Ionizable lipids

Abstract

The present invention generally relates to the field of ionizable (also termed cationic) lipids, and in particular provides a novel type of such lipids as represented by formula (I). The present invention further provides methods for making such lipids as well as uses thereof, in particular in the preparation of nanoparticle compositions, more in particular nanoparticle compositions comprising nucleic acids. It further provides vaccine formulations comprising nanoparticle compositions based on the ionizable lipid disclosed herein.

Description

[0001] IONIZABLE LIPIDS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of ionizable (also termed cationic) lipids, and in particular provides a novel type of such lipids as represented by formula (I). The present invention further provides methods for making such lipids as well as uses thereof, in particular in the preparation of nanoparticle compositions, more in particular nanoparticle compositions comprising nucleic acids. It further provides vaccine formulations comprising nanoparticle compositions based on the ionizable lipid disclosed herein.

[0004] BACKGROUND TO THE INVENTION

[0005] Nucleic acid- based drugs are being explored in a growing number of therapeutic areas. Nonetheless, due to their negative charge, size and instability, the targeted delivery of nucleic acids such as plasmid DNA, messenger RNA, short interfering RNA, single guide RNA and micro-RNAs to tissues and cells poses a major challenge. A plethora of nanoparticulate carrier systems has been explored to encapsulate and deliver nucleic acids. These nanoparticles need to combine efficient and stable encapsulation of the nucleic acid upon storage and in the extracellular environment, with maximum cellular uptake and efficient release of their payload from endosomes into the cytosol.

[0006] Lipid based nanoparticles are clinically used to deliver small interfering RNA and mRNA vaccines and represent the most advanced class of RNA delivery vehicles. Lipid-based nanoparticles are typically composed of a cationic or ionizable lipid that can be protonated at acid pH, a helper phospholipid, a PEGylated lipid and a sterol. Each component has specialized functions in LNP stability and activity. The sterol and the PEGylated lipid are vital for LNP structure and stability, whereas the phospholipid can contribute to stability and endosomal escape. The cationic or ionizable lipid in turn is considered the main driver of activity and tolerability by governing mRNA encapsulation, cellular uptake and endosomal escape. Although effective nucleic acid delivery vehicles, LNPs can induce dose limiting toxicities, such as Complement Activation Related Pseudo-allergy, inflammatory cytokine release and cellular toxicities by accumulation of non-degradable ionizable lipids into cellular membranes. Further improvements in cationic or ionizable lipid chemistries are hence needed to improve efficacy and safety of LNP delivered nucleic acid drugs.

[0007] Accordingly, the present invention relates to a new ionizable lipid as defined by the present set of claims, which have improved characteristics over the currently available ionizable lipids. SUMMARY OF THE INVENTION

[0008] In a first aspect, the present invention provides a lipid, in particular an ionizable lipid represented by formula (I)

[0009] In a further aspect, the present invention provides a lipid nanoparticle or lipid nanoparticle composition comprising a lipid, in particular an ionizable lipid as defined herein. Said nanoparticle composition may further comprise a phospholipid, a sterol and a PEG lipid.

[0010] In yet a further embodiment of the present invention, the lipid nanoparticle or lipid nanoparticle composition as defined herein may further comprise an active agent, in particular a nucleic acid, preferably mRNA.

[0011] In a further aspect, the present invention provides the use of a lipid, in particular an ionizable lipid as defined herein in the manufacture of a lipid nanoparticle or lipid nanoparticle composition.

[0012] In a final aspect, the present invention provides a pharmaceutical composition comprising a lipid nanoparticle or lipid nanoparticle composition as defined herein and a pharmaceutically acceptable agent.

[0013] The invention also provides the pharmaceutical compositions as defined herein for use in human and / or veterinary medicine; in particular for use in vaccination.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0016] Fig. 1 : Viability of different cell types after transfection with LNPs comprising the indicated ionizable lipid, i.e. CT26 (A) and MC38 (B).

[0017] Fig. 2 : Relative Mean Fluorescence Intensity (measured as the fold-increase in eGFP MFI compared to untreated cells) of eGFP expression in CT26 (A) or MC38 (B) cells upon incubation with LNPs comprising the indicated ionizable lipid at an mRNA concentration of 50 ng and 200 ng / well.

