Lipid-like copolymers and method of producing these and the use thereof, pharmaceutical compositions comprising said copolymers, and the use of said compositions

Abstract

The present invention relates to a lipid-like copolymer comprising hydrophilic and hydrophobic units. The present invention further relates to a pharmaceutical composition comprising the lipid-like copolymer and a therapeutic oligonucleotide. The present invention furthermore relates to the use of the pharmaceutical composition as a medicament, in a method of treating or preventing a disease, in a method of treating, preventing or ameliorating cancer, and in a method for inducing an immune response. The present invention moreover relates to producing the pharmaceutical composition. The present invention also relates to the use of the lipid-like copolymer for encapsulation of therapeutic oligonucleotides.

Description

[0001] TITLE Lipid-like copolymers and method of producing these and the use thereof, pharmaceutical compositions comprising said copolymers, and the use of said compositions.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to lipid-like copolymers comprising hydrophilic units and hydrophobic units, a method of producing these and the use of said copolymers. Moreover, the invention relates to a pharmaceutical composition comprising the lipid-like copolymer a therapeutic oligonucleotide, the use thereof and the preparation thereof.

[0004] BACKGROUND

[0005] Therapeutic oligonucleotides are a class of molecules used in the field of molecular medicine to modulate gene expression or protein function for therapeutic purposes. A therapeutic oligonucleotide may encode a peptide or protein replacing or supplementing an endogenous protein or peptide, or may comprise an antigen or epitope, may be complementary to a target gene’s sequence, for instance to reduce or silence the expression of a target gene or protein.

[0006] Therapeutic oligonucleotides are also used to trigger an immune response as part of their mechanism of action. This immune activation can be used strategically to enhance the therapeutic effects or target specific immune-related diseases. By harnessing the immune system’s natural ability to recognize and eliminate pathogens or abnormal cells, oligonucleotides can be used to enhance the efficacy of immunotherapies, stimulate antiviral responses, or boost the immune system’s ability to target cancer cells.

[0007] The formulation of therapeutic oligonucleotides, particularly RNA- based oligonucleotides, plays a critical role in enhancing their effectiveness and overcoming various challenges associated with their delivery, stability, and intracellular uptake.

[0008] Therapeutic oligonucleotides are susceptible to degradation by cellular nucleases present in the body. Formulation strategies aim to protect therapeutic oligonucleotides from enzymatic degradation and increase their stability in

[0009] "SUBSTITUTE SHEETS" biological fluids. This can be achieved by encapsulating the oligonucleotides within protective delivery systems.

[0010] Another major challenge with oligonucleotide therapeutics is efficiently delivering them into target cells. Formulation approaches improve cellular uptake by optimizing the physicochemical properties of oligonucleotides, such as their size, charge, and hydrophobicity. For RNA-based oligonucleotides in particular, delivery systems like lipid nanoparticles or polymer-based carriers can facilitate their efficient uptake by cells and transport across cellular barriers.

[0011] Further, after cellular uptake, oligonucleotides need to be released from the delivery system and reach the appropriate intracellular compartments to exert their therapeutic effects. Formulation approaches ensure the efficient release of oligonucleotides within cells through controlled release mechanisms. This allows oligonucleotides to access their target sites, such as the cytoplasm or specific organelles.

[0012] Formulations aim to protect RNA (and other types of therapeutic oligonucleotides) from degradation and promote its delivery into (target) cells. Lipid- based nanoparticles, polymer-based nanoparticles, and viral vectors are commonly employed as carriers for RNA delivery.

[0013] The formulation process involves optimizing various parameters, such as RNA stability, encapsulation efficiency, release kinetics, and biocompatibility. It may also include steps like encapsulating RNA within protective materials, modifying RNA structure, or incorporating targeting ligands for specific cells or tissues.

[0014] RNA formulation with viral vectors (e.g., adenovirus, lentivirus) may have significant unwanted side effects and unwanted immune responses. To avoid these unwanted effects, non-viral RNA formulations have been developed.

[0015] It is known to use nanoparticle formulations in which the oligonucleotides are embedded or enclosed. For preparing these nanoparticle formulations ionizable lipids may be used having a lipid aliphatic tail. Ionizable lipids have a polar, ionizable head group and one or more aliphatic tails. These lipids selfassembly to form a nanoparticle. It is also known to use ionizable (co)polymers prepared of monomers having ionizable groups. As an example, mRNA vaccines for COVID-19 employ lipid mRNA formulations (WO2019137999A1). Other examples of lipid mRNA formulations are disclosed in US8058069B2 (Lipid formulations for nucleic acid delivery) and W02018081480A1 (Lipid nanoparticle formulations).

[0016] Although an improvement over RNA formulation with viral vectors, lipid RNA formulations have as main disadvantage a decreased RNA delivery efficacy.

[0017] OBJECTS

[0018] It is an object of the present invention to provide a compound for the preparation of improved nanoparticles for therapeutic oligonucleotide delivery. More specifically, it is an object of the present invention to provide an improved lipid RNA formulation, the use of such formulation, as well as a method to produce such a formulation.

[0019] STATEMENT OF THE INVENTION

[0020] In a first aspect, the invention relates to a lipid-like copolymer comprising hydrophilic units and hydrophobic units represented by Formula A wherein:

[0021] Z1 is a chain transfer residue, preferably having the formula s a chain transfer residue, preferably having the formula: wherein each X is independently O or NH, n is the number of hydrophilic units in the copolymer and is an integer between 2 and 150, m is the number of hydrophobic units in the copolymer and is an integer between 2 and 150, preferably n + m is between 4 and 300, more preferably at most 200, even more preferably at most 100, v is an integer between 1 and 4, each R1 is hydrogen or methyl,

[0022] R2a and R2b are each independently selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, 2-ethylhydroxyl, or R2a and R2b together with the nitrogen atom to which they are attached form a 5-, 6- or 7- membered ring, preferably selected from the group consisting of:

[0023] R3 is a linear or branched C12-C20 alkyl or alkenyl group, preferably stearyl, oleoyl, lineoyl or 2-hexyldecyl,

[0024] R4 is a C1-C12 alkyl group, for example n-butyl,

[0025] R5a is hydrogen or methyl; R5b is methyl or CN, and w is 0, 1 or 2 preferably Z2 is selected from the group consisting of:

[0026] C is a carbon atom, O is an oxygen atom, and N is a nitrogen atom, and S is a sulphur atom.

[0027] Corresponding embodiments are also applicable for the method of producing the lipid-like copolymer, the pharmaceutical composition comprising the lipid-like copolymer, the pharmaceutical composition for use as a medicament, the method for producing the pharmaceutical composition and the use of the lipid-like copolymer according to the present invention. In a second aspect, the invention relates to a method of producing the lipid-like copolymer according to any one of the preceding claims, comprising a free radical or RAFT polymerisation of a first hydrophilic monomer according to Formula B and a second hydrophobic monomer according to Formula C and a chain transfer agent (CTA) according to the formula Z1-Z2

[0028] Formula B Formula C wherein Z1 , Z2, X, v, R1 , R2a, R2b, and R3, are as defined above under the first aspect, preferably wherein Z1-Z2 is according to the formula D

[0029] Formula D wherein R4, R5a, R5b and w are as defined above under the first aspect.

[0030] Corresponding embodiments are also applicable for the lipid-like copolymer, the pharmaceutical composition comprising the lipid-like copolymer, the pharmaceutical composition for use as a medicament, the method for producing the pharmaceutical composition and the use of the lipid-like copolymer according to the present invention.

[0031] In a third aspect, the invention relates to a pharmaceutical composition comprising the lipid-like copolymer according to claim 1 and a therapeutic oligonucleotide.

[0032] Corresponding embodiments are also applicable for the lipid-like copolymer, the method of producing the lipid-like copolymer, the pharmaceutical composition for use as a medicament, the method for producing the pharmaceutical composition and the use of the lipid-like copolymer according to the present invention. In a fourth aspect, the invention relates to the pharmaceutical composition according to the third aspect for use as a medicament.

[0033] Corresponding embodiments are also applicable for the lipid-like copolymer, the method of producing the lipid-like copolymer, the pharmaceutical composition comprising the lipid-like copolymer, the method for producing the pharmaceutical composition and the use of the lipid-like copolymer according to the present invention.

[0034] In a fifth aspect, the invention relates to a method for producing the pharmaceutical composition according to the third aspect comprising mixing a lipid- like copolymer, optionally one or more helper lipids, and a therapeutic oligonucleotide in a solvent to obtain a pharmaceutical composition comprising nanoparticles.

[0035] Corresponding embodiments are also applicable for the lipid-like copolymer, the method of producing the lipid-like copolymer, the pharmaceutical composition comprising the lipid-like copolymer, the pharmaceutical composition for use as a medicament, and the use of the lipid-like copolymer according to the present invention.

