Lipid nanoparticles

Abstract

The present invention relates to a composition comprising at least one oligoglycerol-containing lipid, and more particularly to lipid nanoparticles. The composition is particularly suitable for RNA delivery.

Description

[Technical Field]

[0001] 1. Explanation The present invention relates to a composition comprising at least one oligoglycerol-containing lipid, and more particularly to lipid nanoparticles (LNPs). The composition is particularly suitable for RNA delivery. [Background technology]

[0002] In recent years, RNA-based therapies have emerged as a promising new class of drugs. The effectiveness of RNA-based vaccines has already been impressively demonstrated during the COVID-19 pandemic. Various types of RNA molecules, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and microRNAs (miRNAs), crRNAs, tracrRNAs, or sgRNAs, may facilitate the targeting of mRNA or DNA using the Watson-Crick base pairing strategy (Zhu, et al. 2022). In addition to the use of messenger RNA (mRNA) as a preventive vaccine against infectious diseases or a therapeutic vaccine for cancer treatment, RNA can be used as a therapeutic agent to treat chronic or genetic diseases (Li, et al. 2022).

[0003] For parenteral administration, the "naked" mRNA needs to be protected from degradation. This is especially true for nasal administration or administration of mRNA, usually by inhalation or respiration through the nose to the lungs via nebulizer or spray, and particularly for systemic administration of mRNA, such as intravenous, intra-arterial, intramuscular, and subcutaneous injection or infusion. Furthermore, the mRNA needs to be confined to a so-called nonviral delivery system so that it can be transfected, that is, so that the mRNA binds to the surface of the target cell, is subsequently taken up into the cell via endocytosis, and released from the endosome into the cytosol where protein expression occurs.

[0004] To overcome the transfection and degradation problems of "naked" mRNA, polymer-based, particularly lipid nanoparticle (LNP)-based, nonviral delivery systems have been developed over the past few decades. Currently, all approved mRNA-based COVID-19 vaccines, as well as most RNA drug development programs in oncology and non-tumor diseases, utilize LNP technology to facilitate the delivery of mRNA, siRNA, crRNA, tracrRNA, or sgRNA to the cytoplasm of target cells.

[0005] Current LNP technologies for delivering mRNA, siRNA, or RNA species for CRISPR-Cas9-mediated genome editing utilize four main structural components that make up lipid nanoparticles: neutral or amphoteric so-called "helper" phospholipids, cholesterol, polyethylene glycol (PEG) lipids, and ionizable lipids that are neutral or cationic depending on the pH value. Ionizable pH-sensitive lipids contain one or more positively charged ionizable amine groups (at low pH) and are optimized for encapsulation (i.e., particle formation) of anionic RNAs such as mRNA, siRNA, crRNA, tracrRNA, or sgRNA, and for cellular uptake of LNPs (internalization by endocytosis). Furthermore, ionizable lipids play a major role in effective intracellular delivery by promoting membrane fusion during internalization (endocytosis) and facilitating RNA release from endosomes to the cytosol, also known as endosomal exodus (Whitehead, et al. 2014); (Edo Kon 2022); (Sabnis, et al. 2018)). The phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) supports cationizable lipids in the cellular uptake and transport of LNPs and encapsulated RNA (Arteta, et al. 2018). PEG lipids are designed to i) regulate LNP size during production and function as a steric barrier to prevent aggregation during storage, and ii) prevent nonspecific interactions with cellular components after systemic administration.

[0006] Typical examples of lipid nanoparticles (LNPs) include the mRNA-based LNPs Comirnaty (BioNTech / Pfizer's COVID-19 vaccine (Formulation B)), Spikevax (Moderna's COVID-19 vaccine (Formulation A)), and Onpattro (Alnylam, the first interfering RNA (RNAi) drug approved by the FDA). They contain different ionizable cationic lipids, zwitterionic DSPCs as helper lipids, cholesterol, and PEGylated lipids.

[0007] The precise composition and structure of LNPs and their encapsulated RNA molecules are a subject of intense debate. Nevertheless, the most widely accepted model currently assumes that almost all PEG lipids and most DSPC helper lipids reside on the LNP surface. Furthermore, in contrast to classical monolayer liposomes, LNPs contain lipids within them. During the manufacturing process, with a pH shift from acidic to neutral conditions, most of the ionizable lipids separate into a central oil phase. The precise location of RNA within the core differs between small siRNAs and mRNAs up to 100 times larger. It is also widely understood that the composition of LNPs can be influenced by the properties of the RNA or mRNA, their molecular weight, their sequence, and the chemical composition of the nucleotides (see, for example, Schoenmaker, et al. 2021).

[0008] The chemical structure of lipids (ionizable, helper, and PEG), and the formulation composition, i.e., the molar ratio of different components, primarily determine the composition and surface charge of LNPs carrying mRNA or siRNA. PEG lipids play a crucial role in the final LNP size. For example, it has been shown that changing the relative amount of PEG lipids from 3 to 0.25 mol% increased the size of final mRNA-carrying LNPs from 45 nm to 135 nm (number mean diameter) (Arteta, et al. 2018). Smaller relative percentages of PEG lipids allow for a lower surface area-to-volume ratio, resulting in larger particle size. The most important in vivo (and in vitro) performance of mRNA-carrying LNPs is determined by the properties of the ionizable lipids, the chemical structure of the ionizable head group, and the lipid tail, such as the presence of branched or linear lipid tails, the position of ester bonds, and the distance between the head group and other motifs (Whitehead, et al. 2014); (Sabnis, et al. 2018).

[0009] Furthermore, in addition to the properties of the RNA itself (e.g., siRNA vs. mRNA, see above), the RNA-LNP manufacturing procedure influences the final structure of the mature RNA-LNP. The structure, size, charge, and composition of RNA-LNPs are, in turn, major determinants of the potency of the inclusion RNA, measured by its in vivo distribution (specificity of organs, tissues, and / or target cells), as well as by the level and duration of protein expression in the case of mRNA, gene silencing in the case of siRNA, or DNA editing efficiency in the case of CRISPR / Cas-based approaches.

[0010] It is important to note that for systemic administration, each target cell type and administration route requires specific evaluation and optimization of all these parameters. Therefore, there is a high demand for significantly expanding the repertoire of LNP formulations and components to adapt the delivery of therapeutic RNA for the treatment of specific diseases.

[0011] Currently used COVID-19 mRNA vaccines consist of LNPs (Large Polypeptides) with PEGylated lipids incorporated into their surface, which determines the particle size during manufacturing and prevents aggregation in tissues during storage and after IM (intramuscular) or SC (subcutaneous) administration. These vaccines contain diffusible PEG lipids with relatively short lipid chains (C14), which, upon dissociation from the LNPs after administration, generate transfectable LNPs (see, for example, Kulkarni, Witzigmann, and Chen 2019 for a review). COVID-19 mRNA vaccines can be considered overwhelmingly safe. Most recent reports indicate that the rates of allergic reactions and anaphylaxis to mRNA COVID-19 vaccines are similar to those of other vaccines (Jaggers and Wolfson 2023). The estimated rate of anaphylaxis reported by April 2022 was between 2.5 and 4.7 cases per million (Jaggers and Wolfson 2023); (Tenchov, Sasso and Zhou 2023). Some of the symptoms in COVID-19 mRNA vaccine recipients are similar to those in patients who have received systemic administration of Doxil, a PEGylated liposome that carries the cytotoxic drug doxorubicin. However, the frequency of these reactions to mRNA vaccines is much lower than that to Doxil (Szebeni, et al. 2022).

[0012] Polyethylene glycol (PEG) lipids are considered a candidate allergen that may cause rare anaphylactic reactions to mRNA-based COVID-19 vaccines (e.g., Banerji, 2021), but it has been reported that some patients with known PEG allergies may be resistant to mRNA vaccines (Picard, et al. 2022); (Brockow, et al. 2022).