[0018] Fig. 3 : HA specific lgG1 antibody titers upon intramuscular immunization with the LNPs comprising the indicated ionizable lipid. N:P represents the NitrogemPhosphate ratio of the LNP.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0021] Unless a context dictates otherwise, asterisks are used herein to indicate the point at which a mono- or bivalent radical depicted is connected to the structure to which it relates and of which the radical forms part.

[0022] As already mentioned hereinbefore, in a first aspect the present invention provides a lipid, in particular an ionizable lipid represented by formula (I) When describing the compounds / lipids of the present invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise:

[0023] In the context of the present invention, the term lipid is meant to be a chemically defined substance that is insoluble in water but soluble in amongst others alcohol, ether and chloroform. Ionizable or cationic lipids are lipids that are typically composed of three section: an amine head group, a linker moiety and a hydrophobic tail. The term “ionizable” (or alternatively cationic) in the context of a compound or lipid means the presence of any uncharged group in said compound or lipid which is capable of dissociating by yielding an ion (usually an H+ion) and thus itself becoming positively charged. Alternatively, any uncharged group in said compound or lipid may yield an electron and thus becoming negatively charged.

[0024] In a further aspect, the present invention provides a lipid nanoparticle or lipid nanoparticle composition comprising a lipid, in particular an ionizable lipid as defined herein.

[0025] In the context of the present invention, the term lipid nanoparticle (LNP), also termed solid lipid nanoparticles, is meant to be a nanoparticle comprising lipids. They are often used as a pharmaceutical drug delivery system or pharmaceutical formulation. LNPs as drug delivery vehicle were first approved in 2018, and are currently used in several candidate RNA based vaccines. A lipid nanoparticle is typically spherical with an average diameter between 10 and 1000 nanometers, and possesses a lipid core matrix that can solubilize lipophilic molecules. The term lipid is used here in a broader sense and includes triglycerides, diglycerides, monoglycerides, fatty acids, steoids (e.g. cholesterol) and waxes. Biological membrane lipids such as phospholipids, sphingomyelins, bile acids and sterols are typically used as stabilizers in LNPs.

[0026] As used herein, the term "nanoparticle" refers to any particle having a diameter making the particle suitable for systemic, in particular intravenous administration, of, in particular, nucleic acids, typically having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, such as for example between 50 and 200 nm; preferably between 60 and 160 nm.

[0027] Accordingly, in the context of the present invention, the nanoparticles as disclosed herein further comprise one or more additional lipids either or not acting as stabilizers, such as a phospholipid, a sterol and / or a PEG lipid.

[0028] In the context of the present invention, the term “PEG lipid” or alternatively “PEGylated lipid” is meant to be any suitable lipid modified with a PEG (polyethylene glycol) group. Particularly suitable PEG lipids in the context of the present invention are characterized in being C18-PEG lipids, C14-PEG lipids (e.g. DMG-PEG or DMG-PEG2000), C16-PEG lipids, C14-ceramide PEG lipids, C18-ceramide PEG lipids or or C16-ceramide PEG lipids (such as C16 ceramide PEG2000 - N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]).

[0029] C16-ceramide PEG2000

[0030] C18-PEG lipids contain a polyethylene glycol moiety, which defines the molecular weight of the lipids, as well as a fatty acid tail comprising 18 C-atoms. In a particular embodiment, said C18- PEG2000 lipid is selected from the list comprising: a (distearoyl-based)-PEG2000 lipid such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or DSPE- PEG2000 lipid (1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or a (dioleolyl-based)-PEG2000 lipid such as DGG-PEG2000 lipid (1 ,2-Dioleolyl- rac-glycerol) or DGPE-PEG2000 lipid (1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000]).

[0031] C14-PEG lipids contain a polyethylene glycol moiety, which defines the molecular weight of the lipids, as well as a fatty acid tail comprising 14 C-atoms. In a particular embodiment, said C14- PEG2000 lipid is based on dimyristoyl, i.e. having 2 C14 tails, such as selected from the list comprising: a (dimyristoyl-based)-PEG2000 lipid such as DMG-PEG2000 lipid (1 ,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000) or 2-Dimyristoyl-sn-Glycero-3- Phosphoethanolamine glycol-2000 (DMPE-PEG2000).