[0036] In a sixth aspect, the invention relates to use of the lipid-like copolymer according to any one of claims 1 - 3 or prepared according to the method of claims 4 or 5 for encapsulation of therapeutic oligonucleotides.

[0037] Corresponding embodiments are also applicable for the lipid-like copolymer, the method of producing the lipid-like copolymer, the pharmaceutical composition comprising the lipid-like copolymer, the pharmaceutical composition for use as a medicament, and the method for producing the pharmaceutical composition according to the present invention.

[0038] DETAILED DESCRIPTION

[0039] The present invention is elucidated below with a detailed description.

[0040] The present invention is described hereinafter with reference to the accompanying drawings in which embodiments of the present invention are shown. All parameter ranges (such as for n, m, v and 2) include the endpoints of the ranges and all values in between the endpoints, unless otherwise specified. When used in these specification and claims, the terms "comprises" and "comprising" and variations thereof mean that the specified features, steps or integers are included. The terms are not to be interpreted to exclude the presence of other features, steps, or components.

[0041] Unless stated otherwise, when it is stated that any R group is “independently selected from” this means that when several of the same R groups are present in a molecule, they may have the same meaning of they may not have the same meaning.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention is described hereinafter with reference to the accompanying drawings in which embodiments of the present invention are shown.

[0044] Figure 1 : overview of four embodiments of the lipid-like copolymers and cartoons of their structure;

[0045] Figure 2a-b: overview of types of nanoparticles; wherein in figure 2a the individual lipid-like copolymers are shown and in figure 2b the resulting nanoparticles

[0046] Figure 3a: 1 H NMR spectra of the purified lipid tail monomers in deuterated chloroform with key peaks indicated.

[0047] Figure 3b: GPC traces of the resultant polymers (SA-0.5, OA-O.5, LA- 0.5, HDA-0.5) in THF.

[0048] Figure 3c: 1 H NMR spectra of the purified lipid-like copolymers (SA- 0.5, OA-O.5, LA-0.5, HDA-0.5) in deuterated chloroform with key peaks indicated.

[0049] Figure 3d: DSC traces of the purified lipid-like copolymers (SA-0.5, OA-O.5, LA-0.5, HDA-0.5).

[0050] Figure 4a: reaction scheme for formulation of siRNA with lipid-like copolymer and helper lipids.

[0051] Figure 4b: CryoTEM images of nanoparticles from p(DEAEA-co-SA) (SA-0.5) with helper lipids and siRNA.

[0052] Figure 4c: CryoTEM images of nanoparticles from p(DEAEA-co-OA) (OA-O.5) with helper lipids and siRNA.

[0053] Figure 4d: CryoTEM images of nanoparticles from p(DEAEA-co-LA) (LA-0.5) with helper lipids and siRNA. Figure 4e: CryoTEM images of nanoparticles from p(DEAEA-co-HDA) (HDA-0.5) with helper lipids and siRNA.

[0054] Figure 5: TNS fluorescent probe assayfor the determination of nanoparticle apparent pKa.

[0055] Figure 6a: Agarose gel assay showing protection of RNA from benzonase (37 C, 0.5 h), LNPs in component ratio 50 :10 :37.5 :2.5.

[0056] Figure 6b: Agarose gel assay showing protection of RNA from 30 wt% serum (37 C, 4 h), LNPs in component ratio 50 :10 :37.5 :2.5.

[0057] Figure 7a: Cell-uptake in SKOV3 cells of lipid-like polymer nanoparticles encapsulating Cy5-siRNA, and Cy5-siRNA control, was assessed using confocal microscopy.

[0058] Figure 7b: Cell uptake / internalisation was quantified using ImgageJ.

[0059] Figure 7c: SKOV3 cell viability with polymer nanoparticles was assessed using MTS assay.

[0060] Figure 7d: Dual reporter FLuc knockdown assay was carried out across varying concentrations, including DLin-MC3-DMA lipid nanoparticle as positive control.

[0061] Figure 7e: EC50 values were extracted from dose response curve fitting of the knockdown assay

[0062] Figure 8a: Endosomal escape of lipid-like polymer nanoparticle formulations p(DEAEA-co-HDA), p(DEAEA-co-LA), and p(DEAEA-co-OA) in SKOV3 cells was assessed after 3h incubation and 6h incubation.

[0063] Figure 8b: Endosomal escape of Cy5-siRNA control, in SKOV3 cells was assessed after 3h incubation and 6h incubation.

[0064] Figure 8c: Sample colocalization with endo / lysosomes was quantified and assessed with Pearsons coefficient and Manders M2 coefficient, n = 10-20 cells.

[0065] Figure 9: Model lipid bilayer fusion FRET based assay. Proposed mechanism of interaction of lipid-like polymers with bilayers in acidic environments, and degree of lipid bilayer disruption by lipid-like polymer nanoparticle formulations, from FRET assay at pH 6.

[0066] Figure 10: 31 P NMR spectra of endosomal mimic with lamellar phase orientation, and a mixture of endosomal mimic with lipid-like polymer HDA-0.5 . showing lipid mixing induced membrane hexagonal Hll phase formation. Hereafter the invention is described in more detail.

[0067] Lipid-like copolymer

[0068] As discussed above in a first aspect the present invention relates to a lipid-like copolymer comprising hydrophilic and hydrophobic units represented by Formula A wherein:

[0069] Z1 is a chain transfer residue, preferably having the formula each X is independently O or NH, n is the number of hydrophilic units in the copolymer and is an integer between 2 and 150, m is the number of hydrophobic units in the copolymer and is an integer between 2 and 150, preferably n + m is between 4 and 300, more preferably at most 200, even more preferably at most 100, v is an integer between 1 and 4, viz. 1 or 2 or 3 or 4 each R1 is independently hydrogen or methyl, R2a and R2b are each independently selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, 2-ethylhydroxyl, or R2a and R2b together with the nitrogen atom to which they are attached form a 5-, 6- or 7- membered ring, preferably selected from the group consisting of:

[0070] R3 is a linear or branched C12-C20 alkyl or alkenyl group, preferably stearyl, oleoyl, lineoyl or 2-hexyldecyl,

[0071] R4 is a C1-C12 alkyl group, for example n-butyl,

[0072] R5a is hydrogen or methyl; R5b is methyl or CN, and w is 0, 1 or 2 preferably Z2 is selected from the group consisting of:

[0073] C is a carbon atom, O is an oxygen atom, and N is a nitrogen atom, and S is a sulphur atom.

[0074] Said lipid-like copolymer is a copolymer of 1) hydrophilic units (U1) according to formula A1 below having a nitrogen-based structure and 2) hydrophobic units (U2) according to Formula A2 below having a hydrophobic aliphatic tail that is either linear or branched.

[0075] Formula A1 Formula A2 wherein R1 , X, v, R2a, R2b and R3 are as defined above. In the above structure of the lipid-like copolymer, Z1 and Z2 are each a residue of the chain transfer agent and together (Z1-Z2) for the chain transfer agent. This will be discussed below in more detail. In both the hydrophilic units U1 (according to Formula A1) as well as in the hydrophobic units U2 (according to Formula A2) X may be O or may be NH, the former being preferred. In both the hydrophilic units U1 (according to Formula A1) as well as in the hydrophobic units U2 (according to Formula A2) R1 may be hydrogen or methyl, the former being preferred.

[0076] In Formula A, n is the number of hydrophilic units in the copolymer and is an integer between 2 and 150. In Formula A, m is the number of hydrophobic units in the copolymer and is an integer between 2 and 150. The present inventors have observed that a value in this range provides optimal results. In an embodiment, n and m may each individually be an integer between 2 and 100, such as between 2 and 25, such as for example between 3 and 10 or 4 and 7. The total number of monomer is defined by the number of hydrophilic units plus the number of hydrophobic units, viz. n + m. In an embodiment n + m is between 4 and 200, preferably between 4 and 100, such as between 4 and 50 or between 6 and 20 or even 8 and 14.

[0077] The ratio between the hydrophilic and hydrophobic group may be selected by amending the values for n and m but is preferably between 60:40 and 40:60, such as between 55:45 and 45:55, such as between 51 :49 and 49:51 , such 50:50. This ensures that the number of polar, hydrophilic headgroups and the number of hydrophobic tails in the nanoparticles are similar, which is comparable to a molecular lipid structure.

[0078] The spacer between the polymeric backbone and the nitrogencontaining polar group is defined by the number of methylene (-CH2-) groups, defined by v, which may be 1 or 2 or 3 or 4.

