Lipid nanoparticles

Abstract

The present invention relates to compositions, and in particular to lipid nanoparticles, comprising at least one oligoglycerol-containing lipid. The compositions are in particular suitable for delivery of RNA.

Description

[0001] Lipid nanoparticles

[0002] 1 Description

[0003] The present invention relates to compositions, and in particular to lipid nanoparticles (LNP), comprising at least one oligoglycerol-containing lipid. The compositions are in particular suitable for delivery of RNA.

[0004] In the last years RNA based therapeutics have emerged as a promising new drug class. The effectiveness of RNA based vaccine has already been impressively demonstrated in the COVID-19 pandemic. Various types of RNA molecules such as anti-sense oligonucleotides, small interfering RNA (siRNA) & microRNAs (mi-RNAs), crRNA, tracrRNA or sgRNA could facilitate targeting mRNAs or DNA using Watson-Crick base-pairing strategy (Zhu, et al. 2022). Beside the use of messenger RNA (mRNA) as prophylactic vaccine against infectious diseases or therapeutic vaccine for cancer treatment, RNA can be used as therapeutic agent for treatment of chronic or genetic diseases (Li, et al. 2022).

[0005] For parenteral applications “naked” mRNA needs to be protected against degradation. This applies in particular to systemic administrations, e.g., intravenous, intraarterial, intramuscular and subcutaneous injections or infusions of mRNA as well for nasal application or administration of mRNA by breathing into the lungs - usually through the mouth by inhalation or nose by nebulization or spraying. Further the mRNA needs to be entrapped into a so-called non-viral delivery system to make it transfection competent, i.e. , enabling the binding of mRNA to the surface of a target cell of interest, followed by uptake into the cell via endocytosis and release from the endosome into the cytosol, where protein expression takes place.

[0006] To overcome the transfection and degradation problems with “naked” mRNA, polymer- and in particular lipid nanoparticle (LNP) based non-viral delivery systems have been developed in the last decades. Currently, all approved mRNA COVID-19 vaccines and most RNA drug development programs in oncology as well as non-oncology diseases are utilizing LNP technology to facilitate mRNA, siRNA, crRNA, tracrRNA or sgRNA delivery into the cytoplasm of target cells.

[0007] Current LNP technology for delivery of mRNA, siRNA or RNA species for CRISPR-Cas9 mediated genome editing uses four main structural components to build a lipid nanoparticle: a neutral or zwitterionic so-called “helper” phospholipid, cholesterol, a polyethylene-glycol (PEG)-lipid, and an ionizable lipid, which is, depending on the pH value, neutral or cationic. The ionizable, pH-sensitive, lipid contains one or more positively charged ionizable amine groups (at low pH) and has been optimized for encapsulation of the anionic RNA, as for example mRNA, siRNA, crRNA, tracrRNA or sgRNA (i.e., particle formation) and cellular uptake of the LNP (internalization by endocytosis). Further the ionizable lipid is necessary and mainly responsible for effective intracellular delivery by facilitating membrane fusion during internalization (endocytosis) and RNA release from the endosome into the cytosol, the so-called endosomal escape (Whitehead, et al. 2014); (Edo Kon 2022); (Sabnis, et al. 2018). The phospholipid 1 ,2-distearoly-sn-glycero-3-phosphocholine (DSPC) supports the cationizable lipid in cellular uptake and cellular trafficking of the LNP and the encapsulated RNA (Arteta, et al. 2018). The PEG-lipids have been engineered i) to regulate the LNP size during manufacturing and act as a steric barrier to prevent aggregation during storage as well as ii) to prevent unspecific interaction with cellular components after systemic administration.

[0008] Examples for typical lipid nanoparticles (LNPs) are the mRNA based LNPs Comirnaty (BioNTech / Pfizer’s COVID-19 vaccine (Formulation B)), Spikevax (Moderna’s COVID-19 vaccine (Formulation A)) and Onpattro (Alnylam, first interfering RNA (RNAi) drug approved by the FDA). They contain different ionizable cationic lipids, the zwitterionic DSPC as helper lipid, cholesterol, and PEGylated lipids.

[0009] The exact architecture and structure of LNPs and their encapsulated RNA molecules is subject of an intense debate. Nevertheless, currently most accepted models postulate that most if not all PEG lipids and the greater part of the DSPC helper lipids are located at the surface of the LNPs. Further, in contrast to a classical unilamellar liposome, the LNP contains lipids in its interior. During the manufacturing process, which involves a pH shift from acidic to neutral condition, the bulk of the ionizable lipid segregates into a central oil phase. The exact localization of the RNA inside the core differs between the small siRNA and the up to 100-fold larger mRNA. It is also common understanding that the LNP architecture might also be influenced by the nature of the mRNA or siRNA, their molecular weights, their sequence and chemical composition of the nucleotides (for review see e.g.: (Schoenmaker, et al. 2021)).

[0010] The chemical structure of the lipids (ionizable, helper and PEG) and the composition of the formulation, i.e., the molar ratios of the different components, mainly determines the architecture and surface charge of the LNPs carrying mRNA or siRNA. PEG lipids play an important role in the size of the final LNP. For example, it was shown (Arteta, et al. 2018) that by changing the relative amount of PEG lipid from 3 to 0.25 mol% the size of the final mRNA carrying LNP were increased from 45 nm to 135 nm (number average diameter). A smaller relative proportion of PEG lipids enables lower surface-to-volume ratios and consequently higher particle sizes. Most important for the in vivo (and in vitro) performance of the mRNA carrying LNP is the nature of the ionizable lipid, the chemical structure of the ionizable head group and the lipid tails, as there are for example the presence of branched or linear lipid tails, the position of ester bonds and the distance between the head group and the other motifs (Whitehead, et al. 2014); (Sabnis, et al. 2018).

[0011] Further, the manufacturing procedure for the RNA-LNPs beside the nature of the RNA itself (e.g., siRNA versus mRNA, see above) has an impact on the final structure of the mature RNA-LNP. The structure of the RNA-LNP, its size, charge, and composition, in turn are the major determinants for its biodistribution (organ, tissue and / or target cell specificity) and the potency of the entrapped RNA - as measured by level and duration of protein expression in case of mRNA, gene-silencing in case of siRNA or DNA editing efficiency in case of the CRISPR / Cas based approach.

[0012] It is important to note that for systemic administration each target cell type and route of administration requires specific evaluation and optimization of all these parameters. Thus, there is a high need to significantly expand the repertoire of LNP formulations and components to adapt the delivery of the therapeutic RNA for a specific disease treatment.

[0013] Currently used COVID-19 mRNA vaccines are composed of LNPs incorporating PEGylated lipids into their surface, thereby determining particle size during manufacturing, and preventing aggregation during storage and in tissues after IM (intramuscular), or SC (subcutaneous) administration. These vaccines contain diffusible PEG lipids with relatively short lipid chains (C14) which dissociate from the LNP after administration thereby generating transfection competent LNPs (for review see, e.g., (Kulkarni, Witzigmann und Chen 2019)). The COVID-19 mRNA vaccines can be considered as overwhelmingly safe. Most recent reports describe similar rates of allergic reactions and anaphylaxis to mRNA COVID-19 vaccines as to other vaccines (Jaggers and Wolfson 2023). By April 2022, the reported estimated rate for anaphylaxis has been between 2.5 and 4.7 per million (Jaggers and Wolfson 2023); (Tenchov, Sasso und Zhou 2023). Some of the symptoms in recipients of the COVID- 19 mRNA vaccines are similar to that in patients after systematically administered Doxil, which is a PEGylated liposome carrying the cytotoxic drug doxorubicin. However, the frequency of these reactions to the mRNA vaccines has been much lower than that to Doxil (Szebeni, et al. 2022).

[0014] The polyethylene glycol (PEG)-lipids are considered as candidate allergens to cause the rare anaphylactic reactions against the mRNA COVID-19 vaccines (e.g., Banerji, 2021), although it is reported that some patients with known PEG allergy can tolerate the mRNA vaccine (Picard, et al. 2022); (Brockow, et al. 2022).

[0015] Although hypersensitivity is a rare adverse event it can be fatal. Accordingly, there is still a need for improved mRNA vaccines where the PEG-lipid is omitted in the LNP without diminishing the performance of the vaccines. Considering the exceptional importance of the mRNA vaccines technology for the fight against emerging new viral diseases underlines the necessity to avoid undesirable immune reactions and to further improve mRNA vaccines.

[0016] In contrast to the intramuscular injected COVID-19 mRNA vaccines, the use of RNA-based drugs for therapeutic treatment of many diseases will require systemic administration of the LNPs into the blood circulation. Incorporation of diffusible PEG-lipids into the LNPs confer a half-life of 15 min in circulation which is too short to allow extra-hepatic organ targeting (Kulkarni, Witzigmann und Chen 2019). Persistent PEG-lipids with longer lipid chains (e.g., C18) and increased amount could present a possible optimization route to diminish the clearance of LNPs from blood. Further, the size, composition, and charge of the LNPs need to be adapted for in vivo applications, thus improving tissue targeting and efficiency of encapsulated RNA. However, it has been reported that PEGylated nanocarriers do not necessarily escape the detection of the immune system. Unexpected immune reactions against PEGylated nanocarriers, mainly the formation of anti-PEG antibodies after initial injection, might lead to accelerated blood clearance (ABC), i.e., the increased clearance and reduced efficacy of PEGylated nanocarriers following repeated administrations (see for a comprehensive review: (Tenchov, Sasso und Zhou 2023)). Also, the possible presence of pre-existing antibodies in the absence of treatment with a PEGylated nanocarrier is known (Yang und Lai 2015), although the relevance is discussed controversially. The presence of anti-PEG antibodies could also play a role in the occurrence of hypersensitivity reactions like complement activation related pseudoallergy (CARPA). Most of the data have been reported with PEGylated classical nanocarriers (e.g., liposomes) and PEGylated proteins. It is debatable to which extend these observations will also apply for the systemic administration of RNA carrying LNPs. Nevertheless, for repeated administrations of therapeutic RNA LNP drugs, the immunosafety of the RNA carrying LNPs is of exceptional importance. Especially for the sometimes-lifelong treatment of chronic diseases adverse immune reactions must be prevented.

[0017] Unshielded liposomes or LNPs encapsulating RNA possess poor physicochemical characteristics for systemic delivery. Their surface, especially in case of cationic LNPs or liposomes, leads to nonspecific interaction with (mostly anionic) blood components and uptake by macrophages, resulting in rapid clearance by the reticuloendothelial system (RES). Consequently, after administration of unshielded LNPs into the circulation by injection or infusion predominantly so-called first-pass organs are targeted (“first-pass” organs).

[0018] One solution of the problem is the use of hydrophilic polymers attached to the particle's surface. Most commonly PEG has been used, although other polymers have been described (Heyes, et al. 2006), (Nogueira, et al. 2020).

[0019] Further, it is reported that the presence of hydrophilic polymers with a large head group is a prerequisite for the manufacturing of RNA-LNPs with well-defined size suitable for the parenteral administration. RNA-LNPs do not represent highly structured particles, but due to the physicochemical properties of their components they are largely self-assembling systems. The established standard manufacturing processes use a low pH environment for the generation of a heterogeneous population of small LNPs, whereby the positively charged ionizable lipids interact with the negatively charged mRNA or siRNA. Induced by a pH shift to neutral value a reorganization of the different lipids and the RNA occurs. During this process the ionizable lipids diffuse into the center of the LNP particles and the PEG-lipid as well as the helper lipids assemble at the surface of the particle. Accordingly, after this maturation process, different LNP compartments can be detected.

[0020] In the presence of PEG-lipids a defined mRNA-LNP size is achieved when the concentration of PEG-lipid in the outer monolayer is sufficiently high to inhibit further fusion of the smaller LNPs. In the absence of PEG-lipid the LNPs continuously fuse during the manufacturing process to large particles with a heterogeneous size distribution which do not allow any more in vivo administration (Kulkarni, Witzigmann und Chen 2019); (Schoenmaker, et al. 2021). However, it has been shown that the presence of PEG does affect the intracellular delivery and trafficking of non-viral nucleic acid delivery particles. PEG on the surface of RNA carrying LNPs prevents fusion of LNPs with cell membranes thus decreasing cellular uptake and endosomal escape processes. This results into low gene expression (mRNA) or gene silencing potential (siRNA) (Woodie und Lasic 1992); (Heyes, et al. 2006).

[0021] A further disadvantage of the PEGylated mRNA carrying LNPs is that they potentially induce an antibody response against the PEG moiety. This could negatively impact their potential for therapies where repeated administration of PEGylated LNPs would be necessary. This is especially true for lifelong treatment of non-oncology diseases, e.g., protein replacement or substitution therapies in genetically inherited diseases.

[0022] Alternative hydrophilic headgroups which have been described still exhibit these disadvantages. Representative for this class of alternative shielding agents are LNPs functionalized with polysarcosine (pSar). Depending on the molar fraction of pSar lipid at the LNP surface, the LNPs showed one to several orders of magnitude lower transfection in vitro when compared to LNPs containing PEG-DMG, a PEG lipid which is used as shielding agent in the COVID-19 vaccine developed by Moderna Inc. (Nogueira, et al. 2020). To overcome the limitations of these established or proposed alternative shielding agents several approaches have been followed.

[0023] One research approach tries to make the insertion and exposure of PEG lipids at the surface of the LNP transient. In principle this could be achieved by diffusible PEG-lipids that diffuse from the particle surface at a rate determined by the size of their lipid anchors (Ambegia, et al. 2005); (Hattori, et al. 2020). Other research approaches investigate sheddable PEG- lipids that possess a biodegradable bond between the polymer and the lipid anchor that is enzymatically cleaved in the extracellular environment after binding to the target tissue, for example a tumor or damaged tissue in cardiovascular diseases. A strong limitation of this concept is that the bulky headgroup of PEG also inhibits access to the enzymes which would be responsible for the linker cleavage. Alternatively, by introduction of a chemical bond which is sensitive to low pH, cleavage can occur intracellularly in the acidic endosomal compartment. The disadvantage of the latter approach is that the bulky PEG headgroup still negatively interferes with the uptake into the target cell. So, a strategy using diffusible PEG- lipids may be advantageous, rather than those where PEG is permanently bound or not cleaved until entry to the endosome.