[0013] Hypersensitivity, though a rare adverse event, can be fatal. Therefore, there remains a demand for improved mRNA vaccines in which PEG lipids are removed from LNPs without compromising vaccine performance. Considering the extraordinary importance of mRNA vaccine technology in combating new viral diseases, the need to avoid undesirable immune responses and further improve mRNA vaccines is highlighted.

[0014] In contrast to the intramuscularly injected mRNA vaccine for COVID-19, the use of RNA-based drugs for the treatment of many diseases requires systemic administration of LNPs into the bloodstream. Incorporation of diffusible PEG lipids into LNPs confers a half-life of 15 minutes in circulation, which is too short to allow targeting to organs other than the liver (Kulkarni, Witzigmann und Chen 2019). Sustained PEG lipids with longer lipid chains (e.g., C18) and increased quantities may provide a potential optimization pathway to reduce LNP clearance from the blood. Furthermore, the size, composition, and charge of LNPs need to be adjusted for in vivo application, thus improving tissue targeting and the efficiency of inclusion RNA. However, it has been reported that PEGylated nanocarriers do not necessarily evade detection by the immune system. Unexpected immune responses to PEGylated nanocarriers, primarily the formation of anti-PEG antibodies after initial administration, can lead to accelerated blood clearance (ABC), i.e., increased clearance and decreased efficacy of PEGylated nanocarriers after repeated administration (see comprehensive review: (Tenchov, Sasso und Zhou 2023)). While the potential presence of pre-existing antibodies in the absence of PEGylated nanocarrier treatment is known (Yang und Lai 2015), its relevance remains debated. The presence of anti-PEG antibodies may also be involved in the development of hypersensitivity reactions such as complement activation-related pseudoallergies (CARPA). Most data have been reported for classical PEGylated nanocarriers (e.g., liposomes) and PEGylated proteins. The extent to which these observations apply to systemic administration of RNA-carrying LNPs is debatable. Nevertheless, immunosafety of RNA-carrying LNPs is crucial for repeated administration of therapeutic RNA LNP drugs, particularly for the lifelong treatment of chronic diseases, where adverse immune responses must be prevented.

[0015] Unshielded liposomes or LNPs encapsulating RNA possess insufficient physicochemical properties for systemic delivery. Their surfaces, particularly cationic LNPs or liposomes, lead to nonspecific interactions with (primarily anionic) blood components and uptake by macrophages, resulting in rapid clearance by the reticuloendothelial system (RES). Consequently, after administration of unshielded LNPs into circulation via injection or infusion, the primary targets are the so-called first-pass organs.

[0016] One solution to this problem is the use of hydrophilic polymers attached to the particle surface. PEG is the most commonly used, but other polymers have also been described (Heyes, et al. 2006), (Nogueira, et al. 2020).

[0017] Furthermore, the presence of hydrophilic polymers with large head groups has been reported as a prerequisite for producing RNA-LNPs with a clearly defined size suitable for parenteral administration. Although RNA-LNPs are not highly structured particles, due to the physicochemical properties of their components, they are largely self-assembling systems.

[0018] Established standard manufacturing processes utilize a low pH environment to generate a heterogeneous population of small LNPs, where positively charged ionizable lipids interact with negatively charged mRNA or siRNA. A pH shift to neutral induces rearrangement of different lipids and RNAs. During this process, ionizable lipids diffuse to the center of the LNP particle, while PEG lipids and helper lipids aggregate on the particle surface. Therefore, after this maturation process, distinct LNP compartments can be detected.

[0019] In the presence of PEG lipids, the desired mRNA-LNP size is achieved when the PEG lipid concentration in the outer monolayer is sufficiently high to inhibit further fusion of smaller LNPs. In the absence of PEG lipids, LNPs continuously fuse into large particles with a heterogeneous size distribution during the manufacturing process, making in vivo administration impossible (Kulkarni, Witzigmann und Chen 2019); (Schoenmaker, et al. 2021). However, the presence of PEG has been shown to affect the intracellular delivery and transport of nonviral nucleic acid delivery particles. PEG on the surface of RNA-carrying LNPs inhibits fusion of LNPs with the cell membrane, thereby reducing cellular uptake and endosomal escape processes. This results in reduced potential for gene expression (mRNA) or gene silencing (siRNA) (Woodle und Lasic 1992); (Heyes, et al. 2006).

[0020] A further drawback of mRNA-carrying PEGylated LNPs is that they can potentially induce an antibody response against the PEG portion. This can negatively impact their potential in therapies requiring repeated administration of PEGylated LNPs. This is particularly true for lifelong treatment of non-tumor diseases, such as protein exchange or replacement therapies in genetic disorders.

[0021] The alternative hydrophilic head groups described to date still exhibit these drawbacks. A representative of this class of alternative shielding agents is polysarcosine (pSar)-functionalized LNPs. Depending on the mole fraction of pSar lipids on the LNP surface, LNPs showed transfection reductions of one to several orders of magnitude in vitro compared to LNPs containing PEG-DMG, a PEG lipid used as a shielding agent in the COVID-19 vaccine developed by Moderna Inc. (Nogueira, et al. 2020).

[0022] To overcome the limitations of these established or proposed alternative shielding agents, several approaches are being pursued.

[0023] One research approach attempts to make the insertion and exposure of PEG lipids on the LNP surface transient. In principle, this can be achieved by diffusible PEG lipids that diffuse from the particle surface at a rate determined by the size of the lipid anchor (Ambegia, et al. 2005); (Hattori, et al. 2020). Another research approach investigates occludable PEG lipids with biodegradable bonds between the polymer and lipid anchor, which are enzymatically cleaved in the extracellular environment after binding to target tissues, e.g., tumors or damaged tissues in cardiovascular disease. A strong limitation of this concept is that the bulky head group of PEG also inhibits access to the enzymes responsible for linker cleavage. Alternatively, degradation can occur intracellularly in the acidic endosomal compartment by introducing a chemical bond that is sensitive to low pH. The drawback of the latter approach is that the bulky head group of PEG still negatively interferes with uptake into target cells. Therefore, strategies using diffusible PEG lipids may be advantageous over those in which PEG is permanently bound or not degraded until entry into endosomes.

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

[0025] Since PEG dissociation is essential for nucleic acid-containing particles to become transfectable, gene expression is directly related to the rate at which the PEG portion is removed from the particle bilayer. As a result, the in vitro transfection efficiency of the investigated lipid nanoparticles containing complex plasmid DNA was highest with PEG lipids containing the shortest lipid anchors.

[0026] It is important in this context to note that PEG ceramide containing only very short lipid anchors (C8) can significantly extend the circulation time of plasmid DNA:lipid complexes compared to uncoated or "naked" particles, thereby enabling targeting and gene expression at tumor sites (Tam, et al. 2000).

[0027] In summary, there remains a strong demand for shielding agents that can moderately extend the half-life of circulating LNPs, thereby allowing them to bypass "first-pass" organs. Furthermore, such shielding agents must not negatively interfere with the binding of LNPs to their target cells; that is, they must enable efficient uptake into target cells via endocytosis and efficient release of RNA from endosomes into the cytoplasm, mediated by pH-sensitive ionizable lipids. Such novel shielding agents must also maintain the dispersion stability of LNPs (e.g., no fusion or aggregation during storage and / or handling). No such alternatives to currently used PEG lipids have yet been described.