[0079] In an embodiment, the copolymer may be an alternating copolymer comprising alternating hydrophilic units (U1) and hydrophobic units (U2). With “alternating copolymer” is meant that less than around 5 % of hydrophilic units are directly adjacent other hydrophilic units and less than around 5 % of hydrophobic units are directly adjacent other hydrophobic units. This embodiment will have a structure that is resembles U1-LI2- U1-LI2- U1-LI2- U1-LI2. With this embodiment the ratio between n and m is between between 51 :49 and 49:51 , preferably 50:50. The copolymer is preferably an alternating copolymer, since this would lead to a nanoparticle that has a comparable distribution of headgroups and tails as a nanoparticle prepared from molecular lipids.

[0080] In an embodiment, the copolymer may be a random copolymer comprising randomly distributed hydrophilic and hydrophobic units. This embodiment may have very different types of structures, such as for example U1-U1-U2-U1-U2- U2-U1-U2-U2- U1-U2.

[0081] In an embodiment, the copolymer may be a block-copolymer comprising one or more blocks of two or more hydrophilic units and one or more blocks of two or more hydrophobic units, preferably the copolymer is an alternating copolymer. This embodiment may have different types of structures, such as U1-LI1- U1-U1-U1-U2-U2-U2-U2-U2 or U1-U1-U1-U2-U2-U2-U1-U1-U1-U2-U2-U2.

[0082] In a specific embodiment of the lipid-like copolymer, the lipid-like copolymer,

[0083] Z1 has the formula wherein R4 is n-butyl, Z2 has the formula wherein R5a is hydrogen, R5b is methyl, both X are O, v is 2, w isO, both R1 are hydrogen, R2a and R2b are each ethyl, R3 is stearyl, oleoyl, lineoyl or 2-hexyldecyl, and 4 and m are each independently between 3 and 10. In Figure 1 the structures are shown of four specific embodiments of the lipid-like copolymer according to the present invention as well as cartoons of the structure they present. For each of the four structures n equals m and is 5, and n + m is 10. For each of the four structures X is O, v is 2, R1 is hydrogen, and R2a and R2b each are ethyl. These structures differ in the hydrophobic unit. From left to right R3 is stearyl (C18:0), oleoyl (C18:1), lineoyl (C18:2) and 2-hexyldecyl (C16:0).

[0084] Method of producing copolymer

[0085] As discussed above in a second aspect the present invention relates to a method of producing a lipid-like copolymer, comprising a free radical or RAFT polymerisation of a first hydrophilic monomer according to Formula B and a second hydrophobic monomer according to Formula C and a chain transfer agent (CTA) according to the formula Z1-Z2 (discussed below).

[0086] Formula B Formula C wherein Z1 , Z2, X, v, R1 , R2a, R2b, and R3 are as defined as above.

[0087] Type of polymerization

[0088] The present lipid-like copolymer may be produced by chain-growth polymerization. This allows for low polydisperse polymers to be obtained, since there are only a limited number of growing chains present at the same time. There are three key steps in chain growth polymerization, being initiation, propagation, and termination.

[0089] Free-radical polymerization (FRP) is a type of chain-growth polymerization in which unsaturated monomers are one-by-one attached to the active site on a growing polymer chain. With free-radical polymerization (FRP) a polymer is formed by successive addition of free-radical monomer units. In an embodiment, the lipid-like copolymer is produced by FRP. A chain transfer agent (CTA) is used.

[0090] Reversible Addition-Fragmentation chain-Transfer (RAFT) polymerization is type of chain-growth polymerization in which a chain-transfer agent is used. In an embodiment, the lipid-like copolymer is produced by RAFT polymerization.

[0091] Hydrophilic monomer

[0092] As a first monomer (M1) a hydrophilic monomer according to Formula B is used. This will, in the copolymer form hydrophilic unit U1 , which in the final nanoparticles forms the polar headgroup.

[0093] Preferably, the hydrophilic monomer monomer is an acrylate (R1=H and X = O), a methacrylate (R1=methyl and X = O), an acrylamide (R1=H and X = NH), or a methacrylamide (R1=methyl and X = NH). wherein R1 is hydrogen (H) or methyl (CH3) and wherein X is either oxygen (O) or an NH group.

[0094] In an embodiment, the copolymer comprises several types of hydrophilic units. In an embodiment, the copolymer comprises several types of hydrophobic and hydrophilic units.

[0095] Hydrophobic monomer

[0096] As a second monomer (M2) a hydrophobic monomer according to Formula C is used. This will, in the copolymer form hydrophobic unit U2, which in the final nanoparticles forms the hydrophobic, lipid-like tail.

[0097] Preferably, the hydrophobic monomer is an acrylate (R1=H and X = O), a methacrylate (R1=methyl and X = O), an acrylamide (R1=H and X = NH), or a methacrylamide (R1=methyl and X = NH).

[0098] R3 is a linear or branched C12-C20 alkyl or alkenyl group. R3 is preferably a C16-C18 linear or branched alkyl or alkenyl group, most preferably C16:0, C18:0, C18: 1 , or C18:2. Specific examples are stearyl (C18:0), oleoyl (C18: 1), lineoyl (C18:2), and hexadecyl (C16:0). These may be attached at the 1-position which may lead to a linear alkyl group (in case no other branching in the alkyl group) or which may lead to a branched alkyl group (in case branching in the alkyl group), or the alkyl group may be attached at any position to provide branching even in case of a linear alkyl group. For example, hexadecyl may be attached at the 6-, 7- or 8- position (such as at the 7-position) to provide branching and giving respectively hexadecane-6-yl acrylate, hexadecane-7-yl acrylate hexadecane-8-yl acrylate. Other options include 2- butyldodecyl acrylate, 4-butyldodecyl acrylate, 6-butyldodecyl acrylate, 4-hexyldecyl acrylate, and 4-pentylundecyl acrylate.

[0099] Preferred examples of M2 are shown below:

[0100] In an embodiment, the copolymer comprises several types of hydrophobic units. In an embodiment, the copolymer comprises several types of hydrophobic and hydrophilic units.

[0101] Chain transfer agent

[0102] The lipid-like copolymer according to the present invention is prepared using a chain transfer agent CTA (Z1-Z2).

[0103] Examples of CTAs that may be used in the present invention are: thiols (such as dodecanthiol or mercaptoethanol) or halocarbons (such as bromotrichloromethane). Preferably, the lipid-like copolymer is produced using RAFT polymerization using a RAFT CTA, such as a thiocarbonylthio compound, such as dithioesters, (tri)thiocarbamates, and xanthates. These types of CTA allow excellent control over the molecular weight and low polydispersity (narrow molecular weight distribution) of the resulting copolymer.

[0104] In an embodiment, Z1-Z2 is according to the formula D: Formula D wherein R4, R5a, R5b, and w are as discussed above.

[0105] As a specific example of a suitable RAFT CTA 2-{[(Butylsulfanyl)- carbothioyl]sulfanyl}propanoic acid (PABTC) may be used, as shown in the drawing below:

[0106] Initiator

[0107] Optionally an initiator may be used in the polymerization process. A radical initiator is a compound that produces radical species to promote / start radical polymerization. These compounds all have weak bonds that can be dissociated easily. Examples of radical initiators are compounds having a nitrogen-halogen bond, azo compounds, and peroxides. Preferably azo compounds are used, such as 4,4'- azobis(4-cyanopentanoic acid) (ACVA), 2,2'-azobis(2-methylpropionitrile) (AIBN), or 1 , 1 -azobis(cyclohexanecarbonitrile) (ACH N).

[0108] Solvent

[0109] Examples of suitable solvents used during the polymerization are organic solvents, such as dioxane, THF, DMF, isopropanol, DMSO, or mixtures of organic solvents with water. The polymerization may be carried out at a temperature of between 60 and 90 °C. the polymerisation may be carried out for a duration of between 2 and 24 hours. Exact polymerisation times can vary, and depend on the monomer class and its rate of propagation in the chosen polymerisation temperature, solvent, and radical initiator (if used).

[0110] Pharmaceutical composition comprising a lipid-like copolymer and a therapeutic oligonucleotide.

[0111] As discussed above in a third aspect the present invention also relates to a pharmaceutical composition comprising a lipid-like copolymer and a therapeutic oligonucleotide. In an embodiment, the solvent used for the pharmaceutical composition is phosphate buffered saline (PBS). In an embodiment, the pH of the pharmaceutical compositions is between 7 and 8.

