalkenylalkenyl group, an alkylcycloalkenyl group, or an alkylcycloalkylalkenyl group, whereby the preferred carbon atom numbers given in the preceding paragraph also apply to these embodiments. The alkyl and alkenyl moieties of these groups may be linear or branched.

[0173] The unsaturated C4-40 hydrocarbyl group may be straight-chain or branched hydrocarbyl group or may be or may comprise a cyclic group. As examples of the unsaturated C4-40 hydrocarbyl group, 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5-hexenyl, 1 -heptenyl, 2-heptenyl, 3- heptenyl, 4- heptenyl, 5-heptenyl, 6-heptenyl, 1 -octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5- octenyl, 6-octenyl, 7- octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6- nonenyl, 7-nonenyl, 8-nonenyl, 1- decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6- decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1- undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8- undecenyl, 9-undecenyl, 10- undecenyl, 1 -dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5- dodecenyl, 6- dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11 -dodecenyl, and the like may be mentioned. Examples of cyclic groups are cyclopentenyl, cyclohexenyl and cycloheptenyl groups. According to a preferred embodiment, R2is a group selected from pentadecenyl, heptadecenyl, heptadecadienyl, heptadecatrienyl, and nonadecapentaenyl.

[0174] Saturated heterohydrocarbyl group as R2

[0175] Alternatively, R2may be a, preferably non-aromatic, saturated C4-40 heterohydrocarbyl group. The saturated C4-40 heterohydrocarbyl group may contain from 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms. In a preferred embodiment, the divalent group(s) is / are selected from ether, keto, ester, amide, carbamate, and carbonate groups. The number of divalent groups is preferably from 1 to 3, more preferably 1 or 2, and most preferably 1. The number of optional fluorine atoms is preferably from 1 to 3. However, in a preferred embodiment, the saturated C4-40 heterohydrocarbyl group has no fluorine atom substituent. More preferably, it has 1 or 2 of said divalent groups and from 1 to 3 fluorine atoms. Even more preferably, it has 1 or 2 of said divalent groups and no fluorine atom substituent.

[0176] The preferred number or number range of carbon atoms of the saturated C4-40 heterohydrocarbyl group is as described above for the saturated C4-40 hydrocarbyl group. Apart from the divalent groups and optional fluorine atom substituents, the hydrocarbyl moiety or moieties of the saturated C4-40 heterohydrocarbyl group may be as described above for the saturated C4-40 hydrocarbyl group.

[0177] Unsaturated heterohydrocarbyl group as R2

[0178] Alternatively, R2may be a, preferably non-aromatic, unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds. The number of double or triple bonds (i.e. the sum of double and triple bonds) may be from 1 to 4, preferably from 1 to 3, more preferably 1 or 2. Double bonds are preferred. Thus, the unsaturated C4-40 heterohydrocarbyl group may have one or more carbon-carbon double bonds, such as from 1 to 4, preferably from 1 to 3, more preferably 1 or 2 double bonds. If the unsaturated C4-40 heterohydrocarbyl group has more than one double or triple bond, these double bonds are preferably non-conjugated double bonds, e.g. are separated by at least one methylene moiety -CH2-. The unsaturated C4-40 heterohydrocarbyl group may further contain 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 monovalent substituents selected from fluorine atoms. In a preferred embodiment, the divalent group(s) is / are selected from ether, keto, ester, amide, carbamate, and carbonate groups. The number of said divalent groups is preferably from 1 to 3, more preferably 1 or 2, and most preferably 1 . The number of optional fluorine atoms is preferably from 1 to 3. However, in a preferred embodiment, the unsaturated C4-40 heterohydrocarbyl has no fluorine atom substituent. More preferably, it has 1 or 2 of said divalent groups and from 1 to 3 fluorine atoms. Even more preferably, it has 1 or 2 of said divalent groups and no fluorine atom substituent.

[0179] The preferred number or number range of carbon atoms of the unsaturated C4-40 heterohydrocarbyl group is as described above for the saturated C4-40 hydrocarbyl group. Apart from the divalent groups and optional fluorine atom substituents, the unsaturated hydrocarbyl moiety or moieties of the unsaturated C4-40 heterohydrocarbyl group are as described above for the unsaturated C4-40 hydrocarbyl group.

[0180] Preferably, R2is an unsaturated C4-40 heterohydrocarbyl group having one or two nonconjugated carbon-carbon double bonds, and having 1 or 2 divalent groups selected from ether, keto, ester, amide, carbamate, and carbonate groups, more preferably selected from ether, keto, and ester groups. Even more preferred is the case of 1 carbon-carbon double bond and one divalent group selected from ether, keto, and ester groups. Most preferred is an ester group as a divalent group.

[0181] Sterol qroup-containinq R2

[0182] R2may be a saturated or unsaturated C18-40 (preferably C18-35) heterohydrocarbyl group that comprises a (optionally substituted) sterol group or a substituted derivative thereof. The sterol group is a group comprising the fused 4-ring gonane carbon skeleton and a hydroxy group in 3-position (3-hydroxysterol). The fused rings are generally referred to as rings A to D, wherein ring A carries the 3-hydroxy group and ring D is the 5-membered ring. The sterol group may be saturated or may contain one or two carbon-carbon double bonds.

[0183] The 3-hydroxysterol group may have from 1 to 5 alkyl or alkenyl substituents, that each has from 1 to 10 carbon atoms. The substituents on rings A to C are preferably methyl, the substituent on ring D (preferably in 17-position of the sterol group) may be a C1-10 hydrocarbyl group, preferably a C4-10 hydrocarbyl group (such as a linear or branched alkyl or linear or branched alkenyl group). The optionally substituted sterol derivative may have from 1 to 3, preferably 1 or 2 (preferably non-conjugated) carbon-carbon double bonds, one of which is preferably located in ring B (as in cholesterol). Another unsaturated carbon-carbon bond may be present in the C4-10 hydrocarbyl substituent on ring D. The substituent in 17- position may be a 1 ,5-dimethylhexyl group or derivative thereof having one unsaturated carbon-carbon bond.

[0184] The sterol group may be a 3-hydroxycholestane group or an unsaturated derivative thereof having 1 or 2 non-conjugated unsaturated carbon-carbon bonds in a ring and / or the dimethylhexyl moiety thereof. The sterol group or its derivative may be a cholesterol group or a hydrogenated derivative thereof.

[0185] R2groups containing a sterol group (or the substituted derivative thereof) may be a (optionally substituted) sterolcarbonylalkyl group or sterolcarbonylalkenyl group, wherein the alkyl moiety may have from 1 to 5, preferably 2 or 3, carbon atoms, and the alkenyl moiety may have from 2 to 5, preferably 2 or 3, carbon atoms.

[0186] Where R2is or comprises a sterol group, it may be or may comprise a cholesteryl group, and may be a cholesterylesteralkyl group or a cholesterylesteralkenyl group, wherein the alkyl moiety may have from 1 to 5, preferably 2 or 3, carbon atoms, and the alkenyl moiety may have from 2 to 5, preferably 2 or 3, carbon atoms.

[0187] Accordingly, R2may be a saturated or unsaturated C18-40 (preferably C18-35) heterohydrocarbyl group that comprises an (optionally substituted) sterol group, such as a cholesterol group. In a preferred embodiment, it is a cholesterylester-C2-5 alkyl group or a cholesterylester-C2-5 alkenyl group. Specific examples are a cholesterylesterethyl group (shown in Fig. 14, derived from the CholOx monomer, see Example 3), a cholesterylesterpropyl group, and a cholesterylesteralkenyl group, whereby the cholesterylesterethyl group is preferred.

[0188] Embodiments wherein R2contains a sterol group may be combined with the preferred embodiments of R1, notably the latter may be methyl or ethyl. In one embodiment, R2is a group of the following formula (V): wherein

[0189] R6are independently hydrogen atoms or methyl groups,

[0190] R7is a linear or branched C1-10 alkyl group, or a linear or branched C2-10 alkenyl group,

[0191] L is a linear or branched C1-5 alkylene group or a linear or branched C2-5 alkenylene group,

[0192] M is a carbonyl group or single bond linking the adjacent groups, and the dashed line next to the bond (in ring B) means that the bond is either a single bond or a double bond.

[0193] The stereochemistry preferred for the sterol group, notably the cholesterol group, can be taken from Fig. 14.

[0194] The skilled person understands that one or more different monomers of formula (I) may be mixed or that different repeating units of formula (l-a; defined below) may be combined in or for the statistical copolymer.

[0195] Alternatively or additionally, one or more different monomers of formula (II) may be mixed or different repeating units of formula (ll-a; defined below) may be combined in the copolymer. An example is a mixture of monomers SoyOxI , SoyOx2, and SoyOx 3 shown in Fig. 2. In another example, one or more monomers of formula (II) or repeating units of formula (ll-a) having unsaturated C4-40 hydrocarbyl groups as R2may be mixed / combined with monomers of formula (II) or repeating units of formula (ll-a) having saturated C4-40 hydrocarbyl group(s). A specific example is the mixture of monomers referred to as SoyOx in Fig. 2.

[0196] In formula (II), y represents 0 or 1 , whereby 0 is preferred, i.e. the monomer is preferably an oxazoline. It is possible to copolymerize oxazoline and oxazine derivatives having the substituents described above. Thus, the statistical copolymer may be a polyoxazoline or a polyoxazine or a mixed poly(oxazoline-oxazine). However, it is preferred that x and y are both 0 or are both 1 in the statistical copolymer. Most preferably, x and y are both 0.

[0197] According to a preferred embodiment, monomers of formulae (I) and monomers of formulae (II) represent at least 90 mol-%, preferably at least 95 mol-%, even more preferably at least 97 mol-%, and most preferably 100 mol-% based of the total moles of all cationically polymerizable monomers present or added to said mixture. As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of the component(s) of interest to the total number of moles of all components, multiplied by 100.

[0198] The remaining monomers in the mixture may be selected from any other cationically polymerizable monomers as long as the effects of the present invention are not prevented. Accordingly, the mixture may preferably consist essentially of monomers of formulae (I) and monomers of formulae (II).

[0199] Specific monomers other than monomers of formulae (I) and monomers of formulae (II) may be selected from epoxides such as propylene oxide, cyclohexene oxide and oxetanes; tetrahydrofuran; lactones such as e-caprolactone, [3-butyrolactone and y-valerolactone; lactams such as e-caprolactam, and [3-laurolactae; thiiranes such as ethylene sulfide and propylene sulfide; cyclic acetals / ketals such as 1,3-dioxolane and 1,3-dioxepane; vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; N-vinyl compounds such as N- vinylpyrrolidone and N-vinylcarbazole; cyclic siloxanes such as hexamethylcyclotrisiloxane and octamethylcyclotetrasiloxane; allyl ethers such as allyl glycidyl ether. However, preferably, no such other monomers are used in the mixture and / or no such monomer units are present in the copolymer of the invention.

[0200] According to a preferred embodiment, the mole ratio of R1: R2in the statistical copolymer is [R1] : [R2]= 1 to 200 : 1. Preferably, the mole ratio R1: R2in the statistical copolymer is from 1.5 to 100 : 1 , more preferably from 2 to 50 : 1 , even more preferably from 4 to 33 : 1 , and even more preferably from 5 to 33 : 1

[0201] The molar ratio of R2to the total R1and R2in the statistical copolymer, and the ratio of compounds of formula (II) relative to the total of compounds of formula (I) and (II) in the mixture, may be from 0.5 to 50 mol-% preferably from 1 to 40 mol-%, more preferably from 2 to 33 mol-%, even more preferably from 3 to 20 mol-%, even more preferably from 3 to 17 mol-%.

[0202] According to other and more preferred embodiments, the mole ratio of R1: R2in the statistical copolymer is [R1] : [R2]= 1.5 to 200 : 1. Preferably, the mole ratio R1: R2in the statistical copolymer is from 2 to 100 : 1 , more preferably from 4 to 100 : 1 , even more preferably from 6 to 100 : 1 , and even more preferably from 9 to 100 : 1 .

[0203] Accordingly, in more preferred embodiments, the molar ratio of R2to the total R1and R2in the statistical copolymer, and the ratio of compounds of formula (II) relative to the total of compounds of formula (I) and (II) in the mixture, may be from 0.5 to 40 mol-% preferably from 1 to 30 mol-%, more preferably from 1 to 20 mol-%, even more preferably from 1 to 15 mol-%, even more preferably from 1 to 10 mol-%.

[0204] The lipid composition may comprise from 0.2 to 20 % by weight, more preferably 0.5 to 15 % by weight, even more preferably 0.7 to 10% by weight, even more preferably 1 .0 to 7 % by weight, even more preferably 1 .2 to 5 % by weight of the one or more statistical copolymer, based on the total weight of all components (i) to (iv).

[0205] Preferably, the lipid composition comprises, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 0.5 to 25 % by weight, preferably from 1.0 to 10 % by weight, of said one or more statistical copolymer based on the total weight of all components (i) to (iv) in the composition. In another embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 0.2 to 20 % by weight, preferably from 1 .0 to 10 % by weight, of said one or more statistical copolymer based on the total weight of all components (i) to (iv) in the composition.

[0206] Alternatively and preferably, the lipid composition comprises, in an embodiment wherein the lipid composition contains no or little of a sterol (iv), from 0.5 to 25 mol-%, preferably from 1.0 to 10 mol-%, of said one or more statistical copolymer based on the total of all components (i) to (iv) in the composition of 100 mol-%. In another embodiment, wherein the lipid composition contains a sterol (iv), it may contain from 0.2 to 20 mol-%, preferably from 1.0 to 10 mol-%, of said one or more statistical copolymer based on the total of all components (i) to (iv) in the composition of 100 mol-%.

[0207] The statistical copolymer of the invention may be defined in that it comprises repeating units of the following formula (l-a) and repeating units of formula (ll-a): wherein R1and R2are as defined above, x and y independently represent 0 or 1 , preferably in an amount of at least 90 mol-% based of the total moles of repeating units present in the copolymer.

[0208] Regarding R1and R2, the same embodiments, preferred embodiments, as well as combinations thereof apply as described above. This is also the case with regard to the statistical copolymer defined below with respect to formula (III).