[0032] DMPE-PEG2000

[0033] In the context of the present invention, the term “phospholipid” is meant to be a lipid molecule consisting of two hydrophobic fatty acid “tails” and a hydrophilic “head” consisting of a phosphate group. The two components are most often joined together by a glycerol molecule, hence, the phospholipid of the present invention is preferably a glycerol-phospholipid. Furthermore, the phosphate group is often modified with simple organic molecules such as choline (i.e. rendering a phosphocholine) or ethanolamine (i.e. rendering a phosphoethanolamine).

[0034] Suitable phospholipids within the context of the invention can be selected from the list comprising: 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-Dioleoyl-sn-glycero- 3-phosphocholine (DOPC), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPO), 1 -palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPO), 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 (C 16 Lyso PC), 1 ,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1 ,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho- rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, 2-((2,3- bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate) (DOCP), (1 ,2-dioleoyl-sn- glycero-3-phosphate sodium salt (18:1 PA) and mixtures thereof.

[0035] In a more specific embodiment, said phospholipid is selected from the list comprising: 1 ,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and mixtures thereof.

[0036] In the context of the present invention, the term “sterol”, also known as steroid alcohol, is a subgroup of steroids that occur naturally in plants, animal and fungi, or can be produced by some bacteria. In the context of the present invention, any suitable sterol may be used, such as selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.

[0037] In a specific embodiment of the present invention one or more of the following applies:

[0038] - said LNP comprises about and between 25 mol% and 65 mol% of said ionizable lipid;

[0039] - said LNP comprises about and between 5 mol% and 45 mol% of said phospholipid;

[0040] - said LNP comprises about and between 0.5 mol% and 5.0 mol% of said PEG lipid; balanced by the amount of said sterol.

[0041] The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1 , such as 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 12:1 , 14:1 , 16:1 , 18:1 , 20:1 , 22:1 , 24:1 , 26:1 , 28:1 , or 30:1 . In certain embodiments, the N:P ratio may be from about 2:1 to about 10:1. In other embodiments, the N:P ratio is from about 4:1 to about 8:1. For example, the N:P ratio may be about 6:1 , alternatively the N:P ratio may be about 10:1.

[0042] In a very specific embodiment, the present invention provides an LNP having an ionizable lipid / DSPC / cholesterol / DMG-PEG2000 ratio of about 50 / 10 / 38.5 / 1.5 and an mRNA / ionizable lipid molar ratio of 1 :10.

[0043] In yet a further embodiment of the present invention, the lipid nanoparticle or lipid nanoparticle composition as defined herein further comprises a cargo molecule such as a pharmaceutically active agent (e.g. small molecule) or a biomolecule, such as a peptide, protein or a nucleic acid. In a particular embodiment, the cargo may be a nucleic acid, such as DNA or RNA; preferably mRNA. In another particular embodiment, the cargo may be a TLR agonist, such as for example the TLR3 agonist polyl:C, or the TLR9 agonist CpG.

[0044] Prior to being loaded in the lipid nanoparticles, the cargo molecules may further be modified to induce an overall polyanionic nature to the molecules. This can for example be done by bonding them to a Glu10 moiety as exemplified in the examples part. The Glu10 moiety is a moiety of 10 glutamic acids which increases the polyanionic nature of the molecule to which it is attached. Accordingly, the lipid nanoparticles and lipid nanoparticle compositions of the present invention are particularly suitable for the intracellular delivery of their cargo molecules. Hence, the present invention provides the use of the lipid nanoparticles and lipid nanoparticle compositions as defined herein for the intracellular delivery of cargo molecules.

[0045] In a particular embodiment, the lipid nanoparticle or lipid nanoparticle composition as defined herein further comprises a nucleic acid, preferably mRNA.

[0046] A “nucleic acid” in the context of the invention is a deoxyribonucleic acid (DNA) or preferably a ribonucleic acid (RNA), more preferably mRNA. Nucleic acids include according to the invention genomic DNA, cDNA, tRNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic acid can be employed for introduction into, i.e. transfection of cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and / or polyadenylation.

[0047] In the context of the present invention, the term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a 0- D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. Nucleic acids may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial or analogs of naturally-occurring RNA.

[0048] According to the present invention, the term "RNA" includes and preferably relates to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5' untranslated region (5’ -UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.