[0112] Therapeutic oligonucleotides

[0113] Nucleic acids are unstable and require formulation in order to be effective as therapeutics. More specifically, RNA formulation refers to the process of designing and preparing RNA molecules in a form that is suitable for various applications, such as therapeutics, research, or diagnostics. In the context of therapeutics, RNA formulations are developed to enhance the stability, delivery, and efficacy of RNA-based drugs. Types of RNA formulations may include: Messenger RNA (mRNA), RNAi (siRNA, shRNA, miRNA), ASO, and aptamer formulation. mRNA may be formulated to encode specific proteins of interest, which can be introduced into cells to produce therapeutic proteins or elicit an immune response, or for gene editing. Gene editing technology has become a viable therapeutic strategy due to the identification of clustered, regularly interspaced, short palindromic repeat (CRISPR) technology. The invention described herein can be used in combination with CRISPR technology to treat genetic diseases, where the therapeutic cargo is mRNA encoding for a CRISPR associated protein (eg CAS9), and a single guide RNA (sgRNA) used in the CRISPR technology to target specific gene sequences.

[0114] RNAi, such as siRNA, shRNA, miRNA, may be formulated to achieve the silencing of specific genes resulting in a knock down effect. RNAi may inhibit the expression of specific genes by targeting complementary RNA molecules.

[0115] Antisense oligonucleotides (ASO’s) are designed to specifically bind to complementary RNA molecules. They work by hybridizing to the target RNA sequence, thereby preventing the translation of the target mRNA into protein or promoting degradation of the target RNA. ASOs may be formulated to inhibit gene expression at the RNA level by targeting various sites, such as the start codon, splice junctions, or other regulatory regions (but ASOs do not utilize the RNAi pathway or RISC complex like siRNA or shRNA).

[0116] Aptamers are single-stranded DNA or RNA molecules that can bind to specific target molecules, including proteins, peptides, or small molecules. Aptamers fold into unique tertiary structures that allow them to bind to their target molecules with high specificity and affinity. Aptamers may be formulated to interfere with target protein function, disrupt protein-protein interactions, or act as molecular probes for diagnostic or therapeutic applications. Aptamers can modulate gene expression indirectly in some cases, but do not directly induce RNAi-mediated gene knockdown.

[0117] In an embodiment the therapeutical oligonucleotide may be mRNA encoding a peptide or protein replacing or supplementing an endogenous protein or peptide, mRNA comprising an antigen or epitope, mRNA complementary to a target gene’s mRNA sequence, for instance to reduce or silence the expression of a target gene or protein, mRNA encoding for a CRISPR associated protein, CRISPR sgRNA, self-amplifying mRNA, RNAi, an antisense oligonucleotide, an aptamer, and one or more combinations thereof. The RNAi may be an siRNA, miRNA, piRNA, shRNA, and one or more combinations thereof. Therapeutic oligonucleotides may be used to trigger an immune response, for instance to enhance the efficacy of immunotherapies, stimulate antiviral responses, or boost the immune system's ability to target cancer cells. The inventors found that the use of lipid-like polymers in complexing nucleic acid therapeutic molecules, improved stability and efficacy of the therapeutical oligonucleotide. As shown in the examples, the lipid-like copolymer siRNA nanoparticles p(DEAEA-co-OA), p(DEAEA-co-LA), and p(DEAEA-co-HDA), embodiments of the invention, mediated a convincing knock down effect in human epithelial ovarian adenocarcinoma cell line (SKOV3) (Figure 7), compared to DLin- MC3-DMA (dilinoleylmethyl-4-dimethylaminobutyrate) lipid nanoparticle as positive control. Further, endo / lysosome escape of said nanoparticles was persuasively increased compared to Cy5-siRNA as control (figure 8).

[0118] Helper lipids

[0119] For said lipid mRNA formulations natural or synthesized lipid structures may be used. There are several examples of synthesized lipid structures used in the development of lipid-based nanoparticles for RNA delivery:

[0120] 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): DOPE is a commonly used lipid in lipid-based nanoparticle formulations. It is a phospholipid derivative with two oleoyl chains and a phosphoethanolamine headgroup. DOPE is known for its ability to promote efficient endosomal escape of delivered RNA molecules.

[0121] 1.2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC): DOPC is another widely used lipid. It is a zwitterionic phospholipid with two oleoyl chains and a choline headgroup. DOPC-based nanoparticles are known for their stability and biocompatibility.

[0122] 1.2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE): DSPE is a lipid that contains two stearoyl chains and a phosphoethanolamine headgroup. It is often used to incorporate polyethylene glycol (PEG) chains into lipid nanoparticles, which can improve their circulation time and reduce immune response.

[0123] Cationic Lipids: Various cationic lipids, such as 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), are used to prepare positively charged lipid nanoparticles. These cationic lipids can interact with negatively charged RNA molecules and facilitate their cellular uptake.

[0124] One or more helper lipids may be present in the nanoparticles of the present invention. The pharmaceutical composition thus may further comprise a helper lipid selected from the group consisting of a neutral lipid, a sterol, a PEG-lipid and one or more combination thereof, preferably wherein the neutral lipid is selected from the group consisting of DSPC (distearoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPC (dioleolylphosphatidylcholine), POPC (palmitoylphosphatidylcholine), and DOPE (dioleoylglycerophosphoethanolamine), wherein the sterol is cholesterol, and wherein the PEG-lipid is PEG2k-DMG (also called DMG-PEG2000); 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000) or PEG2k-DSPE (also called DSPE-PEG2000; 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) being amphiphilic surface stabilising lipids important for particle size and for increased circulation times in vivo. The PEG-lipid has the effect of reducing particle aggregation.

[0125] In a specific embodiment, as helper lipids a neutral lipid, a sterol and a PEG-lipid are present. The effect thereof is increased stability and delivery efficiency. Neutral helper lipids such as phosphatidylcholine can increase interaction with natural lipid bilayer which is important for in vivo application of LNPs. Cholesterol improves intracellular delivery and LNP stability in vivo. Stabilising PEG-lipid modulates particle size, increases colloidal stability, and increases circulation time in vivo.

[0126] In an embodiment, the molar ratio of the lipid-like copolymer and the helper lipid is between 70:30 and 30:70, preferably between 60:40 and 40:60, more preferably between 55:45 and 45:55, such as 50:50. The effect thereof is stable particles with high nucleic acid encapsulation.

[0127] In an embodiment, the lipid-like copolymer is present in an amount of about 20-70 mol%, the neutral lipid in an amount of 5-30 mol%, the sterol in an amount of 10-60 mol%, and the PEG-lipid in an amount of 0.5- 20 mol%, based on the combined amounts of lipid-like copolymer and helper lipids. The effect thereof is colloidally stable particles of the desired size, with high nucleic acid encapsulation. The molar ratio of lipid-like copolymer is considered from the molecular weight of the combined monomer unit (U1 and U2).

[0128] The ratio between the total amount of lipids and copolymers (forming the self-assembled layers in the nanoparticles, to the oligonucleotide (encapsulated in the nanoparticles) can be determined by the nitrogen to phosphate (N / P) ratio. The N in the N / P ratio stems (mainly) from the polar amine groups on the lipid-like copolymer and other lipids, whereas the P in the N / P ratio stems from the phosphate groups on the oligonucleotides. As the N / P ratio decreases, the relative lipid (copolymer)-to- oligonucleotide mass ratio decreases, reducing the relative ratio of lipid (copolymer) mass. As the N / P ratio increases, the relative lipid (copolymer)-to-oligonucleotide mass ratio increases, increasing the relative ratio of lipid (copolymer) mass. The preferred range of N / P ratio is between 2 and 10, such as 5, to provide an optimal compromise between encapsulation efficiency of oligonucleotide, amount of materials used, and any possible toxicity from excessively cationic materials at high N / P ratios.

[0129] Copolymer

[0130] In an embodiment, the Mn value of the copolymer is between 1400 and 6500 g / mol, preferably between 1400 and 4000 g / mol. In an embodiment, the Mw value of the copolymer is between 2300 and 6700 g / mol, preferably between 2300 and 5000 g / mol. In an embodiment, the polydispersity value of the copolymer is between 1.10 and 1.40, preferably between 1.10 and 1.25. The present inventors have observed that this low molecular weight values allows the copolymer to be incorporated well into lipid nanoparticles, in particular when using in addition to helper lipids. The present inventors also observed that this provides improved encapsulation of e.g. nanoparticle RNA as well as size and morphology of particles obtained.

[0131] In an embodiment, the pKa value of the copolymer is between 5.5 and 7.5, for example between 6.0 and 7.1. In an embodiment, the pKa value of the hydrophilic monomer is between 6.0 and 9.0, for example between 7.0 and 8.0.

[0132] The present copolymer is not merely a combination of hydrophilic and hydrophobic monomers. The invention is a well-considered combination of hydrophilic units having a ionizable head group (with a certain pKa), combined with hydrophobic units having a non-crystalline hydrophobic tail. This unique combination leads to a structure which acts as a poly(lipid). This is different in both structure and function and benefit as prior art copolymers.