[0024] Ambegia et al. systematically investigated PEG-lipids with different lengths of the lipid anchor (Ambegia, et al. 2005). Pharmacokinetic analysis of lipid nanoparticles containing complexed plasmid DNA confirmed a clear correlation between the stability of the PEG- lipids and circulation lifetime of the LNPs. The authors could also clearly show that the particles became more fusogenic over time as the PEG-lipid dissociated from the particle. This was due to the increased exposure of the fusogenic lipid bilayer as the outer PEG coating was removed. The speed of loss was a function of the length of the lipid anchor. The LNPs containing PEG-lipids with longer lipid anchors showed a slower dissociation from the particles and a reduced fusion behavior.

[0025] Since the dissociation of the PEG is necessary for a nucleic acid containing particle to become transfection competent, gene expression is related entirely to the rate at which the PEG-moiety is eliminated from the particle’s bilayer. Consequently, the in vitro transfection efficiency of the investigated lipid nanoparticles containing complex plasmid DNA was highest with the PEG-lipid containing the shortest lipid anchor. In this context it is important to mention that PEG ceramides containing only a very short lipid anchor (08) could sufficiently increase the circulation time of plasmid DNA: lipid complexes compared to uncoated or ‘naked’ particles, thereby allowing targeting and gene expression at tumor sites (Tam, et al. 2000).

[0026] Summarizing, there is still a strong need for a shielding agent which moderately prolongs half-life of the LNPs in circulation, thereby allowing to bypass the “first-pass” organs. Further the shielding agent must not negatively interfere with binding of the LNPs to their target cells, i.e. , it must allow efficient uptake into the target cells via endocytosis and must allow efficient release of the RNA from the endosome into the cytoplasm, mediated by the ionizable, pH-sensitive lipids. Such a novel shielding agent also has to maintain dispersion stability of the LNPs (e.g., no fusion or aggregation during storage and / or handling). Such a substitute for the currently used PEG lipids has not been described yet.

[0027] There is also a strong need to expand the repertoire of helper lipids for the future design of safe and efficacious LNPs. The so-called helper lipids strongly influence the properties of the LNPs, such as particle stability, delivery efficacy, tolerability and biodistribution (for review see, e.g., (Hou, et al. 2021)). Especially helper lipids such as DSPC and cholesterol also play an essential role by participating in the formation of LNPs. For example, DSPC, a zwitterionic phosphatidylcholine with two saturated lipid chains, stabilizes the structure of lipid nanoparticles, whereas 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which is a phosphatidylethanolamine with two unsaturated lipid chains, destabilizes endosomal membranes and facilitates endosomal escape of lipid nanoparticles (Hou, et al. 2021). The biodistribution and structure-function relationship of intravenously administered LNPs was systematically investigated by (Zhang, et al. 2021). By a high-throughput in vivo screening of a LNP library, the group studied how helper lipids influence the protein adsorption and the biodistribution of LNPs. Interestingly LNPs containing the helper lipid DOPE preferentially accumulated in the liver, while LNPs containing DOPC but were otherwise identical preferentially accumulated in the spleen.

[0028] Arteta et al. did a systematic investigation of the relationship between mRNA carrying LNP size, LNP architecture and protein expression after in vitro transfection (Arteta, et al. 2018). Focus of their study was the lipid composition at the surface of the LNP and the architecture of the LNP interior. Surprisingly, the phospholipid DSPC was mainly located at the surface of mRNA-LNPs and strongly influenced the transfection efficacy. Their data clearly showed that both surface composition and size of LNPs determine transfection efficacy, demonstrated in two different cell types in vitro. The effect of DSPC on protein expression was also remarkable, although DSPC promotes lamellar phase bilayers, thereby increasing the bilayer stability and should not be able to promote endosomal escape.

[0029] These investigations clearly show the striking effects of helper lipids on the biological efficacy of the LNPs. Helper lipids can play a central role in modulating the biodistribution, target specificity and protein expression of mRNA encapsulated in LNPs. This clearly shows the high need for a larger repertoire of innovative helper lipids for the future design of LNPs for systemic administration. This is especially true for the future development of LNPs which are designed without conjugation of a tissue specific targeting moiety as for example an antibody. This class of LNPs should preferentially target and specifically accumulate in a tissue or organ after systemic administration. Their inherent affinity to a specific tissue or organ will be determined by their lipid composition, including the helper lipid.

[0030] Thus, there is a great need for lipids which are in particular suitable as excipients for improved drug delivery systems such as improved LNPs.

[0031] The present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0032] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0033] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0034] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from (i) at least one cationic or cationizable lipid;

[0035] (ii) at least one structural lipid;

[0036] (iii) at least one helper lipid; and / or

[0037] (iv) at least one PEG lipid.

[0038] The composition is preferably a lipid nanoparticle (LNP).

[0039] A suitable counter ion for all cationic lipids disclosed herein is Cl Br, mesylate, fumarate; preferably it is Cl’. For all anionic lipids disclosed herein, a suitable counter ion is Na+.K+, Li+, preferably it is Na+.

[0040] In particular, the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0041] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0042] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0043] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from

[0044] (i) at least one cationic or cationizable lipid;

[0045] (ii) at least one structural lipid;

[0046] (iii) at least one helper lipid; and / or

[0047] (iv) at least one PEG lipid.

[0048] In a preferred embodiment the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;

[0049] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0050] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0051] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid.

[0052] In particular, the present invention relates to a composition comprising: b) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0053] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0054] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0055] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid.

[0056] In this embodiment, the at least one oligoglycerol-containing lipid of Formula (I) or (IA) may be comprised in the composition in an amount of about 1 mol% to about 25 mol%, preferably about 1.5 mol% to about 12 mol%, more preferably about 1.5 mol% to about 10 mol%; the least one cationic or cationizable lipid may be comprised in an amount of about 40 mol% to about 55 mol%, preferably about 44 mol% to about 52 mol%, more preferably about 46 mol% to about 50 mol%; and the at least one structural lipid in an amount of about 28 mol% to about 52 mol%, preferably about 32 mol% to about 46 mol%, more preferably about 34 mol% to about 44 mol%, based on the total lipid content of the composition.

[0057] In a further preferred embodiment the present invention relates to a composition comprising: b) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0058] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0059] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0060] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii) at least one structural lipid; and b)(iii) at least one helper lipid.

[0061] In particular, the present invention relates to a composition comprising: b) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5; R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0062] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0063] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii) at least one structural lipid; and b)(iii) at least one helper lipid.

[0064] In this embodiment, the at least one oligoglycerol-containing lipid of Formula (I) or (IA) may be comprised in the composition in an amount of about 1 mol% to about 25 mol%, preferably about 1.5 mol% to about 12 mol%, more preferably about 1.5 mol% to about 10 mol%; the least one cationic or cationizable lipid may be comprised in an amount of about 40 mol% to about 55 mol%, preferably about 44 mol% to about 52 mol%, more preferably about 46 mol% to about 50 mol%; the at least one structural lipid in an amount of about 28 mol% to about 52 mol%, preferably about 32 mol% to about 46 mol%, more preferably about 34 mol% to about 44 mol%; and the at least one helper lipid in an amount of about 5 mol% to about 15 mol%, preferably about 7 mol% to about 12 mol%, more preferably about 9 mol% to about 11 mol%, based on the total lipid content of the composition.

[0065] In this embodiment, the composition may further comprise a PEG lipid. The at least one oligoglycerol-containing lipid of Formula (I) or (IA), preferably NLG2 or CLG2, may be comprised in the composition in an amount of about 10 mol% to about 30 mol%, preferably about 15 mol% to about 25 mol%, more preferably about 18 mol% to about 22 mol%,; the least one cationizable lipid, preferably DODMA, may be comprised in an amount of about 15 mol% to about 35 mol%; more preferably of about 18 mol% to about 30 mol%; the at least one structural lipid, preferably cholesterol, in an amount of about 25 mol% to about 45 mol%, preferably about 30 mol% to about 40 mol%, more preferably about 33 mol% to about 37 mol%; and the at least one helper lipid, preferably DOPE or DSPC, in an amount of about 10 mol% to about 30 mol%, preferably about 13 mol% to about 22 mol%, based on the total lipid content of the composition, and the at least one PEG lipid, preferably DMG- PEG, in an amount of about 0,5 mol% to about 2 mol%. In a further preferred embodiment, the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0066] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0067] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0068] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii) at least one structural lipid; and b)(iv) at least one PEG-lipid.

[0069] In particular, the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;

[0070] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0071] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0072] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii) at least one structural lipid; and b)(iv) at least one PEG-lipid.

[0073] In this embodiment, the at least one oligoglycerol-containing lipid of Formula (I) or (IA) may be comprised in the composition in an amount of about 1 mol% to about 25 mol%, preferably about 5 mol% to about 22 mol%, more preferably about 9 mol% to about 21 mol%; the least one cationic or cationizable lipid may be comprised in an amount of about 40 mol% to about 55 mol%, preferably about 44 mol% to about 52 mol%, more preferably about 46 mol% to about 50 mol%; the at least one structural lipid in an amount of about 28 mol% to about 52 mol%, preferably about 32 mol% to about 46 mol%, more preferably about 34 mol% to about 44 mol%; and the least one PEG-lipid in an amount of about 0,5 mol% to about 4 mol%, preferably about 1 mol% to about 3 mol%, more preferably about 1 ,2 mol% to about 1 ,7 mol%, based on the total lipid content of the composition.

[0074] In a preferred embodiment the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0075] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0076] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0077] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid.

[0078] In particular, the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I A) , wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0079] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0080] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0081] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) (iii) at least one helper lipid.

[0082] In this embodiment, the at least one oligoglycerol-containing lipid of Formula (I) or (IA) may be comprised in the composition in an amount of about 20 mol% to about 40 mol%; and the at least one helper lipid may be comprised in the composition in an amount of about 50 mol% to about 80 mol%; based on the total lipid content of the composition.

[0083] In a preferred embodiment the present invention relates to a composition comprising: a1) at least one oligoglycerol-containing lipid of Formula (I), wherein A = NR3;

[0084] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0085] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0086] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and a2) at least one oligoglycerol-containing lipid of Formula (I), wherein A = N+R4R5;

[0087] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0088] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0089] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) (i) at least one cationizable lipid and .

[0090] (iv) at least one PEG lipid.

[0091] In particular, the present invention relates to a composition comprising: a1) at least one oligoglycerol-containing lipid of Formula (I), wherein A = NR3;

[0092] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0093] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0094] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and a2) at least one oligoglycerol-containing lipid of Formula (I), wherein A = N+R4R5;

[0095] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0096] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; and

[0097] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) (i) at least one cationizable lipid and .

[0098] (ii) at least one structural lipid; and

[0099] (iv) at least one PEG lipid.

[0100] In this embodiment, the at least one oligoglycerol-containing lipid of Formula (I) or (IA) wherein A = NR3, preferably NLG2, may be comprised in the composition in an amount of about 10 mol% to about 30 mol%; the at least one oligoglycerol-containing lipid of Formula (I) or (IA) wherein A = N+R4R5, preferably CLG2, may be comprised in the composition in an amount of about 10 mol% to about 30 mol%; and the at least one cationizable lipid, preferably DODMA, may be comprised in the composition in an amount of about 10 mol% to about 30 mol%; based on the total lipid content of the composition, the at least one PEG lipid, preferably DMG-PEG, may be comprised in the composition in an amount of about 2 mol% to about 4 mol%; based on the total lipid content of the composition; and the at least one structural lipid, preferably cholesterol, may be comprised in the composition in an amount of about 30 mol% to about 45 mol%; preferably in an amount of about 34 mol% to about 38 mol% based on the total lipid content of the composition.

[0101] In some embodiments in the oligoglycerol-containing lipid of Formula (I) or (IA), B = a linear or branched oligoglycerol group having from 2 to 15 glycerol units, from 2 to 10 glycerol units, from 2 to 5 glycerol units.

[0102] In a preferred embodiment the oligoglycerol-containing lipid of Formula (I) or (IA) is a phosphatidyloligoglycerol, wherein A = O-P(-O')(=O)-O. Such compounds are also termed DAPGx (diacyl-phospho-oligoglycerol; x = number of glycerol units) and in particular DPPGX(dipalmitoyl-phospho-oligoglycerol; x = number of glycerol units) or DPPG2 (dipalmitoyl- phospho-diglycerol) or DPPG3 (dipalmitoyl-phospho-triglycerol) herein In a further preferred embodiment, the oligoglycerol-containing lipid of Formula (I) or (IA) is a cationic oligoglycerol-containing lipid, wherein A = N+R4R5. Such compounds are also termed CLGX(cationic lipid containing oligoglycerol; x=number of glycerol units) and in particular CLG2 (cationic lipid containing diglycerol) herein. Preferably R4and R5each independently are C1-C5-alkyl, and in particular R4= R5= CH3.

[0103] In a further preferred the oligoglycerol-containing lipid of Formula (I) or (IA) is a cationizable oligoglycerol containing lipid, wherein A = NR3. Such compounds are also termed NLGX(cationizable lipid containing oligoglycol; x=number of glycerol units) and in particular NLG2 (cationizable lipid containing diglycerol) herein. Preferably R3= C1-C5-alkyl, in particular R3= CH3.

[0104] According to embodiments of the invention the composition can contain two or more oligoglycerol-containing lipid of Formula (I) and / or (IA), preferably at least phosphatidyloligoglycerol, wherein A = 0-P(-0')(=0)-0 and at least one cationic oligoglycerol containing lipid, wherein A = N+R4R5.

[0105] Preferably, the at least one phosphatidyloligoglycerol wherin A = 0-P(-0')(=0)-0 may be comprised in the composition of the previous embodiments in an amount of about 2 mol% to about 15 mol%, preferably about 4 mol% to about 12 mol%, and the at least one cationic oligoglycerol containing lipid, wherein A = N+R4R5is comprised in an amount of about 5 mol% to about 15 mol%, preferably about 8 mol% to about 12 mol% based on the total lipid content of the composition.

[0106] Preferred is an oligoglycerol-containing lipid of Formula (I) or (IA), which comprises from 2 to 4 glycerol units, preferably 2 or 3 glycerol units and more preferably 2 glycerol units.

[0107] In the oligoglycerol-containing lipids of Formula (I) or (IA), residues R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms. Preferred is an oligoglycerol-containing lipid, wherein R1and R2independently are a hydrocarbon group having 13 to 19 C-atoms. The hydrocarbon groups can be saturated or mono- or polyunsaturated. The hydrocarbon groups can be linear or branched. Preferably R1and R2independently are a linear or branched saturated C13- to C19-alkyl group, more preferably a linear saturated C13- to C19-alkyl group. More preferred, R1and R2independently are a linear saturated C15 or C17 alkyl group, most preferred a linear saturated C15 alkyl group.