[0028] There is also a strong need to expand the repertoire of helper lipids for the future design of safe and effective LNPs. So-called helper lipids strongly influence LNP properties such as particle stability, delivery efficiency, tolerability, and biodistribution (see, for example, Hou, et al. 2021). Helper lipids such as DSPC and cholesterol, in particular, play an essential role by participating in LNP formation. For example, DSPC, a zwitterionic phosphatidylcholine with two saturated lipid chains, stabilizes the structure of lipid nanoparticles, while 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a phosphatidylethanolamine with two unsaturated lipid chains, destabilizes the endosomal membrane and promotes the endosomal escape of lipid nanoparticles (Hou, et al. 2021). The biodistribution and structure-function relationships of intravenously administered LNPs were systematically investigated by (Zhang, et al. 2021). The group studied how helper lipids affect LNP protein adsorption and in vivo distribution through high-throughput in vivo screening of an LNP library. Interestingly, LNPs containing the helper lipid DOPE preferentially accumulated in the liver, while LNPs containing DOPC but otherwise identical preferentially accumulated in the spleen.

[0029] Arteta et al. systematically investigated the relationship between the size of mRNA-carrying LNPs, the composition of LNPs, and protein expression after in vitro transfection (Arteta, et al. 2018). Their study focused on the lipid composition of the LNP surface and the composition of the LNP interior. Surprisingly, phospholipids, specifically DSPCs, were primarily present on the mRNA-LNP surface and strongly influenced transfection efficiency. Their data, demonstrated in vitro in two different cell types, clearly showed that both the surface composition and size of LNPs, as demonstrated in vitro in two different cell types, determine transfection efficiency. While DSPCs should not promote lamellar bilayer formation, thereby increasing bilayer stability and facilitating endosomal escape, their effect on protein expression was also significant.

[0030] These studies clearly demonstrate the significant influence of helper lipids on the biological efficacy of LNPs. Helper lipids can play a central role in regulating the in vivo distribution, target specificity, and protein expression of mRNA encapsulated in LNPs. This clearly indicates a high demand for a large repertoire of innovative helper lipids for the future design of LNPs for systemic administration. This is particularly true for the future development of LNPs designed without tissue-specific target moieties, such as antibodies. This class of LNPs should preferentially target tissues or organs after systemic administration and accumulate specifically within them. Their intrinsic affinity for specific tissues or organs is determined by their lipid composition, including helper lipids.

[0031] Therefore, there is a great demand for lipids that are particularly suitable as excipients for improved drug delivery systems, such as improved LNPs. [Overview of the project]

[0032] This invention is: a) at least one equation (I)

[0033] [ka]

[0034] (wherein, A = O-P(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 are each independently a hydrocarbon group having 12 to 24 C atoms; R 3 , R 4 , and R 5 are each independently hydrogen or a hydrocarbon group having 1 to 5 C atoms; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units) the oligoglycerol-containing lipid; and b)(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 at least one further lipid selected from The present invention relates to a composition comprising The composition is preferably a lipid nanoparticle (LNP).

[0035] Suitable counterions for all cationic lipids disclosed herein are Cl - , Br - , mesylate, fumarate; preferably it is Cl - . Suitable counterions for all anionic lipids disclosed herein are Na + , K + , Li + , preferably it is Na + .

[0036] In particular, the present invention: a) At least one formula (IA)

[0037] [ka]

[0038] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (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 at least one additional lipid selected from This relates to compositions containing the following:

[0039] In a preferred embodiment, the present invention is: a) at least one equation (I)

[0040] [ka]

[0041] (In the formula, A = OP(-O -)(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid This relates to compositions containing the following:

[0042] In particular, the present invention: b) at least one equation (IA)

[0043] [ka]

[0044] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid This relates to compositions containing the following:

[0045] In this embodiment, the total lipid content of the composition is used as a reference. At least one oligoglycerol-containing lipid of formula (I) or (IA) may be included 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%; At least one cationic or cationizable lipid may be present 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 At least one structural lipid is present in an amount of about 28 mol% to about 52 mol%, preferably about 32 mol% to about 46 mol%, and more preferably about 34 mol% to about 44 mol%.

[0046] In a further preferred embodiment, the present invention is: b) at least one equation (I)

[0047] [ka]

[0048] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5Each is a hydrocarbon group having either hydrogen or 1 to 5 carbon atoms independently: y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 This relates to compositions containing the following:

[0049] In particular, the present invention: b) at least one equation (IA)

[0050] [ka]

[0051] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 This relates to compositions containing the following:

[0052] In this embodiment, the total lipid content of the composition is used as a reference. At least one oligoglycerol-containing lipid of formula (I) or (IA) may be included 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%; At least one cationic or cationizable lipid may be present 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%; At least one structural lipid is present 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 At least one helper lipid is present in an amount of about 5 mol% to about 15 mol%, preferably about 7 mol% to about 12 mol%, and more preferably about 9 mol% to about 11 mol%.

[0053] In this embodiment, the composition may further contain PEG lipids. Based on the total lipid content of the composition, at least one oligoglycerol-containing lipid of formula (I) or (IA), preferably NLG2 or CLG2, may be present 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%; At least one cationizable lipid, preferably DODMA, may be present in an amount of about 15 mol% to about 35 mol%, preferably about 18 mol% to about 30 mol%; At least one structural lipid, preferably cholesterol, is present in an amount of about 25 mol% to about 45 mol%, preferably about 30 mol% to about 40 mol%, and more preferably about 33 mol% to about 37 mol%; At least one helper lipid, preferably DOPE or DSPC, is present in an amount of about 10 mol% to about 30 mol%, preferably about 13 mol% to about 22 mol%, and At least one PEG lipid, preferably DMG-PEG, is present in an amount of about 0.5 mol% to about 2 mol%.

[0054] In a further preferred embodiment, the present invention is: a) at least one equation (I)

[0055] [ka]

[0056] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 This relates to compositions containing the following:

[0057] In particular, the present invention: a) At least one formula (IA)

[0058] [ka]

[0059] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 This relates to compositions containing the following:

[0060] In this embodiment, the total lipid content of the composition is used as a reference. At least one oligoglycerol-containing lipid of formula (I) or (IA) may be included 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%; At least one cationic or cationizable lipid may be present 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%; At least one structural lipid is present 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 At least one PEG lipid is present in an amount of about 0.5 mol% to about 4 mol%, preferably about 1 mol% to about 3 mol%, and more preferably about 1.2 mol% to about 1.2 mol%.

[0061] In a preferred embodiment, the present invention: a) at least one of formula (I)

[0062]

化

[0063] (wherein, A = O-P(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 are each independently a hydrocarbon group having 12 to 24 C atoms; R 3 , R 4 , and R 5 are each independently hydrogen or a hydrocarbon group having 1 to 5 C atoms; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units) of the oligoglycerol-containing lipid; and b)(iii) relates to a composition comprising at least one helper lipid .

[0064] In particular, the present invention: a) at least one of formula (IA)

[0065]

化

[0066] (wherein, A = O-P(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b)(iii) at least one helper lipid This relates to compositions containing the following:

[0067] In this embodiment, based on the total lipid content of the composition, at least one oligoglycerol-containing lipid of formula (I) or (IA) may be present in the composition in an amount of about 20 mol% to about 40 mol%; and at least one helper lipid may be present in the composition in an amount of about 50 mol% to about 80 mol%.