[0133] Moreover, the present inventors have provided a copolymer that is easy to synthesize and for which manufacturing can be easily scaled compared to molecular ionizable lipids according to the prior art. As can be observed from the experiments, the present copolymers also provide excellent protection of RNA from degradation compared to molecular ionizable lipid based nanoparticle formulations.

[0134] Nanoparticles

[0135] In an embodiment, the present invention relates to nanoparticles formed of a therapeutic oligonucleotide and a lipid-like copolymer.

[0136] These nanoparticles may have different forms, such as the form of a vesicle (such as polymersomes) or filled nanoparticles encapsulating said therapeutic oligonucleotide. These examples are shown in the form of cartoons in Figures 2a and 2b.

[0137] Polymersomes are vesicles having a self-assembled bilayer of lipid- like copolymers formed when the polymer structure is an amphiphilic block copolymer, where the hydrophobic aliphatic tail block assembles associates to avoid water. Polymer micelles formed from block copolymers have a single layer of lipid-like copolymers where the hydrophobic aliphatic tail block forms the particle interior. Whether a block copolymer forms polymersomes or micelles depends on the relative fractions of hydrophilic and hydrophobic blocks.

[0138] Nanoparticles formed from random copolymers, without amphiphilic block-like nature but with an overall slightly hydrophobic nature, form structures mainly with one internal phase. In the case of nanoparticles formed from the assembly of random copolymers combined with other formulation components (such as RNA, cholesterol, neutral helper lipid, and PEG-lipid) the particles have a complex internal morphology.

[0139] It should be noted that these structures are all formed by selfassembly and that the final form depends on several factors, such as the amphiphilic structure, the length of the copolymer, the n to m ratio, the assembly conditions as well as the ratio of lipid-like copolymers to helper lipids and the overall ratio of lipids / copolymers to oligonucleotides (determined by N / P ratio discussed below) as well as the type and size of the oligonucleotides.

[0140] In an embodiment a lipid conjugated targeting ligand is added to the pharmaceutical composition in an amount of about 0.5 - 10 mol%, preferably 1 - 5 mol%, based on the combined amounts of lipid-like copolymer, helper lipids, and lipid conjugated targeting ligand. The conjugated targeting ligand may be one or more of antibodies, transferrin, folate, GalNAc, integrin derived ligands, arginine based cell penetrating peptides, virus derived peptides.

[0141] In an embodiment, the nanoparticles have an average diameter in the range of from about 50 nm to about 300 nm, as measured by dynamic light scattering.

[0142] In an embodiment, the concentration of nanoparticles in the pharmaceutical composition is between 105- 1012particles per mL.

[0143] The pharmaceutical composition for use as a medicament

[0144] As discussed above in a fourth aspect the present invention relates to the pharmaceutical composition for use as a medicament.

[0145] In an embodiment the pharmaceutical composition is for use in a method of treating, preventing or ameliorating cancer, preferably wherein the cancer is selected from breast cancer, ovarian cancer, lung cancer, leukaemia, melanoma, prostate cancer, glioblastoma.

[0146] In another embodiment the pharmaceutical composition is for use in a method of treating or preventing a disease, chosen from the group comprising metabolic diseases, angiogenisis related diseases, neurodevelopmental disorders, neurodegenerative disorders, viral infections, cardiovascular diseases, or other genetic diseases.

[0147] In yet another embodiment the pharmaceutical composition is for use in a method for inducing an immune response, preferably an immune response against tumour growth. Immunomodulator targets include, i) TNF, ii) cytokines, such as growth factors, for example TGF-a, TGF-b, and IGF, iii) interleukins, for example TL-2, IL-4, IL-12, IL-15, IL-18, and IL-20, and iv) interferons, for example IFN-a, IFN-b, IFN-g.

[0148] Method for producing the pharmaceutical composition.

[0149] EAs discussed above in a fifth aspect the present invention also relates to a method for producing the pharmaceutical composition by mixing a lipid- like copolymer, optionally one or more helper lipids, and a therapeutic oligonucleotide in a solvent to obtain a pharmaceutical composition comprising nanoparticles.

[0150] In an embodiment, a first solvent may be used to dissolve lipid-like copolymer and optionally one or more helper lipids (e.g., ethanol) and the oligonucleotide may be dissolved in a second solvent, e.g., an aqueous buffer (e.g., of pH 4) and mixed together to form a mixture of copolymer, optionally one or more helper lipids, and oligonucleotide in a solvent being a mixture of the first and second solvent.

[0151] In an embodiment formulation of siRNA with lipid-like copolymer is carried out using a rapid mixing procedure of an ethanol lipid solution with nucleic acids in an acetate buffer (figure 4a). Solvents that may be used as first solvent are alcohols, such as ethanol, and isopropyl alcohol, or other water miscible organic solvents such as THF. Other solvents that may be used as second solvent are water or aqueous buffers, when an aqueous buffer is used this preferably has a pH of between 3-5 and this is preferably an acetate buffer.

[0152] A mixing devise may be used to mix the components together, such as a T-junction microfluidic device with separate volume ratios for the first flow comprising the copolymer (and helper lipids) and the second flow comprising the oligonucleotides. By adapting the first and second flow rates the N / P ratio can be set to the predetermined desired value.

[0153] The flow ratio between first flow and second flow may vary between 1 : 1 and 1 :10. The total flow rate can be varied between 1-25 mL / min which can influence the final size of the nanoparticles, wherein a low flow rate favors larger nanoparticles.

[0154] The total concentration of copolymer and helper lipids in the first solvent may be between 0.5-10 mg / mL. The concentration of oligonucleotide in the second solvent may be between 10-1000 pg / mL.

[0155] The temperature during the process is preferably between 15 and 30 °C. The duration of the mixing is preferably between 5 and 60 seconds depending on batch size.

[0156] Use of copolymer

[0157] As discussed above in a sixth aspect the present invention also relates to the use of the lipid-like copolymer for encapsulation of therapeutic oligonucleotides.

[0158] Effects of the invention

[0159] The lipid-like copolymers, nanoparticles thereof with the therapeutic oligonucleotides, and pharmaceutical compositions thereof have been shown to have a significant impact on RNA stability, delivery efficacy and knockdown efficacy. The multivalent nature of the lipid-like polymer (compared to mono amine ionizable lipids) gives the nanoparticles high stability and protects cargo from degradative enzymes. Further, without wanting to be bound by theory, the inventors hypothesize that lipid- like copolymers help the nanoparticles escape intracellular endosomes more effectively due to the lipids bilayer membrane disrupting properties.

[0160] The present invention has the effect of combining the advantages of (molecular) lipids with the advantages of polymers, it was shown by the present inventors to have both lipid properties in the sense that the lipid-like copolymer of the present invention is able to be formed into lipid nanoparticles (preferably co-formulated with one or more helper lipids). Prior art ionizable co-polymers cannot integrate in the same level in lipid nanoparticles because they have less lipid-like character or too high molecular weights. The lipid-like copolymer of the present invention however still maintains the advantages of polymers that include multivalent interaction with therapeutic oligonucleotides, such as RNA to form more stable nanoparticles, increasing protection from RNAses. This multivalent nature of the lipid-like copolymer according to the present invention could potentially increase stability compared to nanoparticles constructed of molecular lipids, such as during lyophilization processes, because of the polymeric linkage. The present invention also allows for variation in the properties of the lipid nanoparticles by variations in the hydrophilic and hydrophobic monomer functionality, the ratios of monomers, and the molecular weight with relative ease.

[0161] The present inventors found that the lipid-like copolymers help the nanoparticles to escape intracellular endosomes more effectively due to the lipid bilayer membrane disrupting properties. In particular, the lipid-like copolymers help to destabilise endosomal membranes, and has high knockdown efficacy. While the multivalent nature of the lipid-like copolymer (compared to mono amine ionizable lipids) gives the formulation high stability and protects cargo from degradative enzymes.

[0162] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. The scope of the present invention is defined by the appended claims. One or more of the objects of the invention are achieved by the appended claims.

[0163] EXAMPLES

[0164] The present invention is further elucidated based on the Examples below which are illustrative only and not considered limiting to the present invention.

[0165] Preparation of lipid-like copolymer monomer synthesis

[0166] Linoleyl acrylate (LA), and hexyldecyl acrylate (HDA) were synthesised following the same general protocol, which is described below for oleoyl acrylate (OA), the others can be prepared in the same matter, the only difference being the starting alcohol. Stearyl acrylate was commercially obtained. To a 100 mL double neck round bottom flask equipped with a magnetic stir bar, oleoyl alcohol (0.022 mol, 6.95 ml), dichloromethane (30 mL), and triethylamine (0.055 mol, 7.67 mL) were added under a nitrogen atmosphere, and the reaction was cooled to 0 °C. Acryloyl chloride (0.022 mol, 1.79 mL) in dichloromethane (10 mL) was subsequently added dropwise over an hour with stirring. After the addition of the acryloyl chloride, the reaction proceeded at room temperature for approximately 16 hours with continued stirring. Upon completion, the reaction mixture was washed first with hydrochloric acid (HCI) (1 M), second with sodium hydroxide (1 M) and last with brine. The organic layer was dried over anhydrous MgSC and filtered, and the solvent was removed via rotary evaporation. The product was purified by flash column chromatography (DCM / hexane, 50 / 50) to give oleoyl acrylate as a clear colourless oily liquid. Structure and purity were confirmed by 1 H-NMR spectroscopy (Brucker Advance 400 MHz spectrometer in deuterated chloroform) (figure 3a).