[0108] In a further embodiment R1and R2independently are preferably a linear saturated or unsaturated C15 or C17 alkyl group, most preferably a saturated or unsaturated C17 alkyl group.

[0109] Most preferred are oligoglycerol-containing lipids of Formula (II) or of Formula (HA), A) wherein n = 2 to 20; and in particular n = 2 or 3; and wherein A = O-P(-O')(=O)-O; or NR3; or N+R4R5;

[0110] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0111] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1.

[0112] Such oligoglycerol-containing lipids of Formula (II) or (HA), have a linear oligoglycerol group and in particular a di- or triglycerol group.

[0113] Especially preferred are oligoglycerol-containing lipids of Formula (HA) wherein n = 2 or 3; wherein A = O-P(-O')(=O)-O; or NR3; or N+R4R5; and R3, R4and R5each independently are a hydrocarbon group having 1-5 C-atoms; and R1and R2each independently are a linear saturated or unsaturated hydrocarbon group having 15 or 17 C-atoms.

[0114] Most preferred is an oligoglycerol-containing lipid having the Formula which is termed DPPG2 herein.

[0115] Further most preferred is an oligoglycerol-containing lipid having the Formula which is termed CLG2 herein.

[0116] Further most preferred is an oligoglycerol-containing lipid having the Formula which is termed NLG2 herein.

[0117] Most preferred is an oligoglycerol-containing lipid having the Formula which is termed DSPG2 herein.

[0118] Further most preferred is an oligoglycerol-containing lipid having the Formula which is termed DSAmG2 herein.

[0119] Further most preferred is an oligoglycerol-containing lipid having the Formula which is termed DSAG2 herein.

[0120] A further preferred composition comprises:

[0121] (a) at least one oligoglycerol-containing lipid of Formula (I) or (IA), optionally

[0122] (i) at least one phosphatidyloligoglycerol of Formula (I) or (IA), wherein A = O-P(-O')(=O)-O; and

[0123] (ii) at least one cationic oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = N+R4R5; and

[0124] (b)(i) at least one cationizable lipid; and (b)(ii) at least one structural lipid.

[0125] A further preferred composition comprises:

[0126] (a) at least one oligoglycerol-containing lipid of Formula (I) or (IA); and

[0127] (b)(iii) at least one helper lipid, preferably at least two helper lipids.

[0128] Particularly preferred the composition is a lipid nanoparticle also referred to as LNP herein.

[0129] The term „lipid nanoparticle(s)“ or„LNP(s)“ as used herein relates to particles characterized by a diameter between 1 and 1000 nm having an outer lipid shell enclosing inner lipid phases. The outer lipid shell may be in the form of a lipid mono- or bilayer. The inner lipid phases may be in the form of inverted micelles, and / or solid lipid phases. In a preferred embodiment the lipid nanoparticle comprises at least an outer lipid shell comprised of one lipid monolayer and inner lipid phases comprising inverted micelles (also known as reverse micelles). The term „lipid nanoparticle(s)“ or „LNP(s)“ according to the present invention does not encompass liposomes characterized by one or several concentric lipid bilayers surrounding an aqueous lumen. In one embodiment, the term „lipid nanoparticle(s)“ or „LNP(s)“ relates to particles comprising ionizable lipids combined with membrane forming lipids, cholesterol, cationic lipids and / or anionic lipids.

[0130] The term „lipid nanoparticle" or „LNP“ as used herein does not encompass mono-bilayer liposomes comprising only a single lipid bilayer encapsulating an aqueous lumen or liposome in liposome vesicles.

[0131] The LNPs according to the invention may have a diameter defined by the Z-average size defined by the intensity weighted mean hydrodynamic size of particles measured by dynamic light scattering (Z-Av) of between about 50 nm to about 1000 nm, between about 70 nm and 700 nm, In one embodiment the diameter may be between about 250 nm and about 550 nm. In another embodiment the diameter may be between about 70 and about 180 nm, or between about 80 nm and 150 nm.

[0132] The LNPs according to the invention may have a negative zeta potential, preferably between about -1 and -30 mV.

[0133] The compositions according to the present invention comprise diacyl-phospho- oligoglycerols (DAPGX), in particular, diacyl-phospho-oligoglycerols having two or three glycerol units, such as dipalmitoyl-phospho-diglycerol (DPPG2) and dipalmitoylphosphotriglycerol (DPPG3), as anionic phospholipids as well as their ionizable (NLGX, in particular NLG2) and cationic derivatives (CLGX, in particular CLG2) as a new class of lipids for providing LNPs for encapsulating RNA molecules. The most preferred lipids carry two or three glycerol moieties as head groups, respectively, favourable for design and manufacturing of RNA containing LNPs.

[0134] The RNA molecules preferentially comprise protein coding mRNA, non-coding RNA e.g., as immunoadjuvant, siRNA for gene silencing as well as RNA for in vivo gene editing, as for example by the CRISPR / Cas technology. So, diacyl-phospho-oligoglycerols having two or three glycerol units, and in particular DPPG2 and DPPG3 as well as their cationic derivatives and their derivatives with the same headgroup but different fatty acid chain lengths, present a new class of lipids (the G2- and G3- lipid class) for replacement of PEG lipids or other shielding agents in RNA-LNP formulations. These G2- and G3- lipids allow the design of novel next generation RNA-LNP formulations with improved in vivo and in vitro transfection after parenteral administration, showing low aggregation and high chemical and physical shelf-life stability.

[0135] In some embodiments, a further advantage of LNPs functionalized with a member of the G2- or Gs-lipid family may be an increased storage stability which may facilitate increased transfection after storage.

[0136] A further advantage of LNPs functionalized with a member of the G2- or Gs-lipid family is their well-defined and controlled hydrodynamic diameter. For in vivo applications the controllable size and charge of the G2- and Gs-lipid nanoparticles offers the potential to optimize the biodistribution of LNPs.

[0137] In some embodiments, a further advantage of LNPs functionalized with a member of the G2- or Gs-lipid family may be an increased by-pass of liver accumulation compared to the conventional PEG-containing LNPs.

[0138] Further, in vivo some target cells are not easily accessible. These target cells might be covered by extracellular matrix molecules, cellular debris, or mucus in the case of pulmonary administration. A small size is also a prerequisite to reach target cells behind the endothelial basement membrane as for example smooth muscle cells or tumor cells by diffusion through pores in the tumor blood vessel system (enhanced permeability and retention [EPR] effect).

[0139] A further advantage of the G2- and Gs-lipids is the potential of their cationic derivatives (e.g., CLG2) or anionic derivative (e.g., DPPG2 or DPPGs) to modulate the overall charge of LNPs, including PEGylated LNPs, as measured by the zeta potential. The charge of LNPs is known to directly modulate their target specificity, e.g., cationic LNPs preferentially accumulate in proliferating endothelial cells. Further the so-called protein corona of the LNPs can be modified by modulating their surface charge, thus indirectly changing their target specificity. So, the G2- and Gs-lipids represent novel tools for LNP engineering and optimization of their biological activity for treatment of diseases.

[0140] In summary, LNPs comprising lipids with G2- or G3 functionalized head groups are characterized by advantageous properties over LNPs comprising solely lipids with prior art headgroups as disclosed for example by Jorgensen et al, 2023.

[0141] In addition to the biophysical findings described above, we have shown that LNPs containing DPPG2 allow successful entrapment of RNA (e.g., mRNA, siRNA) into uniform and small LNP, and that these LNPs are mechanically and chemically stable, tightly packed vesicles, most probable due to the cooperative effects between the individual lipid bilayers (see examples).

[0142] This particular property of G2 lipid and therefore of nucleic acid-containing LNPs is most likely also based on the property of G2 or G3 lipids to displace water from the membranes’ interstitial space through interaction with other G2 or G3 lipids, especially at stronger curvature. The reduced amount of water between the membranes is expected to result in reduced hydrolysis of the nucleic acid, which is structurally weakened by the electrostatic interaction (the electrostatic interaction between phosphate and a positively charged function destabilizes nucleic acids, since the natural conformation, which is based on repulsion of the phosphate groups, among other things, is thus no longer given). Thus, LNPs with G2 or G3 lipids are most likely more stable and could simplify the development of a lyophilized RNA LNP product.

[0143] This stabilizing effect is probably further supported by a mechanical effect, the low-water environment enhancing a direct polar interaction of the nucleic acid with the G2 / G3 groups, so that mechanical influences become less important during LNP production (tight, less flexible wrapping of the nucleic acids).

[0144] A further advantage of the G2- and Gs-lipid containing RNA-LNPs is the avoidance of the potential risk of anaphylactic reactions. There is an ongoing discussion as to whether PEG is causing the few instances of anaphylactic reactions that have been observed with both the Moderna and the Pfizer COVID-19 vaccines. Although the contribution of PEG to the anaphylactic reactions is speculative it is important to investigate whether a next generation of “PEG-free” LNPs will result in different outcomes.

[0145] A further advantage of the G2- and Gs-lipid containing RNA LNPs is the lower potential risk of accelerated blood clearance after repeated administrations. It has been reported that DPPG2 containing liposomes were not able to induce an immune response against DPPG2 even after several intravenous administrations (Lokerse, et al. 2021).

[0146] The inventive composition preferable comprises at least one cationic or cationizable lipid.

[0147] A cationic lipid is typically a lipid having a net positive charge, for example in some embodiments at a certain pH. In some embodiments, a cationic lipid may comprise one or more amine group(s) which bear a positive charge. In some embodiments, a cationic lipid may comprise a cationic, meaning positively charged, headgroup. In some embodiments, a cationic lipid may have a hydrophobic domain (e.g., one or more domains of a neutral lipid or an anionic lipid) provided that the cationic lipid has a net positive charge. In some embodiments, a cationic lipid comprises a polar headgroup, which in some embodiments may comprise one or more amine derivatives such as primary, secondary, and / or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and / or imidazole groups as well as pyridinium, piperizine and amino acid headgroups such as lysine, arginine, ornithine and / or tryptophan. In some embodiments, a polar headgroup of a cationic lipid comprises one or more amine derivatives. In some embodiments, a polar headgroup of a cationic lipid comprises a quaternary ammonium. In some embodiments, a headgroup of a cationic lipid may comprise multiple cationic charges. In some embodiments, a headgroup of a cationic lipid comprises one cationic charge.

[0148] Preferred cationic lipids are selected from the group comprising DOSPA (2,3-dioleyloxy- N- [2-(sperminecarboxamido)ethyl]- N,N- dimethyl-1-propanaminium trifluoroacetate); DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane); DMEPC (1 ,2-dimyristoyl-sn-glycero-3- ethylphosphocholine); DOTMA (1 ,2-di-O-octadecenyl-3-trimethylammonium propane); DOTAP (1 ,2-dioleoyl-3-trimethylammonium propane); DMTAP (1 ,2-dimyristoyl-3- trimethylammonium propane); DMRIE (2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)- dimethylazanium bromide); DDAB (didodecyl(dimethyl)azanium bromide); DORIE (1 ,2- dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide); DC-Choi (3P-[N-(N\N'- dimethylamino-ethane)carbamoyl]cholesterol); DOEPC (dioleyl ether phosphatidylcholine), ePC (ethylphosphatidylcholine).

[0149] A cationizable lipid is a lipid that can exist in a positively charged form or neutral form depending on pH. The cationizable lipid may have a pKa value between 5 and 7.5, preferably between 6 and 7. This means that the lipid is uncharged at a pH above the pKa value and positively charged below the pKa value.

[0150] Preferred cationizable lipids are selected from the group comprising DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate); ALC-0315 ((4-hydroxybutyl)azanediyl) bis(hexane-6,1-diyl)bis(2-hexyldecanoate); SM-102 (also termed Lipid H) (heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6- (undecyloxy)hexyl)amino) octanoate); A2-lso5-2DC18 (ethyl 5,5-di((Z)-heptadec-8-en-1- yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1 H-imidazole-2-carboxylate); 9A1P9 (decyl (2- (dioctylammonio)ethyl) phosphate); BAME-O16B (bis(2-(dodecyldisulfa-nyl)ethyl) 3,3'-((3- methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate); C12-200 (1 , 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl) amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin-1 -yl)ethyl)azanediyl) bis(dodecan-2-ol); CKK-E12 (3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione); OF-Deg-Lin ((((3,6-dioxopiperazine-2, 5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9"Z,9"Z, 12Z,12'Z,12"Z,12"Z)- tetrakis (octadeca-9,12-dienoate)); 3060i10 ((tetrakis(8-methylnonyl) 3,3',3",3"-(((methylazanediyl) bis(propane-3,1diyl))bis (azanetriyl))tetrapropionate)); TT3 ( N1,N3,N5- tris(3-(didodecylamino)propyl)benzene-1 ,3,5- tricarboxamide); FTT5 ( hexa(octan-3- yl) 9,9',9",9",9'"',9""-((((benzene-1 ,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3, 1-diyl)) tris(azanetriyl)) hexanonanoate) and 1 ,2-Dioleyloxy-3- dimethylaminopropane (DODMA). Preferably the cationizable lipid is selected from DODMA, ALC-0315 or SM-102.

[0151] The inventive composition preferable comprises a structural lipid which is a sterol, preferably selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomadidine, ursolic acid and a- tocopherol; and in particular the structural lipid is cholesterol. The inventive composition preferable further comprises at least one helper lipid. A helper lipid is a lipid capable of increasing the effectiveness of delivery of lipid-based particles to a target, preferably into a cell. The helper lipid can be neutral or negatively charged. Particular suitable helper lipids are selected from hydrogenated soy L-a-phosphatidylcholine (HSPC);

[0152] 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0153] 1.2-diolioyl-sn-glycero-3-phosphoethanolamin (DOPE);

[0154] 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);

[0155] 1.2-dimyristoyl-sn-glycero-phosphocholine (DM PC);

[0156] 1.2-diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-palmitoyl-2-olioyl-sn-glycero-3-phosphocholine (POPC).

[0157] 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is most preferred.

[0158] In some embodiments of the invention the composition does not comprise a helper lipid. It was found that helper lipids, such as DSPC, contained in LNPs can be replaced by oligoglycerol-containing lipids of Formula (I) resulting in stable LNPs.

[0159] The composition of the invention may further comprise e) a PEG lipid.