[0068] In a preferred embodiment, the present invention is: a1) At least one equation (I)

[0069] [ka]

[0070] (where A = NR 3 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and a2) At least one equation (I)

[0071] [ka]

[0072] (where A = N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationizable lipid, and (iv) at least one PEG lipid This relates to compositions containing the following:

[0073] In particular, the present invention: a1) At least one equation (I)

[0074] [ka]

[0075] (where A = NR 3 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and a2) At least one equation (I)

[0076] [ka]

[0077] (where A = N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationizable lipid and. (ii) at least one structural lipid; and (iv) at least one PEG lipid This relates to compositions containing the following:

[0078] In this embodiment, A = NR is defined as the total lipid content of the composition. 3 At least one oligoglycerol-containing lipid of formula (I) or (IA), preferably NLG2, may be included in the composition in an amount of about 10 mol% to about 30 mol%; A = N + R 4 R 5At least one oligoglycerol-containing lipid of formula (I) or (IA), preferably CLG2, may be included in the composition in an amount of about 10 mol% to about 30 mol%; and at least one cationizable lipid, preferably DODMA, may be included in the composition in an amount of about 10 mol% to about 30 mol%; and at least one PEG lipid, preferably DMG-PEG, may be included in the composition in an amount of about 2 mol% to about 4 mol% based on the total lipid content of the composition; and at least one structural lipid, preferably cholesterol, may be included in the composition in an amount of about 30 mol% to about 45 mol%, preferably about 34 mol% to about 38 mol%, based on the total lipid content of the composition.

[0079] In some embodiments of the oligoglycerol-containing lipid of formula (I) or (IA), B = a linear or branched oligoglycerol group having 2 to 15 glycerol units, 2 to 10 glycerol units, and 2 to 5 glycerol units.

[0080] In a preferred embodiment, the oligoglycerol-containing lipid of formula (I) or (IA) is A = OP(-O - This is a phosphatidyl oligoglycerol that is )(=O)-O. Such compounds are referred to herein as DAPG x (Diacyl-phospho-oligoglycerol; x = number of glycerol units), and especially DPPG x It is also called (dipalmitoyl-phospho-oligoglycerol; x = number of glycerol units), DPPG2 (dipalmitoyl-phospho-diglycerol), or DPPG3 (dipalmitoyl-phospho-triglycerol).

[0081] In further embodiments, the oligoglycerol-containing lipid of formula (I) or (IA) is A = N + R 4 R 5 It is a cationic oligoglycerol-containing lipid. Such compounds are referred to herein as CLG x(Cationic oligoglycerol-containing lipid; x = number of glycerol units), and also specifically called CLG2 (cationic diglycerol-containing lipid). Preferably, R 4 and R 5 Each of these is independently a C1-C5 alkyl group, and in particular R 4 =R 5 =CH3

[0082] Further preferred oligoglycerol-containing lipids of formula (I) or (IA) are given by A = NR 3 It is a cationizable oligoglycerol-containing lipid. Such compounds are referred to herein as NLG x (Cationizable oligoglycol-containing lipid; x = number of glycerol units), and also known in particular as NLG2 (cationizable diglycerol-containing lipid). Preferably, R 3 =C1~C5 alkyl, especially R 3 =CH3

[0083] According to embodiments of the present invention, the composition contains two or more oligoglycerol-containing lipids of formula (I) and / or (IA), preferably A = OP(-O - )(=O)-O is at least one phosphatidyl oligoglycerol and A = N + R 4 R 5 It may contain at least one cationic oligoglycerol-containing lipid.

[0084] Based on the total lipid content of the composition, preferably A = OP(-O - At least one phosphatidyl oligoglycerol that is )(=O)-O may be included in the composition of the embodiment in an amount of about 2 mol% to about 15 mol%, preferably about 4 mol% to about 12 mol%, and A = N + R 4 R 5 At least one cationic oligoglycerol-containing lipid is present in an amount of about 5 mol% to about 15 mol%, preferably about 8 mol% to about 12 mol%.

[0085] Preferred are oligoglycerol-containing lipids of formula (I) or (IA) containing 2 to 4 glycerol units, preferably 2 or 3 glycerol units, and more preferably 2 glycerol units.

[0086] In an oligoglycerol-containing lipid of formula (I) or (IA), residue R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms. Preferred is R 1 and R 2 However, it is an oligoglycerol-containing lipid, which is a hydrocarbon group having 13 to 19 C atoms independently. The hydrocarbon group can be saturated or monounsaturated or polyunsaturated. The hydrocarbon group can be linear or branched. Preferably R 1 and R 2 R is independently a linear or branched saturated C13-C19 alkyl group, more preferably a linear saturated C13-C19 alkyl group. 1 and R 2 These are independently linear saturated C15 or C17 alkyl groups, with the most preferred being linear saturated C15 alkyl groups.

[0087] In a further embodiment, R 1 and R 2 These are independently preferably linear saturated or unsaturated C15 or C17 alkyl groups, most preferably saturated or unsaturated C17 alkyl groups.

[0088] The most preferred is formula (II) or formula (IIA).

[0089] [ka]

[0090] [ka]

[0091] (wherein the formula n=2 to 20; and in particular n=2 or 3; and A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each of these is a hydrocarbon group having either hydrogen or 1 to 5 carbon atoms independently; (y=0 or 1; and z=0 or 1) It is an oligoglycerol-containing lipid.

[0092] Such oligoglycerol-containing lipids of formula (II) or (IIA) have linear oligoglycerol groups and, in particular, di or triglycerol groups.

[0093] What is particularly preferable? n=2 or 3; A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; and R 3 , R 4 , and R 5 However, each is an independent hydrocarbon group having 1 to 5 carbon atoms; and R 1 and R 2 However, each is a linear saturated or unsaturated hydrocarbon group having 15 or 17 carbon atoms independently. It is an oligoglycerol-containing lipid of formula (IIA).

[0094] The most preferred is the formula

[0095]

Chem.

[0096] It is an oligoglycerol-containing lipid having the following structure, which is referred to herein as DPPG2.

[0097] Further most preferred are those of formula

[0098]

Chem.

[0099] It is an oligoglycerol-containing lipid having the following structure, which is referred to herein as CLG2.

[0100] Further most preferred are those of formula

[0101]

Chem.

[0102] It is an oligoglycerol-containing lipid having the following structure, which is referred to herein as NLG2.

[0103] The most preferred are those of formula

[0104]

Chem.

[0105] It is an oligoglycerol-containing lipid having the following structure, which is referred to herein as DSPG2.

[0106] Further most preferred are those of formula

[0107]

Chem.

[0108] This is an oligoglycerol-containing lipid having the following properties, which is referred to herein as DSAmG2.

[0109] The most preferable of all is the formula

[0110] [ka]

[0111] This is an oligoglycerol-containing lipid having the following properties, which is referred to herein as DSAG2.

[0112] Further preferred compositions: (a) At least one oligoglycerol-containing lipid of formula (I) or (IA), optionally (i) A = OP(-O - Phosphatidyl oligoglycerol of formula (I) or (IA) such that )(=O)-O; and (ii) A = N + R 4 R 5 A cationic oligoglycerol-containing lipid of formula (I) or (IA) that is at least one such lipid; and (b)(i) at least one cationizable lipid; and (b)(ii) at least one structural lipid Includes.

[0113] Further preferred compositions: (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 Includes.

[0114] Particularly preferred compositions are lipid nanoparticles, also known as LNPs in this specification. As used herein, the term "lipid nanoparticle" or "LNP" relates to a particle having an outer lipid shell enclosing an inner lipid phase, and characterized by a diameter between 1 and 1000 nm. The outer lipid shell may be in the form of a lipid monolayer or bilayer. The inner lipid phase may be in the form of inverted micelles and / or a solid lipid phase. In a preferred embodiment, the lipid nanoparticle comprises an outer lipid shell composed of one lipid monolayer, and an inner lipid phase comprising at least inverted micelles (also known as reverse micelles). The term "lipid nanoparticle" or "LNP" according to the present invention does not encompass liposomes characterized by one or more concentric lipid bilayers surrounding an aqueous lumen. In one embodiment, the term "lipid nanoparticle" or "LNP" refers to a particle comprising ionizable lipids in combination with membrane forming lipids, cholesterol, cationic lipids and / or anionic lipids.