[0167] Polymerisation

[0168] All polymerisations were carried out with the same general protocol, which is described below for stearyl acrylate, the others can be prepared in the same matter, the only difference being the M2, as described below. In a typical polymerisation the molar ratios of the hydrophobic monomer (M1) to the hydrophobic monomer (M2) to the chain transfer agent (CTA) to the initiator (I) is = 10: 10: 1 : 0.1.

[0169] As chain transfer agent 2-propionic acidyl butyl trithiocarbonate (PABTC) is used (97.48 mg, 0.409 mmol), as hydrophilic monomer (M1) 2-(diethylamino)ethyl acrylate (DEAEA: 0.700 g, 4.09 mmol) is used, as hydrophilic monomer (M2) stearyi acrylate (SA: 1.327 g, 4.09 mmol), as initiator, 4,4'-azobis(4-cyanopentanoic acid) is used (ACVA: 11.45 mg, 0.0409 mmol), and as polymerization solvent dioxane is used (1.303 mL). These were all added to a vial deoxygenated by bubbling with argon and left to stir in an oil bath at 70 °C. After 24 h, the solution was removed from the oil bath and the polymer dialysed against THF for 48 hours (1 kDa membrane), concentrated, and dried under vacuum. 1 H-nuclear magnetic resonance (NMR) spectra were conducted on a Bruker Advance 400 MHz spectrometer. Deuterated chloroform (CDCI3) is used as the solvent and tetramethyl silane (TMS) as the chemical shift reference. Gel permeation chromatography (GPC) was recorded using a Shimadzu Prominence-i GPC system. The system was configured with a PLgel mixed D column and a Shimadzu RID-20A differential refractive index detector. The used eluent was tetrahydrofuran (THF) with a flow rate of 1 mL / min a polystyrene calibration standards. Figure 3b shows the GPC traces of the lipid-like copolymers in THF. The copolymers are respectively named p(DEAEA-co-SA) when stearyi acrylate is used, p(DEAEA-co- OA) when oleyl acrylate is used, p(DEAEA-co-LA) when linoleyl acrylate (LA), is used and p(DEAEA-co-HDA) when hexyldecyl acrylate (HDA) is used. Different ratios of DEAEA and lipid tail monomer have been used, and different degrees of polymerisation targeted (Table 1). Figure 3c shows the 1 H NMR spectra of the purified lipid-like copolymers in deuterated chloroform with key peaks indicated and Figure 3c shows the chemical structures of these molecules with the key peaks assigned to specific chemical moieties.

[0170] Potentiometric titration of the polymers was performed in a 20mL vial with Mettler Toledo pH meter and general-purpose probe. Solutions of copolymer (1 mg / mL) were prepared with 50:50 ethanol to deionised water (at room temperature), 1M NaOH was added to take starting pH to approximately 12. Titrant HCI (0.01M) was gradually added with micropipettes under gentle stirring, after reaching a stable pH value, base again added. The titration curve and the first derivative of the curve were 5 used to determine the equivalence points and the pKa taken as the half equivalence point (see table 1).

[0171] Differential scanning calorimetry (DSC) data were recorded on a DSC Q2000, TA instruments, with an indium standard calibration. The thoroughly dried polymer samples, all of 2-3 mg were weighed directly into tzero hermetic aluminum pans and0 sealed. Samples were initially heated to 150 °C, subsequently two cooling / heating cycles between -90 °C to 150 °C with a rate of 10 °C per min were performed. The data presented, represents the second heating cycle (Figure 3d).

[0172] Table 1. Characterisation data from the synthesised lipid-like copolymers, from 1 H5 NMR spectroscopy, GPC, pH titration, and DSC.

[0173] SA-0.75 5: 15 65 3670 3990 1.15 - -18.7

[0174] 3-SA-0.5 30: 30 52 7680 6150 1.15 6.8 -15.8

[0175] OA-0.5 p(DEAEA-co-OA) 10: 10 42 2770 2650 1.19 6.3

[0176] OA-O.75 5: 15 64 4800 3340 1.26 6.7

[0177] 3-OA-0.5 30: 30 52 6910 5010 1.38 6.6

[0178] LA-0.5 p(DEAEA-co-LA) 10: 10 33 2350 1440 1.23 6.4

[0179] LA-0.75 5: 15 34 3630 2520 1.20 7.1

[0180] 3-LA-0.5 30: 30 36 5650 3640 1.32 6.7

[0181] HDA-0.5 p(DEAEA-co-HDA) 10: 10 60 3630 2430 1.14 6.1

[0182] HDA-0.75 5: 15 61 4140 2170 1.14 6.9 -HDA-0.5 30: 30 57 5830 3560 1.25 6.7 a) Conversion obtained from 1 H-NMR using combined monomer peaks at ~6 ppm and polymer peaks at 4-4.2 ppm, b) Mn obtained from 1 H-NMR spectra comparing peak at 3.35 ppm of CH2 adjacent to trithiocarbonate, and polymer peaks at 4-4.2 ppm, c) Mn obtained from GPC in THF with polystyrene standards, d) Dispersity Mw / Mn obtained from GPC in THF with polystyrene standards.

[0183] Formulation of pharmaceutical composition

[0184] For the formulation of oligonucleotides using lipid-like copolymers, either Cy5-siRNA (Merck MISSION® siRNA Fluorescent Universal Negative Control #1), or dual reporter FLuc siRNA was used. In addition to the lipid-like copolymer three helper lipids were used, being DSPC, cholesterol, and DMG-PEG2000.

[0185] Formulation screening was performed using a pipette mixing method, and variation of molar ratios of lipid-like copolymer: DSPC: cholesterol: PEG-lipid. An optimal formulation of 50:10:37.5:2.5 was found. Formulations were prepared at N / P ratio 5. Microfluidic mixing of nanoparticle formulation components was then used for the optimal formulation using a modified procedure of a method previously described for siRNA. In brief, copolymer and lipids were dissolved in ethanol at molar ratios of 50:10:37.5:2.5 (lipid-like-copolymer: DSPC: cholesterol: DMG-PEG2000). The lipid mixture was combined with a 2 mM sodium acetate buffer (pH 4) containing siRNA at a ratio of 3:1 (aqueous: ethanol) using rapid microfluidic mixing (figure 4a). Formulations were buffer exchanged to 1x PBS (by dialysis over 24h in 1x PBS), and stored at 4 C. siRNA encapsulation (loading % and recovery %) was determined via Quant-iT Ribogreen RNA assay (Thermo Fisher) by comparing lysed LNPs in 1% Triton X-100 with LNPs in TE buffer (according to manufacturer’s recommendations). Recovery % of siRNA was calculated with: amount of siRNA (retained + unretained) / (total amount of siRNA (used in the formulation)) x 100%, and the encapsulation % of siRNA was calculated with: 1-(amount of siRNA (unretained) / amount of siRNA (retained + unretained)) x 100%.

[0186] Dynamic light scattering (DLS) in PBS, and zeta-potential measurements in water (pH 7.4), were conducted on a Malvern Zetasizer Nano and the manufacturers software was used for processing and analyzing the data. Measurements were performed after 1 week and 4 weeks after formulation while being stored at 4 degrees Celsius. Cryogenic Transmission Electron Microscopy (cryo-TEM) experiments were performed using the CryoTitan (Thermo Fisher Scientific) equipped with a field emission gun and an autoloader and operated at a 300 kV acceleration voltage. Samples for cryo-TEM were prepared by plasma treating the grids (Lacey carbon coated, R2 / 2, Cu, 200 mesh, EM Sciences) in a Cressington 208 carbon coater for 40 s. Then, 4 pL of the particle solution was pipetted on the grid and blotted in a Vitrobot MARK III at room temperature and 100% humidity. The grid was blotted for 3 s (offset -3) and directly plunged and frozen in liquid ethane (figure 4b - e).