[0209] According to a preferred embodiment, the lipid composition comprises monomer units of formula (ll-a) relative to the total content of all monomer units of formulae (l-a) and (ll-a) in an amount of from 0.5 to 50 mol-%, preferably from 1 to 40 mol-%, more preferably from 2 to 33 mol-%, even more preferably from 3 to 20 mol-%, and still more preferably from 3 to 17 mol- %.

[0210] According to more preferred embodiments, the lipid composition comprises monomer units of formula (ll-a) relative to the total content of all monomer units of formulae (l-a) and (ll-a) in an amount of from 0.5 to 40 mol-%, preferably from 1 to 30 mol-%, more preferably from 1 to 20 mol-%, even more preferably from 1 to 15 mol-%, and still more preferably from 1 to 10 mol- %.

[0211] The polymerization initiator generally introduces an end group into the statistical copolymer. This end-group may be chemically modified. The end-group is preferably selected from hydrogen, benzyl, Ci-3-alkyl or a Cs-alkenyl group, more preferably methyl or ethyl. This end- group corresponds to group T1in formula (III) below. This end-group preferably contains less than 10, preferably less 5 carbon atoms.

[0212] The statistical copolymer is generally end-capped. The end-cap may correspond to group T2in formula (III) below. This end-group preferably contains less than 10, preferably less than 5 carbon atoms. More preferably, both end groups T1and T2contain less than 10, preferably less than 5 carbon atoms.

[0213] The statistical copolymer of the invention preferably has the following general formula (III):

[0214] T1— {[A]m, [B]n}staf- T2(III) wherein

[0215] T1is an end-group derived from a polymerization initiator,

[0216] T2is an end-group (end-cap) obtained by termination of the cationic polymerization using a nucleophile, or an end group obtainable by modification thereof,

[0217] A are repeating units of the formula (l-a) which may be the same or different;

[0218] B are repeating units of the formula (I l-a) which may be the same or different; m is an integer of at least 5 defining the number of repeating units A in the copolymer, n is an integer of at least 1 defining the number of repeating units B in the copolymer, stat indicates that repeating units A and B are statistically distributed in the copolymer backbone indicated by curly brackets { }, wherein n+m is from 10 to 400, preferably from 20 to 170, more preferably from 30 to 150, even more preferably from 40 to 120. In another preferred embodiment, n+m is from 45 to 90. The repeating units of the formula (l-a) and (ll-b) are as defined above.

[0219] According to a preferred embodiment, T2is selected from a halogen atom, such as fluorine, chlorine, bromine or iodine; an azide group -N3; a fluoro(Ci-6-alkyl)sulfonic acid ester group such as a nonaflate group -OSO2C4F9 or a trifluoromethane sulfonate group -OSO2CF3, a fluorosulfonate group -OSO2F; a Ci-w-aryl- or Ci-8-alkylsulfonic acid group such as a tosyl group CH3-C6H4-SO2- or the mesyl group CH3-SO2-; an unsubstituted, mono- or disubstituted amino group -NH2, -NHR’ or -NR’2, a hydroxyl group -OH, a thiol group -SH, an ester group -OCOR’, a thioester group -SCOR’; a phthalimide group or a cyano group -CN, or functional groups obtainable by modification of these end groups, wherein R’ is a monovalent organic radical of 1 to 6 carbon atoms. According to a preferred embodiment, the statistical copolymer has a number average molecular weight Mnof from 1.000 to 250.000, preferably from 3.000 to 50.000, more preferably from 3.000 to 15.000. The molecular weight is determined by size exclusion chromatography, as described in Example 3 (SEC).

[0220] According to a preferred embodiment, the lipid composition according to the present invention does not contain PEG (polyethylene glycol) and none of said components (i) to (iii) and, if present (iv) is PEGylated.

[0221] The sterol (iv)

[0222] It is pointed out that the sterol as component (iv) of the lipid composition is independent and independently defined herein from a sterol group that may be part of R2. The following section relates to sterol as component (iv) of the lipid composition.

[0223] The lipid composition according to the present invention may, and preferably does, contain a sterol. The sterol may be a phytosterol or cholesterol or combination of a phytosterol and cholesterol. In one embodiment, the sterol is a phytosterol selected from the group consisting of b-sitosterol, stigmasterol, b-sitostanol, campesterol, brassicasterol, and combinations thereof. In one embodiment, the phytosterol is selected from the group consisting of b- sitosterol, b-sitostanol, campesterol, brassicasterol.

[0224] Preferably, the sterol is cholesterol or a cholesterol ester. Examples of cholesterol esters are cholesterol succinic acid, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, and cholesteryl phosphorylcholine.

[0225] The sterol, preferably cholesterol, may be present in an amount of from 0 to 55 % by weight, preferably from 10 to 45 % by weight, more preferably 12 to 40 % by weight, still more preferably 15 to 30 % by weight, based on the total weight of all components (i) to (iv).

[0226] Alternatively, on a molar basis (preferred), the sterol, preferably cholesterol, may be present in an amount of from 0 to 55 mol-%, preferably from 10 to 45 mol-%, more preferably 12 to 40 mol-%, still more preferably 15 to 30 mol-%, based on the total of all components (i) to (iv) of 100 mol-%. The lipid composition of the invention may or may not contain a cholesterol. In one embodiment, wherein the lipid composition may contain little or no of a sterol, the lipid composition may comprise

[0227] (i) from 50 to 90 %, preferably 60 to 80 %, by weight of said one or more cationic lipids, preferably said one or more cationically ionizable lipids,

[0228] (ii) from 8 to 30 %, preferably 12 to 20 %, by weight of said one or more phospholipids,

[0229] (iii) from 0.5 to 25 %, preferably from 1.0 to 10 %, by weight of one or more statistical copolymers, and

[0230] (iv) from 0 to 20 %, preferably from 0 to 10 %, by weight of a sterol, preferably of cholesterol, based on the total weight of all components (i) to (iv).

[0231] On a mole basis, in an embodiment wherein the lipid composition contains little or no of a sterol, the lipid composition may comprise

[0232] (i) from 50 to 90 mol-%, preferably 60 to 80 mol-%, of said one or more cationic lipids, preferably said one or more cationically ionizable lipids,

[0233] (ii) from 8 to 30 mol-%, preferably 12 to 20 mol-%, of said one or more phospholipids,

[0234] (iii) from 0.5 to 25 mol-%, preferably from 1.0 to 10 mol-%, of one or more statistical copolymers, and

[0235] (iv) from 0 to 20 mol-%, preferably from 0 to 10 mol-%, of a sterol, preferably of cholesterol, based on the total of all components (i) to (iv) of 100 mol-%.

[0236] In another embodiment wherein the lipid composition contains substantial amounts of a sterol, the lipid composition may comprise

[0237] (i) from 30 to 60 %, preferably 40 to 55 %, by weight of said one or more cationic lipids, preferably said one or more cationically ionizable, lipid,

[0238] (ii) from 3 to 20 %, preferably 5 to 15 %, by weight of said one or more phospholipids,

[0239] (iii) from 0.2 to 20 %, preferably from 1.0 to 10 %, by weight of said one or more statistical copolymer, and

[0240] (iv) from 20 to 55 %, preferably from 30 to 45 %, by weight of a sterol, preferably of cholesterol, based on the total weight of all components (i) to (iv).

[0241] On a (preferred) mole basis, in another embodiment wherein the lipid composition contains substantial amounts of a sterol, the lipid composition may comprise (i) from 30 to 60 mol-%, preferably 40 to 55 mol-%, of said one or more cationic lipids, preferably said one or more cationically ionizable, lipid,

[0242] (ii) from 3 to 20 mol-%, preferably 5 to 15 mol-%, of said one or more phospholipids,

[0243] (iii) from 0.2 to 20 mol-%, preferably from 1.0 to 10 mol-%, of said one or more statistical copolymer, and

[0244] (iv) from 20 to 55 mol-%, preferably from 30 to 45 mol-%, of a sterol, preferably of cholesterol, based on the total of all components (i) to (iv) of 100 mol-%.

[0245] Possible further components of the lipid composition

[0246] The lipid composition may comprise a solvent, preferably an organic solvent. The organic solvent may be selected from alcohols such as ethanol, iso-propanol or tert.-butanol. The lipid composition may also contain water and may thus be aqueous. The skilled person understands that the composition generally contains an organic solvent before LNP formation and is generally aqueous after LNP formation.

[0247] According to a preferred embodiment, the lipid composition according to the present invention further comprises a payload that may be encapsulated in LNPs. The payload may be a peptide, but preferably a nucleic acid, such as an RNA or a DNA molecule. RNA is preferred. The concentration of the payload may be 0.1 mg / ml or less in the lipid composition. In some embodiments, the concentration of the payload is 10 pg / ml to 200 pg / ml in the lipid composition. In some embodiments, the concentration of the payload is 30 pg / ml to 100 pg / ml in the lipid composition. The type of payload depends on the use of the composition or LNP. The payload may be the active ingredient of a pharmaceutical composition or medicine, see further below.

[0248] The peptide may have a size of from 5 to 200 amino acid residues, preferably from 10 to 100 amino acid residues. The RNA or DNA may have a size of from 10 nucleotides to 10 kb, preferably from 18 nucleotides to 5 kb.

[0249] According to a preferred embodiment, the composition does not comprise PEG or a PEGylated component.

[0250] In the present invention, the lipid composition contains components (i) to (iii) and, if present (iv) and any payload in the form of LNP. Accordingly, the lipid composition may comprise the LNP of the invention or may be a composition comprising the LNPs of the invention. The LNP is described next. The lipid nanoparticle (LNP) and LNP composition

[0251] As used in the present disclosure, "nanoparticle" refers to a particle having an average size or diameter suitable for parenteral administration. Accordingly, the LNP of the present invention may be used as or in a medicine or in a pharmaceutical composition.

[0252] The lipid composition according to the present invention may be provided as LNPs or take the form of LNPs. In some embodiments, the largest dimension of a nanoparticle is 1 pm or smaller (e.g. <1 pm, <900 nm, <800 nm, <700 nm, <600 nm, <500 nm, <400 nm, <300 nm, <200 nm, <175 nm, <150 nm, <125 nm, <100 nm, <75 nm, <50 nm), when measured by dynamic light scattering (DLS). The particle size of the LNP is preferably measured and indicated in terms of the commonly used Z-average diameter and may be determined as given in Example 4.

[0253] The particle size of the LNP in terms of the Z-average diameter may range from about 40 nm to about 700 nm, from about 50 nm to about 500 nm, from about 60 nm to about 400 nm, from about 70 nm to about 300 nm, or from about 80 to 200 nm, in this order of increasing preference.

[0254] The present invention provides an LNP obtained or obtainable from the lipid composition and optionally comprising a payload. The payload may be, as described above, a peptide or a nucleic acid contained in or encapsulated in said LNP. RNA and DNA are preferred payloads and RNA is most preferred in view of the medical application of the LNP or lipid composition. The size of the peptide, RNA and DNA may be as described above. The Z-average diameter values and ranges given above also apply to the LNP containing a payload, independent from the type of payload.

[0255] The size or size range of the LNP containing a payload is generally as given above for the LNP. The composition of the LNP in terms of components (i) to (iii) and, if present, (iv) may be as given above for the lipid composition.

[0256] The LNP of the invention should have a polydispersity index (PDI) of at most 0.5, preferably at most 0.4, more preferably of at most 0.3. The PDI may be determined as described in Example 4.

[0257] If the payload is a nucleic acid, the ratio N / P of the LNP, indicating the molar ratio of cationic amine groups (N = nitrogen) of the cationic lipid to negatively-charged nucleic acid phosphate groups (P) of the nucleic acid, may be from 1 to 25, preferably from 2 to 20, more preferably from 3 to 17.

[0258] A composition comprising the LNP of the invention is also referred to herein as LNP composition. The LNP composition generally contains a solvent, usually an aqueous solvent. Thus, the composition is generally a liquid composition, usually a liquid aqueous composition. Alternatively, the LNP composition may be frozen. The composition generally contains a buffer to stabilize the pH in the desired range, such as from 5 to 8, preferably from 6 to 7.5. The buffer may be, depending on the intended application or use of the composition and the target pH, for example an acetate buffer or a phosphate buffer.

[0259] Regarding the composition of the LNP of the invention with regard to components (i) to (iii) and, if present, (iv), the same definitions as given above in the context of the lipid composition applies. Notably, depending on whether the LNPs contain little or no sterol (iv) or contains substantial contents of sterol (iv), the two embodiments of compositions given for the lipid composition apply also to the LNP and LNPs in the LNP composition.

[0260] According to a preferred embodiment, the composition does not comprise PEG or a PEGylated component.

[0261] Payload of the LNPs

[0262] As mentioned above, the payload may be a peptide, but is preferably a nucleic acid, such as an RNA or a DNA. RNA is preferred. The peptide may have a size of from 5 to 200 amino acid residues, preferably from 10 to 100 amino acid residues. The nucleic acid may have a size of from 10 nucleotides to 10 kb, preferably from 18 nucleotides to 5 kb.

[0263] The RNA may be a single-stranded RNA, especially mRNA. In some embodiments, the RNA comprises one or more modified nucleosides in place of uridine (modRNA), wherein the modified nucleoside is preferably selected from pseudouridine (qj), N-methyl-pseudouridine (m1qj), and 5-methyl-uridine (m5U). In some embodiments, the RNA comprises at least one of the following, preferably all of the following: a 5’ cap; a 5’ UTR; a 3’ UTR; and a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides. The 5’ cap may be a cap1 or cap2 structure.

[0264] The type of payload depends on the use of the composition or LNP. The payload may be the active ingredient of a pharmaceutical composition or medicine, or it may be a nucleic acid encoding a peptide or protein that may be expressed in cells, preferably eukaryotic cells, in a subject (patient), or in cells of a subject, see further below.

[0265] Examples of peptides and proteins include, but are not limited to, immunostimulants, e.g., cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins. More specific examples are cytokines, such as interferon-alpha (IFN-a) or interferon-gamma (IFN-y), interleukins, such as IL2, IL7, I L12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. Further, said peptide may be an antigen used for vaccination by administering to a patient the LNP of the invention.