[0049] In a further aspect, the present invention provides a pharmaceutical composition comprising one or more LNP’s as defined herein and a pharmaceutically acceptable agent, such as a carrier, excipient,.... Such pharmaceutical compositions are particularly suitable as a vaccine. Thus, the invention also provides a vaccine comprising one or more LNP’s according to the present invention.

[0050] In the context of the present invention, the term “vaccine” as used herein is meant to be any preparation intended to provide adaptive immunity (antibodies and / or T cell responses) against a disease. To that end, a vaccine as meant herein contains at least one nucleic acid molecule, e.g. mRNA molecule encoding an antigen to which an adaptive immune response is mounted. This antigen can be present in the format of a weakened or killed form of a microbe, a protein or peptide, or an antigen encoding a nucleic acid. An antigen in the context of this invention is meant to be a protein or peptide recognized by the immune system of a host as being foreign, thereby stimulating the production of antibodies against is, with the purpose of combating such antigens. Vaccines can be prophylactic (example: to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (example, to actively treat or reduce the symptoms of an ongoing disease). The administration of vaccines is called vaccination.

[0051] The vaccine of the invention may be used for inducing an immune response, in particular an immune response against a disease-associated antigen or cells expressing a disease- associated antigen, such as an immune response against cancer. Accordingly, the vaccine may be used for prophylactic and / or therapeutic treatment of a disease involving a disease- associated antigen or cells expressing a disease- associated antigen, such as cancer. Preferably said immune response is a T cell response. In one embodiment, the disease- associated antigen is a tumor antigen. The antigen encoded by the RNA comprised in the nanoparticles described herein preferably is a disease-associated antigen or elicits an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.

[0052] The present invention also provides the LNP’s, pharmaceutical compositions and vaccines according to this invention for use in human or veterinary medicine. The use of the LNP’s, pharmaceutical compositions and vaccines according to this invention for human or veterinary medicine is also intended. Finally, the invention provides a method for the prophylaxis and treatment of human and veterinary disorders, by administering the LNP’s, pharmaceutical compositions and vaccines according to this invention to a subject in need thereof. Such pharmaceutical compositions are particularly suitable in various fields such as prophylactic vaccines, therapeutic vaccines, protein replacement therapies, gene editing, gene silencing, small molecule delivery, etc.

[0053] The present invention further provides the use of an LNP, a pharmaceutical composition or a vaccine according to the present invention for the immunogenic delivery of said one or more nucleic acid molecules. As such the LNP’s, pharmaceutical compositions and vaccine of the present invention are highly useful in the treatment several human and veterinary disorders. Thus, the present invention provides the LNP’s, pharmaceutical compositions and vaccines of the present invention for use in the treatment of cancer or infectious diseases.

[0054] The lipid nanoparticles of the present invention may be prepared in accordance with the protocols as specified in the Examples part. More generally, the LNP’s may be prepared using a method comprising:

[0055] - preparing a first alcoholic composition comprising said ionizable lipid, said phospholipid, said sterol, said PEG lipid, and a suitable alcoholic solvent;

[0056] - preparing a second aqueous composition comprising said one or more nucleic acids and an aqueous solvent;

[0057] - mixing said first and second composition in a microfluidic mixing device.

[0058] In further detail, the lipid components are combined in suitable concentrations in an alcoholic vehicle such as ethanol. Thereto, an aqueous composition comprising the nucleic acid is added, and subsequently loaded in a microfluidic mixing device.

[0059] The aim of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (i.e. lipid phase and nucleic acid phase) in a microscale device. Such sample mixing is typically achieved by enhancing the diffusion effect between the different species flows. Thereto several microfluidic mixing devices can be used, such as for example reviewed in Lee et al., 2011. A particularly suitable microfluidic mixing device according to the present invention is the NanoAssemblr from Precision Nanosystems.

[0060] Other technologies suitable for preparing the LNP’s of the present invention include dispersing the components in a suitable dispersing medium, for example, aqueous solvent and alcoholic solvent, and applying one or more of the following methods: ethanol dilution method, a simple hydration method, sonication, heating, vortex, an ether injecting method, a French press method, a cholic acid method, a Ca2+fusion method, a freeze-thaw method, a reversed-phase evaporation method, T-junction mixing, Microfluidic Hydrodynamic Focusing, Staggered Herringbone Mixing, and the like. The ionizable lipid of the present invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention and that the compounds of this invention can be prepared by any standard synthetic process commonly used by one skilled in the art of organic chemistry.