[0187] Apparent pKa of the particles was determined with TNS assays. 6-(p- Toluidino)-2-naphthalenesulfonic acid sodium salt (TNS reagent) is a compound that electrostatically interacts with the cationic lipid membrane, resulting in fluorescence. Master buffer stock was prepared(10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, 150 mM sodium chloride). Using 1 M sodium hydroxide and 1 M hydro-chloric acid, 16 unique buffers from the master buffer stock at different pH values between 4 and 10 were prepared. A stock of 300 mM TNS in DMSO was prepared. 90 uL of each buffer in the pH range in triplicate was added to a black- bottomed 96-well plate. 5 uL particle sample at a concentration of 0.05 mg / mL mRNA was added to each well. Then, add 2 uL of 300 mM TNS reagent solution was added to each well. TNS only and particle only, were included as control wells. Fluorescence intensity was measured (ex. 325 nm, em. 445 nm) and data was fitted with a sigmoidal curve to obtain the apparent pKa (pH at which fluorescence is half maximum) of the formulation (figure 5).

[0188] To determine the ability of lipid-like copolymer nanoparticles to protect their nucleic acid cargo from degradation, siRNA loaded nanoparticles were incubated at 37 degrees Celsius with either benzonase for 0.5 hours (figure 6a), or 30% fetal bovine serum for 4 hours (figure 6b). After incubation, 10% triton-X was added to the samples to dissociate the particles, and an agarose gel electrophoresis assay run. Protected siRNA bands were visualized using SYBR-gold fluorescence. 1 % agarose gels were prepared in TBE buffer containing SYBR Gold. Samples were mixed with gel loading dye and run for 40 minutes at 50 V. Gels were imaged with a GE Image Quant 350 (GE healthcare Europa GmbH). Table 2. Characterisation data from formulated RNA nanoparticles, comparing lipid- like polymer component on nanoparticle physicochemical properties, with the same lipid molar ratios and T-junction formulation mixing method.

[0189] RNA RNA

[0190] Polymer3Z-av. size (d.nm)bPDI Zetapotential (mV)c

[0191] E.E. (%)dRec. (%)

[0192] SA-0.5 148.8 ± 5.73 0.331 ± 0.019 0.45 ± 1.221 78.0 50.0

[0193] SA-0.75 219.0 ± 3.667 0.158 ± 0.027 7.34 ± 0.892 48.0 22.8

[0194] 3-SA-0.5 286.4 ± 50.45 0.297 ± 0.028 2.77 ± 0.651 76.7 20.9

[0195] OA-0.5 108.6 ± 2.452 0.098 ± 0.025 0.38 ± 0.598 86.5 96.3

[0196] OA-O.75 183.3 ± 1.650 0.132 ± 0.055 5.39 ± 0.851 67.2 59.1

[0197] 3-OA-0.5 199.7 ± 1.779 0.058 ± 0.019 -1.10 ± 0.628 84.2 51.4

[0198] LA-0.5 126.2 ± 0.265 0.071 ± 0.027 -0.89 ± 1.022 81.1 92.4

[0199] LA-0.75 168.1 ± 7.267 0.217 ± 0.047 3.72 ± 0.621 61.0 48.8

[0200] 3-LA-0.5 200.4 ± 4.417 0.164 ± 0.077 -3.75 10.600 87.6 52.8

[0201] HDA-0.5 92.41 1.200 0.19710.008 -1.261 0.821 87.1 95.7

[0202] HDA-0.75 160.0 1 2.46 0.193 1 0.064 4.411 0.767 55.7 60.8

[0203] 3-HDA-0.5 194.9 1 6.865 0.21010.022 -4.67 10.382 81.5 53.8 a) Molar ratio of lipid components, [lipid-like polymer] :[DSPC] :[Cholesterol] :[DMG- PEG2kDa], 50: 10: 37.5: 2.5. b) Z-Average size from dynamic light scattering in PBS, c) Zetapotential measured in 0.05X PBS, d) Encapsulation efficiency % of siRNA was calculated with: 1-(amount of siRNA (unretained) / amount of siRNA (retained + unretained)) x 100%. e) Recovery % of siRNA was calculated with: amount of siRNA (retained + unretained) / (total amount of siRNA (used in the formulation)) x 100%.

[0204] In Vitro studies

[0205] Human epithelial ovarian adenocarcinoma cell line SKOV3 were maintained in a humidified incubator at 37 °C with 5% CO2. Cells were cultured in RPMI medium containing 10% FBS, 1 % penicillin / streptomycin. For uptake studies, cells were seeded in ibidi 8 well microscopy slides in RPMI medium and cultured for 24h, cells were washed, and samples (100 nM) were incubated with cells for 16 h. Cells were washed with PBS to remove free nanoparticles and live cell imaging solution added. Nuclei were stained with Hoechst 33342, cell membrane with WGA- AF488, and red signal represents Cy5-labeled samples. Cells were imaged with a Leica TCS SP8X (63* oil immersion objective) equipped with microscope incubator (Okolab, 37 oC, 5% CO2) for live cell imaging. Cell boundary regions of interest were drawn in Imaged software and fluorescent signal determined for a minimum of n = 40 cells. Cell-uptake in SKOV3 cells of lipid-like polymer nanoparticles encapsulating Cy5-siRNA, and Cy5-siRNA control, was visualized using confocal microscopy (Figure 7a).

[0206] For knockdown studies, SKOV3 cells stably expressing dual luciferase reporter genes were seeded in 96-well plates (10 000 cells per well) in RPMI medium and allowed to attach overnight. The medium was removed and replaced with lipid-like copolymer siRNA nanoparticles (dual reporter FLuc siRNA), at varying concentrations in triplicate. The cells and particles were incubated for 24 h, then the medium was removed and replaced by fresh medium and incubated for another 48 h. Gene knock-down was evaluated using the luciferase bioluminescence assay using a Tecan Spark multimode microplate reader. Immediately following Flue readout, an MTS cell viability assay was carried out to confirm the cell viability after exposure to the lipid-like polymer nanoparticles. The assay was performed according to the manufacturers guidelines. Cell-uptake in SKOV3 cells of lipid-like polymer nanoparticles encapsulating Cy5-siRNA, and Cy5-siRNA control, was assessed using confocal microscopy (Figure 7a). Cell uptake / internalisation was quantified using ImgageJ (Figure 7b). SKOV3 cell viability with polymer nanoparticles was assessed using MTS assay (Figure 7c). Dual reporter FLuc knockdown assay was carried out across varying concentrations, including DLin-MC3-DMA lipid nanoparticle as positive control (Figure 7d). EC50 values were extracted from dose response curve fitting of the knockdown assay (Figure 7e).

[0207] To assess endo / lysosome colocalization, cells were seeded in ibidi 8 well microscopy slides in RPMI medium and cultured for 24h, cells were washed and samples (200 nM) were incubated with cells for 3 h and 6 h. Cells were washed with PBS to remove free nanoparticles and live cell imaging solution added. Nuclei were stained with Hoechst 33342, endo / lysosomes with LysoTracker Green DND-26, and red signal represents Cy5-labeled samples. Quantification of sample colocalization with endo / lysosomes inside cells was carried out with coloc2 plugin for Imaged software and expressed as Pearson’s coefficient and Mander’s M2 coefficient on n = 10-20 cells. Endosomal escape of lipid-like polymer nanoparticle formulations p(DEAEA-co-HDA), p(DEAEA-co-LA), p(DEAEA-co-OA), and Cy5-siRNA control, in SKOV3 cells was assessed after 3h incubation and 6h incubation (figure 8a and 8b). Sample colocalization with endo / lysosomes was quantified and assessed with Pearsons coefficient and Manders M2 coefficient, n = 10-20 cells (figure 8c).

[0208] FRET membrane fusion assay. Endosome bilayer mimicking liposomes were used to measure LNP fusion to endosome lipid bilayers. The DOPE-conjugated FRET probes DOPE-NBD and DOPE-Rho were added into liposomes. Endosomal mimicking anionic liposomes were prepared by mixing DOPC: DOPE: Choi: DOPE-NBD: DOPE-Rho (molar ratio = 58: 20: 20: 1 : 1) in chloroform followed by rotary evaporation to obtain a thin lipid film. The dried film was subsequently hydrated in PBS (pH 7.4) and sonicated for 20 min at final total lipid concentration of 10 mM. PBS (pH 6) was added to black 96- well plates (100 pL / well), and 1 pL liposomes were added to each well. Polymer LNPs were added at a 10:1 weight ratio LNP to liposomes. Upon incubating for 10 min, fluorescence intensity measurements (I) were determined on Tecan plate reader (Excitation (DOPE-NBD) = 465 nm, Emission (DOPE-Rho) = 595 nm). Lipid membrane fusion was monitored by the decrease of the FRET 595 emission. Liposomes incubated in PBS (pH 6) were used as negative control (I0). liposomes in PBS Triton X-100 solutions (2 wt.%) were used as positive control (11). The lipid membrane fusion activity (%) was calculated as 1- [(1-11) / (I0-11 )] x 100%, and can be seen in figure 9.