[0160] The term “PEG lipid” or “PEGylated lipid” refers to a lipid which is covalently bound to polyethylene glycol (PEG). Polyethylene glycol (PEG) is one of the most widely applied nonionic, hydrophilic polymer that provides the stealth effect for the LNPs improving their circulation time in the body (Knop, et al. 2010) (Albertsen, et al. 2022).

[0161] In an embodiment of the invention comprising a PEG lipid only part of PEG lipid included in conventional LNP formulations is replaced by at least one oligoglycerol-containing lipid of Formula (I) or (IA), and in particular by at least one oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = O-P(-O’)(=O)-O.

[0162] In a further embodiment of the invention comprising a PEG lipid, at least one oligoglycerolcontaining lipid of Formula (I) or (IA), and in particular at least one oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = NR3; or N+R4R5is present in addition to the PEG lipid. In those embodiments the at least one oligoglycerol-containing lipid of Formula (I) or (I A) , and in particular at least one oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = NR3; or N+R4R5is used to modulate the LNP composition.

[0163] In a most preferred embodiment the composition of the invention does not comprise a PEG lipid.

[0164] According to the invention empty carrier systems and in particular empty lipid nanoparticles (LNPs) can be provided. Preferred however are compositions, further comprising c) a therapeutic and / or prophylactic agent.

[0165] Most preferred, the therapeutic and / or prophylactic agent is a nucleic acid, in particular a ribonucleic acid (RNA). The RNA is preferably selected from the group consisting of messenger RNA (mRNA), antisense RNA (aRNA), small interfering RNA (siRNA), CRISPR- RNA (crRNA, tracrRNA or sgRNA), asymmetrical interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof.

[0166] In a further preferred embodiment the therapeutic and / or prophylactic agent is a small molecule, in particular a cytostatic. Preferably the inventive composition comprises doxorubicin (DOX) which is a cytotoxic anthracycline. Further suitable cytostatic anthracyclines presently employed in cancer treatment include epirubicin, idarubicin, daunorubicin and mitoxantrone. In further embodiments of the invention other cytostatics such as mitomycin C, gemcitabine, trabectedin, etc. or platinum derivatives such as cisplatin, carboplatin or oxaliplatin or other agents disclosed below can be included.

[0167] In a further preferred embodiment the composition comprises an alternative of additional active pharmaceutical ingredient which is selected from the group consisting of further anthracyclines such as aclarubicin, amrubicin, pirarubicin, valrubicin and zorubicin; anthracenediones such as mitoxantrone and pixantrone; antineoplastic antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine and vinorelbine; alkylating agents such as cyclophosphamide and mechlorethamine hydrochloride: campthothecins such as topotecan, irinotecan (CPT-11), lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin; purine and pyrimidine derivatives such as 5-fluorouracil, gemcitabine (2,2'-difluoro-2-deoxycytidine, dFdC), floxuridine (FLIDR), cytarabine (cytosine arabinoside), 6-azauracil (6-All); oxazaphosphorines such as cyclophosphamide, ifosfamide and trofosfamide; taxanes such as paclitaxel and docetaxel; podophyllotoxin derivatives such as etopside and teniposide; platinum-based compounds such as cisplatin, carboplatin, oxaliplatin, nedaplatin; methotrexate; tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, adavosertib and lapatinib; and cytarabines such as cytosine arabinoside.

[0168] Most preferred according to the invention is a composition comprising: a) at least one cationic or cationizable lipid; b) cholesterol; c) at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol and 1 ,2- dipalmitoyl-sn-glycero-3-phosphotriglycerol; d) 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and e) optionally a therapeutic and / or prophylactic agent.

[0169] This composition preferably does not contain a PEG lipid.

[0170] Further most preferred according to the invention is a composition comprising: a) at least one cationic or cationizable lipid; b) cholesterol; c) at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol and 1 ,2- dipalmitoyl-sn-glycero-3-phosphotriglycerol; d) CLG2; and e) optionally a therapeutic and / or prophylactic agent.

[0171] This composition preferably does not contain a PEG lipid.

[0172] Further most preferred according to the invention is a composition comprising: a) at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol and 1 ,2-dipalmitoyl- sn-glycero-3-phosphotriglycerol; b) at least one of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); and c) optionally a therapeutic and / or prophylactic agent.

[0173] This composition preferably does not contain a PEG lipid.

[0174] Further most preferred according to the invention is a composition comprising: a) at least one cationic or cationizable lipid; b) cholesterol. c) CLG2; and d) a PEG lipid, and e) optionally a therapeutic and / or prophylactic agent. The inventive compositions and in particular inventive DPPG2 or DPPG3 based LNPs are particularly suitable for drug delivery.

[0175] In a further preferred embodiment, the present invention relates to a composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I) or (I A) , A) wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0176] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0177] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C- atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0178] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid; b)(ii) at least one structural lipid; and b)(iv) at least one PEG-lipid; wherein the oligoglycerol-containing lipid of Formula (I) is NLG2 or CLG2.

[0179] Preferably the structural lipid is cholesterol, the helper lipid is DSPC and the PEG-lipid is DSPE-PEG. Preferably NLG2 / cholesterol / DSPC / DSPE-PEG in a molar range of about 35 mol% to about 55 mol% NLG2, about 30 mol% to about 50 mol% cholesterol, about 5 mol% to about 15 mol% DSPC and about 0 mol% to about 6.5 mol% DSPE-PEG, more preferably about 42 mol% to about 50 mol% NLG2, about 39 mol% to about 47 mol% cholesterol, about 8 mol% to about 12 mol% DSPC and about 1 mol% to about 2 mol% DSPE-PEG, based on the total lipid content of the composition.

[0180] Preparation of LNPs so far bases on a microfluidic approach in which an aqueous phase, typically with a low pH and containing a nucleic acid, is rapidly mixed with an ethanolic solution the necessary lipids, typically including PEG-lipids, cholesterol, DSPC and an ionizable lipid, which has a positively charge at low pH-values. The resulting primary dispersion of smaller liposome-like vesicles containing the nucleic acids are then dialyzed against a buffer having neutral pH which neutralizes the positive charge of the vesicles. Therefore, electrostatic repulsion no longer keeps the vesicles at a distance and causes vesicle fusion. This fusion stop when a particle size is reached with the maximum density of PEG-chains on its surface. Thus, the ratio of PEG-lipid in the lipid mixture determined the final LNP sizes.

[0181] The LNPs may be produced with prior art methods including for example microfluidic methods or organic solvent injection or using dual centrifugation (DC) with or without organic solvents (e.g., ethanol).

[0182] The invention also allows for the preparation of patient-specific LNPs (e.g., with patientspecific mRNA to support cancer immune therapy) using DC. Bedside DC-preparation allows the application of LNPs that may not be perfectly storage stable, furthermore kits containing the necessary vials, lipids and drugs can be made available.

[0183] 2 Figures

[0184] The present invention is further illustrated by the enclosed figures.

[0185] Figure 1 : Potency of NLG2 containing LNPs is compared to transfection (luminescence units) of the PEG containing Formulation B (positive control). Luminescence was measured 24 h after transfection of HEK 293 cells in serum containing medium. Luciferase mRNA was encapsulated into LNPs containing varying ratios of NLG2.

[0186] Figure 2: Agarose gel electrophoresis for detection of the mRNA encapsulation efficiency of different LNPs. PEG-containing LNP Formulation B was compared to the RNA-LNPs with DPPG2 as the shielding lipid. (+ T = Triton treated)

[0187] Figure 3: Potency of DPPG2 containing LNPs (based on Formulation B w / o PEG) is presented as x-fold transfection (luminescence units) of the LNP Formulation B which acts as positive control. GFP mRNA was encapsulated into LNPs with varying DPPG2 contents. Luminescence was measured 24 h after transfection of HEK 293 cells in serum containing medium. Error bars are SD.

[0188] Figure 4: Potency of DPPG2 containing LNPs (based on Formulation B w / o PEG) without DMG-PEG is presented as absolute luminescence units compared to Formulation B. Luminescence measured 24 h after transfection of HEK 293 cells with LNPs containing GFP mRNA. Error bars are SD.

[0189] Figure 5: Luminescence of the different DPPG2 LNPs containing 150, 300 and 500 ng mRNA is presented as x-fold transfection (luminescence units) of the respective DPPG2 formulations containing 50 ng mRNA. Luminescence was measured 24 h after transfection of HEK 293 cells with GFP mRNA encapsulated into LNPs with varying DPPG2 contents. DPPG2 formulations were based on Formulation B without PEG. Formulation B served as positive control. Error bars are SD.

[0190] Figure 6: Potency of DPPG3 containing LNPs is presented as x-fold transfection (luminescence units) of the LNP Formulation B which acts as positive control. Luminescence was measured 24 h after transfection of HEK 293 cells with serum containing medium. Luciferase mRNA was encapsulated into LNPs with varying DPPG3 contents. Error bars are SD. Figure 7: Potency of CLG2 containing LNPs is presented as x-fold transfection (luminescence units) of the Formulation B (positive control). Luminescence was measured 24 h after transfection of HEK 293 cells with serum containing medium. Luciferase mRNA was encapsulated into LNPs with varying CLG2 contents. LNPs compositions were based on Formulation A and Formulation B, respectively (see text for details). Error bars are SD.

[0191] Figure 8: Potency of CLG2 and DPPG2 containing LNPs is presented as x-fold transfection (luminescence units) of Formulation B (positive control). Formulation B contains DSPC as helper lipid and DMG-PEG as shielding lipid. Luminescence was measured 24 h after transfection of HEK 293 cells with serum containing medium. Luciferase mRNA was encapsulated into LNPs with varying DPPG2 contents. Error bars are SD.

[0192] Figure 9: Transfection efficiency in HEK-293 cell line for NLG2 & CLG2 containing LNPs formulated with DODMA as ionizable lipids. Chemiluminescence data compared to the conventional DMG-PEG containing Formulation B as control. The data presented were obtained from at least three independent experiments. Columns from left to right: Control formulation LNPs; DODMA containing LNPs; DODMA containing LNP w / NLG2 (20%); DODMA containing LNP w / NLG2 (20%) & CLG2 (20%); DODMA containing LNP w / CLG2 (20%).

[0193] Figure 10: Transfection efficiencies in HEK 293 cell line for NLG2 containing LNPs represented as chemiluminescence compared to the conventional DMG-PEG containing Formulation B as control. Columns from left to right: Control Formulation B; LNPs like B w / NLG2(25%); LNPs like B w / NLG2 (21 %).

[0194] Figure 11 : Transfection efficiencies in HEK 293 cell line for DPPG2 containing LNPs represented by chemiluminescence, compared to the DMG-PEG-containing Formulation B as control. Evaluation after one week of storage

[0195] Figure 12: DPPG2 containing LNPs could by-pass liver accumulation compared to the conventional DMG-PEG-containing Formulation B (measured by Luciferase mRNA expression 4 hours after intravenous application).

[0196] Figure 13: Transfection efficiencies measured in HEK-293 cell line immediately after manufacturing (0 week), one and three weeks storage with LNPs like A W / DPPG3 (10%) represented as x fold increase compared to control LNPs like B with PEG. Columns from left to right: 0 week; 1 week; 3 weeks.

[0197] 3 List of references

[0198] Albertsen, Camilla Hald, Jayesh Kulkarni, Dominik Witzigmann, Marianne Lind, 'Karsten Pertersson, und JensB. Simonsen. „The role of lipid components in lipid nanoparticles for vaccines and gene therapy." Advanced drug delivery reviews 188 (2022): 114416-114428.

[0199] Ambegia, E., S. Ansell, P. Cullis, J. Heyes, L. Palmer, und I. MacLachlan. ..Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression." Biochimica et Biophysica Acta 1669 (2005): 155-163.

[0200] Arteta, Yanez, et al. ..Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles." PNAS 115 (2018): E3351- E3360.

[0201] Brockow, Knut, et al. ..Experience with polyethylene glycol allergy-guided." Allergy 77 (2022): 2200-2210.

[0202] Carrasco, Manuel J., et al. onization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration." Communications Biology, 2021.

[0203] Edo Kon, Uri Elia, Dan Peer,. ..Principles for designing an optimal mRNA lipid nanoparticle vaccine,." Current Opinion in Biotechnology, 2022.

[0204] Garcia, Josep, Carles Felip, Svea Stephan, and Paul Pietsch. Development and optimization of LN P formulations- using Knauer Nanoscaler White paper - Curapath, 2022. https: / / www.knauer.net / Application / application_notesA / PH0076_Development_and _Optimization_of_LNP.pdf

[0205] Hattori, Yoshiyuki, Kyoko Tamaki, Sho Sakasai, Kei-lchi Ozaki, und Hiraku Onishi. ..Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro genbe silencinf effects and siRNA biodistribution in mice." Molecular Medicine Reports 22 (2020): 4183- 4196.

[0206] Heyes, James, Kim Hall, Vicky Tailor, Richard Lenz, und Ian MacLachlan. ..Synthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug delivery." Journal of controlled release 112 (2006): 280-290.

[0207] Hou, Xucheng, Tai Zaks, Robert Langer, und Yizhou Dong. „Lipid nanoparticles for mRNA delivery." Nature Reviews Materials 6 (2021): 1078-1094. Jaggers, Jordon, and Anna R. Wolfson. "mRNA COVID-19 Vaccine Anaphylaxis: Epidemiology, Risk Factors,." Current Allergy and Asthma Reports 23 (2023): 195- 200.

[0208] Knop, Katrin, Richard Hoogenboom, Dagmar Fischer Prof. Dr., und Ulrich S. Schubert Prof. „Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives." Angewandte Chemie International Edition 49 (2010): 6288-6308.

[0209] Koehler, Jonas K., et al. ..Tailoring the Lamellarity of Liposomes Prepared by Dual Centrifugation." pharmaceutics 15 (2023): 706-727.

[0210] Kulkarni, Jayesh, Dominik Witzigmann, und Sam Chen. „Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics." Accounts of Chemical Research, 2019.

[0211] Li, Q., et al. "RNA editing underlies genetic risk of common inflammatory diseases." Nature 608 (2022): 569-577.

[0212] Lokerse, Wouter J.M., et al. ..Mechanistic investigation of thermosensitive liposome immunogenicity and understanding the drivers for circulation half-life: A polyethylene glycol versus 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol study." Journal of controlled release 333 (2021): 1-15.

[0213] Nogueira, Sara S., et al. „Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA delivery." ACS Applied Nano Materials 3 (2020): 10634-10645.