[0266] The amount of payload that may be incorporated into the LNPs, in the case of nucleic acid, was defined above with respect to the N / P ratio.

[0267] If the payload is a protein or peptide, the amount of protein or peptide may be from 0.5 to 10 % by weight, preferably from 1 to 7 % by weight, more preferably from 1.5 to 5 % by weight based on the total weight of components (i) to (iv) and the weight of the protein or peptide.

[0268] A method of producing LNPs

[0269] The present invention provides a method of producing the LNP of the invention using the statistical copolymer of the invention. The method may comprise mixing (i) one or more cationic lipids, (ii) one or more phospholipid, (iii) one or more statistical copolymer of the present invention, and (iv) optionally a sterol.

[0270] Methods of forming LNP are generally known in the art, e.g. by rapid mixing of two or three different solutions. A first solution may contain components (i), (ii), (iii) and, if present, (iv) in the concentrations given above in an organic solvent. A second solution may contain the payload, generally in an aqueous solution. A possible third solution may be an aqueous buffer solution. Apparatuses for rapid mixing are commercially available, and several different ones are used in the Examples. Thereby, LNP compositions as described above can be obtained. The LNPs may be stored refrigerated or in frozen state.

[0271] Methods of treatment and medical uses The present invention provides LNP or a lipid composition of the present invention for use in a method of treatment or prevention of a disease or disorder in a subject. In particular, a LNP composition may be used in a method for human medical purposes and / or for veterinary medical purposes, preferably for human medical purposes.

[0272] The disease or disorder to be treated or prevented generally depends on the payload present in the LNP of the invention. The payload can be considered the active pharmaceutical ingredient of the invention. The payload may be a peptide or a nucleic acid (generally RNA or DNA). Thus, any disease or disorder that may be treated or prevented by delivering a peptide or nucleic acid may be treated or prevented. Among preventions, vaccinations are preferred. A widely known vaccination is the Covid-19 vaccination, wherein a modRNA encoding the SARS-CoV-2 virus spike protein or a portion thereof may be used as the payload of the LNP of the invention for delivery of the modRNA into cells of a subject.

[0273] Moreover, the present invention provides a method for delivering nucleic acid to cells of a subject, the method comprising administering to the subject the LNP composition or LNP according to the present invention, wherein said LNP contains said nucleic acid.

[0274] Furthermore, the present invention provides a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to a subject the LNP composition or LNP according to the present invention, wherein the LNP contains a nucleic acid that encodes the therapeutic peptide or protein.

[0275] The present invention also provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject the LNP composition or LNP according to the present invention, wherein delivering a nucleic acid contained or encapsulated in said LNP to cells of the subject is beneficial in treating or preventing the disease or disorder.

[0276] The present invention provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject LNP composition or LNP according to the present invention, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

[0277] In preferred embodiments, the present invention provides a method of treating or preventing a disorder or condition, wherein the method comprises applying or administering to a subject in need thereof an LNP composition or LNP according to the present invention. Accordingly, the LNP composition, may be used in a method of treating or preventing a disorder or condition, wherein the disorder or condition can be any disorder, disease, or condition which can be treated or prevented by use of an antibody or fragment thereof, in particular cancer, cardiovascular diseases, neurological diseases, infectious diseases, autoimmune diseases, virus diseases, bacterial diseases, genetic diseases or disorder and diseases or disorders related thereto.

[0278] In preferred embodiments, the invention provides a method of treating or preventing a disorder or condition, wherein the method comprises applying or administering to a subject in need thereof an LNP composition or LNP according to the invention, wherein the disorder or condition is an infection with a pathogen, a cardiovascular disease or condition, a neurological disease or condition, an infectious disease or condition, an autoimmune diseases or condition, a cancer or tumor disease or condition, an eye or ophthalmic disease or condition, a lung or pulmonary disease or condition, a neurological disease or condition, a genetic disease or condition, or a lung disease or condition.

[0279] In preferred embodiments, the subject in need thereof is a mammalian subject, preferably a human subject.

[0280] In preferred embodiments, the method of treatment is a treatment of a chronic disease, whereby administration is performed more than once, for example once or more than once a day, once or more than once a week, once or more than once a month.

[0281] In preferred embodiments, the method of treatment comprises a step of applying or administering to a subject, wherein applying or administering may be parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intraocular, intravitreal, subretinal, intranasal or intratumoral administration. Preferred administration routes are parenteral, such as subcutaneous, intravenous, or intramuscular. In particularly preferred embodiments, the administration is intravenous or intramuscular.

[0282] The Statistical Copolymer

[0283] The present invention also relates to a specific statistical copolymer. The statistical copolymer of the present invention is a copolymer obtainable by co-polymerizing two or more types of monomers having different substituents in 2-position of the monomer heterocycle by cationic polymerization. In this kind of copolymer, the arrangement of the repeating units along the polymer chain may be of random, alternating, periodic, or gradient manner.

[0284] The present invention provides a statistical copolymer obtained or obtainable by a method comprising cationic ring-opening polymerization (CROP) of a mixture of (a) and (b) in the presence of a polymerization initiator, as described herein. The invention also provides a statistical copolymer, wherein the statistical copolymer comprises repeating units of formula (l-a) and repeating units of formula (ll-a), as defined herein. The invention also provides a statistical copolymer of the general formula (III) described herein. The preferred embodiments described above also apply to the statistical copolymer of the invention.

[0285] In a preferred embodiment of the statistical copolymer, R1is hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group (with preferred embodiments as described above) and R2is an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double bonds (generally non-conjugated), and having 1 to 5, preferably 1 to 3 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents. Regarding the preferred number and types of the one or more carbon-carbon double bonds, reference is made to the disclosure above.

[0286] Further preferred is the case where R2is an unsaturated C4-40 heterohydrocarbyl group having one or two non-conjugated carbon-carbon double bonds, and having 1 or 2 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate and carbonate groups, preferably selected from ether, ester and amide groups. Even more preferred is the case of 1 carbon-carbon double bond and one divalent group selected from ether, amide, and ester groups, preferably ester groups.

[0287] In a specific embodiment, group R2is a saturated or unsaturated C18-40 (preferably C18-35) heterohydrocarbyl group that contains a sterol group, wherein the sterol group as well as its preferred embodiments are as described above. Where R2is or comprises a sterol group, it may be or comprise a cholesteryl group, and may be a cholesterylesteralkyl group or cholesterylesteralkenyl group (the former being preferred), wherein the alkyl or alkenyl moiety may have from 1 to 5, preferably 2 or 3, carbon atoms. This embodiment may be combined with the preferred embodiments of R1, notably wherein R1is methyl or ethyl. The skilled person understands that these preferred embodiments are at the same time preferred embodiments for the lipid composition and LNP of the invention. In one embodiment, the statistical copolymer of the invention is one wherein

[0288] R1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group, and

[0289] R2is a group of the following formula (V): wherein

[0290] R6are independently hydrogen atoms or methyl groups, preferably the R6are the same, more preferably both R6are methyl groups,

[0291] R7is a linear or branched C1-10 alkyl group, or a linear or branched C2-10 alkenyl group, preferably it is a branched C3-8 alkyl group, or a branched C3-8 alkenyl group,

[0292] L is a linear or branched C1-5 alkylene group or a linear or branched C2-5 alkenylene group, preferably a linear C2-3 alkylene group,

[0293] M is a carbonyl group or single bond linking the adjacent groups (L and O), and the dashed line next to the bond (in ring B) means that the bond is either a single bond or a double bond, preferably a double bond.

[0294] The embodiments and preferred embodiments regarding R1and the combination with R2, the ratio of groups of formula (l-a) and (I l-a) and the ratios of groups of formula (I) and (II) in said mixture, also apply to the statistical copolymer. This also applies to the polymerization degrees indicated by the number n+m in the polymer and the preferred molecular weight ranges.

[0295] Selected preferred embodiments

[0296] A lipid composition or an LNP composition comprising:

[0297] (i) from 30 to 60 % by weight of the one or more cationic lipid,

[0298] (ii) from 3 to 20 % by weight of the one or more phospholipid,

[0299] (iii) from 0.7 to 10 % by weight, preferably from 1.0 to 7 % by weight, of the one or more statistical copolymers as defined herein, and

[0300] (iv) from 20 to 55 % by weight of cholesterol, wherein

[0301] R1is methyl or ethyl,

[0302] R2is as defined herein, x and y are both 0,

[0303] A, B, m, n, T1, and T2, as far as applicable, are as defined herein, the molar ratio of R2to the total R1and R2in the one or more statistical copolymers, and / or the ratio of compounds of formula (II) relative to the total of compounds of formula (I) and (II) present in or added to the mixture, is 3 to 15 mol-%, preferably from 3.5 to 12 mol-%, more preferably from 3.5 to 10 mol-%, and even more preferably from 4.0 to 7.0 mol-%.

[0304] Even more preferable, the molar ratio of R2to the total R1and R2in the one or more statistical copolymers, and / or the ratio of compounds of formula (II) relative to the total of compounds of formula (I) and (II) present in or added to the mixture, is 1 to 15 mol-%, preferably from 1 to 10 mol-%.

[0305] List of abbreviations used EXAMPLES

[0306] Introduction: Production of statistical polymers of the invention

[0307] Prior art lipid nanoparticle (LNP) formulations generally comprise a PEGylated lipid to ensure particle stability and to provide the LNP with a certain "stealth effect". However, the overuse of PEG-based materials, e.g. in cosmetics, has resulted in a steadily increasing proportion of patients expressing antibodies against PEG. In this invention, the "PEG dilemma" is overcome by replacing the hydrophilic PEG by the statistical copolymers of the invention, such as certain poly(2-oxazoline)s (POX) or poly(2-oxazine)s (POZ).

[0308] The PEGylated lipids in prior art LNP formulations comprise a hydrophobic end group attached at the hydrophilic polymer to enable an anchoring in the LNP, thus representing amphiphiles (Fig. 1A). In the present invention, the hydrophilicity / lipophilicity is tuned by variation of the substituents in 2-position of the monomer heterocycle (oxazoline or oxazine). POX with aliphatic substituents comprising four or more carbon atoms are considered hydrophobic. Amphiphilic statistical POX or POZ copolymers can be obtained by copolymerization of hydrophilic and hydrophobic monomers. A small fraction of a monomer comprising a long aliphatic substituent provides anchoring points in a hydrophobic LNP, whereas the large fraction of hydrophilic repeating units tends to arrange in "loops" on the LNP surface (Fig. 1B). The length of the lipophilic side chain derived from the hydrophobic monomers influences the (strength of the) anchoring in the LNP. In addition, the hydrophobic / hydrophilic monomer fraction in the polymer affects the size of the "loops" at the LNP surface (Fig. 1C).

[0309] For that purpose, the hydrophobic monomers are copolymerized with the hydrophilic monomers, such as 2-ethyl-2-oxazoline (EtOx) and / or 2-methyl-2-oxazoline (MeOx). Thereby, a variation of the monomer feed ratios is targeted to adjust the hydrophilicity of the materials (5 mol% to 40 mol% hydrophobic 2-oxazoline). In particular, copolymers featuring a certain (low) content of the hydrophobic monomers behave as amphiphiles and thus were considered to represent alternatives to PEG-based lipids. Literature data describing synthesis and several properties exist for several monomer combinations (e.g. Colloid Polym. Sci. 2006, 284, 1313- 1318; J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 515-522). The final copolymer composition of the purified polymers can be assessed by means of NMR spectroscopy.

[0310] The first two hydrophobic monomers were specified based on literature research. 2-n-Nonyl- 2-oxazoline (NonOx) was selected because the properties of statistical and block copolymers comprising NonOx were studied earlier. In contrast, little was known regarding the polymerization of SoyOx and properties of SoyOx containing copolymers. SoyOx is obtained from fatty acids in soybean oil (C18-fraction), thus representing a mixture of 2-oxazolines based on stearic acid, oleic acid and linoleic acid The SoyOx used here contained 2-oxazolines based on stearic acid (4 mol%), oleic acid (21 mol%) and linoleic acid (75 mol%) (Fig. 2).

[0311] The microwave-assisted cationic ring-opening polymerization (CROP) of SoyOx in bulk as well as in acetonitrile as a solvent was first reported in 2005 (R. Hoogenboom, U. S. Schubert, Green Chem. 2006, 8, 895-899.). Further publications describe the assembly of SoyOx- containing copolymers, from which it was considered statistical copolymers of SoyOx and EtOx could perform likewise (R. Hoogenboom, M. A. M. Leenen, H. Huang, C.-A. Fustin, J.-F. Gohy, U. S. Schubert, Colloid Polym. Sci. 2006, 284, 1313-1318; H. Huang, R. Hoogenboom, M. A. M. Leenen, P. Guillet, A. M. Jonas, U. S. Schubert, J.-F. Gohy, J. Am. Chem. Soc. 2006, 128, 3784-3788). In addition, the copolymerization parameters are known for this monomer pair, confirming an even distribution of both repeating units along the polymer chain. These known polymerization conditions were adapted for the CROP of the other monomer pairs (SoyOx / MeOx, NonOx / EtOx, NonOx / MeOx) to enable a straightforward comparison of the individual copolymers with each other.

[0312] Contact angle measurements of a series of films of statistical copolymers P(EtOx-sfaf-SoyOx) were reported (R. Hoogenboom, H. M. L. Thijs, M. W. M. Fijten, U. S. Schubert, J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 5371-5379), which was useful to estimate suitable copolymer compositions. Molar SoyOx fractions between 5 mol% und 40 mol% were considered promising. As low molar fractions of SoyOx seem particularly promising, these molar fractions were also targeted for the other copolymer pairs, although MeOx (instead of EtOx) as well as NonOx (replacing SoyOx) was considered to increase the overall hydrophilicity of the copolymers.