[0061] EXAMPLES

[0062] EXAMPLE 1: PREPARATION OF THE IONIZABLE LIPID

[0063] 1. General information

[0064] Unless otherwise stated, all glassware was oven dried before use and all reactions were carried out under an argon atmosphere using standard Schlenk-techniques. Dry solvents were purchased from Acros Organics or Sigma-Aldrich and used without further purification. All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. Reaction progress was monitored by thin layer chromatography (TLC) performed on aluminum plates coated with Kieselgel F254 with 0.2 mm thickness. Visualization was achieved by ultraviolet light (254 nm) or by staining with potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck ans co.). Mass spectra were obtained using a Finnigan MAT 8200 (70 eV), an Agilent 5973 (70 eV), using electrospray ionization (ESI) or electron impact ionization (El). All 1 H NMR, 13C NMR NMR were recorded on a BrukerAV-400 in Chloroform-d1 or DMSO-d6. Chemical shifts are given in parts per million (ppm), referenced to tetramethylsilane using the solvent peak as internal standard (CDCI3:1H = 7.26 ppm,13C = 77.16 ppm; CD3SOCD3:1H = 2.50 ppm,13C = 39.52 ppm). Coupling constants were quoted in Hz.1H NMR splitting patterns were designated as singlet (s), broad (brd), doublet (d), triplet (t), quartet (q), pentet (p), sextet (se), septet (sep), octet (o) or combinations thereof. Splitting patterns that could not be interpreted were designated as multiplet (m).

[0065] 2. Synthesis of the ionizable lipid IL3 EXAMPLE 2: IN VITRO EXPERIMENTS

[0066] Materials and methods: mRNA synthesis: mRNAs encoding eGFP and FireFly luciferase were prepared in vitro by T7-mediated transcription from linearized DNA templates (peTheRNAvs3 vector), which incorporates 5’ and 3’ UTRs and a polyA tail. The final mRNA utilizes Cap1 and 100% replacement of uridine with N1-methyl-pseudo-uridine.

[0067] LNP synthesis:

[0068] Lipid based nanoparticles are produced by microfluidic mixing of an mRNA solution in sodium acetate buffer (100mM, pH4) and lipid solution in a 2:1 volume ratio at a speed of 9mL / min using the NanoAssemblr Benchtop (Precision Nanosystems). The lipid solution contained a mixture of the ionizable lipid of interest, DSPC, DOPC or DOPE (Avanti), Cholesterol (Sigma) and DMG- PEG2000 (Sunbright GM-020, NOF corporation. Three ionizable lipids were tested with formula:

[0069] (IL1 ) S-Ac7-Dog [Comparative example 1 ] - see synthesis route in W02020182993

[0070] (IL2) S-Ac7-DHDa [Comparative example 2] - see synthesis route in W02020182993

[0071] (IL3) S-Ade-DHDa (formula I according to the invention) - see synthesis route in example 1 IL1 and IL2 are ionizable lipids considered to be known in the state-of-the art. LNPs were dialyzed against TBS (10000 times more TBS volume than LNP volume) using slide-a-lyzer dialysis cassettes (20K MWCO, 3mL, ThermoFisher). Size, polydispersity and zeta potential were measured with a Zetasizer Nano (Malvern). mRNA encapsulation was measured by standard Ribogreen RNA assay (Invitrogen).

[0072] Cell lines: CT26 - MC38

[0073] The most optimal culturing conditions per cell type including growth medium, subcultivation ratio, and medium renewal recommendations are summarized below in table 1. To harvest adherent cells, used-up growth medium was discarded and cells were rinsed twice with phosphate buffered saline (PBS) (Sigma) before addition of trypsine-EDTA (0.05%) (Gibco, Thermo Fisher Scientific) to loosen the cells. Medium renewal needs to occur every 2 to 3 days, whenever cells reached confluency of approximately 70%. Cell viability was determined using the Vi-Cell XR Cell Viability Analyzer (Beckman Coulter).