[0214] Petrini, Matteo, Wouter JM Lokerse, Agnieszka Mach, Martin Hossann, Olivia M Merkel, und Lars Lindner. ..Effect of surface charge, pegylation and functionalization with Dipalmitoylphosphatidyldiglycerol on Liposome- Cells interactions and Local drug delivery to solid tumors via thermosensitive liposomes." International journal of nanomedicines 16 (2021): 14045-14061.

[0215] Picard, Matthieu, et al. ..Safety of COVID-19 vaccination in patients with polyethylene glycol allergy: A case series." The Journal of Allergy and Clinical Immunology: In Practice 10 (2022): P620-P625.

[0216] Sabnis, Staci, et al. „A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates." The American Society of Gene and Cell Therapy. 26 (2018): 1509-1519.

[0217] Schoenmaker, Linde, et al. „mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability." International Journal of Pharmaceutics 601 (2021): 120585-120597.

[0218] Song, L.Y., Q.F. Ahkong, Z. Wang Q. Rong, S. Ansell, M.J. Hope, und B. Mui. ..Characterization of the inhibitory eject of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes." Biochimica et Biophysica Acta 1558 (2001): 1-13.

[0219] Szebeni, Janos, et al. ..Applying lessons learned from nanomedicines to understand rare hypeersensitivity reactions to mRNA-based SARS-CoV-2 vaccines." Nature Nanotechnology 17 (2022): 337-346.

[0220] Tam, P, et al. ..Stabilized plasmid-lipid particles for systemic gene therapy." Gene Therapy 7 (2000): 1867-1874.

[0221] Tenchov, Rumiana, Janet M. Sasso, und Qiongqiong Angela Zhou. ..PEGylated Lipid Nanoparticle Formulations: Immunological Safety." 34 (2023): 941-960. llrits, Ivan, et al. „A Review of Patisiran (ONPATTRO®) for the Treatment of Polyneuropathy in People with Hereditary Transthyretin Amyloidosi." Neurology and Therapy 9 (2020): 301-315.

[0222] Wang, X., Liu, S., Sun, Y. et al. ..Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery." Nature Protocols, 2022: 265-291.

[0223] Whitehead, Kathryn A., et al. "Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity." Nature Communications 5 (2014): 1-10.

[0224] Woodie, Martin C., und Danilo D. Lasic. „Sterically stabilized liposomes." Biochimica et Biophysica Acta, 1113 (1992): 171-199.

[0225] Yang, Qi, und Samuel K. Lai. „Anti-PEG immunity: emergence, characteristics, and unaddressed questions." WIREs Nanomed Nanobiotechnology 7 (2015): 655-677.

[0226] Zhang, Rui, et al. ..Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver." Biomaterials Science 9 (2021): 1449-14463.

[0227] Zhu, Yiran, Liyuan Zhu, Xian Wang, und Hongchuan Jin. „RNA-based therapeutics: an overview and prospectus." Cell Death Dis 13 (2022): 644-658. 4 Examples

[0228] 4.1 Example 1 : Method for formulation of RNA-LNPs

[0229] The RNA-LNPs of the present invention were formulated using a NanoScaler (KNAUER Wissenschaftliche Gerate GmbH, Berlin, Germany) integrated with impingement jet mixing system (Garcia, et al. 2022). The invention involves the formulation of RNA-LNPs through the integration of an organic phase (comprising lipids) and an aqueous phase (containing mRNA payload and dilution buffer) within the NanoScaler.

[0230] The organic phase was prepared with a lipid mix consisting of an ionizable lipid, helper lipid, cholesterol and a shielding lipid in defined molar ratio. The aqueous phase consists of diluted mRNA (GFP or Luciferase (Luc)) in 25 mM or 6.25 mM of acetate buffer, depending on the defined formulation maintaining the pH between 4.0 &.5.O. The amine:phosphate ratio (N / P) was maintained at between 6.20 and 5.13. The flow ratio between the organic and aqueous phases was maintained at 1 :3, with a flow rate of 4.5 mL / min, comprising 3 mL / min undiluted LNPs and 1.5 mL / min undergoing in-line dilution with PBS.

[0231] Following the formulation stage, the LNPs were further diluted in a 10 mM phosphate- buffered saline (PBS) at pH 7.4 to reduce ethanol concentration. Subsequently, purification was achieved using centrifugal concentrators (Vivaspin™ 500, Merck KGaA, Darmstadt, Germany) effectively eliminating residual ethanol, thus ensuring the safety and efficacy of the formulation.

[0232] The lipid composition of known mRNA based COVID-19 vaccines were used as control (Table 1). The formulation containing the lipids of the invention were prepared analogously with the difference that DPPGXor CLGXor NLGXlipids were used replacing the corresponding lipid in the lipid mix with similar or varying lipid ratios (see following examples). Table 1 : Description of the mRNA based COVID-19 vaccines which are used as control formulations. 4.2 Example 2: Synthesis routes of cationic / cationizable lipids of Formula (II)

[0233] Example 2a: Ester variants

[0234] The synthesis route for NLG2 and CLG2 is depicted below starting from glycidol and 1 ,2- isopropylideneglycerol. The synthesis proceeds in a stereospecific manner, therefore if needed also distinct stereoisomers can be generated by choice of the enantiomerically pure starting materials in step 1 and 2 (R- or S-glycidol and R- or S-1 ,2-isopropylideneglycerol), as well as step 9 (R- or S-glycidol). The cationizable NLG2 was obtained from glycidol and 1 ,2-isopropylideneglycerol in 11 synthesis steps. The cationic CLG2 was obtained in 2 additional steps from NLG2. The synthesis route is applicable to other oligo-containing lipids of Formula (II) with ester linked fatty acid chains.

[0235]

[0236] Step 1 :

[0237] 3.8 kg glycidol are dissolved in 22.8 L NMP, then 8.02 kg para-methoxybenzyl chloride (PMBCI) are added followed by 2.26 kg NaH at 0-5 °C. After 4 hours at RT, the reaction mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) and 8.0 kg of 1.1 are obtained as colorless oil.

[0238] Step 2+3: 8.0 kg of 1.1 are dissolved in 64 L DMF, then 8.16 kg 1 ,2-isopropylideneglycerol is added, followed by 6.94 kg tBuOK at 0-5 °C and the resulting reaction mixture stirred for 12 h at RT to obtain 2.1. Subsequently, without work up or further purification additional 6.91 kg tBuOK and 12.6 kg benzyl bromide are added at 0-5 °C and the resulting reaction mixture stirred at 4 h at RT. Then, the reaction mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SC>4 and concentrated under reduced pressure. 7.0 kg of pure compound 3.1 is obtained as yellow oil after purification by silica gel chromatography (n-Heptane / Ethyl acetate).

[0239] Step 4:

[0240] 379 g 3.1 are dissolved in 2.65 L MeOH and then cooled to 0 °C, where 2 L 1M HCI are slowly added. The reaction mixture is stirred at 25 °C for 4 h. Then, saturated NaHCCh solution is added to adjust the mixture to pH = 7 at 0-5 °C, followed by extracted with EtOAc. The organic phase is washed brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 310 g of crude 5.1 as yellow oil.

[0241] Step 5:

[0242] 310 g of 5.1 are dissolved in 2.5 L DMF and then cooled to 0 °C, where 369 g tBuOK and 422 g BnBr are added. The reaction mixture is stirred at 25 °C for 4 h, before being poured into ice water followed by extraction with EtOAc. The organic phase is washed brine, dried over Na2SO4, filtered and concentrated under reduced pressure. After silica column chromatography (n-heptane / EtOAc) 115 g of pure 6.1 are obtained as yellow oil.

[0243] Step 6:

[0244] 331 g of 6.1 are dissolved in 3 L AON and 332 ml H2O and then cooled to 0 °C, where 652 g cerium ammonium nitrate (CAN) are added. The reaction mixture is stirred at 15 °C for 8 h, before being poured into ice water followed by extraction with EtOAc. The organic phase is washed brine, dried over Na2SO4, filtered and concentrated under reduced pressure. After silica column chromatography (n-heptane / EtOAc) 168 g of pure 7.1 are obtained as yellow oil.

[0245] Step 7:

[0246] 153 g 7.1 are dissolved in 765 ml DCM, then 71 g triethylamine added, followed by dropwise addition of 50.1 g MsCI. The reaction mixture is stirred at 20 °C for 2 h, before being poured into water followed by extraction with DCM. The organic phase is dried over Na2SC>4, filtered and concentrated under reduced pressure to obtain 183 g of crude 25.1 as yellow oil.

[0247] Step 8:

[0248] 183 g 25.1 are dissolved in 366 ml EtOH and the resulting solution is added to 1.48 kg of a MeNH2 solution (33% wt) in EtOH. The reaction mixture is warmed to 55°C and stirred for 48 h, before evaporation to dryness. The residue is taken up in MeOH / toluene and washed with 10 % aqueous Na2COs solution, before concentration under reduced pressure to obtain 151 g of crude 26.1 as yellow oil.

[0249] Step 9:

[0250] 43.6 g 26.1 are dissolved in 109 ml toluene and then a solution of 9 g glycidol in 109 ml toluene added. The reaction mixture is warmed to 100°C and stirred for 8 h. 3 batches are run in parallel, then combined and evaporated to dryness. The residue is taken up in MTBE and adjusted to pH5 with HCI and then phases separated. The aqueous phase is adjusted to pH8 with saturated NaHCCh solution and extracted with ethyl acetate, before concentration of the organic phase under reduced pressure to obtain 161 g of crude 35.1 as yellow oil.

[0251] Step 10:

[0252] 161 g 35.1 are dissolved in 800 ml DCM and then 62.2 g triethylamine, 11.2 g DMAP and 169 g palmitoyl chloride added subsequently. The resulting reaction mixture is stirred at 20°C for 1 h, before pouring into water. After separation the organic phase is concentrated under reduced pressure and then purified by silica column chromatography (n- heptane / EtOAc) to obtain 146 g of pure 36.1 as yellow oil.

[0253] Step 11 :

[0254] 6 g Pd / C is suspended in 300 ml AcOH and then 67 g of 36.1 are added and the reactor flushed with 30 psi H2. The resulting reaction mixture is stirred at 30°C for 24 h. 2 batches are run in parallel and worked up together. After filtration through celite, the filtrate is poured into water and the pH adjusted to 7 with saturated NaHCOs solution. After extraction with ethyl acetate, the organic phase is concentrated under reduced pressure. The residue is recrystallized from ACN, followed by purification via silica column chromatography (n- heptane / EtOAc / MeOH) to obtain 38.6 g of pure NLG2 as white solid. Step 12:

[0255] 20 g NLG2 are suspended in 100 ml ACN and then 7.8 g methyl iodide is added. The resulting reaction mixture is stirred at 40°C for 16 h. After filtration and concentration under reduced pressure 21 g of crude 38.1 are obtained as white solid.

[0256] Step 13:

[0257] 21 g 38.1 are added to a 315 ml 1 :1 :1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C for 48 h. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain 10 g pure CLG2 as white solid. Characterization by ICP- MS confirms CLG2 as chloride salt.

[0258] Example 2b: Ether variants

[0259] The synthesis route for oligoglycerol-containing lipids of Formula (II) with ether linkages 84.1 and 85.1 is depicted below starting from 2.1 and 1 ,2-isopropylideneglycerol. The synthesis proceeds in a stereospecific manner, therefore if needed also distinct stereoisomers can be generated by choice of the enantiomerically pure starting materials for synthesis of 2.1 and in step 6 (R- or S-glycidol and R- or S-1 ,2-isopropylideneglycerol). The synthesis route is applicable to other oligo-containing lipids of Formula (II) with ether linked fatty acid chains.

[0260]

[0261] Step 1 :

[0262] Dihydropyran is added to a solution of 2.1 and PPTS in DCM and the resulting reaction mixture is stirred at RT until completion of the reaction. The mixture is diluted with diethylether and washed with brine, followed by drying over Na2SC>4 and concentration under reduced pressure to obtain crude 3.4.

[0263] Step 2:

[0264] 3.4 is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro-5,6- dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCO3 and water. The organic phase is dried over Na2SC>4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n- Heptane / Ethyl acetate) to obtain 4.2.

[0265] 4.2 is dissolved in DCM, then triethylamine added, followed by dropwise addition of MsCI. The reaction mixture is stirred at 20 °C until completion, before being poured into water followed by extraction with DCM. The organic phase is dried over Na2SC>4, filtered and concentrated under reduced pressure to obtain crude 27.2.

[0266] Step 4:

[0267] 27.2 is dissolved in EtOH and the resulting solution is added to a MeNH2 solution (33% wt) in EtOH. The reaction mixture is warmed to 55°C and stirred until completion before evaporation to dryness. The residue is taken up in MeOH / toluene and washed with 10 % aqueous Na2COs solution, before concentration under reduced pressure to obtain crude 28.2.

[0268] Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of palmityl alcohol 70.1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSCU, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71 .1 as a waxy white solid.

[0269] 1 ,2-lsopropylidene-rac-glycerol is dissolved in NMP, then PMBCI is added and the solution cooled to 0 °C. NaH is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SC>4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n- Heptane / Ethyl acetate) to obtain crude 31.2. Step 7:

[0270] 31.2 is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SC>4 and concentrated under reduced pressure to obtain crude 32.2.

[0271] Step 8:

[0272] DCM is added to a mixture of 71.1 , 32.2 and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72.1.

[0273] Step 9:

[0274] 72.1 is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCOs and water. The organic phase is dried over Na2SC>4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 73.1.

[0275] Step 10:

[0276] 73.1 , triphenylphosphine, tetrabutylammonium iodide and anhydrous 1 ,2-dibromoethane are combined in a flask and heated to 60 °C. The reaction mixture is stirred at 60 °C until the reaction is complete. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (n- heptane / ethyl acetate) to afford 74.1.

[0277] Step 11 :

[0278] 28.1 , N,N-diisopropylethylamine and 74.1 are dissolved in acetonitrile and stirred at room temperature under nitrogen until completion of the reaction. Then the solvent is removed under reduced pressure. The residue is dissolved in DCM and washed with water. The organic fractions are dried over MgSC>4 and the solvent is removed under reduced pressure to yield 82.2.

[0279] Step 12:

[0280] 82.2 is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is diluted with brine and extracted with MeTHF. The organic phase is separated, dried over Na2SC>4 and concentrated under reduced pressure. The crude residue is dissolved in THF and cooled to 0 °C. A mixture of TBAF and acetic acid is added at 0 °C and the reaction mixture stirred at 30 °C until completion of the reaction. The solution is poured into a mixture of EtOAc and saturated NaHCOs solution, and the organic phase separated. The organic phase is washed with saturated NaHCCh and brine, dried over Na2SC>4 and concentrated under reduced pressure. The residue is purified by silica column chromatography (n-heptane / EtOAc / MeOH) to obtain pure 83.1.