[0313] The lipophilic monomer was further varied. 2-(1-Ethylpentyl)-2-oxazoline (EtPentOx, Fig. 3) comprises a branched alkyl substituent, resulting in homopolymers featuring a low glass transition temperature (K. Kempe, E. F. J. Rettler, R. M. Paulus, A. Kuse, R. Hoogenboom, U. S. Schubert, Polymer 2013, 54, 2036-2042.). This is advantageous with respect to the self-assembly of amphiphilic block copolymers (U. Mansfeld, S. Hoeppener, K. Kempe, J.-M. Schumers, J.-F. Gohy, U. S. Schubert, Soft Matter 2013, 9, 5966-5974) as well as in amphiphilic co-networks (S. A. Wilhelm, M. Maricanov, V. Brandt, F. Katzenberg, J. C. Tiller, Polymer 2022, 242, 124582). Similar effects come into play during the formulation of LNP. The branching of EtPentOx at C1 of the substituent impacts the polymerization kinetics: the polymerization rate constant kpdecreases when compared to monomers with linear substituents, i.e. the CROP proceeds slower (K. Kempe, A. Baumgaertel, R. Hoogenboom, U. S. Schubert, J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 5100-5108).

[0314] Example 1 : Production and characterization of poly(2-oxazoline)s

[0315] Materials

[0316] EtOx (Acros Organics, 99%+) and MeOx (Sigma-Aldrich, 98%) were dried over barium oxide or calcium hydride and distilled under argon atmosphere. SoyOx (> 99%), NonOx (> 99%) and EtPentOx (> 99%) were obtained from NGP Polymers and used as received. Methyl tosylate (abcr, 98%) was distilled under reduced pressure and stored under argon atmosphere. Acetonitrile (99,9%, extra dry over molecular sieve) and Ambersep 900-OH’ were purchased from Thermo Scientific. All other chemicals were obtained from common suppliers and used as received.

[0317] Synthesis

[0318] The reaction solutions were prepared in a glovebox ( BRAUN equipped with an UNIIab gas purification system) under nitrogen atmosphere. Each solution was based on an initial overall monomer concentration [M]o of 1.5 mol L1in acetonitrile and a molar ratio of [MeTos]o to [M]o of 1 : 60. The hydrophobic monomer fraction (i.e. SoyOx, NonOx, or EtPentOx) was varied (5 mol%; 10 mol%; 15 mol%; 20 mol%; 25 mol%; 30 mol%; 40 mol%). In an exemplary copolymerization of 60 mol% EtOx with 40 mol% EtPentOx, 1.34 g (13.5 mmol) EtOx, 1.52 g (9 mmol) EtPentOx and 70 mg (0.375 mmol) MeTos were dissolved in 12.7 mL acetonitrile using an appropriate microwave reaction vial. The vials were capped inside the glovebox, and subsequently removed from it. The CROPs were conducted using a Biotage lnitiator+ microwave at the following settings: Temperature control, 140 °C, absorption level "very high". CROP comprising SoyOx and NonOx were conducted for 30 min polymerization time, whereas CROP comprising EtPentOx were performed for 45 min. Subsequently, all CROP were terminated by addition of 100 pL of water. Samples were withdrawn from the reaction solutions for analysis by means of SEC and NMR spectroscopy.

[0319] Except for copolymers with an increased MeOx fraction, the solutions were concentrated under reduced pressure, diluted with chloroform, and washed with aqueous NaHCOs solution and brine. The organic phases were dried over Na2SO4 and filtered. The solvent was removed under reduced pressure and the purified polymers were dried in vacuo.

[0320] Polymers JC791 to JC793, JC801 , and JC818 to JC821 were purified as follows: Acetonitrile was removed under reduced pressure. The polymers were then dissolved in water and eluted over a short column filled with Ambersep 900-OH’. The solutions were concentrated under reduced pressure and the residual water was removed via lyophilization.

[0321] JC800 was first extracted as described above and subsequently treated with Ambersep 900- OH’. All purified polymers were analyzed by means of SEC and1H NMR-spectroscopy.

[0322] Characterization

[0323] Proton (1H) nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AC 300 MHz spectrometer. The measurements were performed at room temperature using deuterated chloroform as a solvent. Chemical shifts (6) are given in parts per million (ppm) relative to the residual non-deuterated solvent resonance signal.

[0324] Size exclusion chromatography of MeOx-containing polymers (SEC9) was measured on an Agilent 1200 series system equipped with a PSS degasser, a G1310A pump, a G1329A auto sampler, a Techlab oven at 40 °C, a G1362A refractive index detector (RID) and a PSS GRAM guard / 30 / 1000 A column (10 pm particle size). / V, / V-Dimethylacetamide (DMAc) with 0.21 wt% LiCI was used as an eluent at a flow rate of 1 mL min"1. Polystyrene (PS) standards (400 to 1 ,000,000 g mol"1) were used to calculate the molar masses.

[0325] Size exclusion chromatography of EtOx-containing polymers (SEC1) was measured on a Shimadzu system equipped with a CBM-20A system controller, a LC-10AD VP pump, a RID-10A refractive index detector, a SPD-10AD VP UV detector, and a SDV linear S column from PSS at 40 °C using CHC^EtjN: / so-propanol (94:4:2) as eluent at a flow rate of 1 mL min-1. The calibration was made of ten separate polystyrene polymer standards of narrow molar mass distribution (Supplier: Polymer Standards Service GmbH, PSS, Mainz, Germany, Mp= 370 to 128 000 g mol’1).

[0326] Example 2: Results of production and characterization of poly(2-oxazoline)s

[0327] Seven compositions were selected for each monomer pair, i.e. MeOx / SoyOx, EtOx / SoyOx, MeOx / NonOx, EtOx / NonOx, MeOx / EtPentOx, EtOx / EtPentOx leading to a library of 42 statistical copolymers (Table 1).

[0328] In agreement with kinetic studies conducted initially, the monomer conversion was (nearly) quantitative after 30 min polymerization time of the CROP conducted at 140 °C in acetonitrile using methyl tosylate as an initiator ([M]o / [l]o = 60, [M]o= 1.5 M) for the copolymerizations of MeOx / SoyOx, EtOx / SoyOx, MeOx / NonOx and EtOx / NonOx, respectively. Accordingly, the composition of the final copolymers determined by means of1H NMR spectroscopy closely resembled the initial monomer feed ratio. To identify suitable polymerization conditions for the copolymerizations involving EtPentOx, two kinetic studies were conducted (40% EtPentOx 160% EtOx and 10% EtPentOx 190% EtOx). Based on the obtained results, a polymerization time of 45 min was selected, whereas all other reaction parameters and the targeted copolymer compositions were kept as described above. Accordingly, the monomer conversions of those 14 CROPs were (nearly) quantitative also in these cases (Table 1).

[0329] Table 1 : Selected characterization data of the statistical copolymers.

[0330] aOverall monomer (M) conversion determined by1H-NMR spectroscopy (CDCI3, 300 MHz) of the reaction solution.bdetermined by SEC1 (CHCI3, RID, PS calibration) for EtOx-containing copolymers and by SEC9 (DMAc, RID, PS calibration) for MeOx-containing copolymers.cdetermined by1H-NMR spectroscopy (CDCI3, 300 MHz) of the purified copolymers by integration of signals assigned to the POx-backbone and to the methyl protons of NonOx, SoyOx or EtPentOx.

[0331] Analysis of the copolymers by means of size exclusion chromatography (SEC) indicated narrow molar mass distributions. With a few exceptions, dispersity values D remained below 1.15 when comprising NonOx or SoyOx and below 1.2 when comprising EtPentOx (Fig. 4 to Fig. 6). Except for the comonomer combination EtOx / EtPentOx, an increased molar fraction of the hydrophobic monomer resulted in an earlier elution of the copolymers, which is in agreement with the increase in molar masses Mn.

[0332] 1H NMR spectroscopy confirmed that the purified copolymers did not contain any toluene sulfonic acid resulting from the counter ion of the CROP. The spectra were also used to estimate the copolymer compositions. The signal integrals assigned to the POx-backbone at 3.5 ppm (signal B) and those assigned to the methyl protons of the hydrophobic repeating units at 0.85 ppm (signal A) were used for that purpose as they did not overlap with any other signals irrespective of the monomer combination. As is evident from Fig. 7 to Fig. 12, the signal intensity of "A" increased with increased content of the hydrophobic monomer (NonOx, SoyOx, or EtPentPOx) after normalization according to signal B. All other signals in the spectra can be assigned as well, which is not depicted in the figures for clarity. The double bond content in the SoyOx-containing copolymers remained unaffected by the CROP, as confirmed by integration of the vinyl proton signals at 5.4 ppm.

[0333] Example 3: Synthesis of statistical poly(2-oxazoline)-based copolymers comprising hydrophilic monomers and a novel 2-oxazoline functionalized with cholesterol

[0334] Cholesterol usually forms a large portion of a lipid nanoparticle. The inventors conceived that a statistical copolymer comprising cholesterol-containing units as the hydrophobic comonomer units could be anchored in an LNP. A 2-oxazoline that is functionalized with cholesterol in 2- position of the heterocycle was designed (Fig. 13). The synthesis procedure relied on the formation of a chloroethyl amide derivative formed through ethyl chloroformate-mediated reaction of cholesterol hydrogen succinate and 2-chloroethylamine. In a second step, the 2- oxazoline ring was formed using potassium carbonate as a base. The novel monomer CholOx was obtained in overall yields of 67% and fully characterized via1H and13C NMR spectroscopy, high-resolution electrospray mass spectrometry (HR-ESI MS) as well as elemental analysis (see experimental section).

[0335] As the good performers among the other statistical copolymers generally contained on the order of 5 mol% units of the hydrophobic monomer, we targeted statistical copolymers based on the monomer combinations CholOx / MeOx and CholOx / EtOx with a molar fraction of 5% CholOx. As CholOx could not be solubilized in acetonitrile, the polymerization conditions of the CROP had to be modified compared to those of the other statistical copolymers. Benzonitrile was used as solvent at an initial total monomer concentration [M]oof 1 mol L1. Higher monomer concentrations were prohibited by the restricted solubility of CholOx. Methyl tosylate was used a CROP initiator at an initial [M]o / [l]o of 60 (Fig. 14). The required polymerization times were estimated from kinetic studies performed for the homopolymerizations of MeOx and EtOx, respectively, at 140 °C. Accordingly, the monomer conversions of both CROPs were quantitative (Table 2).

[0336] Experimental The materials were as described in Example 1. Benzonitrile (> 99%) and 2-chloroethylamine hydrochloride (> 99%) were purchased from Sigma-Aldrich. Ethyl chloroformate (97%) was obtained from AlfaAesar. Cholesterol hydrogen succinate (97%) was purchased from TCI.

[0337] Synthesis of CholOx

[0338] (3S,8S,9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-

[0339] 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro- 1H-cyclopenta[a]phenanthren-3-yl 4-( (2- chloroethyl)amino)-4-oxobutanoate (1); see Fig. 13, first step

[0340] Triethyl amine (3.6 ml, 2.62 g, 0.026 mol) was added to a solution of cholesterol hydrogen succinate (10 g, 0.021 mol) in THF (180 ml). The mixture was cooled in an ice bath for around 20 min. Ethyl chloroformate (2.5 ml, 2.85 g, 0.026 mol) was added slowly. The reaction was allowed to reach room temperature and kept for 1 h. A slurry of 2-chloroethylamine hydrochloride (2.5 g, 0.022 mol) and triethylamine (3.4 ml, 2.47 g, 0.024 mol) in DMF (40 ml) was added. The mixture was stirred at room temperature for two days. The volatiles were evaporated. The residue was dissolved in dichloromethane and washed with water and aq. sodium hydrogencarbonate solution. The organic phase was dried over sodium sulphate and filtered. The solvent was evaporated. Yield: 10.63 g (94%).1H NMR (CDCI3, 300 MHz): 5 (ppm) 6.18 (s, broad, N / 7, 1 H), 5.38 (d, J = 4.3 Hz, C=C-H, 1 H), 4.70-4.54 (m, CH-OC=O, 1 H), 3.62 (s, (C / 72)2-CI, 4H), 2.71-2.60 (m, CH2-CH2-C=O, 2H), 2.56-2.48 (m, -CH2-CH2-C=O, 2H), 2.36- 2.30 (m, C=C-CH2-CH, cholesterol, 2H), 2.07-1.93 (m, CH2, cholesterol, 2H), 1.93-1.79 (m, CH2, cholesterol, 3H), 1.68-0.79 (m, CH2, CH+5s CH3, cholesterol, 34H), 0.69 (s, CH3, 3H).13C NMR (CDCh, 75 MHz): 5 (ppm) 172.34 (Cq), 171.80 (Cq), 139.55 (Cq), 122.72 (C=C-H), 74.52 (CH-OC=O), 56.68 (CH), 56.14 (CH), 50.02 (CH), 43.96 (CH2-CH2-CI), 42.3, 41.27 (CH2-CH2- Cl), 39.73 (CH2), 39.51 (CH2), 38.06 (C=C-CH2-CH), 36.96 (CH2), 36.58 (CH2), 36.18 (CH2), 35.78 (CH), 31.89 (CH2), 31.85 (CH), 31.07 (CH2-CH2-C=O), 29.88 (CH2-CH2-C=O), 28.21 (CH2), 28.00 (CH), 27.73 (CH2), 24.27 (CH2), 23.83 (CH2), 22.81 (CH3), 22.55 (CH3), 21.02 (CH2), 19.30 (CH3), 18.71 (CH3), 11.85 (CH3). HR-ESI MS [m / z]: calcd. for C35H54CINO3: [M+Na]+, 570.3684; found: 570.3648, error 4.9 ppm. EA: calcd. for: C35H54CINO3; C, 72.30; H, 9.93; N, 2.55; Found: C, 72.88; H, 10.03; N, 2.53.