[0074] Table 1 : Cell type specific culturing conditions

[0075] Abbreviations: DMEM: Dulbecco’s Modified Eagle Medium; HEPES: 4-(2-hydroxyethyl)-&- piperazineethanesulfonic acid; RPMI: Roswell Park Memorial Institute; P / S: Penicillin / Streptomycin; FBS: Foetal Bovine Serum

[0076] Transfection

[0077] Cells were plated in a 96-well plate at a density of 20-30x10e4 cells / 1 OOpi complete growth medium (specific per cell type). Transfection was performed when cells reached 70-90% confluency. The positive control Lipofectamine (MessengerMAX, Invitrogen) was diluted in OptiMEM (serum reduced, Gibco) and incubated for 10 minutes. In the meantime, eGFP mRNA and LNPs encapsulating eGFP mRNA were diluted in OptiMEM to get to a concentration of the mRNA content of 200 and 50ng / well. mRNA : lipid complexes were incubated in a 1 : 1 ratio for 5 minutes and were added to each condition in quadruplicate. Cells were incubated for 24 hours at 37°C 5% CO2. Afterwards cells were harvested using 1 x TrypLE select enzyme (Gibco) and stained with a live / dead marker SYTOX blue (Life Technologies) in FACS buffer (PBS supplemented with 1 % bovin serum albumin (BSA) and 0.09% azide (all from Sigma)). Cells were immediately acquired after addition of the live dead marker using the Attune Nxt Flow Cytometer (ThermoFisher Scientific).

[0078] For assessment of eGFP expression, cells were stained with SYTOX blue. Within the gate of SYTOX blue negative cells, expression levels of eGFP were determined. The relative mean fluorescence intensity (rel MFI) was calculated as the MFI value of the expression marker divided by that of untransfected cells.

[0079] Data was acquired on an Attune Nxt cytometer and analyzed with Flow Jo Software. Flow cytometric data were analyzed using the Flowjo version 10 software.

[0080] Results - Expression levels of reporter eGFP mRNA upon in vitro transfection of CT26 or MC38 cells with the indicated LNP compositions.

[0081] LNPs were produced at a standard molar ratio ionizable lipid / DSPC / cholesterol / DMG-PEG2000 of about 50 / 10 / 38.5 / 1.5. eGFP mRNA was encapsulated in all LNPs as reporter mRNA, at a mRNA / ionizable lipid molar ratio of 1 / 10.

[0082] All LNPs showed a high encapsulation efficiency, as measured by the RiboGreen assay (data not shown). Figure 1A reveals that none of the LNPs comprising the tested ionizable lipids have a significant impact on the viability of the transfected CT26. The viability of transfected MC38 cells is slightly lower for LNPs comprising the ionizable lipid IL3 compared to LNPs comprising IL1 or IL2 at a concentration of 50 ng / well, but this difference is not observed at a concentration of 200ng / well (Figure 1 B).

[0083] Figure 2A and B show the Relative Mean Fluorescence Intensity (measured as the fold-increase in eGFP MFI compared to untreated cells) of eGFP expression in CT26 and MC38 cells respectively upon incubation with the indicated LNPs at mRNA concentration of 50 ng and 200 ng / well. As evident from this figure, incubation LNPs at a concentration of 50 ng / well was not efficient in transfecting cells. However, at a concentration of 200 ng / well, the LNPs comprising IL1 and IL3 outperform LNPs comprising IL2. Specifically, in CT26 cells (A) IL1 and IL3 LNPs perform equally well while in MC38 cells (B), LNPs with an ionizable lipid according to formula I of the invention (IL3) were most efficient and even superior in transfecting cells.

[0084] EXAMPLE 3: IN VIVO EXPERIMENTS

[0085] Induction of anti-HA (hemagglutinin) immune responses upon intramuscular mRNA vaccination

[0086] Materials and methods:

[0087] Assessment of mouse endpoint Immunoglobulin titers

[0088] 10Oul of mouse whole blood was collected on d21 and d35 in serum gel tubes (SarsTedt). Serum was separated from the blood clot by centrifugation at 10 000 g for 10 min at 4C.