[0281] Step 13:

[0282] 83.1 is suspended in ACN and then methyl iodide is added. The resulting reaction mixture is stirred at 40°C until completion of the reaction. After filtration and concentration under reduced pressure crude 84.1 is obtained.

[0283] Step 14:

[0284] 84.1 is added to a 1 : 1 :1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C until completion of the reaction. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain pure 85.1. Characterization by ICP-MS confirms 85.1 as chloride salt. 4.3 Example 3: Synthesis routes for anionic lipids of Formula (II)

[0285] Example 3a: Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phospho-diglycerol and 1,2- dipalmitoyl-sn-glycero-3-phospho-triglycerol

[0286] The synthesis route for manufacturing of DPPG2 and DPPG3 was described in patent application WO 97 / 30058 A1 as part of the synthesis of glycerophospholipids of the molecule class of phosphatidyloligoglycerols (PGX). This synthesis is applicable to other anionic oligo-containing lipids of Formula (II) with ester linked, saturated fatty acid chains.

[0287] Example 3b: Synthesis of unsaturated ester linked anionic lipids of Formula (II) The synthesis route depicted below is for suited mono- and polyunsaturated ester-linked anionic lipids of Formula (II). In the example below in particular the synthesis of DOPG2 with oleyl lipid chains is shown. The synthesis proceeds via an intermediate of formula 4.2. DOPG2 is obtained in 13 synthesis steps in total and 4 steps from 4.2 and 34.1. The synthesis proceeds in a stereospecific manner, therefore if needed also distinct stereoisomers can be generated by choice of the enantiomerically pure starting materials in step 1 , 2 and 5 (R- or S-glycidol and R- or S-1 ,2-isopropylideneglycerol).

[0288]

[0289] Step 1 :

[0290] Glycidol is added to a suspension of NaH in DMF at 0 °C, followed by BnBr. The resulting reaction mixture is warmed to RT and stirred until completion of the reaction. Afterward, water is added, and the mixture extracted with DOM. The organic layer is washed with water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) to obtain

[0291] 1.2.

[0292] Step 2:

[0293] 1.2 is dissolved in DMF, then sn-1 ,2-IPG is added, followed by tBuOK at 0-5 °C and the resulting reaction mixture is stirred at RT until completion of the reaction. Then, the reaction mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain crude

[0294] 2.2. Step 3:

[0295] Dihydropyran is added to a solution of 2.2 and PPTS in DCM and the resulting reaction mixture is stirred at RT until completion of the reaction. The mixture is diluted with diethylether and washed with brine, followed by drying over Na2SO4 and concentration under reduced pressure to obtain crude 3.2.

[0296] Step 4:

[0297] 3.2 is added to a suspension of Pd / C in EtOH / DCM, followed by degassing. The resulting mixture is stirred under H2 (0.5 MPa) at RT until completion of the reaction. The mixture is filtered and the filtrate concentrated under reduced pressure. Crude 4.2 is purified by silica gel chromatography (n-Heptane / Ethyl acetate).

[0298] Step 5:

[0299] 1 ,2-lsopropylideneglycerol is dissolved in D F, then BnBr added and the solution cooled to 0 °C. tBuONa is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and the pH adjusted to 6-7 with aqueous HCI, followed by extraction with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain crude 31.1.

[0300] Step 6:

[0301] 31.1 is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SO4 and concentrated under reduced pressure to obtain crude 32.1 .

[0302] Step 7:

[0303] Pyridine and DMAP are added to a solution of 32.1 in DCM, which is then cooled to 5 °C. Acetyl chloride is carefully added, and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 33.1. Step 8:

[0304] Pd / C is suspended in EtOH and then 33.1 and DCM are added and the reactor flushed with H2. The resulting reaction mixture is stirred under H2 (5 bar) at 25°C until completion of the reaction. The mixture is filtered and the filtrate is concentrated under reduced pressure to obtain crude 34.1 , which is recrystallized several times from n-heptane.

[0305] Step 9:

[0306] DIPEA is added to a suspension of 34.1 in MeTHF and DCM, then mixture is cooled to -15 °C. 2-Cyanethyl-N,N-diisopropylchlorphosphoramidite (PAMCI) is dissolved in DCM and the solution cooled to -20 °C, before addition to the reaction mixture. The resulting mixture is stirred for 1 h at-15 °C, followed by 1.5 h at O°C and 1 h at 20 °C. Then 4,5-dicyanoimidazole (DCI) and 4.2 are dissolved in MeTHF and the solution slowly added at 20 °C to the reaction mixture. If necessary, an additional amount of DCI is added as solution in DCM after stirring for 1 h, then the whole mixture is stirred overnight at 20 °C. Then aqueous 35% w / w H2O2 solution is added at 0 °C over 1 h and then stirred at 20 °C for another 1 h. The reaction mixture is diluted with 5 wt% aq. Na2S20s solution and extracted with MeTHF. The organic phase is dried over Na2SO4 and the solvents are removed under reduced pressure. The crude intermediate 55.1 is purified by silica chromatography (DCM / acetone).

[0307] Step 10:

[0308] DBU is added to a solution of 55.1 dissolved in MeTHF and the reaction mixture stirred at RT for 2 h. Then Pd / C is added, and the obtained reaction suspension is stirred under H2 atmosphere (1.2 bar) for 1 h. The reaction mixture is filtered over celite and washed with 1M aq. citric acid solution and organic phase separated. The organic phase is washed with 5 wt%. aq. NaHCO3 solution, dried over Na2SO4 and concentrated under reduced pressure to obtain 58.1.

[0309] Step 11 :

[0310] A mixture of 58.1 , K2CO3 and 2:1 MeOH / H2O was stirred at RT until completion of the reaction. MeOH is removed under reduced pressure, then the aqueous phase extracted with MeTHF. The organic phase is separated, dried over Na2SO4 and concentrated under reduced to obtain crude 61.1. Step 12:

[0311] Pyridine and DMAP are added to a solution of 61.1 in DCM, which is then cooled to 5 °C. Oleyl chloride is carefully added, and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 64.1.

[0312] Step 13:

[0313] 64.1 is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is diluted with brine and extracted with MeTHF. The organic phase is separated, dried over Na2SO4 and concentrated under reduced pressure. The crude residue is purified by silica column chromatography (n-heptane / EtOAc / MeOH) to obtain pure 67.1.

[0314] Example 3c: Synthesis of ether linked anionic lipids of Formula (II)

[0315] The synthesis route depicted below is suited for saturated, mono- and polyunsaturated ether-linked anionic lipids of Formula (II). In the example below in particular the synthesis of 1O4.w1 with oleyl lipid chains is shown. The synthesis proceeds via an intermediate of formula 4.2. 104. )1 is obtained in 8 synthesis steps in total and 4 steps from 4.2 and 73.0)1. The synthesis proceeds in a stereospecific manner, therefore if needed also distinct stereoisomers can be generated by choice of the enantiomerically pure starting materials for synthesis of 4.2 and in step 2 (R- or S-1 ,2-isopropylideneglycerol).

[0316]

[0317] Step 1 :

[0318] Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of oleyl alcohol 70. w1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSO4, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71. w1 as a waxy white solid.

[0319] Step 2:

[0320] 1 ,2-lsopropylideneglycerol is dissolved in NMP, then PMBCI is added and the solution cooled to 0 °C. NaH is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n- Heptane / Ethyl acetate) to obtain crude 31.2.

[0321] Step 3:

[0322] 31.2 is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SO4 and concentrated under reduced pressure to obtain crude 32.2.

[0323] Step 4:

[0324] DCM is added to a mixture of 71. w1 , 32.2 and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72. w1.

[0325] Step 5:

[0326] 72.w1 is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCO3 and water. The organic phase is dried over Na2SO4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 73. w1.

[0327] Step 6:

[0328] DIPEA is added to a suspension of 73. w1 in MeTHF and DCM, then then mixture is cooled to -15 °C. 2-Cyanethyl-N,N-diisopropylchlorphosphoramidite (PAMCI) is dissolved in DCM and the solution cooled to -20 °C, before addition to the reaction mixture. The resulting mixture is stirred for 1 h at -15 °C, followed by 1.5 h at 0°C and 1 h at 20 °C. Then 4,5- dicyanoimidazole (DCI) and 4.2 are dissolved in MeTHF and the solution slowly added at 20 °C to the reaction mixture. If necessary, an additional amount of DCI is added as solution in DCM after stirring for 1 h, then the whole mixture is stirred overnight at 20 °C. Then aqueous 35% w / w H2O2 solution is added at 0 °C over 1 h and then stirred at 20 °C for another 1 h. The reaction mixture is diluted with 5 wt% aq. Na2S20s solution and extracted with MeTHF. The organic phase is dried over Na2SO4 and the solvents are removed under reduced pressure. The crude intermediate 98. w1 is purified by silica chromatography (DCM / acetone).

[0329] Step 7 :

[0330] DBU is added to a solution of 98. w1 dissolved in MeTHF and the reaction mixture stirred at RT for 2 h. The reaction mixture is filtered over celite and washed with 1 aq. citric acid solution and organic phase separated. The organic phase is washed with 5 wt%. aq. NaHCOs solution, dried over Na2SO4 and concentrated under reduced pressure to obtain 101.w1.

[0331] Step 8:

[0332] 101.0)1 is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is diluted with brine and extracted with MeTHF. The organic phase is separated, dried over Na2SO4 and concentrated under reduced pressure. The crude residue is purified by silica column chromatography (n-heptane / EtOAc / MeOH) to obtain pure 104.0)1.

[0333] 4.4 Example 4: Potency of NLG2 used as ionizable lipid

[0334] Lipids described herein were used to prepare LNPs by the UM method (Garcia, et al. 2022) in accordance with Example 1. The UM NanoScaler (Knauer) consisted of 5 impingement jets mixers (UM), 3 pumps and 2 Valves. The UM NanoScaler (Knauer) was configured for LNP production with proper configuration with the operating at a total flow rate (TFR) 4.5 ml / min and maintaining an aqueous-to-organic phase ratio of 3:1. Following the formulation, the resulting LNPs were diluted in PBS at pH 7.4. To remove excess organic phase, Vivaspin™ 500 (Sigma) was employed in the subsequent purification step.

[0335] The cationizable lipid NLG2 can be used in RNA-LNP formulations to form particles with desirable particle size and PDI but showed low (between 15-25%) mRNA encapsulation efficiency (measured by Ribogreen) (Carrasco, et al. 2021) (Table 2). This was tested by replacing the cationizable lipid in the Formulation B based LNP formulation (46.3% ALC- 0315) as well as in the Formulation A formulation (50% SM-102) with NLG2. This was not unexpected since NLG2 does consist of linear and not branched alkyl chains in the lipid tail which are described as necessary for successfully complex mRNA or siRNA. Therefore, encapsulation efficacy can be probably notably improved by using branched fatty acid chains in NLGX. The NLG2-containing RNA-LNPs further possess a pKa value of -5.2 (Table 2) which is lower than observed for known RNA-LNP (6.2-6.6).

[0336] Table 2: Formulation containing NLG2 as ionizable lipid replacing ionizable lipid in Formulation A & Formulation B

[0337] Consequently, the NLG2 containing LNPs showed only neglectable in-vitro transfection efficiency when compared to LNP Formulation B (< 1 %) (Figure 1). This was the case irrespective of the type of helper lipid (DSPC or DOPE) present in the composition.

[0338] Furthermore, increasing the amount of DOPE helper lipid ascendingly from 20% to 30% or varying the N / P ratio in the range from 3.0 to 10.0 resulted in encapsulation efficiencies of less than 25% (data not shown). Additionally, when compared to the LNP Formulation B as control, these NLG2-containing LNPs had in vitro transfection efficiency < 1%.

[0339] 4.5 Example 5: Potency of DPPG2 lipid as shielding lipid in mRNA-LNP formulation

[0340] The effect of three different concentrations of DPPG2 (2%, 5% and 10%) in RNA-LNP by replacing the PEG-DMG as shielding lipid in the Formulation B was investigated next.

[0341] Surprisingly, using DPPG2 as only shielding lipid resulted in dispersion-stable particles with a size of 130 to 160 nm with a narrow size distribution demonstrated by a PDI < 0.20 (Table 2). Notably, the formulations containing 5% and 10% anionic DPPG2, exhibited excellent mRNA encapsulation efficiencies (> 80%) (Table 3). This data is indeed remarkable since literature suggests that PEG lipids are mandatory to produce LNPs with an acceptable size range.

[0342] Table 3: Effect of replacing DMG-PEG with DPPG2 in mRNA based LNPs

[0343] It is also observed that with increasing DPPG2 content in the PEG-free RNA-LNPs the zeta potential of the particles is notably decreasing from -12 mV (2 mol% DPPG2) to -23 mV (10 mol% DPPG2) (Table 3). Formulation B RNA-LNPs had a zeta potential of -6.2±0.6 mV. This data suggests that DPPG2 as a negatively charged phospholipid, has a significant impact on the surface charge indicating that DPPG2, as well as DPPG3, is preferentially located in the LNP surface monolayer.

[0344] When tested for the stability of mRNA encapsulated in all the DPPG2 containing RNA-LNPs, it was proved that the mRNA was still intact after 48 hours, among which 10% ratio of DPPG2 showed comparatively the best performance (Figure 2). The data demonstrated the dispersion stability of DPPG2-based, PEG free RNA-LNPs.

[0345] Interestingly, the presence of serum-containing medium showed a significantly positively influence on the in vitro transfection efficiency of DPPG2-containing LNPs (based on Formulation B) resulting in substantial increase when compared to the PEGylated Formulation B (Figure 3). The difference in transfection can be explained by an improved interaction of the DPPG2-based RNA-LNPs with the surface of the target cell and / or improved intracellular uptake and / or facilitated release from the endosome. This effect could be majorly attributed due to considerable smaller glycerol headgroups of G2- and G3- lipids compared to PEG lipids that are known to sterically hinder such interactions.