[0341] (3S,8S,9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-

[0342] 2,3,4, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1 H-cyclopenta[a]phenanthren-3-yl 3- (4,5-dihydrooxazol-2-yl)propanoate (CholOx); see Fig. 13, second step 1 (10 g, 0.019 mol) was dissolved in DMF (230 ml) and potassium carbonate (2.9 g, 0.021 mol) was added. The mixture was stirred overnight at 70 °C. After cooling to room temperature, the reaction mixture was extracted with n-heptane. The combined n-heptane layers were washed with water and aq. sodium hydrogencarbonate solution and dried over sodium sulphate. After filtration, the solution was concentrated until a white precipitate appeared. Subsequent to storage at -20 °C overnight, the product was collected by centrifugation and dried in vacuo. Yield: 6.97 g (71%).1H NMR (CDCI3, 300 MHz): 5 (ppm) 5.38 (d, J = 4.6 Hz, C=C-H 1 H), 4.71- 4.56 (m, C / 7-OC=O, 1 H), 4.3-4.22 (t,3J = 9.44 Hz, CH2, oxazoline, 2H), 3.86-3.79 (t,3J = 9.45 Hz, CH2, oxazoline, 2H), 2.71-2.48 (m, CH2-CH2-C-O, 2H), 2.37-2.29 (m, CH2-CH2-C-O, 2H), 2.07-1.93 (m, CH2, cholesterol, 2H), 1.93-1.77 (m, CH2, cholesterol, 3H), 1.69-0.78 (m CH2, CH+5s CH3, cholesterol, 34H), 0.69 (s, CH3, 3H).13C NMR (CDCI3, 75 MHz): 5 (ppm) 171.65 (Cq), 167.13 (Cq, oxazoline), 139.64 (Cq), 122.64 (C=C-H), 74.25 (CH-OC=O), 67.45 (CH2, oxazoline), 56.69 (CH), 56.14 (CH), 54.38 (CH2, oxazoline), 50.02 (CH), 42.31 , 39.73 (CH2), 39.51 (CH2), 38.05 (C=C-CH2-CH), 36.97 (CH2), 36.59 (CH2), 36.18 (CH2), 35.78 (CH), 31.90 (CH2), 31.86 (CH), 30.70 (CH2-CH2-C=O), 28.21 (CH2), 28.00 (CH), 27.72 (CH2), 24.27 (CH2), 23.82 (CH2), 23.24 (CH2-CH2-C=O), 22.80 (CH3), 22.55 (CH3), 21.02 (CH2), 19.31 (CH3), 18.71 (CH3), 11.85 (CH3). HR-ESI MS: calcd. for C33H53NO3: [M+H]+, 512.4098; found: 512.4070, error 5.4 ppm. C, 77.45; H, 10.44; N, 2.74; Found: C, 77.78; H, 10.49; N, 2.77.

[0343] Cationic ring-opening polymerization

[0344] CROP was done as described in Example 1 , except the following. CROP comprising CholOx were based on 5 mol% hydrophobic monomer and performed in benzonitrile an initial overall monomer concentration [M]o of 1 mol L’1. Polymerization times were 33 min for the CROP of CholOx / MeOx and 43 min for the CROP of CholOx / EtOx, respectively.

[0345] Polymer JC847 was purified as follows: the reaction solution was precipitated into diethyl ether, re-dissolved in water and eluted over a short column filled with Ambersep 900-OH’. The solution was concentrated under reduced pressure and the residual water was removed via lyophilization. Polymer JC845 was purified as follows: The volatiles were removed at reduced pressure. The residue was dissolved in chloroform and washed with water and aq. sodium hydrogencarbonate solution. The organic phase was dried over sodium sulphate and filtered. The solution was concentrated and precipitated into cold diethyl ether.

[0346] Proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra were obtained as described in Example 1. High-resolution electrospray mass spectrometry (HR-ESI MS) was performed in positive ion mode on a MicroQTOF mass spectrometer from Bruker. The instrument was calibrated with a standard (m / z = 50 to 3,000, ESI-L Low Concentration Tuning Mix, Agilent Technologies). Elemental analyses (EA) were measured using a Eurovector EA3000 (Leintech) analyzer. Size exclusion chromatography was done as described in Example 1.

[0347] Results

[0348] Analysis of the copolymers by means of size exclusion chromatography indicated narrow molar mass distributions with dispersity values D < 1.15 (Fig. 15).1H NMR spectroscopy confirmed that the purified copolymers did not contain any toluene sulfonic acid resulting from the counter ion of the CROP. Due to the quantitative monomer conversions, the copolymer compositions estimated from the spectra were in line with the feed ratios. The signal integrals assigned to the POx-backbone at 3.5 ppm (signal B) and those assigned to the methyl protons at C13 of the cholesterol moieties at 0.67 ppm (signal A) were used for that purpose as depicted in Fig. 16 to Fig. 17. In addition, the vinylic proton and the methine proton at C3 of the cholesterol moieties were clearly evident from the spectra, thereby confirming the incorporation of the novel monomer into the statistical copolymers.

[0349] Table 2: Selected characterization data of the statistical copolymers comprising CholOx. aOverall monomer conversion determined by1H NMR spectroscopy (CDCI3, 300 MHz) of the reaction solution.bdetermined by SEC1 (CHCI3, RID, PS calibration) and by SEC9 (DMAc, RID, PS calibration), respectively.

[0350] Example 4: Screening and production of LNPs

[0351] Suitable POX polymers were subjected to a design of experiments (DoE) for stable manufacturing (Rampado, R.; Peer, D., Design of experiments in the optimization of nanoparticle-based drug delivery systems. Journal of Controlled Release 2023, 358, 398- 419.). DoE is a statistical method employed for planning, conducting, and analyzing experiments. It involves systematically and efficiently designing an experiment to study and analyze the effects of various input variables (factors) on the outcome variables (target values). The factors involved in a design of experiment include both critical process parameters (CPP) (e.g. microfluidics setup) and critical material parameters (CMP) (e.g. polymer candidate). Regarding the evaluation of the lipid nanoparticles obtained, critical quality attributes (CQA) are defined to determine the quality of the particles produced. These include size, polydispersity index (PDI), encapsulation, and transfection efficiency (EE). A specific mathematical design is chosen depending on the purpose, either screening or optimization. For this purpose, a response surface method was employed, focusing on modeling the interaction between CPPs and CQAs. This creates a function that can be visualized as a multidimensional surface for each outcome, where the independent variables are CPPs.

[0352] Materials

[0353] PolyA was supplied by Sigma Aldrich (Darmstadt, Germany). EGFP mRNA was supplied by Ribopro (Noord-Brabant, Netherlands), CleanCap® EGFP mRNA was supplied by Trilink Bio Technologies (San Diego, CA, USA). DSPC and Cholesterol were from Sigma Aldrich (Darmstadt, Germany). PhytoChoi®, ALC-0315, ALC-0159 were from Evonik (Essen, Germany).

[0354] Formulation of lipid nanoparticles

[0355] The LNP formulation utilized in the Biontech / Pfizer vaccine Comirnaty® served as a benchmark for developing optimized LNP formulations. The vaccine's overall composition includes a neutral phospholipid, cholesterol, a polyethylene glycol (PEG) lipid, and an ionizable cationic lipid. More precisely, in optimized formulations, the PEG lipid is substituted with various statistical polyoxazoline copolymers in different ratios.

[0356] A Quality by Design (QbD) approach was employed to develop optimized LNP formulations. Initially, a screening process was conducted to identify the most suitable copolymer candidates. Subsequently, an experimental design was established to systematically explore and examine the formulation space surrounding LNPs, with a specific focus on the most promising polymer candidates.

[0357] The software used was MODDE® Version 10.1. and the experimental design applied was Box Behnken design. The experimental plan included 4 factors: Polymer % ratio, lipid concentration, total flow rate (TFR) and flow rate ratio (FRR) regarding the microfluidics production.

[0358] The LNPs are prepared using the FLUIGENT (Paris, France) Microfluidic flow controller

[0359] LineUp Flow EZ™ along with flow rate sensor FLOW UNIT L and L+ as well as the MFCS™ series, using the Flowboard hub. The software used for flow rate control is OxyGEN. ALC- 0315, cholesterol, DSPC, and ALC-0159 / statistical copolymers are mixed in ethanol with a molar ratio of 46.3, 42.7, 9.4, and 1.6, respectively. Different total lipid concentrations are required individually for each formulation as the lipid phase. PolyA or EGFP mRNA is dissolved in a 50 mM pH 4 acetate buffer, considering the required concentration for each individual formulation as the aqueous phase. The lipid and aqueous phases are mixed in a staggered Herringbone mixing microfluidic chip (Fluidic 187, Microfluidic ChipShop, Jena, Germany) with different flow rate ratio (FRR) and total flow rate (TFR) according to the Design of Experiments (DoE). The particles loaded with PolyA were diluted 10-fold in 1x PBS buffer, while particles loaded with mRNA were dialysized overnight against 1x PBS buffer with Slide-A-Lyzer MINI Dialysis Devices (10k WCO).

[0360] Particle size, size distribution, and mRNA encapsulation efficiency

[0361] Particle size was measured as the hydrodynamic diameter (intensity-averaged particle size, Z-average) and polydispersity index (PDI), respectively, with ZetaSizer Ultra dynamic light scattering (DLS) instrument from Malvern Panalytical (Malvern, UK). Samples are measured in triplicate through backscatter detection the day after production and subsequent dilution procedure in 1x PBS.

[0362] Specifically, particle size was measured within the following instrument setting. Temperature was set at 25°C with return to set default temperature after each measurement. Equilibration time was 10 (s), Data processing was set with Automatic Size display limit mode and General purpose analysis model. Regarding advanced settings options, data was collected in back scatter (angle of detection), with optimal positioning method, automatic attenuation and measurement process. No pause after sub-runs and no optical filter was applied. Samples after microfluidics production and dilution in PBSxl buffer were placed in the polystyrene cuvettes DTS0012 without further dilution. Results are given as Z-average diameter [nm],

[0363] PDI is defined as polydispersity index which is a dimensionless measure of the broadness of the size distribution calculated from the cumulants analysis. PDI ranges from 0 to 1 in the Zetasizer software and values above 0.3 indicate that the distribution is polydisperse. Results are automatically calculated during the measurement without necessity to set further indications to the software. The calculations for these parameters are defined in the ISO standard document 13321 :1996 E and ISO 22412:2008.

[0364] The mRNA encapsulation efficiency (EE) is determined by Quant-it™ Ribogreen (Invitrogen) assay as a percentage of free to total mRNA concentration in LNP. The LNP samples and standard mRNA are diluted in 1 x buffer pH 7. To measure free mRNA, the sample is only diluted in 1x TE buffer, while the quantification of total RNA involved dilution in 2% Triton X- 100 in TE buffer. Following a 10-minute incubation at 37°C, 100 pL of RiboGreen reagent (diluted 1 :200 in 1x TE buffer) is added to each sample. The plate was then incubated for 5 minutes in the dark, and the fluorescence signal is measured using a microplate reader Infinite® M PLEX (Tecan Trading AG, Switzerland) at 37°C with excitation and emission wavelength of 480 and 520 nm, respectively. Total and free RNA are quantified using a standard curve prepared with a range from 0.05 pg / mL to 2 pg / mL mRNA. Measurements are done in triplicates and the results EE are given as an average of percentage.

[0365] Transfection

[0366] HEK293T-cells are seeded in 96-well plate 24 h prior to transfection, attaining a confluency of 80-90% (20,000 cells per well). mRNA-LNP were diluted in DMEM in low-glucose medium (10% FCS, 1% P / S) to a final dose of 100 / 200 ng mRNA in 100 pl volume, determined based on the encapsulation efficiency results from the Ribogreen assay. Following incubation at 37°C, 5 % CO2 for 24h, cells were analyzed for EGFP protein expression level under the Axio Observer.ZI fluorescence microscope (Fig. 23). The microscope is equipped with an A- Plan 10x / 0.25 Ph1 objective (Zeiss), filters were applied with a 38 HE Green Fluorescent Protein channel (Aex = 450-490 nm Aem = 500-550 nm), and a mercury vapor short-arc lamp. Image acquisition is set to a 10-second exposure time. The data evaluation is performed with ZEISS ZEN 3.9 software.

[0367] Results: screening of polymers

[0368] Statistical copolymers (produced as described in Examples 1 and 2) with varying hydrophilic / hydrophobic ratios were selected from the POX provided (Table 1), including those with high, intermediate, and low hydrophilic / hydrophobic ratios.

[0369] The selected POX polymers were used to prepare LNPs based on the Comirnaty™ (Biontech) formulation (Table 3) using a Fluigent Microfluidics system (Jena, Germany). The setup for each batch production is reported in Fig. 31, 32, and 33. Table 3: Comirnaty™ formulation for reference

[0370] Batch: POX-LNP

[0371] Information about the batch production.

[0372] Date: 10 / 07 / 2023

[0373] Formulation: benchmark Comirnaty™, substituting the PEGylated lipid with Polyoxazoline polymers [Polymers MW Mn].

[0374] Payload: PolyA

[0375] Microfluidics setup: 0 (Fig. 31)

[0376] The first polymer screening included the following microfluidic formulation parameters (Table 4) and polymers containing hydrophilic [EtOx, MeOx] and hydrophobic monomers [SoyOx, NonOx] in ratios of 40 / 60, 20 / 80 and 5 / 95 (Table 5).

[0377] Table 4: Microfluidics formulation parameters

[0378] Table 5: Characterization of LNPs regarding Z-average diameter and PDI [Date: 12 / 07 / 2023], The ID number of most favorable polymers is underlined. LNPs with most favorable quality characteristics, specifically with PDI smaller than 0.3 were obtained for candidates JC801 (ID9) and JC793 (ID12), see Fig. 18 and Table 5. These polymers include higher amount of hydrophilic component, therefore further screening proceeded by investigating candidates with higher content of hydrophilic monomer.

[0379] Batch: POX-LNP2

[0380] Information about the batch production.

[0381] Date: 31 / 07 / 2023

[0382] Formulation reference: benchmark ComirnatyTM, substituting the PEGylated lipid with Polyoxazoline polymers [Polymers MW Mn].

[0383] Payload: PolyA

[0384] Microfluidics setup: 0 (Fig. 31)

[0385] Microfluidics process parameters: Table 6

[0386] Table 6: Microfluidics formulation parameters

[0387] The second polymer screening investigated copolymers containing higher ratios between hydrophilic and hydrophobic monomers e.g. ratios 15 / 85, 10 / 90 and 5 / 95 (Table 7).

[0388] Table 7: Characterization of LNPs regarding Z-average diameter value and PDI [Date: 01 / 08 / / 2023], The ID number of the most favorable polymers is underlined. LNPs with most favorable quality characteristics, specifically with PDI smaller than 0.3 were obtained for candidates CMN052 (ID 2), CMN053 (ID 3), JC800 (ID 8), JC801 (ID 9), JC792 (ID 11), and JC793 (ID 12), see Fig. 19.