[0115] As used herein, the term "lipid nanoparticle" or "LNP" does not encompass unilamellar liposomes that contain only a single lipid bilayer enclosing an aqueous lumen, or liposomes within liposome vesicles.

[0116] The LNP according to the present invention may have a diameter between about 50 nm and about 1000 nm, or between about 70 nm and 700 nm, as defined by the Z-average size (Z-Av), which is defined as the intensity-weighted average hydrodynamic size of the particles measured by dynamic light scattering. 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 nm and about 180 nm, or between about 80 nm and 150 nm.

[0117] The LNP according to the present invention may have a negative zeta potential (preferably between about -1 mV and -30 mV).

[0118] The composition according to the present invention includes diacyl-phospho-oligoglycerol (DAPG) as anionic phospholipid, such as dipalmitoyl-phospho-diglycerol (DPPG2) and dipalmitoyl-phosphotriglycerol (DPPG3). x ), particularly diacyl-phospho-oligoglycerols having two or three glycerol units, and novel classes of lipids for providing LNPs for encapsulating RNA molecules, their ionizable derivatives (NLG x , especially NLG2) and cationic derivatives (CLG x This includes, in particular, CLG2. The most preferred lipids have two or three glycerol moieties as head groups, which are advantageous for the design and production of RNA-containing LNPs.

[0119] RNA molecules preferentially include protein-coding mRNA, non-coding RNA such as immunoadjuvants, siRNA for gene silencing, and RNA for in vivo gene editing by technologies such as CRISPR / Cas.

[0120] Thus, diacyl-phospho-oligoglycerols having two or three glycerol units, particularly DPPG2 and DPPG3, as well as their cationic derivatives, and their derivatives having the same head group but different fatty acid chain lengths, present a novel lipid class (G2 and G3 lipid classes) for use as alternatives to PEG lipids or other shielding agents in RNA-LNP formulations. These G2 and G3 lipids enable the design of new next-generation RNA-LNP formulations that exhibit improved in vivo and in vitro transfection after parenteral administration, low aggregation, and high chemical and physical shelf-life stability.

[0121] In some embodiments, a further advantage of LNPs functionalized with members of the G2 or G3 lipid family may be improved storage stability, which can facilitate enhanced transfection after storage.

[0122] A further advantage of LNPs functionalized with members of the G2 or G3 lipid families is their clearly defined and controlled hydrodynamic diameter. For in vivo applications, the controllable size and charge of G2 and G3 lipid nanoparticles offer the potential to optimize the in vivo distribution of LNPs.

[0123] In some embodiments, a further advantage of LNPs functionalized with members of the G2 or G3 lipid family may be the increased bypass of hepatic accumulation compared to conventional PEG-containing LNPs.

[0124] Furthermore, in vivo, some target cells are not easily reachable. These target cells may be covered by extracellular matrix molecules, cell debris, or mucus, especially in the case of lung administration. Small size is also a prerequisite for reaching target cells behind the endothelial basement membrane, such as smooth muscle cells or tumor cells, by diffusion through the pores of the tumor vascular system (enhanced permeability and retention [EPR] effect).

[0125] A further advantage of G2 and G3 lipids is the potential of their cationic (e.g., CLG2) or anionic (e.g., DPPG2 or DPPG3) derivatives to modulate the overall charge of LNPs (including PEGylated LNPs) as measured by zeta potential. The charge of LNPs is known to directly modulate their target specificity; for example, cationic LNPs preferentially accumulate in proliferating endothelial cells. Furthermore, the so-called protein corona of LNPs can be altered by modifying their surface charge, thus indirectly altering their target specificity. Therefore, G2 and G3 lipids represent novel tools for LNP engineering for disease treatment and for optimizing their biological activity.

[0126] In summary, LNPs containing lipids with G2 or G3 functionalized head groups are characterized by advantageous properties compared to NPs containing only lipids with prior art head groups, as disclosed, for example, by Joergensen et al, 2023.

[0127] In addition to the biophysical findings described above, we demonstrated that DPPG2-containing LNPs can successfully encapsulate RNA (e.g., mRNA, siRNA) within uniform, small LNPs, and that these LNPs are mechanically and chemically stable, densely packed vesicles, most likely due to the synergistic effects between individual lipid bilayers (see examples).

[0128] This particular property of G2 lipids, and therefore nucleic acid-containing LNPs, is most likely based on the property of G2 or G3 lipids to displace water from the membrane gap through interaction with other G2 or G3 lipids, particularly with strong curvature. The reduced amount of water in the membrane is expected to result in reduced hydrolysis of nucleic acids, which is structurally weakened by electrostatic interactions (among others, electrostatic interactions between phosphate and positively charged functions destabilize nucleic acids because they no longer provide natural conformation based on phosphate group repulsion, etc.). Therefore, LNPs containing G2 or G3 lipids are most likely to be more stable and may simplify the development of LNP products of lyophilized RNA.

[0129] This stabilizing effect is likely further supported by mechanical effects, where a low-water environment enhances direct polar interactions between nucleic acids and G2 / G3 groups, thus reducing the importance of mechanical influences (tight, inflexible wrapping of nucleic acids) during LNP production.

[0130] A further advantage of RNA-LNPs containing G2 and G3 lipids is the avoidance of the potential risk of anaphylactic reactions. Currently, there is ongoing debate as to whether PEG is responsible for the lower incidence of anaphylactic reactions observed with both Moderna and Pfizer's COVID-19 vaccines. While PEG's contribution to anaphylactic reactions is speculative, it is important to investigate whether next-generation "PEG-free" LNPs yield different results.

[0131] A further advantage of RNA LNPs containing G2 and G3 lipids is the low potential risk of accelerated blood clearance after repeated administration. Liposomes containing DPPG2 have been reported to fail to induce an immune response to DPPG2 even after several intravenous administrations (Lokerse, et al. 2021).

[0132] The composition of the present invention preferably comprises at least one cationic or cationizable lipid.

[0133] Cationic lipids are lipids that, for example in some embodiments, typically have a positive net charge at a specific pH. In some embodiments, cationic lipids may contain one or more amine groups that have a positive charge. In some embodiments, cationic lipids may contain cationic, i.e., positively charged head groups. In some embodiments, when cationic lipids have a positive net charge, cationic lipids may have hydrophobic regions (e.g., one or more regions of neutral or anionic lipids). In some embodiments, cationic lipids contain polar head groups, which in some embodiments may include one or more amine derivatives such as primary, secondary, and / or tertiary amines, quaternary ammoniums, amines, amidinium salts, or various combinations of guanidine and / or imidazole groups, as well as amino acid head groups such as pyridinium, piperidine, and lysine, arginine, ornithine, and / or tryptophan. In some embodiments, the polar head groups of cationic lipids contain one or more amine derivatives. In some embodiments, the polar head group of the cationic lipid contains a quaternary ammonium compound. In some embodiments, the head group of the cationic lipid may contain multiple cationic charges. In some embodiments, the head group of the cationic lipid contains a single cationic charge.

[0134] Preferred cationic lipids include DOSPA(2,3-dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate); DOTAP(1,2-dioleoyl-3-trimethylammonium-propane); DMEPC(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine); DOTMA(1,2-di-O-octadecenyl-3-trimethylammoniumpropane); DOTAP(1,2-dioleoyl-3-trimethylammonium Dimyristoyl-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-hydroxyethylammonium bromide); DC-Choi (3P-[N-(N / N'-dimethylaminoethane)carbamoyl]cholesterol);

[0135] JPEG2026534185000024.jpg9135

[0136] The group is selected from those including DOEPC (dioleyl ether phosphatidylcholine) and ePC (ethyl phosphatidylcholine).