[0209] Membrane phase 31 P NMR spectroscopy. Endosomal mimicking anionic liposomes were prepared by mixing DOPC: DOPE: Choi (molar ratio = 60: 20: 20) in chloroform followed by rotary evaporation to obtain a thin lipid film. The dried film was subsequently hydrated in D2O (20mM HEPES, pH 6) and sonicated for 10 min at final total lipid concentration of 20 mM. 31 P NMR spectra of these endosomal mimics was acquired on a Bruker 400 MHz spectrometer at 25 °C, with 0.5 s acquisition time, 1 s relaxation delay and 8000 scans (figure 10). Lipid-like polymer was dissolved in chloroform (1 mM), and a thin film formed by rotary evaporation. Subsequently the cloudy white liposomes in D2O were added to the polymer film and vortexed for 10 min, and another 31 P NMR spectra acquired with the same settings (figure 10).

[0210] The above experiments clearly show that the copolymers according to the invention provide better thermal behaviour, as specifically visible from the DSC data provided above. This improvement can be found in the lack of melting and crystallisation peaks in the DSC. The liquid-disordered phase behaviour of the non-crystalline polymers allows homogenous incorporation of all formulation components. The above experiments clearly show that the copolymers according to the invention provide better endosome escape because of disruption of lipid membrane, as specifically visible from the microscopy data, membrane fusion data, and 31 P NMR data provided above. This improvement can be found in higher cytosolic delivery of RNA cargo, and thus higher bioavailability of RNA.

Claims

CLAIMS1. A lipid-like copolymer comprising hydrophilic and hydrophobic units represented by Formula AFormula A wherein:Z1 is a chain transfer residue, preferably having the formula is a chain transfer residue, preferably having the formula:each X is independently O or NH, n is the number of hydrophilic units in the copolymer and is an integer between 2 and 150, m is the number of hydrophobic units in the copolymer and is an integer between 2 and 150, preferably n + m is between 4 and 300, more preferably at most 200, even more preferably at most 100, v is an integer between 1 and 4, each R1 is independently hydrogen or methyl,R2a and R2b are each independently selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, 2-ethylhydroxyl, or R2a and R2b together with the nitrogen atom to which they are attached form a 5-, 6- or 7- membered ring, preferably selected from the group consisting of:R3 is a linear or branched C12-C20 alkyl or alkenyl group, preferably stearyl, oleoyl, lineoyl or 2-hexyldecyl,R4 is a C1-C12 alkyl group, for example n-butyl,R5a is hydrogen or methyl; R5b is methyl or CN, and w is 0, 1 or 2 preferably Z2 is selected from the group consisting of:C is a carbon atom, O is an oxygen atom, and N is a nitrogen atom, and S is a sulphur atom.

2. The lipid-like copolymer according to claim 1 , wherein the copolymer may be an alternating copolymer comprising alternating hydrophilic and hydrophobic units, a random copolymer comprising randomly distributed hydrophilic and hydrophobic units, or a block-copolymer comprising one or more blocks of two or more hydrophilic units and one or more blocks of two or more hydrophobic units, preferably the copolymer is an alternating copolymer.

3. The lipid-like copolymer according to claim 1 or claim 2, wherein Z1 has the formulawherein R4 is n-butyl, Z2 has the formulawherein R5a is hydrogen, R5b is methyl, w is 0, both X are O, v is 2, both R1 are hydrogen, R2a and R2b are each ethyl, R3 is stearyl, oleoyl, lineoyl or 2-hexyldecyl, and n and m are each independently between 3 and 10.

4. A method of producing the lipid-like copolymer according to any one of the preceding claims, comprising a free radical or RAFT polymerisation of a first hydrophilic monomer according to Formula B and a second hydrophobic monomeraccording to Formula C and a chain transfer agent (CTA) according to the formula Z1-Z2Formula B Formula C wherein Z1 , Z2, X, v, R1 , R2a, R2b, and R3 are as defined in claim 1.

5. The method according to claim 4, wherein Z1-Z2 is according to the formula DFormula D wherein R4, R5a, R5b and w are as defined in claim 1.

6. A pharmaceutical composition comprising the lipid-like copolymer according to claim 1 and a therapeutic oligonucleotide.

7. The pharmaceutical composition according to claim 6, wherein the therapeutic oligonucleotide is selected from the group consisting of mRNA encoding a peptide or protein replacing or supplementing an endogenous protein or peptide, mRNA comprising an antigen or epitope, mRNA complementary to a target gene’s mRNA sequence, mRNA encoding for a CRISPR associated protein, CRISPR sgRNA, self-amplifying mRNA, RNAi, an antisense oligonucleotide, an aptamer, and one or more combinations thereof.

8. The pharmaceutical composition according to any one of claim 6 or 7, wherein the RNAi is selected from the group consisting of siRNA, miRNA, piRNA, shRNA, and one or more combinations thereof.

9. The pharmaceutical composition according to any one of claims 6 - 8, wherein the pharmaceutical composition further comprises a helper lipid selected from the group consisting of a neutral lipid, a sterol, a PEG-lipid and one or more combination thereof, preferably wherein the neutral lipid is selected from the groupconsisting of DSPC, DPPC, DOPC, POPC, and DOPE, wherein the sterol is cholesterol, and wherein the PEG-lipid is PEG2k-DMG or PEG2k-DSPE.

10. The pharmaceutical composition according to any one of claims 6 - 9, wherein as helper lipids a neutral lipid, a sterol and a PEG-lipid are present.

11. The pharmaceutical composition according to any one of claims 6 -10, comprising nanoparticles formed of said therapeutic oligonucleotide and lipid-like copolymer, preferably said nanoparticle having the form of a liposome, polymersomes, lipid nanoparticle or polymer micelle encapsulating said therapeutic oligonucleotide12. The pharmaceutical composition according to any one of claims 6 -11 , wherein said nanoparticles have an average diameter in the range of from about 50 nm to about 300 nm, as measured by dynamic light scattering.

13. The pharmaceutical composition according to any one of claims 6 -12, wherein the molar ratio of the lipid-like copolymer and the one or more helper lipids is between 70:30 and 30:70, preferably between 60:40 and 40:60, more preferably between 55:45 and 45:55, such as 50:50.

14. The pharmaceutical composition according to claim 12, wherein the lipid-like copolymer is present in an amount of about 20-70 mol%, the neutral lipid in an amount of 5-30 mol%, the sterol in an amount of 10-60 mol%, and the PEG-lipid in an amount of 0.5-20 mol%, based on the combined amounts of lipid-like copolymer and helper lipids.

15. The pharmaceutical composition according to any one of claims 6 - 14, wherein the N / P ratio is between 2 and 10.

16. The pharmaceutical composition according to claim 14 or 15, wherein a lipid conjugated targeting ligand is added in an amount of about 0.5 - 10 mol%, preferably 1 - 5 mol%, based on the combined amounts of lipid-like copolymer, helper lipids, and lipid conjugated targeting ligand, preferably wherein the conjugated targeting ligand is selected from the group consisting of antibodies, transferrin, folate, GalNAc, integrin derived ligands, arginine based cell penetrating peptides, virus derived peptides.

17. The pharmaceutical composition according to any one of claims 6 -16 for use as a medicament.

18. The pharmaceutical composition according to any one of claims 6 -17 for use in a method of treating or preventing a disease, chosen from the groupcomprising cancer, metabolic diseases, angiogenisis related diseases, inflammatory and autoimmune diseases, neurodevelopmental disorders, neurodegenerative disorders, viral infections, cardiovascular diseases, or other genetic diseases.

19. The pharmaceutical composition according to any one of claims 6 -18 for use in a method of treating, preventing, or ameliorating cancer, preferably wherein the cancer is selected from breast cancer, ovarian cancer, lung cancer, leukaemia, melanoma, prostate cancer, glioblastoma.

20. The pharmaceutical composition according to any one of claims 6 -19 for use in a method for inducing an immune response, preferably an immune response against tumour growth, in a subject, wherein an immunomodulator target is selected from i) TNF, ii) cytokines, such as growth factors, for example TGF-a, TGF- b, and IGF, iii) interleukins, for example TL-2, IL-4, IL-12, IL-15, IL-18, and IL-20, and iv) interferons, for example IFN-a, IFN-b, IFN-g.

21. A method for producing the pharmaceutical composition according to any one of claims 6 - 20 comprising mixing a lipid-like copolymer, optionally one or more helper lipids, and a therapeutic oligonucleotide in a solvent to obtain a pharmaceutical composition comprising nanoparticles.

22. Use of the lipid-like copolymer according to any one of claims 1 - 3 or prepared according to the method of claims 4 or 5 for encapsulation of therapeutic oligonucleotides.

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