[0389] The screening step highlighted that LNPs containing polymers with high content of hydrophilic monomer were able to form particles with the best / good size and PDI.

[0390] Example 5: Optimizing LNPs based on POX polymers JC801 and JC793

[0391] The second step of the strategy aimed to improve the formulation of the favored POx polymers JC801 and JC793 by developing a DoE to explore the formulation space and select candidates with increased encapsulation efficiency (EE) and transfection rate.

[0392] DOE: JC801-BNT-DoE1

[0393] Date: 12 / 12 / 2023

[0394] Formulation reference: benchmark Comirnaty™, substituting the PEGylated lipid with polyoxazoline polymers [Polymers Mn,theor].

[0395] Payload: PolyA

[0396] Design of experiment: Box Behnken design, created with MODDE Software

[0397] Fixed factors: Microfluidics set up 1 (Fig. 32)

[0398] To improve the CQAs (size, PDI and EE), a DoE was developed for the polymer candidate JC801 containing the following experimental factors. (Table 8, Table 9). The DoE included 27 formulations with varying POx ratios (0.5%, 1.6%, and 2.7%), different lipid solution concentrations (5 mM, 12.5 mM, and 20 mM), different Flow Rate Ratios (Organic / Aqueous: 0.1 , 0.175, and 0.25), and different Total Flow Rates (1 mL / min, 3 mL / min, and 5 mL / min).

[0399] Table 8: Design of experiment factors Table 9: Design of experiment sample run. Total flow rate (TFR), Flow rate ratio (FRR). The ID numbers of most favorable candidates are underlined.

[0400] Table 10: Characterization of LNPs. ID numbers of most favorable candidates are underlined.

[0401] The design of the experiment reveals that LNPs with a 0.5% polymer ratio exhibit lower quality, whereas superior results are achieved with higher polymer amounts, such as 1.6% and 2.7% (Table 10, Fig. 20, Fig. 21).

[0402] DOE: JC793-BNT-DOE

[0403] Formulation reference: benchmark Comirnaty™, substituting the PEGylated lipid with polyoxazoline polymers [Polymers Mn,theor]-

[0404] Payload: PolyA

[0405] Design of experiment: Box Behnken design, created with MODDE Software

[0406] Fixed factors: Microfluidics set up 1 (Fig. 32)

[0407] To improve the CQAs (size, PDI and EE), a DoE was developed for the polymer candidate JC793 containing the following experimental factors (Table 8.1, Table 9.1). The DoE included 15 formulations with varying POx ratios (1 %, 5.5 %, and 10 %), different Lipid solution concentrations (1 mM, 10.5 mM, and 20 mM), different Flow Rate Ratios (Organic / Aqueous: 0.1 , 0.175, and 0.25) and consistent Total Flow Rate (2ml / min)

[0408] Table 8.1 : Design of experiment factors Table 9.1 : Design of experiment sample run. Total flow rate (TFR), Flow rate ratio (FRR).

[0409] The ID numbers of most favorable candidates are underlined.

[0410] Table 10.1: Characterization of LNPs. ID numbers of most favorable candidates are underlined.

[0411] The obtained particles influenced by production parameters (Flow Rate Ratio, Lipid solution concentration) induced various N / P ratios along with different POx ratio, which consequently affected CQAs (size, PDI and EE) (Table 10.1, Fig. 62 (A) and (B)). LNPs were subsequently stored at 5°C for one week, after which their size and PDI were assessed (Fig. 62 (C)). The selected particles were further evaluated for their EE% (Fig. 62 (D)). A heat map (Fig. 63) generated based on the measured data from (Table 10.1, Fig. 62) illustrates a predictive model of CQAs as influenced by the production parameters and POx ratios. LNPs showed the smallest hydrodynamic particle sizes and PDI by increasing the lipid concentration to 20 mM and by increasing the polymer ratio to 10.0%. The same trend is observed for encapsulation efficiency, specifically LNPs showed higher levels of payload encapsulation at higher values of Pox ratio and lipid concentration.

[0412] Example 6: Testing LNPs for their ability to transfect mRNA

[0413] The third step of the formulation strategy aimed at testing LNPs for their ability to transfect mRNA. According to literature, LNPs which prove to have best cell culture transfection results show N / P ratio approximately of 6 (Schoenmaker, L.; Witzigmann, D.; Kulkarni, J. A.; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, D. J. A., mRNA-lipid nanoparticle COVID- 19 vaccines: Structure and stability. International Journal of Pharmaceutics 2021 , 601 , 120586.). In association with the data collected from previous batch productions and parallel data from other experiments, the polymer candidates JC801 and JC793 were selected for further investigation.

[0414] Batch: POX-BNT-mRNA

[0415] Information about the batch production.

[0416] Date: 17 / 01 / 2024

[0417] Formulation reference: benchmark Comirnaty™, substituting the PEGylated lipid with polyoxazoline polymers [Polymers Mn ,theor ]■

[0418] Payload: mRNA eGFP RiboPro

[0419] Microfluidics setup: 1 (Fig. 32)

[0420] LNPs with the following microfluidic formulation parameters (Table 11) and the candidates JC793 and JC801 were prepared with mRNA expressing eGFP protein. Comirnaty™ formulation containing ALC-0159 (benchmark) was prepared as control. Table 11: Microfluidics formulation parameters

[0421] Table 12: Characterization of LNPs regarding Z-average diameter and PDI [Date: 18 / 01 / / 2024] and encapsulation efficiency EE [Date: 18 / 01 / / 2024]

[0422] Polymer candidates JC793 and JC801 proved to form LNPs, with favorable size and PDI and initially good EE for eGFP mRNA using the following microfluidic formulation parameters, Table 11 and Table 12, Fig. 22. A photograph of transfection of HEK291 cells is shown in Fig. 23.

[0423] Example 7: Testing LNPs fortheir ability to transfect mRNA

[0424] Candidate JC821 (see Table 1) was subjected to testing (Table 13).

[0425] Samples: 231212 JC821-BNT produced 12.12.2023, 240109 JC821-BNT produced 09.01.2024

[0426] Formulation reference: benchmark Comirnaty™, lipid nanoparticles formulated by substituting the PEGylated lipid with polyoxazoline polymer in 5% lipid ratio.

[0427] Payload: PolyA

[0428] Microfluidics set up: 1 for sample 231212 JC821-BNT (Fig. 32), 2 for sample 240109 JC821- BNT (Fig. 33)

[0429] Table 13: Microfluidics formulation parameters Table 14: Characterization of LNPs regarding Z-average diameter, PDI, and encapsulation efficiency

[0430] Initial results for the candidate JC821 show a favourable quality in terms of size, PDI and EE (Table 14).

[0431] Example 8: Production of mRNA

[0432] In vitro mRNA synthesis

[0433] The mRNA used for the formulation of the LNP was either bought from TriLink (eGFP) or prepared in house using in vitro transcription (IVT) of a template DNA. For this the HiScribe T7 mRNA Kit with CleanCap Reagent AG from NEB - an optimized RNA synthesis formulation and trinucleotide cap analog technology for co-transcriptional capping of mRNAs was used.

[0434] After the IVT, an additional enzymatic A-tailing was performed to ensure the stability of the mRNA.

[0435] The linearized plasmid (here Green Lantern GFP linearized with Xba1) and all kit components apart from the enzymes were thawed to room temperature. For one reaction 1 pg of template DNA, each 2 pL of Reaction Buffer (10x), CleanCap Reagent AG (40 mM) and NTPs (50 mM) were mixed in a PCR tube. Here, UTP was substituted with 1N- Methylpseudouridine (CrystalChem). Subsequently, 2 pL of T7 RNA Polymerase was added and the reaction incubated at 37 °C for 2 h in a thermocycler (846-X-070-311 , AnalytikJena). Afterwards 2 pL DNAsel was added to the reaction and incubated for 15 min at 37 °C.

[0436] For the A-tailing, 63 pL of nuclease free water, 10 pL 10x PolyA Reaction Buffer (B0276S, NEB) and 5 pL PolyA Polymerase (ML0276L, NEB) was added to the 20 pL reaction and incubated for 30 min at 37 °C. Subsequently, the mRNA was cleaned using the Monarch® RNA Cleanup Kit (T2050L, NEB). mRNA purification The synthesised mRNA was purified using the Monarch® RNA Cleanup Kit 500 pg (T2050L, NEB). To 100 pL mRNA reaction, 200 pL Binding Buffer and 300 pL Ethanol absolute (20821.330, VWR) were added and mixed by pipetting up and down. The sample was then loaded onto the column which was inserted in the collection tube and centrifuged at 16,000 x g for 1 min. The flow through was discarded and the column washed twice with 500 pL RNA Cleanup Wash Buffer (16,000 x g, 1 min). The column was transferred into an RNase-free 1.5 mL microfuge tube and the cleaned mRNA eluted with 50 pL nuclease free water after an incubation time of 5 min at room temperature by centrifuging the tube for 1 min at 16,000 x g. The purified mRNA was stored at -80 °C until further use.

[0437] Example 9: LNP production and formulation

[0438] Prior art LNPs which are used in current COVID-19 vaccines in particular, consist of four main components: a neutral phospholipid, cholesterol, a polyethylene glycol (PEG) lipid and an ionizable cationic lipid. The latter contains positively charged ionizable amino groups that can interact with and stabilize anionic mRNAs during particle formation. The use of PEG lipids makes it possible to control particle size and prevent aggregation during storage. Together with the mRNA, these components form particles of about 60-150 nm in diameter. Optimization of the transfection and expression efficiency of LNP can be achieved through the ratios of lipid components used, the production process itself, as well as the selection of lipids. In addition, the use of specific lipid components can influence the toxicity, inflammation-activating properties and also the desired cell specificity of the LNP.

[0439] In the present invention, the PEG lipid components in LNP formulations are replaced by the polymers of the invention. As a starting point for the lipid composition, the lipid mixtures used in the Moderna mRNA vaccine Spikevax® and Biontech / Pfizer mRNA vaccine Comirnaty® were used.

[0440] Microfluidics were applied for the self-assembly of uniform particles, allowing for the reproducible production of 25 - 250 pL nanoparticles. This allowed for a yield of practically 100% and thus effective screening to compare nanoparticle formulations.

[0441] The LNP production processes were optimized on a technical scale and the process parameters for upscaling were determined. LNPs were produced on a laboratory scale using the NanoAssemblr® Spark and on a technical scale using NanoAssemblr® Ignite™. Optimization of transfection and expression efficiency of LNP was achieved via the ratios of lipid components used. Extensive quality controls, including zeta sizer and mRNA quantification were performed.

[0442] Spark formation of LNPs

[0443] The lipid nanoparticles (LNP-mRNA) were prepared using NanoAssemblr® Spark (Precision Nanosystems) microfluidic. The Spark instrument enables a rapid low volume production of nanoparticles using the Spark NxGen single-use microfluidic cartridges. The formulation method uses a pressure-driven mechanism, and the procedure was carried out according to the Spark user guide. Three input solutions were filled into different channels of the Spark cartridges: 48 pL aqueous 100 mM acetate buffer pH 4.0 with 0.43 pg / pL mRNA, 24 pL of a mix of 25 mM lipids in ethanol and 72 pL PBS buffer. The mixing process was carried out by using formulation mode setting 3. After formulation, the outlet volume of 96 pL was diluted with 144 pL PBS und stored at 4 °C, ready for downstream cell assays and size analysis via Zetasizer Ultra (Malvern Panalytical Ltd).

[0444] Ignite formation of LNPs

[0445] The lipid nanoparticle (LNP-mRNA) was prepared using NanoAssemblr® Ignite (Precision Nanosystems) microfluidic mixing technology. The method is designed to produce reproducible, homogenous, and high-quality nanoparticles. The mixing cartridges can be shared with other NanoAssemblr systems enables scale up of seamless formulations. The method was carried out according to the Ignite user guide. In order to mix the mRNA with the lipids, two syringes were filled with the corresponding solutions and connected to the mixing cartridges. In detail, 1.3 mL of an aqueous solution containing the mRNA at a concentration of 174 pg / ml in aqueous 70 mM acetate buffer, pH 4.0, was mixed with 0.7 ml aqueous ethanolic lipid solution containing 12.5 mM lipids. The flow rate ratio between the aqueous solution and the aqueous ethanolic lipid solution was 3:1, and the total flow rate was 12 ml / min.

[0446] As an example for the Comirnaty formulation as a reference, the aqueous ethanolic lipid solution was prepared by dissolving ALC-0315, [(4-hydroxybutyl)azandiyl]bis (hexan-6,1- diyl)bis(2-hexyldecanoat) (obtained from Evonik), DSPC, 2-distearoyl-sn-glycero-3 phosphocholine (Sigma-Aldrich), cholesterol (Evonik), and ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (Evonik) at a molar ratio of 46.3 : 9.4 : 42.7 : 1.6 in ethanol. For this purpose, separate 12.5 mM solutions of the individual lipids were prepared in ethanol and the solutions were mixed in the ratio indicated above to obtain the aqueous ethanolic lipid solution. In the formulation of the POX-LNP, the PEG lipid (ALC-0159) was replaced by the POX lipid using the same mixture of the other components. This approach of only replacing the PEG lipid was also used for other lipid mixtures, e.g. that of Spikevax. 1.3 ml of the obtained LNP- mRNA product was immediately diluted with 64 ml PBS (1x) and concentrated to approximately 1.0 ml at 2000 x g for 30 minutes at 20°C using Amicon® Ultra-15 centrifugal filtration tubes. Finally, LNP samples were sterilised with a 0.2 pm syringe filter and stored at 4 °C for further applications. Size distribution of particles was analysed using the Zetasizer Ultra (Malvern Panalytical Ltd).

[0447] Example 10: Quality control of LNP

[0448] Specifications of the polydispersity index, particle size, particle concentration and zeta potential are determined on batches using the Malvern's Zetasizer after adaptation of the method. The percentage of formulated and entrapped mRNA in the LNP is analyzed using the Quant-it™ RiboGreen RNA Assay Kit. In addition, the expression efficiency is determined using reporter genes such as GFP or RFP in a selected cell line. The stability of the LNP and the influence of the buffer composition at 4 °C, -20 °C and -80 °C were analyzed by extensive series of measurements.