[0137] Cationizable lipids are lipids that can exist in a positively charged or neutral form depending on the pH. Cationizable lipids may have a pKa value between 5 and 7.5, preferably between 6 and 7. This means that the lipid is not charged at pH above its pKa value, and is positively charged at pH below its pKa value.

[0138] Preferred cationizable lipids include DLin-MC3-DMA,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate); ALC-0315((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); SM-102 (also called Lipid H)(heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate); A2-Iso5-2DC18(ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate);9A1P9(decyl(2-(dioctylammonio)ethyl)phosphate);BAME-O16B(bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo -10-Oxa-13,14-Dithia-3,6-Diazahexacosyl)Azandiyl)Dipropionate);C12-200(1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)Azandiyl)Bis(dodecane-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(axantriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9”Z,12Z,12'Z,12”Z,12”Z)-tetra Kiss(octadeca-9,12-dienoate));306Oi10((tetrakiss(8-methylnonyl)3,3',3",3”-(((methylazandiyl)bis(propane-3,1diyl))bis(azantriyl))tetrapropionate));TT3(N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide);A lipid is selected from the group comprising 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.

[0139] The composition of the present invention preferably contains a structural lipid selected from the group consisting of sterols, preferably cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomadidine, ursolic acid, and α-tocopherol; and the structural lipid is cholesterol in particular.

[0140] The composition of the present invention preferably further comprises at least one helper lipid. The helper lipid is a lipid that can enhance the delivery efficacy of lipid-based particles to a target, preferably into cells. The helper lipid may be neutral or negatively charged. Particularly suitable helper lipids are: Hydrogenated soybean L-α-phosphatidylcholine (HSPC); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-Diolioyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-Dimiristoyl-sn-glycerophosphocholine (DMPC); 1,2-Diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-palmitoyl-2-olioyl-sn-glycero-3-phosphocholine (POPC) Selected from the following, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is most preferred.

[0141] In some embodiments of the present invention, the composition does not contain helper lipids. We have found that helper lipids such as DSPC contained in LNP can be replaced with oligoglycerol-containing lipids of formula (I), resulting in the production of stable LNP.

[0142] The composition of the present invention, e) PEG lipids This may further include:

[0143] The term "PEG lipid" or "PEGylated lipid" refers to lipids covalently bonded to polyethylene glycol (PEG). Polyethylene glycol (PEG) is one of the most widely applied nonionic hydrophilic polymers that provides stealth to LNPs and improves their circulation time in the body (Knop, et al. 2010) (Albertsen, et al. 2022).

[0144] In embodiments of the present invention that include PEG lipids, only a portion of the PEG lipids contained in conventional LNP formulations are composed of at least one oligoglycerol-containing lipid of formula (I) or (IA), particularly A = OP(-O - It is replaced by an oligoglycerol-containing lipid of formula (I) or (IA) such that )(=O)-O).

[0145] Further embodiments of the present invention, including PEG lipids, include oligoglycerol-containing lipids of at least one formula (I) or (IA), particularly A = NR 3 ; or N + R 4 R 5 In addition to the PEG lipids, at least one oligoglycerol-containing lipid of formula (I) or (IA) is present. In those embodiments, at least one oligoglycerol-containing lipid of formula (I) or (IA), particularly A = NR 3 ; or N + R 4 R 5 At least one oligoglycerol-containing lipid of formula (I) or (IA) is used to modulate the LNP composition.

[0146] In its most preferred embodiment, the composition of the present invention does not contain PEG lipids.

[0147] According to the present invention, empty carrier systems, particularly empty lipid nanoparticles (LNPs), can be provided. However, preferred are: c) Therapeutic and / or prophylactic agents The composition further comprises [the specified element].

[0148] Most preferably, therapeutic and / or prophylactic agents are nucleic acids, particularly ribonucleic acid (RNA). 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), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof.

[0149] In a further preferred embodiment, the therapeutic and / or prophylactic agent is a small molecule, particularly a cell growth inhibitor. Preferably, the composition of the present invention comprises doxorubicin (DOX), which is a cytotoxic anthracycline. Further suitable cytostatic anthracyclines currently used in cancer treatment include epirubicin, idarubicin, daunorubicin, and mitoxantrone. In a further embodiment of the present invention, other cell growth inhibitors such as mitomycin C, gemcitabine, trabectedin, or platinum derivatives such as cisplatin, carboplatin, or oxaliplatin, or other agents disclosed below, may be included.

[0150] In a further preferred embodiment, the composition includes: further anthracyclines such as aracrubicin, amrubicin, pirarubicin, barurubicin, and solubicin; anthracendions such as mitoxantrone and picantrone; antitumor antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine, and vinorelbine; alkylating agents such as cyclophosphamide and mechloretamine hydrochloride; camptothecins such as topotecan, irinotecan (CPT-11), lulutotecan, 9-aminocamptothecin, 9-nitrocampotecin, and 10-hydroxycampotecin; 5-fluorouracil, gemcitabine (2,2'-difluoro-2-deoxycytidine, dFd C) Includes substitutes for additional active ingredients selected from the group consisting of purines and pyrimidine derivatives such as phloxlysine (FUDR), cytarabine (cytosine arabinoside), and 6-azauracil (6-AU); oxazaphosphorines such as cyclophosphamide, ifosfamide, and trophosfamide; taxanes such as paclitaxel and docetaxel; podophyllotoxin derivatives such as etopside and teniposide; platinum compounds such as cisplatin, carboplatin, oxaliplatin, and nedaplatin; methotrexate; tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, adavocertib, and lapatinib; and cytarabines such as cytosine arabinoside.

[0151] The most preferred aspect of this invention is: 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) Optional therapeutic and / or prophylactic agents It is a composition containing [the specified ingredient]. This composition preferably does not contain PEG lipids.

[0152] Further most preferred aspects of the present invention are: 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) Optional therapeutic and / or prophylactic agents It is a composition containing [the specified ingredient]. This composition preferably does not contain PEG lipids.

[0153] Further most preferred aspects of the present invention are: 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) Optional therapeutic and / or prophylactic agents. It is a composition containing [the specified ingredient]. This composition preferably does not contain PEG lipids.

[0154] Further most preferred aspects of the present invention are: a) At least one cationic or cationizable lipid; b) Cholesterol. c)CLG2; and d) PEG lipids, and e) Optional therapeutic and / or prophylactic agents It is a composition containing [the specified ingredient].

[0155] The compositions of the present invention, particularly DPPG2 or DPPG3-based LNPs, are especially suitable for drug delivery.

[0156] In a further preferred embodiment, the present invention is: a) at least one formula (I) or (IA)

[0157] [ka]

[0158] [ka]

[0159] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b)(iii) at least one helper lipid; b)(ii) at least one structural lipid; and b)(iv) at least one PEG lipid; The present invention relates to a composition comprising the oligoglycerol-containing lipid of formula (I), wherein the oligoglycerol-containing lipid is NLG2 or CLG2.

[0160] Preferably, the structural lipid is cholesterol, the helper lipid is DSPC, and the PEG lipid is DSPE-PEG. Preferably, the NLG2 / cholesterol / DSPC / DSPE-PEG is in the 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, based on the total lipid content of the composition. More preferably, the molar range is 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.

[0161] Conventional LNP preparations are based on a microfluidic approach, in which a typically low-pH aqueous phase containing nucleic acids is rapidly mixed with an ethanolic solution of the necessary lipids (typically containing PEG lipids, cholesterol, DSPC, and ionizable lipids that have a positive charge at low pH values). The resulting primary dispersion of small liposome-like vesicles containing nucleic acids is then dialyzed with a buffer solution having a neutral pH, which neutralizes the positive charge of the vesicles. As a result, electrostatic repulsion forces can no longer maintain the distance between the vesicles, causing vesicle fusion. This fusion stops when the particle size reaches the maximum PEG chain density on its surface. Therefore, the ratio of PEG lipids in the lipid mixture determines the final LNP size.