[0089] Black flat bottom maxisorp 96 well plates (437111 , Life Technologies) were coated overnight at 4C with 100 pl 1 g / ml of recombinant H1 N1 (A / Puerto Rico / 8 / 1934) HA protein (Sino Biological, 11684-V08H) in carbonate / bicarbonate buffer (0.1 M, pH 9.6). Plates were subsequently blocked with 100 pl of 3%BSA (05479-250g, Sigma) in PBS (w / v) for 2h. Subsequently, plates were washed 3 times with PBS / 0.1 %Tween (101 13103, Fisher Scientific). A serial dilution of serum samples was added to the plates (initial 100X dilution of serum for d21 and initial 1000x serum dilution for d35; 5X dilution steps). After 2h incubation at RT plates were washed 5 times and a solution of rabbit anti-mouse lgG1 conjugated with HRP (1 :15 000, Biorad, OBT1508P) were added for another 1 h. After a final wash, the fluorescent Amplex UltraRed Reagent (A36006, Invitrogen) was used to develop plates according to the manufacturers’ instructions. Plates were read on a Tecan Infinite 200 Pro with ex=540nm, em=590nrn. The dilutions of serum of TBS treated mice served for cut-off determination, being the average fluorescence measured in the TBS samples plus 3 standard deviations. All points beyond cut-off were considered to be below quantification limit. The 5PL curves were fit to the dilution data then the endpoint titer was calculated at cross point of the modeled curve with the cut off.

[0090] LNP production:

[0091] To assess the impact of the ionizable lipid on the LNPs’ physicochemical properties and capacity on the endpoint Immunoglobulin titers, the LNPs comprising the ionizable lipids under investigation (IL1 , IL2 and IL3) were generated in 2 different N:P ratio’s (N:P ratio of 6 and N:P ratio of 10). LNP1 and LNP2 were based on respectively S-Ac-7-Dog and S-Ac7-DHDa as ionizable lipid, while LNP3 was based on S-Ade-DHDa as ionizable lipid (for structures see LNP production in Example 2).

[0092] LNP formulations were prepared as indicated above.. All formulations were characterized for size and PDI using Dynamic Light Scattering Zetasizer Nano-ZS (Malvern Pananlytical Ltd., Malvern, UK) and stored afterwards at 4°C. The physico-chemical characteristics of each formulation can be found in Table 2.

[0093] Table 2: List of relevant physico-chemical properties of different LNP formulations. These include the type of ionizable lipid, the lipid composition and the mol fraction of each component, the size and PDI (as determined by dynamic light scattering) and the encapsulation efficiency. Encapsulation efficiency has been measured by Ribogreen assay.

[0094] Note: EE% is defined as the encapsulation efficiency of the mRNA inside the LNP nanoparticle.

[0095] Results:

[0096] The respective mRNA LNP vaccines induced anti-HA antibody titers. A clear elevation in titers was observed after boosting with a LNP formulation comprising the ionizable lipid S-Ade-DHDa (IL3) compared with LNP formulation comprising the ionizable lipids S-Ac-7-Dog (IL1 ) and S- Ac7-DHDa (IL2), which are known in the art. Moreover, a LNP formulation with a N:P ratio of 10 was more efficient compared to a LNP formulation with a N:P ratio of 6 (Fig. 3).

[0097] REFERENCES

[0098] 1. Lee CY, Chang CL, Wang YN, Fu LM. Microfluidic mixing: a review. Int J Mol Sci. 2011;72(5):3263-87.

Claims

CLAIMS1 . An ionizable lipid of formula (I)2. A lipid nanoparticle or lipid nanoparticle composition comprising an ionizable lipid as defined in claim 1 .

3. The lipid nanoparticle or lipid nanoparticle composition according to claim 2, further comprising a phospholipid, a sterol and / or a PEG lipid.

4. The lipid nanoparticle or lipid nanoparticle composition according to anyone of claims 2 to 3, further comprising an active agent, in particular a nucleic acid, preferably mRNA.

5. Use of an ionizable lipid as defined in claim 1 in the manufacture of a lipid nanoparticle or lipid nanoparticle composition.

6. Use of claim 5, wherein said lipid nanoparticle or lipid nanoparticle composition further comprises a phospholipid, a sterol and / or a PEG lipid.

7. Use of claim 5 or 6, wherein said lipid nanoparticle or lipid nanoparticle composition further comprises an active agent, in particular a nucleic acid, preferably mRNA.

8. A pharmaceutical composition comprising a lipid nanoparticle or lipid nanoparticle composition as defined in anyone of claims 2 to 4, and a pharmaceutically acceptable agent.

9. A pharmaceutical composition as defined in claim 8 for use in medicine.

10. A pharmaceutical composition as defined in claim 8 for use in vaccination.

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