[0449] Ribo Green Assay

[0450] To determine the mRNA concentration and packaging of the LNP, a Quant-iT™ RiboGreen™ assay (R11490, Invitrogen) was performed, where free RNA was fluorescently labeled with an intercalating dye (RiboGreen) and quantified using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, United States). The LNP samples were diluted 1 :50 and 1 :500 in 1x TE buffer pH 7. Free mRNA was measured by diluting the sample in 1x TE (Tris-EDTA buffer) while the total RNA amount was quantified by diluting the sample in 2% Triton X-100 in TE buffer (28817.295, VWR). After incubating the samples at 37 °C for 10 min, 100 pL RiboGreen reagent (diluted 1:50 in 1x TE buffer) was added to each sample, the plate incubated for 5 min in the dark, followed by measuring the fluorescence signal using the microplate reader. For the quantification of total and free RNA a standard curve was prepared ranging from 3 ng / pL and 3 pg / pL mRNA.

[0451] Example 11 : Cell treatments with LNP

[0452] The functionality and toxicity of the GFP mRNA containing LNP was determined by cell culture experiments using human embryonic kidney 293 cells (HEK293) and Jurkat cells for the first screenings of various LNP. Promising candidates were later used for detailed cell culture experiments using human induced pluripotent stem cell (hiPSC)-derived cell types.

[0453] HEK293 cells

[0454] HEK293 cells are an immortalized cell line derived from embryonic kidney cells of a human fetus in 1973 and are widely used in cell biology due to their reliable growth and proneness to transfection. HEK293 DSMZ cells were seeded into a cell culture treated 96-well plate in a concentration of 5x105cells / well in DMEM (41965-039-500 mL, Gibco) + 10% FCS (A3840401, Gibco) + 1% Pen / Strep (15140122, Gibco). The cells were incubated at 37 °C, 5% CO2 for 24 h before the transfection. The respective Green Lantern GFP mRNA containing LNP were diluted in PBS (21-040-CVR, Corning) and added to the cells in various concentrations. The GFP expression of the HEK293 DSMZ cells was measured in the IncuCyte® S3 (Sartorius, Gottingen, Germany) while simultaneously incubating the plate at 37 °C, 5% CO2. For a period of 24 h, a picture was taken hourly of each well (10x, green + phase). The data evaluation was performed with the Incucyte software “Incucyte 2022B Rev2”.

[0455] Jurkat cells

[0456] Jurkat cells are an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry. Jurkat cells can produce interleukin 2 and are used in research involving the susceptibility of T lymphocytes for gene transfer. Hence, Jurkat cells are considered to be excellent predictors of LNP transduction into differentiated T cells.

[0457] Jurkat cells were cultivated in RPMI + GlutaMAX medium (72400-021, Gibco) + 10% FCS (A3840401; Gibco) + 1% Pen / Strep (15140122, Gibco) + beta-mercaptoethanol (21985-023, Gibco). For the transfection with LNP, the cells were harvested, washed in PBS (21-040- CVR, Corning) and seeded into a cell culture treated 96-well plate at a concentration of 5x105cells / well using ImmunoCult-XF T Cell Expansion Medium (10981, StemCell Technologies), + 100 ng / pL human IL-2, (130-097-743, Miltenyi Biotec) + 1 g / mL human ApoE3 (350-02- 500UG, Peprotech). The respective Green Lantern GFP mRNA containing LNP were diluted in PBS (21-040-CVR, Corning) and added to the cells in various concentrations. The GFP expression of the Jurkat cells was measured in the IncuCyte® S3 (Sartorius, Gottingen, Germany) while simultaneously incubating the plate at 37 °C, 5% CO2. For a period of 24 h, a picture was taken hourly of each well (10x, green + phase). The data evaluation was performed with the Incucyte software “Incucyte 2022B Rev2”. Human T lymphocytes were isolated from whole blood as peripheral blood mononuclear cells (PBMCs) and purified by magnetic cell sorting. They were cultured in vitro and finally transfected with LNP. Depending on the LNP, various parameters such as viability, proliferation, cell type-specific immune responses (e.g. CD69 and CD62L for T cells, CD25 and CD30 for B cells), stimulus-dependent cytokine release (e.g. TNF-a, IL-10, IL-6, IFN-y) and immune cell effector functions (e.g. cell killing assay) were investigated and analyzed.

[0458] Generation of differentiated human macrophages from human induced pluripotent cells (hiPSC) via hematopoietic progenitor cells (HPC)

[0459] Human macrophages were generated from hiPSC-derived HPC in cell culture using M-CSF (macrophage colony stimulating factor) at concentrations of 60 to 150 ng / mL. The expression of CD68 was examined in these cells. The expression profile of the cells, which were initially characterized by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)+, phalloidi n+, octamer-binding transcription factor 4 (OCT4)+ and PU1-, initially changed to DAPI+, phalloidin+, OCT4- and PU1+. The particularly relevant macrophages then expressed the markers CD11b, PU1, CD45, F4 / 80, 25F9 and Sigled.

[0460] The differentiated macrophages were treated with LNP and the efficiency of transduction and, subsequently, their functions were examined, in particular for migration, e.g. in an in vitro wound healing ("scratch") assay, and for phagocytosis using red pHrodo bacterial particles and contrast microscopic observation over 2 - 24 h, in each case comparing exposure to IL-4 and IL-10.

[0461] Human neurons and microglia are differentiated from hiPSC and neuronal progenitor cells (NPCs) and hematopoietic progenitor cells (HPCs)

[0462] Microglia cells were also differentiated from HPCs by adding the growth factors monocyte colony-stimulating factor (M-CSF), interleukin (I L)-34 and transforming growth factor (TGF)B1 , and characterized using the surface proteins TREM2 and CD11b and the cytoplasmic protein IBA1.

[0463] For cortical neurons, NPCs were differentiated from hiPSCs in a first step. Based on dual SMAD inhibition, NPCs were generated within 6 weeks. In a second step, NPCs were differentiated into cortical neurons with the addition of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) and analyzed for expression of the markers hexaribonucleotide binding protein-3 (NEUN), microtubule-associated protein-2 (MAP2) and beta-3-tubulin in order to correctly assign the cells. Differentiated human (cardiac) muscle cells from hiPSC

[0464] Ventricular myocardial precursor cells (cardiac progenitors) were differentiated in a six-day protocol by modulation of the Wingless-related integration site (Wnt) signaling cascade. The modulation of the Wnt signaling cascade was achieved by the addition of the small molecules CHIR99021 (Wnt signaling pathway activator) and Wnt-C59 (Wnt signaling pathway inhibitor). The ventricular progenitor cells were then differentiated into cardiomyocytes using a specific medium. Differentiated cardiomyocytes were identified after 20 days by staining and detection of cardiac troponin T and ventricular myosin light chain (vMLC2).

[0465] The resulting contracting cardiomyocytes were treated with LNP and their morphology and contractility were tested. The basic medium in the differentiation was RPMI1640+L-glutamine + B27 (up to day 6 HVPs without insulin, from then on with insulin).

[0466] Since one of the most frequent and relevant side effects of the previous mRNA vaccines is the occurrence of myocarditis, particular importance was assigned to this aspect with detailed examination and characterization of the cardiomyocytes.

[0467] Example 12: Cytotoxicity of LNP

[0468] Plasmid-based expression of mRNAs was induced in test cells by nucleofection to investigate the toxicity of individual transcripts independent of LNP transduction. The effect on cellular viability and toxic effects of the respective RNAs was then investigated, in particular by vital staining and measurement for adenosine triphosphate (ATP).

[0469] For the assessment of cytotoxicity by ATP measurement, the CellTiter-GLO Luminescent Cell Viability assay (Promega) was used. The assay was conducted according to the manufacturer’s protocol and data were evaluated as relative viabilities indicated by ATP content normalized to untreated cells.

[0470] Example 13: Cytokine measurement by ELISA

[0471] Release of cytokines (interleukin-1 B (IL-1 B), IL-6, tumor necrosis factor (TNF) and interferon (IFN)) was tested in the cellular supernatant of various differentiated cells using enzyme- linked immuno-sorbent assay (ELISA). In hiPSC-derived cells, the immune activation after treatment with LNP was checked. This served as an important marker for complications that could occur with LNP and is an indication for inflammation.

[0472] Results of Examples 8 to 13 LNP formulation

[0473] Different statistical POx copolymers were used for the substitution of PEG components in the formulation of LNP. Two previously tested formulations, shown in Table 15, were varied and benchmarked against the initial PEG containing formulation.

[0474] Table 15: Components of benchmark LNP in % by weight.

[0475] For the inclusion of the POX copolymers, PEG-lipid was completely substituted by a POX lipid taken from the selection presented in Table 16. Concentrations of the respective PEG replacement varied between 1.5 and 10% and formulation was done by one of the procedures detailed in the methods section, yielding the LNPs presented in Table 17.

[0476] Table 16: Overview over available POX copolymers.

[0477] Table 17: Formulated LNP using different POx copolymers as PEG replacement (italic indicates reference LNP containing PEG compounds, bold LNP were considered most promising for future applications, SP = Spark formulation, S = Ignite formulation).

[0478] Quality Control of LNP

[0479] Analysis of the synthesized LNP yielded diameters and polydispersity indices in a broad range as indicated in Table 18. For LNPs SP243 - 277, the correlation of size and polydispersity index with the percentage of PEG substitute is additionally depicted in Fig. 48, showing the tunability of the physical LNP properties by variation of the amount of PEG substitute.

[0480] Table 18: Z-average diameters and polydispersity indices of formulated LNP RNA content and packaging were analyzed after synthesis for LNPs produced on bigger scale (S134 - S271), results are shown in Table 19, showing a consistently high packaging efficiency and mRNA concentrations in a similar range for all LNP. Slight deviations in RNA concentration were caused by different final volumes after centrifugal ultrafiltration.

[0481] Table 19: RNA content and packaging percentage of LNP.

[0482] Cell treatment with LNP

[0483] LNP with severely increased diameters as well as LNP with visible precipitation after formulation and dialysis were discarded and not tested on cell lines. All other LNP were tested either on HEK293, Jurkat or both cell lines as indicated in the methods section. Most LNP with promising expression results compared to PEG-containing benchmark LNP were formulated freshly after the initial experiments using either the same mRNA or a similar, in-house produced mRNA to check for reproducibility and applicability for different mRNAs. Fig. 24 and 25 show the GFP expression of the two tested cell lines after LNP treatment for a selection of small batch LNP that passed quality control by size and optical analysis. Further LNPs are depicted in separate figures, Fig. 49 for LNPs SP243 - 277 and Fig. 50 for LNPs SP460 - 502. LNP concentrations were not tested and adjusted after synthesis for these LNPs, explaining slightly different expressions of equal formulations.

[0484] The most promising formulations were produced in bigger scale to enable further analysis. Selected LNP were SP185 - SP189 (Comirnaty-like LNP formulation with different PEG replacements), their labels in the bigger scale batch were S134 - 138 (for details see Table 17), LNP SP191 - 195 (Spikevax-like LNP formulation with different PEG replacements), labelled as S146 - 150 and LNPs SP477, 478, 494, 497, 498 (labelled as S262, S263, S268, S270, S271 , respectively, in bigger batch). Cell tests were then repeated using LNP S134 - 138, S146 - S150, 262, 263, 268, 270, 271 in comparison to previously formulated benchmark LNP (Comirnaty and Spikevax without PEG-replacement). HEK293 and Jurkat cells that were treated with 1 pg / mL of the respective LNP were incubated in the IncuCyte for 24 h while a picture (1 Ox, green + phase) was taken every hour to track the GFP expression of the cells. Fig. 26 and Fig. 27 show images of HEK293 and Jurkat cells, respectively, 24 h after transfection, while Fig. 28 and Fig. 29 show the corresponding green fluorescence intensities after 24 h for LNPs containing JC793, JC820 or JC821 as PEG substitute, while Fig. 51 shows intensities for LNPs with JC892 and 896.

[0485] Four promising LNP candidates, namely S134, S135, S136 and S146 were chosen for more detailed assessment on various cell types. Astrocytes, Cardiomyocytes, Neurons and Macrophages, which were differentiated from hiPSC, as well as T cells isolated from Buffy Coat PBMCs, HEK293 and Jurkat cells were treated with 1 pg / ml of the respective LNP. Their GFP expression was measured in the IncuCyte for 24°h while a picture (1 Ox, green + phase) was taken every 2 hours. Figs. 34-40 show the total green object integrated intensity over time of each cell type after the LNP transfection. Nearly all tested cell types showed an increased GFP expression compared to the untreated control. However, cell specific differences in the transfection efficiency of the LNPs could be observed. T cells for example could best be transfected by LNP S146 (see Fig. 39), while Macrophages showed the highest GFP expression after the transfection with LNP S134 and the lowest with S146 (see Fig. 37). In addition, for neurons, no relevant expression could be observed, further indicating the cell specificity of the tested LNP.

[0486] Viability / Toxicity

[0487] Viability / Toxicity of selected LNP (S134 - S138) was assessed by the CellTiter-GLO Luminescent Cell Viability Assay (Promega). The results are shown in Figs. 41-47. For most cell types, no significant drop in ATP content could be observed upon LNP treatment, but minor cell specific toxicity could be observed in some cases, i. e. for S146 on macrophages and S134 on astrocytes.

[0488] Example 14: Production of further statistical copolymers

[0489] Additional statistical copolymers were synthesized subsequent to submission of the patent application. These comprise the following:

[0490] Statistical copolymers that contain 2-n-tridecyl-2-oxazoline (MyrOx), i.e. a 2-oxazoline derivative of myristic acid.

[0491] Statistical copolymers that contain 2-n-heptyl-2-oxazoline (HeptOx), i.e. a 2-oxazoline with a linear C7 substituent as a non-branched analog to EtPentOx.