[0346] The negative influence of the large PEG headgroup on nucleic acid uptake in target cells in vivo and in vitro has been described (Song, et al. 2001). Interestingly, when investigating the liposome-cell interactions of classical drug-loaded anionic liposomes containing 5% DPPG2 or 5% DSPE-PEG, respectively, the anionic DPPG2 liposome showed higher liposome-cell interaction (2-fold in FACS analysis) when compared to its PEGylated counterpart, but, importantly, only in serum-free media, i.e. , in the absence of proteins and other serum components. As expected, both anionic liposomal formulations (which had comparable zeta potentials) containing either 5% DPPG2 or 5% DSPE-PEG2000 showed nearly undetectable cell targeting after protein adsorption (Petrini, et al. 2021).

[0347] Importantly, all DPPG2 containing LNPs showed a clear dose-response relationship over the tested range from 50 ng to 500 ng. This is an important prerequisite for the development of a therapeutic RNA-LNP for treatment of patients. This contrasts with the PEGylated Formulation B formulation which showed a constant low transfection efficiency (Figure 4 and Figure 5)

[0348] To investigate whether the addition of G2- or Gs-lipids to the LNP formulation can prevent aggregation with blood components such as erythrocytes after systemic injection, G2-lipid or Gs-lipid containing and PEGylated RNA-LNPs were added into erythrocyte suspensions. All the G2- or Gs-lipid modified RNA-LNPs were able to prevent aggregation with erythrocytes.

[0349] The influence of DPPG2 and the effect of PEG lipid as shielding component also plays a significant role on LNP size. This was demonstrated by experiments with selected RNA- LNPs (Table 4). As expected, the size of a PEG-free Formulation B was higher (134 nm) compared to the respective PEG-containing formulation (77 nm) (Table 4). Importantly the size of all three LNPs was in the range of ~77 to 130 nm, which would allow systemic administration in patients, with the DPPG2 formulation having a particle size of 128 nm and a favourable PDI of 0.15. It is worth mentioning that they had transfection efficiencies of more than 70 %. The PEG free Formulation A increased in particle size from 116 to 145 nm after 24 hours at 4°C (see Table 4).

[0350] Table 4: LNPs containing DPPG2 as shielding lipid replacing DMG-PEG and the effect of absence of DMG-PEG on Formulation B based LNP

[0351] To further assess the shelf-life stability of different RNA-LNPs, we conducted a comparison between two formulations containing either DPPG2 or DMG-PEG as shielding lipid (Table 5). The DPPG2-containing RNA-LNP had a lipid composition of ALC- 0315 / DSPC / Chol / DPPG246.3:9.4:34.3:10 (mol / mol) while the PEGylated RNA-LNP were composed of ALC-0315 / DSPC / Chol / DMG-PEG 46.3:9.4:42.7:1.6 (mol / mol). As buffer, 25 mM acetate pH 4.0 was used as buffer for both formulations with or without 10% sucrose as cryoprotectant. Formulations were stored for 48 hours and 2 weeks 2-8°C or -20°C. Remarkably, both formulations demonstrated sustained stability, maintaining constant particle size even after one week of storage at 2-8°C. However, when stored at -20°C, both formulations exhibited a comparable increase in particle size. Apparently, the presence of sucrose as model cryoprotectant did not have an influence on the increase in size. The encapsulation efficiency for the GFP mRNA remained good for all formulations as analysed by agarose gel electrophoresis (data not shown).

[0352] Transfection efficacy of the different formulations stored under different conditions was determined in accordance with Example 4. As shown in Figure 11 , surprisingly, DPPG2 containing formulations showed highest transfection efficiency when stored at 4°C without any cryoprotectant.

[0353] Table 5: Stability of LNPs containing DPPG2 replacing DMG-PEG compared to formulation B based LNP containing DMG-PEG as shielding lipid with varying storage conditions.

[0354] 4.6 Example 6: Potency of DPPG3 lipid as shielding lipid in mRNA-LNP formulation / DPPG3 containing formulations with complete substitution of PEG lipid

[0355] In a next step, ability of DPPG3 to replace PEG-DMG as shielding lipid in RNA-LNP was investigated. The lipid compositions and characterization results of the formulations are shown in Table 6.

[0356] DPPGs (5 or 10 mol%) was successfully used in RNA-LNP to replace the shielding lipid DMG-PEG in both Formulation A and Formulation B like LNPs. RNA-LNPs with well-defined size, a narrow size distribution with a PDI <0.2), and a good mRNA encapsulation efficiency (70-83%) were produced.

[0357] Table 6: Incorporation of 5% and 10% DPPG3 into the Formulation A & B based formulations

[0358] Also, the in vitro transfection efficiency (in serum-containing medium) of DPPGs-containing RNA-LNPs was increased compared to the PEGylated Formulation B LNP (Figure 6). A striking increase in transfection efficiency was observed especially for the PEG-free Formulation A RNA-LNPs applying SM-102 and DPPG3 as cationizable and shielding lipid, respectively (Figure 6), respectively.

[0359] Table 7: Effect on zeta potential by replacing DPPG3 as shielding lipid with Formulation A & B based LNPs

[0360] To assess the transfection efficiency of different RNA-LNPs stored for up to 3 weeks, a comparison between two formulations as shown in Table 14 containing either DPPG3 or DMG-PEG as shielding lipid were produced as disclosed above. The DPPG3-containing RNA-LNP (LNPs like A with DPPG3) had a lipid composition of SM-102 / DSPC / Chol / DPPG3 50:10:30:10 (mol / mol) while the PEGylated RNA-LNP like formulation A were composed of SM-102 / DSPC / Chol / DMG-PEG 50:10:38.5:1.5 (mol / mol).

[0361] Table 14: DPPGs-containing and PEGylated RNA-LNPs

[0362] Transfection efficacy for the different LNPs in in H EK-293 cell using comprising 50, 150 and 300 ng of RNA was determined immediately after manufacturing (0 week), one and three weeks storage at 4°C. As shown in Figure 13, Remarkably, compared to the control (LNP like B) all RNA-LNPs formulations containing DPPG3 demonstrated superior transfection efficiencies, even after 3 weeks.

[0363] 4.7 Example 7: Effect of cationic CLG2 lipid in mRNA-LNPs

[0364] The possibility of increasing the zeta potential of PEGylated RNA-LNPs based on Formulation A and B while achieving a well-controlled particle size and maintaining a satisfying transfection efficiency was investigated next. For this purpose, the new lipid CLG2, a cationic member of the G2 lipid family, which carries a permanent positive charge was used.

[0365] Initial experiments were for testing whether the cationic CLG2 could replace the neutral charged helper lipid DSPC in Formulation A & B. Final lipid compositions and characterization results are shown in Table 8. The PDI of the CLG2-containing PEGylated RNA-LNPs (Table 8) were comparable to the control Formulation B (Table 6). However, particle size was larger for the former. The encapsulation efficiencies were > 80% for the CLG2-containing PEGylated RNA-LNPs. The pKa values (as measured by TNS assay) were in the range of 6.2 to 6.3 for all three formulations. It is important to mention that all formulations had still a negative zeta potential which is favourable for in vivo administration but was slightly higher than for Formulation B (Table 6). However, since PEG is known to successfully shield the charge of membrane forming lipids, the effect of CLG2 on the surface charge might be underrepresented by the zeta potential results. So, surprisingly, the cationic CLG2 can replace the neutral DSPC as helper lipid in PEGylated RNA-LNP formulations, thereby modulating the zeta potential and potentially also influence the protein corona of the resulting LNPs in vivo after systemic administration.

[0366] Importantly, the particle size of PEGylated CLG2-containg RNA-LNPs was affected by the structure of the cationizable lipid and the amount of CLG2 used (Table 8). Particle size was 170 nm (PDI 0.19) and 218 nm (PDI 0.19) for RNA-LNP using SM-102 (Formulation A like) and ALC-0315 (Formulation B like) for RNA-LNP using ~10 mol% CLG2 as helper lipid, respectively. When increasing CLG2 content from 10 mol% to 20 mol% in RNA-LNP using 50 mol% SM-102 (Formulation A like) particle size was decreased from 170 nm (PDI 0.19) to 148 nm (PDI 0.17), respectively. Thus, it can be assumed that smaller LNPs can be generated by incorporation of higher levels of CLG2 even in the total absence of PEG-lipid. The mRNA in the CLG2 formulations was not degraded after 48 hours (data not shown). Table 8: Potency of CLG2 as helper lipid

[0367] In the presence of a serum-containing medium, the in vitro transfection efficiency of PEGylated RNA-LNPs containing CLG2 as a helper lipid exhibited a marked improvement compared to the PEGylated LNPs found in Formulation B (Figure 7). Table 9: Potency of CLG2 to replace the ionizable lipid in Formulation A based LNPs

[0368] As expected, CLG2 containing Formulations A & B could only form micro-particles with wide size distribution (>0.25) when CLG2 having linear acyl chains was used to replace the respective ionizable lipid with branched alkyl chains in a LNP formulation (Table 9).

[0369] Interestingly, CLG2 could also not stabilize RNA-LNPs in the absence of PEG-DMG or DPPG2 (see Table 10) resulting in unfavourable particle sizes and PDIs even after buffer exchange. Table 10: Potency of CLG2 as shielding lipid by replacing DMG-PEG in mRNA-LNPs

[0370] 4.8 Example 8: Potency of CLG2 (cationic lipids) & DPPG2 as helper lipid in mRNA- LNPs

[0371] In a next step we combined two members of the G2-lipid family, the anionic DPPG2 and the cationic CLG2 in RNA-LNPs to replace the helper lipid DSPC and PEGylated stabilizer lipids, respectively. Table 11 depicts the used lipid compositions and characterization results of the prepared formulations. Both RNA-LNP formulations had good pKa values and good encapsulation efficiencies. Further, the potential of CLG2 to modulate the zeta potential of the RNA-LNP was again demonstrated. This is clearly shown by comparison of the RNA-LNPs containing 9.4% CLG2 and 10% DPPG2 (zeta potential: -6.5±0.4 mV; Table 11), or RNA-LNPs containing 9.4% CLG2and 1.6% DMG-PEG (-1.010.5 mV; Table 8), versus RNA-LNPs containing 0% CLG2, 9.4% DSPC and 10% DPPG2 (-2311.8 mV; Table 3).

[0372] The LNP formulation, which contains nearly equimolar amounts of the negatively charged DPPG2 (10%) and the positively charged CLG2 (9.4%) has similar zeta potential (-6.510.4 mV) as compared to the “standard” LNP Formulation B which contains 9.4% of the zwitterionic DSPC (zeta potential -6.210.4 V).

[0373] Remarkably, RNA-LNP consisting of CLG2 and DPPG2 showed up to 10-fold higher in vitro transfection efficiency than the PEGylated RNA-LNPs (see Figure 8).

[0374] Thus, by combining cationic and anionic members of the G2- and Gs-lipid family it is possible to design and produce PEG free mRNA LNPs which share several features (including the almost same zeta potential) with the approved COVID-19 vaccines (Formulation B and Formulation A) and the approved siRNA LNP Onpattro® (Urits, et al. 2020).

[0375] CLG2 could also facilitate charge (zeta potential) modulation for LNP formulations in the presence of a shielding agent, e.g., a PEGylated lipid or an anionic member of the G2- or Gs-lipid family.

[0376] Tablel 1 : Substitution with G2 family lipids as shielding and helper lipids

[0377] Therefore, the presence of G2- or Gs-lipids in RNA LNP formulations may have the potential to ensure systemic stability without the loss of transfection activity by PEGylation.

[0378] Using DPPG2 or DPPG3 as a replacement for PEG-lipids in LNPs would offer a promising solution for the application of RNA-LNPs for in vivo applications. Moreover, cationic G2- or Gs-lipids which also appears to be LNP compatible, could serve as a cationic component to modulate the zeta potential, if necessary.

[0379] 4.9 Example 9: NLG2 & CLG2 compatibility with classical DODMA / DOPE lipids

[0380] The miscibility of the oligoglycerol lipids NLG2 and CLG2, respectively, with DODMA (1 ,2- dioleyloxy-3-dimethylaminopropane) as a representative member of classical ionizable lipids was tested. In contrast to ALC-0315 and SM-102, the hydrophobic moieties in DODMA are two non-branched unsaturated Cis fatty acids linked to a propane backbone by ether bonds. Further a variation of the identity of the zwitterionic phospholipid was tested by replacing DSPC with DOPE in the LNPs according to the compositions of Table 12. The LNPs were prepared using the described formulation method utilizing the impingement jet technology (UM) as described above.

[0381] As shown in Table 12, LNPs with a size range of 100-190 nm, narrow polydispersity index (PDI), and encapsulation efficiency of at least 95% were successfully generated. Their potential for generating LNPs with a size of approximately 100 nm was demonstrated (both, NLG2 and CLG2, containing LNP).

[0382] Table 12: Formulation details of NLG2 & CLG2 containing LNPs with DODMA as ionizable lipid component

[0383] Transfection efficiencies were measured in HEK 293 cell line. All three formulations which contained NLG2, CLG2 or both, CLG2 and NLG2, in addition to DODMA as ionizable lipid, showed superior transfection efficiency compared to the Formulation B based (with ALC- 0315 as ionizable lipid) formulations (with varying mRNA amount ranging from 50 ng, 150ng and 300 ng). Further, compared to the 4-component formulation comprised of DODMA (50 %) : DSPC (10 %) : Cholesterol (38.5 %) : DMG-PEG (1.5 %), the addition of CLG2or (especially) NLG2 resulted in a 5-component formulation with at least comparable, at lower (50 ng) mRNA amount clearly improved transfection efficiency (Figure 9). The formulation containing both, DOPE phospholipid and NLG2, was most efficient for in vitro mRNA delivery - probably by improved endosomal escape compared to the DSPC containing LNPs.

[0384] In conclusion, the miscibility of NLG2 and CLG2 with DOPE and DODMA as alternative to DSPC and branched ionizable lipids, respectively, allows the generation of LNPs with high in vitro potency.

[0385] Further, the compatibility of NLG2 and CLG2 with DODMA shows that oligoglycerol lipids containing fatty acids bound via ester bonds to a glycerol backbone can synergistically work with a lipid where the fatty acid chains are bound via ether bonds to a propane backbone. These results clearly demonstrate the broad and universal applicability of the new class of oligoglycerol lipids for rational design of LNPs.

[0386] 4.10 Example 10:NLG2 in 3-component LNPs

[0387] It was investigated whether NLG2 can completely replace the PEG shielding lipid as well as the zwitterionic DSPC as helper lipid during LNP formation. This approach allowed the generation of 3-component LNP formulations with -100 nm diameter and narrow size distribution. LNPs were generated and analysed as described above.