[0162] LNPs can be produced by prior art methods, including, for example, microfluidic methods, or organic solvent injection, or by using or not using a double centrifugation (DC) with or without an organic solvent (e.g., ethanol).

[0163] The present invention also enables the preparation of patient-specific LNPs (e.g., patient-specific mRNAs to support cancer immunotherapy) using DCs. Bedside DC preparation allows for the application of LNPs that may not have complete storage stability, and further kits containing the necessary vials, lipids, and drugs become available.

[0164] Figure 2 The present invention will be further explained with reference to the accompanying drawings. [Brief explanation of the drawing]

[0165] [Figure 1] Figure 1: The efficacy of NLG2-containing LNPs is compared with that of transfection (luminescent units) of PEG-containing preparation B (positive control). Luminescence was measured 24 hours after transfection of HEK293 cells in serum-containing medium. Luciferase mRNA was encapsulated in LNPs containing various ratios of NLG2. [Figure 2] Figure 2: Agarose gel electrophoresis to detect mRNA inclusion efficiency of different LNPs. LNP formulation B containing PEG was compared with RNA-LNP containing DPPG2 as a shielding lipid. (+T = Triton treatment) [Figure 3] Figure 3: The efficacy of DPPG2-containing LNPs (based on Formulation B w / o PEG) is presented as x times the transfection (luminescent units) of LNP Formulation B, which serves as a positive control. GFP mRNA was encapsulated in LNPs with various DPPG2 content. Luminescence was measured 24 hours after transfection of HEK293 cells in serum-containing medium. Error bars represent SD. [Figure 4] Figure 4: The efficacy of DMG-PEG-free DPPG2-containing LNPs (based on formulation B, which does not contain PEG) is shown in absolute luminescence units compared to formulation B. Luminescence was measured 24 hours after transfection of HEK293 cells with LNPs containing GFP mRNA. Error bars represent standard deviation (SD). [Figure 5]Figure 5: Luminescence of LNPs of different DPPG2 formulations containing 150, 300, and 500 ng of mRNA is presented as x-fold transfection (luminescent units) for each DPPG2 formulation containing 50 ng of mRNA. Luminescence was measured 24 hours after transfection of HEK293 cells with GFP mRNA encapsulated in LNPs with various DPPG2 contents. The DPPG2 formulations were based on formulation B, which does not contain PEG. Formulation B served as a positive control. Error bars represent SD. [Figure 6] Figure 6: The efficacy of DPPG3-containing LNPs is presented as x-fold transfection (luminescence units) of LNP preparation B, which serves as a positive control. Luminescence was measured 24 hours after transfection of HEK293 cells in serum-containing medium. Luciferase mRNA was encapsulated in LNPs with various DPPG3 content. Error bars represent SD. [Figure 7] Figure 7: The efficacy of CLG2-containing LNPs is presented as x times the transfection (luminescence unit) compared to formulation B (positive control). Luminescence was measured 24 hours after transfection of HEK293 cells in serum-containing medium. Luciferase mRNA was encapsulated in LNPs with different CLG2 content. The LNP compositions were based on formulations A and B, respectively (see text for details). Error bars represent SD. [Figure 8] Figure 8: The efficacy of CLG2 and DPPG2-containing LNPs is shown as x-fold transfection (luminescence units) compared to formulation B (positive control). Formulation B contains DSPC as a helper lipid and DMG-PEG as a shielding lipid. Luminescence was measured 24 hours after transfection of HEK293 cells in serum-containing medium. Luciferase mRNA was encapsulated in LNPs with varying DPPG2 content. Error bars represent standard deviation (SD). [Figure 9]Figure 9: Transfection efficiency of NLG2 and CLG2-containing LNPs containing DODMA as an ionizable lipid in the HEK-293 cell line. Chemiluminescence data compared to conventional DMG-PEG-containing formulation B as a control. The presented data were obtained from at least three independent experiments. Columns from left to right: control formulation LNP; DODMA-containing LNP; DODMA-containing LNP with NLG2 (20%); DODMA-containing LNP with NLG2 (20%) and CLG2 (20%); DODMA-containing LNP with CLG2 (20%). [Figure 10] Figure 10: Transfection efficiency of NLG2-containing LNPs in the HEK293 cell line, expressed as chemiluminescence, compared to conventional DMG-PEG-containing formulation B as a control. Columns from left to right: Control formulation B; LNPs like B with NLG2 (25%); LNPs like B with NLG2 (21%). [Figure 11] Figure 11: Transfection efficiency of DPPG2-containing LNP in the HEK293 cell line, expressed by chemiluminescence, compared to DMG-PEG-containing formulation B as a control. Evaluation after 1 week of storage. [Figure 12] Figure 12: DPPG2-containing LNP avoided liver accumulation compared to conventional DMG-PEG-containing preparation B (measured by luciferase mRNA expression 4 hours after intravenous administration). [Figure 13] Figure 13: Transfection efficiency measured in HEK-293 cell lines immediately after production (week 0), 1, and 3 weeks of storage, using LNPs like A w / DPPG3 (10%) containing DPPG3 (10%), expressed as an x-fold increase compared to control LNPs like B with PEG.

[0166] 3 List of References Albertsen, Camilla Hald, Jayesh Kulkarni, Dominik Witzigmann, Marianne Lind, Karsten Pertersson, and Jens B. Simonsen. "The role of lipid components in lipid nanoparticles for vaccines and gene therapy." Advanced drug delivery reviews 188 (2022): 114416-114428.

[0167] Ambegia, E., S. Ansell, P. Cullis, J. Heyes, L. Palmer, and 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.

[0168] Arteta, Yanez, et al. "Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles." PNAS 115 (2018): E3351-E3360.

[0169] Brockow, Knut, et al. "Experience with polyethylene glycol allergy-guided." Allergy 77 (2022): 2200-2210.

[0170] Carrasco, Manuel J., et al. "Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration." Communications Biology, 2021.

[0171] Edo Kon, Uri Elia, Dan Peer,. "Principles for designing an optimal mRNA lipid nanoparticle vaccine,." Current Opinion in Biotechnology, 2022.

[0172] Garcia, Josep, Carles Felip, Svea Stephan, and Paul Pietsch. Development and optimization of LNP formulations- using Knauer Nanoscaler. White paper -Curapath, 2022. https: / / www.knauer.net / Application / application_notes / VPH0076_Development_and_Optimization_of_LNP.pdf

[0173] Hattori, Yoshiyuki, Kyoko Tamaki, Sho Sakasai, Kei-Ichi 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.

[0174] 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.

[0175] Hou, Xucheng, Tal Zaks, Robert Langer, und Yizhou Dong. "Lipid nanoparticles for mRNA delivery." Nature Reviews Materials 6 (2021): 1078-1094.

[0176] Jaggers, Jordon, and Anna R. Wolfson. "mRNA COVID-19 Vaccine Anaphylaxis: Epidemiology, Risk Factors,." Current Allergy and Asthma Reports 23 (2023): 195-200.

[0177] 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.

[0178] Koehler, Jonas K., et al. "Tailoring the Lamellarity of Liposomes Prepared by Dual Centrifugation." pharmaceutics 15 (2023): 706-727.

[0179] Kulkarni, Jayesh, Dominik Witzigmann, und Sam Chen. "Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics." Accounts of Chemical Research, 2019.

[0180] Li, Q., et al. "RNA editing underlies genetic risk of common inflammatory diseases." Nature 608 (2022): 569-577.

[0181] 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.

[0182] Nogueira, Sara S., et al. "Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA delivery." ACS Applied Nano Materials 3 (2020): 10634-10645.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Schoenmaker, Linde, et al. "mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability." International Journal of Pharmaceutics 601 (2021): 120585-120597.