[0492] Statistical copolymers featuring a degree of polymerization (DP) of 120. The additional monomers MyrOx and HeptOx were copolymerized with the hydrophilic monomers EtOx and MeOx. Thereby, the polymerization mixtures contained 5, 10 and 15 mol% MyrOx or HeptOx, respectively (Table 20). The polymerization conditions (Fig. 52) were the same as described for the statistical copolymers reported earlier.

[0493] Table 20: Selected characterization data of the statistical copolymers comprising HeptOx and MyrOx as hydrophobic repeating units. aOverall conversion determined by1H NMR spectroscopy (CDCh, 300 MHz) of the reaction solutions.bdetermined by SEC1 (CHCh, RID, PS calibration) for copolymers comprising EtOx and by SEC9 (DMAc, RID, PS calibration) for copolymers comprising MeOx.cdetermined by1H NMR spectroscopy (CDCh, 300 MHz) of the purified copolymers by integration of the signals assigned to the POx backbone and the HeptOx Oder MyrOx methyl proton signals.dTheoretical molar mass estimated according to: Mn theor= DP(M1) x M(M1) + DP(M2) x M(M2). The DP values were obtained from the molar factions of initiator and monomer and the overall monomer conversion.

[0494] Analysis by means of size exclusion chromatography (SEC) revealed narrow molar mass distributions of the copolymers with dispersity values D around 1.1 (Fig. 53 and Fig. 54).

[0495] According to1H NMR spectroscopy (Figs. 55 to 58), none of the purified copolymers contained any toluene sulfonic acid formed from the CROP counter ion. In addition, the copolymer composition was estimated from the spectra based on integration of signals assigned to the POx backbone at 3.5 ppm (signal B) and the methyl proton signal of the HeptOx or MyrOx repeating units at 0.85 ppm (signal A).

[0496] Statistical copolymers based on NonOx / MeOx with a DP value of 120 were obtained by copolymerisation of a mixture of 3 mol% NonOx and 97 mol% MeOx, as well as 5 mol% NonOx and 95 mol% MeOx (Fig. 59). The CROP was conducted in acetonitrile using methyl tosylate as an initiator. The [monomer] to [initiator] and polymerization time were adjusted according to the polymerization rate constant. All other polymerization conditions were kept as described for the other copolymerizations.

[0497] The characterization data obtained from1H NMR-spectroscopy und SEC are summarized in Table 21.

[0498] Table 21 : Selected characterization data of the statistical copolymers with degree of polymerization DP = 120. aOverall conversion determined by1H NMR spectroscopy (CDCh, 300 MHz) of the reaction solutions.bdetermined by SEC9 (CHCh, RID, PS calibration).cdetermined by1H NMR spectroscopy (CDCh, 300 MHz) of the purified copolymers by integration of the signals assigned to the POx backbone and the NonOx methyl proton signals.dTheoretical molar mass estimated according to: Mn theor= DP(M1) x M(M1) + DP(M2) x M(M2). The DP values were obtained from the molar factions of initiator and monomer and the overall monomer conversion.

[0499] Due to the near quantitative monomer conversions, the monomer feed ratios are in accordance with those determined from the1H-NMR spectra of the purified copolymers (Fig. 60). SEC (Fig. 61) revealed unimodal molar mass distributions with a slight low molar mass tailing, which is due to a known chain transfer reaction occurring during the CROP. Dispersity values were slightly above 1.2, as typically observed for polymers based on mainly MeOx at the targeted molar masses.

[0500] Experimental section:

[0501] EtOx (Acros Organics, 99%+) and MeOx (Sigma-Aldrich, 98%) were dried over barium oxide or calcium hydride and distilled in argon atmosphere prior to usage. MyrOx (> 97%) and NonOx (> 99%) were obtained from NGP Polymers and used as received. HeptOx was synthesized from octane nitrile and 2-amino-ethanol using cadmium(ll)acetate as a catalyst according to the Witte-Seel iger-method (https: / / onlinelibrary.wiley.com / doi / epdf / 10.1002 / anie.197202871). Methyl tosylate (abcr, 98%) was distilled at reduced pressure and stored in argon atmosphere. Acetonitrile (99.9%, extry dry) and Ambersep 900-OH’ were purchased from Thermo Scientific. All other chemicals were obtained from common suppliers and used as received.

[0502] The reaction solutions were prepared in a glove box (MBRAUN, UNIIab gas purification system) in nitrogen atmosphere. For most solutions, the total monomer concentration [M]o was 1.5 mol L’1in acetonitrile and the molar ratio of [MeTos]o to [M]o was 1 : 60. Deviating from that, the molar ratio of [MeTos]o to [M]o was 1 : 120 for JC896 und JC892 , i.e. the copolymers with a targeted DP value of 120.

[0503] The fraction of the hydrophobic monomer (i.e. MyrOx or HeptOx) was varied (5 mol%; 10 mol%; 15 mol%; 3 mol% or 5 mol% for NonOx). For an exemplary copolymerization of 90 mol% EtOx with 10 mol% MyrOx, 2.01 g (20.3 mmol) EtOx, 380 mg (2.25 mmol) HeptOx and 69 mg (0.37 mmol) MeTos were dissolved in 12.7 mL acetonitrile using a suitable microwave reaction vessel. All vessels were capped inside the glovebox and subsequently removed from it. The CROP were conducted in a Biotage Initiator microwave synthesizer using the following settings: Temperature control, 140 °C, 30 min, absorption level „very high". Deviating from that, the reaction time was 55 min for JC896 and 50 min for JC892. Subsequently, the CROPs were terminated through addition of 100 pL water, and samples were withdrawn for analytics by means of SEC and NMR spectroscopy.

[0504] For polymers based on EtOx, solutions were concentrated at reduced pressure, diluted with chloroform and washed with sat. aq. NaHCChsolution and brine. The organic phases were dried over Na2SO4, and filtered. The solvent was removed at reduced pressure, and the purified polymers were dried in vacuo.

[0505] For polymers based on MeOx, solutions were concentrated at reduced pressure, diluted with water, and eluted through a short column filled with Ambersep 900-OH’. Subsequently, the aqueous solutions were concentrated at reduced pressure and lyophilized.

[0506] All purified polymers were analyzed by means of SEC and NMR spectroscopy.

Claims

Claims1 . A lipid composition comprising:(i) one or more cationic lipids, preferably cationically ionizable lipids,(ii) one or more phospholipids, and(iii) one or more statistical copolymers obtainable by a method comprising cationic ring-opening polymerization of a mixture comprising:(a) one or more monomers of the following formula (I),whereinR1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group, x represents 0 or 1 ; and(b) one or more monomers of the following formula (II)whereinR2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds, andcontaining 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; y represents 0 or 1 ; in the presence of a polymerization initiator; and (iv) optionally further comprising a sterol, preferably cholesterol.

2. The lipid composition according to claim 1 , wherein the mole ratio of R1: R2in the statistical copolymer is [R1] : [R2]= 1 to 200 : 1.

3. The lipid composition according to any one of the preceding claims, wherein R2contains a sterol group, preferably a cholesterol group.

4. The lipid composition according to any one of the preceding claims, comprising:(i) from 30 to 60 mol-% of the one or more cationic lipid,(ii) from 3 to 20 mol-% of the one or more phospholipid,(iii) from 0.2 to 20 mol-% of the one or more statistical copolymer, and(iv) from 20 to 55 mol-% of a sterol, such as cholesterol, wherein the sum of all components (i) to (iv) is 100 mol-%.

5. The lipid composition according to any one of claims 1 to 3, comprising:(i) from 50 to 90 mol-% of said one or more cationic lipids,(ii) from 8 to 30 mol-% of said one or more phospholipids,(iii) from 0.5 to 25 mol-% of one or more statistical copolymers, and(iv) from 0 to 20 mol-%, preferably from 0 to 10 mol-%, of a sterol, such as cholesterol, based on the total of all components (i) to (iv) of 100 mol-%.

6. The lipid composition according to any one of the preceding claims, wherein monomers of formulae (I) and monomers of formulae (II) represent at least 90 mol-%, preferably at least 95 mol-%, and most preferably at least 97 mol-% based of the total moles of all cationically polymerizable monomers present or added to said mixture.

7. A lipid composition, in particular according to any one of the preceding claims, comprising:(i) one or more cationic lipids, preferably cationically ionizable lipids,(ii) one or more phospholipids, and(iii) one or more statistical copolymers comprising repeating units of the following formula (l-a) and repeating units of formula (ll-a):(l-a) (ll-a) whereinR1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group;R2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carboncarbon double and / or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; and an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds, and containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; x and y independently represent 0 or 1 , preferably in an amount of at least 90 mol-% based of the total moles of repeating units present in the statistical copolymer.

8. The lipid composition according to claim 7, comprising monomer units of formula (ll-a) relative to the total content of all monomer units of formulae (l-a) and (ll-a) in an amount of: from 0.5 to 50 mol-%, preferably from 1 to 40 mol-%, more preferably from 2 to 33 mol-%, still more preferably from 3 to 20 mol-%, and still more preferably from 3 to 17 mol-%.

9. The lipid composition according to claim 7, comprising monomer units of formula (I l-a) relative to the total content of all monomer units of formulae (l-a) and (I l-a) in an amount of: from 0.5 to 40 mol-%, preferably from 1 to 30 mol-%, more preferably from 1 to 20 mol-%, still more preferably from 1 to 15 mol-%, and still more preferably from 1 to 10 mol-%.

10. The lipid composition according to claim 7, 8 or 9, comprising at least 90 mol-%, preferably at least 95 mol-%, and most preferably at least 97 mol-%, repeating units of formula (la) and formula (Ila) among all monomer units (repeating units) of said statistical copolymer.

11. A lipid composition, in particular according to any one of the preceding claims, comprising:(i) one or more cationic lipids, preferably cationically ionizable lipids,(ii) one or more phospholipids, and(iii) one or more statistical copolymers, wherein the one or more statistical copolymer has the following general formula (III):T1— {[A]m, [B]n}staf- T2(III) whereinT1is an end group derived from a polymerization initiator,T2is an end group obtained by termination of the cationic polymerization using a nucleophile, or an end group obtainable by modification thereof,A are repeating units of the formula (l-a) which may be the same or different, as defined in claim 6;B are repeating units of the formula (I l-a) which may be the same or different, as defined in claim 6; m is an integer of at least 5 defining the number of repeating units A in the copolymer, n is an integer of at least 1 defining the number of repeating units B in the copolymer, stat indicates that repeating units A and B are statistically distributed in the copolymer backbone indicated by curly brackets { }, wherein n+m is preferably from 10 to 200, preferably from 20 to 170, more preferably from 30 to 150;wherein T2may be selected from a halogen atom, such as fluorine, chlorine, bromine or iodine; an azide group -N3; a fluoro(Ci-6-alkyl)sulfonic acid ester group such as a nonaflate group -OSO2C4F9 or a trifluoromethane sulfonate group - OSO2CF3, a fluorosulfonate group -OSO2F; a Ci-s-aryl- or Ci-8-alkylsulfonic acid group such as a tosyl group CH3-C6H4-SO2- or the mesyl group CH3-SO2-; an unsubstituted, mono- or di-substituted amino group -NH2, -NHR’ or -NR’2, a hydroxyl group -OH, a thiol group -SH, an ester group -OCOR’, a thioester group -SCOR’; a phthalimide group or a cyano group -CN, or functional groups obtainable by modification of these end groups, wherein R’ is a monovalent organic radical of 1 to 6 carbon atoms.

12. The lipid composition according to any one of the preceding claims, further comprising a nucleic acid, such as RNA and / or DNA.

13. A lipid nanoparticle (LNP) obtained or obtainable from the lipid composition as defined in any one of the preceding claims and optionally comprising a peptide or a nucleic acid encapsulated in said LNP; and / or an LNP composition comprising said LNP and an aqueous solvent; said LNP and / or said LNP composition being preferably for use in medicine.

14. The LNP or the LNP composition according to claim 13, said LNP or said composition not comprising PEG or PEGylated components, preferably for use in medicine.

15. The LNP composition or said LNP according to claim 13 or 14 for use in the treatment or prevention of a disease or disorder in a subject.

16. The LNP composition or said LNP for the use according to claim 15, wherein the use is vaccination using said peptide as an antigen or using, as said nucleic acid, mRNA encoding an antigen.

17. A statistical polymer obtainable by a method comprising cationic ring-opening polymerization of a mixture comprising:(a) one or more monomers of the following formula (I),whereinR1represents a hydrogen atom or a group selected from a C1-3 alkyl group, a cyclopropyl group, and a C3 alkenyl group, x represents 0 or 1 ; and(b) one or more monomers of the following formula (II)whereinR2represents a, preferably non-aromatic, group selected from a saturated C4-40 hydrocarbyl group; an unsaturated C4-40 hydrocarbyl group having one or more carbon-carbon double and / or triple bonds; a saturated C4-40 heterohydrocarbyl group containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; an unsaturated C4-40 heterohydrocarbyl group having one or more carbon-carbon double and / or triple bonds, and containing 1 to 5 divalent groups selected from ether, thioether, sulfoxide, sulfone, keto, ester, amide, carbamate, and carbonate groups, and / or 1 to 5 fluorine atoms as substituents; y represents 0 or 1 ; in the presence of a polymerization initiator; or a statistical copolymer comprising repeating units of the following formula (l-a) and repeating units of formula (ll-a):wherein x, y, R1and R2are as defined above.

18. The statistical copolymer according to claim 17, wherein R2is a group of the following formula (V):whereinR6are independently hydrogen atoms or methyl groups,R7is a linear or branched C1-10 alkyl group, or a linear or branched C2-10 alkenyl group,L is a linear or branched C1.5 alkylene group or a linear or branched C2-5 alkenylene group,M is a carbonyl group or single bond linking the adjacent groups, and the dashed line next to the bond means that the bond is either a single bond or a double bond.

19. The statistical copolymer according to claim 17 or 18, wherein R1is a methyl or ethyl group, preferably a methyl group.

20. The statistical copolymer according to any one of claims 17 to 19, comprising monomer units of formula (I l-a) relative to the total content of all monomer units of formulae (l-a) and (I l-a) in an amount of from 1 to 15 mol-%, preferably from 1 to 10 mol-%.

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