[0388] Formulations according to Table 13 completely replacing PEG lipid and DSPC helper lipid (and part of ALC-0315) by NLG2 with molar ratios of 21% and 25% were found to form LNPs with a vesicle size of - 100 nm and monodisperse distribution (PDI 0.12 -0.17) with - 80 % mRNA encapsulation efficiency (Table 13). The successful encapsulation of mRNA in Formulation B based, NLG2 containing 3 component formulations was further confirmed using agarose gel electrophoresis (data not shown).

[0389] Table 13: Formulation details of NLG2 containing 3-component LNPs in absence of helper lipid and shielding lipid component

[0390] Both NLG2 containing formulations showed superior in vitro transfection efficiency in comparison to a Formulation B like control formulation (Figure 10).

[0391] These results clearly demonstrate the versatile potential of the oligoglycerol lipids in the formation of LNPs. In these experiments their dual function, working as shielding agent and as helper lipid, reduced the complexity of the LNP formulation by making a fourth component unnecessary - an advantage not to be underestimated during manufacturing and storage of a licensed vaccine for human use.

[0392] 4.11 Example 11 :Potency of (10%) DPPG2 containing LNPs: in vivo in mice

[0393] To understand the biodistribution of LNPs containing DPPG2 as a shielding lipid, BALB / c mice (male, 6 weeks old, -20 g) were intravenously injected with LNPs containing Luc- mRNA (for formulation details see Table 14). Groups were treated with LNPs like B (W / DPPG2), LNP formulation B (w / PEG) and saline as control (n = 5 per group). Each experimental group received a single dose of 20 pg (100 pL) of Luciferase-mRNA (Luc- mRNA) encapsulated in LNPs. Ex vivo imaging for luminescence was performed for 4 hours post-administration using an in vivo imaging system (I VIS) (Wang, et al. 2023). Ex vivo analysis was performed on various organs, including the liver, lungs, heart, kidneys, spleen, lymph node) as well as blood samples. Notably, as shown in Figure 12, the accumulation of LNPs containing DPPG2 was significantly reduced in the liver compared to conventional LNPs containing PEG. This result clearly demonstrates the liver-bypassing effect of DPPG2 containing LNPs and the potential of oligoglycerol lipids to modify the biodistribution of mRNA carrying LNPs.

[0394] Table 14: Formulation details of DPPG2 containing LNPs replacing shielding lipid

[0395] Items

[0396] The present application also discloses the following items:

[0397] Item 1. A composition, preferably being a lipid nanoparticle (LNP), comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0398] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0399] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0400] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from

[0401] (i) at least one cationic or cationizable lipid;

[0402] (ii) at least one structural lipid;

[0403] (iii) at least one helper lipid; and / or

[0404] (iv) at least one PEG lipid.

[0405] Item 2. The composition of item 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0406] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0407] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; and

[0408] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from (i) at least one cationic or cationizable lipid;

[0409] (ii) at least one structural lipid;

[0410] (iii) at least one helper lipid; and / or

[0411] (iv) at least one PEG lipid.

[0412] Item 3. The composition of item 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0413] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0414] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0415] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii)at least one structural lipid.

[0416] Item 4. The composition of any one of items 1-3 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0417] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0418] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; and

[0419] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii)at least one structural lipid. Item 5. The composition of item 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0420] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0421] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0422] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iii) at least one helper lipid.

[0423] Item 6. The composition of any one of items 1 , 2 or 5 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0424] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0425] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; and

[0426] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iii) at least one helper lipid.

[0427] Item 7. The composition of item 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;

[0428] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0429] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5

[0430] C-atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0431] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iv) at least one PEG-lipid.

[0432] Item 8. The composition of any one of items 1 , 2 or 7 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0433] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0434] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; and

[0435] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iv) at least one PEG-lipid.

[0436] Item 9. The composition of item 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0437] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0438] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; and

[0439] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid.

[0440] Item 10. The composition of any one of items 1 , 2 or 9 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA), wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;

[0441] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0442] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; and

[0443] B = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid.

[0444] Item 11. The composition of any one of items 1 - 10, wherein the at least one oligoglycerolcontaining lipid of Formula (I) or (IA) comprises a phosphatidyloligoglycerol, wherein A = O-P(-O-)(=O)-O.

[0445] Item 12. The composition of any one of items 1 - 11 , wherein the at least one oligoglycerol-containing lipid of Formula (I) or (IA) comprises a cationic oligoglycerol containing lipid, wherein A = N+R4R5. Item 13. The composition of any one of items 1 - 12, wherein the at least one oligoglycerol-containing lipid of Formula (I) or (IA) comprises a cationizable oligoglycerol containing lipid, wherein A = NR3.

[0446] Item 14. The composition of any one of items 1 - 13, wherein the at least one oligoglycerol-containing lipid of Formula (I) or (IA) comprises a phosphatidyloligoglycerol, wherein A = 0-P(-0')(=0)-0 and a cationic oligoglycerol containing lipid of Formula (I) or (IA), wherein A = N+R4R5.

[0447] Item 15. The composition of any one of items 1 - 14, wherein the at least one oligoglycerol-containing lipid of Formula (I) or (IA) comprises from 2 to 4 glycerol units, preferably 2 or 3 glycerol units and more preferably 2 glycerol units.

[0448] Item 16. The composition of any one of items 1 - 15, wherein R1and R2independently are a linear or branched hydrocarbon group.

[0449] Item 17. The composition of any one of items 1 - 16, wherein R1and R2independently are a saturated or a monounsaturated or a polyunsaturated hydrocarbon group.

[0450] Item 18. The composition of any one of items 1 - 17, wherein R1and R2independently are a hydrocarbon group having 13 to 19 C-atoms and, in particular 15 or 17 C- atoms.

[0451] Item 19. The composition of any one of items 1 - 18, wherein R1and R2are a linear saturated C13- to C19- alkyl group.

[0452] Item 20. The composition of any one of items 1 - 19, wherein R3= Ci-Cs-alkyl, in particular R3= CH3, and / or R4and R5each independently are Ci-Cs-alkyl, and in particular R4= R5= CH3.

[0453] Item 21. The composition of any one of items 1-20, wherein the at least one oligoglycerol-containing lipid is of Formula (I I A), wherein = 2 to 20; and in particular n = 2 or 3; and wherein A = O-P(-O')(=O)-O; or NR3; or N+R4R5;

[0454] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0455] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1.

[0456] Item 22. The composition of any one of items 1-21 , wherein the at least one oligoglycerol-containing lipid is of Formula (II), wherein = 2 to 20; and in particular n = 2 or 3; and wherein A = O-P(-O')(=O)-O; or NR3; or N+R4R5;

[0457] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0458] R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1.

[0459] Item 23. The composition of any one of items 1-22, comprising an oligoglycerolcontaining lipid having the Formula

[0460] Item 24. The composition of any one of items 1-22, comprising an oligoglycerol- containing lipid having the Formula Item 25. The composition of any one of items 1-22, comprising an oligoglycerolcontaining lipid having the Formula

[0461] Item 26. The composition of any one of items 1-22, comprising an oligoglycerolcontaining lipid having the Formula

[0462] Item 27. The composition of any one of items 1-22, comprising an oligoglycerol- containing lipid having the Formula

[0463] Item 28. The composition of any one of items 1-22, comprising an oligoglycerol- containing lipid having the Formula

[0464] Item 29. The composition of any one of items 1 to 28, comprising,

[0465] (a) at least one oligoglycerol-containing lipid of Formula (I) or (IA), optionally (i) at least one phosphatidyloligoglycerol of Formula (I) or (I A) , wherein A = O-P(-O')(=O)-O; and

[0466] (ii) at least one cationic oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = N+R4R5; and

[0467] (b)(i) at least one cationizable lipid; and (b)(ii) at least one structural lipid.

[0468] Item 30. The composition of any one of items 1 to 29, comprising

[0469] (a) at least one oligoglycerol-containing lipid of Formula (I) or (IA); and

[0470] (b)(iii) at least one helper lipid, preferably at least two helper lipids.

[0471] Item 31. The composition of any one of items 1 - 30 being a lipid nanoparticle (LNP).

[0472] Item 32. The composition of any one of items 1 to 31, wherein the structural lipid is a sterol, preferably selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomadidine, ursolic acid and a-tocopherol.

[0473] Item 33. The composition of any one of items 1 to 32, wherein the structural lipid is cholesterol.

[0474] Item 34. The composition of any one of items 1-33, wherein the helper lipid is selected from the group consisting of hydrogenated soy L-a-phosphatidylcholine (HSPC);

[0475] 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0476] 1.2-diolioyl-sn-glycero-3-phosphoethanolamin (DOPE);

[0477] 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);

[0478] 1.2-dimyristoyl-sn-glycero-phosphocholine (DM PC);

[0479] 1.2-diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-palmitoyl-2-olioyl-sn-glycero-3-phosphocholine (POPC); and in particular wherein the helper lipid is

[0480] 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0481] Item 35. The composition of any one of items 1-6 and 9-34, comprising b)(iv) a PEG lipid.

[0482] Item 36. The composition of any one of items 1-6 and 9-34, wherein the composition does not comprise a PEG lipid. Item 37. The composition of any one of items 1-4, 7-8, 11-28 and 31-36 wherein the composition does not comprise a helper lipid.

[0483] Item 38. The composition of any one of items 1-4, 7-8, 11-28, 31-33 and 36-37, wherein the composition does not comprise a helper lipid and does not comprise a PEG lipid.

[0484] Item 39. The composition of any one of items 1-38, further comprising c) a therapeutic and / or prophylactic agent.

[0485] Item 40. The composition of item 39, wherein the therapeutic agent is a nucleic acid, in particular a ribonucleic acid (RNA).

[0486] Item 41. The composition of item 40, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), antisense RNA (aRNA), small interfering RNA (siRNA), CRISPR-RNA (crRNA), asymmetrical interfering RNA (aiRNA), micro- RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof.

[0487] Item 42. The composition of item 39, wherein the therapeutic and / or prophylactic agent is a small molecule.

[0488] Item 43. The composition of any one of items 1-42, comprising: a) at least one cationizable lipid; b) cholesterol; c) at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol and 1 ,2- dipalmitoyl-sn-glycero-3-phosphotriglycerol; d) at least one of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and CLG2 according to item 25 or 26; and e) optionally a therapeutic and / or prophylactic agent.

[0489] Item 44. The composition of item 43, wherein the composition does not contain a PEG lipid.

Claims

Claims1. A composition comprising: a) at least one oligoglycerol-containing lipid of Formula (I),wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from(i) at least one cationic or cationizable lipid;(ii) at least one structural lipid;(iii) at least one helper lipid; and / or(iv) at least one PEG lipid, wherein the composition is preferably a lipid nanoparticle (LNP).

2. The composition of claim 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5C-atoms; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b) at least one further lipid selected from(i) at least one cationic or cationizable lipid;(ii) at least one structural lipid;(iii) at least one helper lipid; and / or(iv) at least one PEG lipid.

3. The composition of claim 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii)at least one structural lipid.

4. The composition of any one of claims 1-3 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA),wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; and b)(ii)at least one structural lipid.

5. The composition of claim 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iii) at least one helper lipid.

6. The composition of any one of claims 1 , 2 or 5 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA),wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iii) at least one helper lipid.

7. The composition of claim 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iv) at least one PEG-lipid.

8. The composition of any one of claims 1 , 2 or 7 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA),wherein A = 0-P(-0’)(=0)-0; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(i) at least one cationic or cationizable lipid; b)(ii)at least one structural lipid; and b)(iv) at least one PEG-lipid.

9. The composition of claim 1 comprising: a) at least one oligoglycerol-containing lipid of Formula (I),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; y = 0 or 1 ; and z = 0 or 1 ; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid.

10. The composition of any one of claims 1 , 2 or 9 comprising: a) at least one oligoglycerol-containing lipid of Formula (IA),wherein A = O-P(-O’)(=O)-O; or NR3; or N+R4R5;R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;R3, R4and R5each independently are hydrogen or a hydrocarbon group having 1-5 C-atoms; andB = a linear or branched oligoglycerol group having from 2 to 20 glycerol units; and b)(iii) at least one helper lipid.

11. The composition of any one of claims 1-10, comprising an oligoglycerol-containing lipid having the Formulaand / or comprising an oligoglycerol-containing lipid having the Formulaand / or comprising an oligoglycerol-containing lipid having the Formula12. The composition of any one of claims 1 to 11 , comprising,(a) at least one oligoglycerol-containing lipid of Formula (I) or (IA), optionally(i) at least one phosphatidyloligoglycerol of Formula (I) or (IA), wherein A = O-P(-O')(=O)-O; and(ii) at least one cationic oligoglycerol-containing lipid of Formula (I) or (IA), wherein A = N+R4R5; and(b)(i) at least one cationizable lipid; and(b)(ii) at least one structural lipid.

13. The composition of any one of claims 1 to 12, comprising(a) at least one oligoglycerol-containing lipid of Formula (I) or (IA); and(b) (iii) at least one helper lipid, preferably at least two helper lipids.

14. The composition of any one of claims 1 - 13 being a lipid nanoparticle (LNP).

15. The composition of any one of claims 1 to 14, wherein the structural lipid is a sterol, preferably selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomadidine, ursolic acid and a-tocopherol; and in particular wherein the structural lipid is cholesterol.

16. The composition of any one of claims 1-15, wherein the helper lipid is selected from the group consisting of hydrogenated soy L-a-phosphatidylcholine (HSPC);1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);1.2-diolioyl-sn-glycero-3-phosphoethanolamin (DOPE);1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);1.2-dimyristoyl-sn-glycero-phosphocholine (DM PC);1.2-diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-palmitoyl-2-olioyl-sn-glycero-3-phosphocholine (POPC); and in particular wherein the helper lipid is1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC);17. The composition of any one of claims 1-16, further comprising c) a therapeutic and / or prophylactic agent.

18. The composition of claim 17, wherein the therapeutic agent is a nucleic acid or a small molecule, in particular a ribonucleic acid (RNA), preferably selected from the group consisting of messenger RNA (mRNA), antisense RNA (aRNA), small interfering RNA (siRNA), CRISPR-RNA (crRNA), asymmetrical interfering RNA(aiRNA), micro-RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof.

19. The composition of any one of claims 1-18, comprising: a) at least one cationizable lipid; b) cholesterol; c) at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol and 1 ,2-dipalmitoyl- sn-glycero-3-phosphotriglycerol; d) at least one of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and CLG2; and e) optionally a therapeutic and / or prophylactic agent.

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