[0187] Song, L.Y., Q.F. Ahkong, Z. Wang Q. Rong, S. Ansell, M.J. Hope, und B. Mui. "Characterization of the inhibitory eiect 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.

[0188] 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.

[0189] Tam, P, et al. "Stabilized plasmid-lipid particles for systemic gene therapy." Gene Therapy 7 (2000): 1867-1874.

[0190] Tenchov, Rumiana, Janet M. Sasso, and Qiongqiong Angela Zhou. "PEGylated Lipid Nanoparticle Formulations: Immunological Safety." 34 (2023): 941-960.

[0191] Urits, Ivan, et al. "A Review of Patisiran (ONPATTRO®) for the Treatment of Polyneuropathy in People with Hereditary Transthyretin Amyloidosis." Neurology and Therapy 9 (2020): 301-315.

[0192] 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.

[0193] Whitehead, Kathryn A., et al. "Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity." Nature Communications 5 (2014): 1-10.

[0194] Woodle, Martin C., and Danilo D. Lasic. "Sterically stabilized liposomes." Biochimica et Biophysica Acta, 1113 (1992): 171-199.

[0195] Yang, Qi, and Samuel K. Lai. "Anti-PEG immunity: emergence, characteristics, and unaddressed questions." WIREs Nanomed Nanobiotechnology 7 (2015): 655-677.

[0196] Zhang, Rui, et al. "Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver." Biomaterials Science 9 (2021): 1449-1463.

[0197] Zhu, Yiran, Liyuan Zhu, Xian Wang, and Hongchuan Jin. "RNA-based therapeutics: an overview and prospectus." Cell Death Dis 13 (2022): 644-658. [Examples]

[0198] 4 Examples 4.1 Example 1: Method for formulating RNA-LNP The RNA-LNPs of the present invention were formulated using a NanoScaler (Knauer Wissenschaftliche Geraete GmbH, Berlin, Germany) integrated with a collision jet mixing system (Garcia, et al. 2022). The present invention includes the formulation of RNA-LNPs by integration of an organic phase (containing lipids) and an aqueous phase (containing an mRNA payload and dilution buffer) within the NanoScaler.

[0199] The organic phase was prepared with a lipid mixture consisting of ionizable lipids, helper lipids, cholesterol, and shielding lipids in predetermined molar ratios. The aqueous phase consisted of diluted mRNA (GFP or luciferase (Luc)) in 25 mM or 6.25 mM acetate buffer, depending on the predetermined formulation, maintaining the pH between 4.0 and 5.0. The amine:phosphate ratio (N / P) was maintained between 6.20 and 5.13. The flow rate ratio between the organic phase and the aqueous phase was maintained at 1:3 with a flow rate of 4.5 mL / min, including 3 mL / min of undiluted LNP and 1.5 mL / min of empirical in-line dilution with PBS.

[0200] After the formulation stage, the LNP was further diluted with 10 mM phosphate-buffered saline (PBS) at pH 7.4 to reduce the ethanol concentration. Subsequently, it was subjected to centrifugal concentrating (Vivaspin). TM Purification was performed using Merck KGaA (500, Darmstadt, Germany) to effectively remove residual ethanol, thereby ensuring the safety and efficacy of the formulation.

[0201] Lipid compositions of known mRNA-based COVID-19 vaccines were used as controls (Table 1). DPPG x or CLG x or NLG x Formulations containing the lipids of the present invention were prepared in the same manner, except that lipids were used to replace the corresponding lipids in the lipid mixture in similar or different lipid ratios (see Examples below).

[0202] [Table 1]

[0203] 4.2 Example 2: Synthesis route of cationic / cationizable lipid of formula (II) Example 2a: Ester variant The synthetic pathways for NLG2 and CLG2 starting from glycidol and 1,2-isopropylideneglycerol are shown below. The synthesis proceeds stereospecifically, and therefore, different stereoisomers can also be produced by selecting enantiomerically pure starting materials in steps 1 and 2 (R- or S-glycidol and R- or S-1,2-isopropylideneglycerol) and step 9 (R- or S-glycidol), as needed. Cationizable NLG2 was obtained from glycidol and 1,2-isopropylideneglycerol in 11 synthetic steps. Cationic CLG2 was obtained from NLG2 in two additional steps. This synthetic pathway can be applied to other oligo-containing lipids of formula (II) having esterified fatty acid chains.

[0204] [ka]

[0205] Step 1: 3.8 kg of glycidol was dissolved in 22.8 L of NMP, then 8.02 kg of paramethoxybenzyl chloride (PMBCl) was added, followed by the addition of 2.26 kg of NaH at 0-5°C. After 4 hours at RT, the reaction mixture was poured into ice water and extracted with siRNA. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The isolated residue was purified by silica gel chromatography (n-heptane / ethyl acetate) to obtain 8.0 kg of 1.1 as a colorless oil.

[0206] Steps 2+3: 8.0 kg of compound 1.1 was dissolved in 64 L of DMF, then 8.16 kg of 1,2-isopropylideneglycerol was added, followed by the addition of 6.94 kg of tBuOK at 0-5°C. The resulting reaction mixture was stirred at RT for 12 hours to obtain compound 2.1. Subsequently, without workup or further purification, an additional 6.91 kg of tBuOK and 12.6 kg of benzyl bromide were added at 0-5°C, and the resulting reaction mixture was stirred at RT for 4 hours. The reaction mixture was then poured into ice water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. After purification by silica gel chromatography (n-heptane / ethyl acetate), 7.0 kg of pure compound 3.1 was obtained as a yellow oil.

[0207] Step 4 : Dissolve 379g of 3.1 in 2.65L of MeOH, then cool to 0°C, and slowly add 2L of 1M HCl. Stir the reaction mixture at 25°C for 4 hours. Then, at 0-5°C, add saturated NaHCO3 solution to adjust the mixture to pH=7, and then extract with siRNA. Wash the organic phase with brine, dry over Na2SO4, filter, and concentrate under reduced pressure to obtain 310g of crude 5.1 as yellow oil.

[0208] Step 5 : 310 g of 5.1 is dissolved in 2.5 L of DMF, then cooled to 0°C, and 369 g of tBuOK and 422 g of BnBr are added. The reaction mixture is stirred at 25°C for 4 hours, then poured into ice water and subsequently extracted with ethyl acetate. The organic phase is washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. After silica column chromatography (n-heptane / ethyl acetate), 115 g of pure 6.1 is obtained as a yellow oil.

[0209] Step 6 : 331 g of 6.1 is dissolved in 3 L of ACN and 332 ml of H2O, then cooled to 0°C, and 652 g of cerium ammonium nitrate (CAN) is added. The reaction mixture is stirred at 15°C for 8 hours, then poured into ice water and subsequently extracted with siRNA. The organic phase is washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. After silica column chromatography (n-heptane / siRNA), 168 g of pure 7.1 is obtained as a yellow oil.

[0210] Step 7 : Dissolve 153 g of 7.1 in 765 ml of DCM, then add 71 g of triethylamine, followed by the dropwise addition of 50.1 g of MsCl. Stir the reaction mixture at 20°C for 2 hours, then pour into water and extract with DCM. Dry the organic phase over Na2SO4, filter, and concentrate under reduced pressure to obtain 183 g of crude 25.1 as yellow oil.

[0211] Step 8 : Dissolve 183 g of 25.1 in 366 ml of EtOH, and add the resulting solution to a 33% wt MeNH2 solution (1.48 kg) in EtOH. The reaction mixture is heated to 55°C, stirred for 48 hours, and then dried by evaporation. The residue is added to MeOH / toluene, washed with a 10% Na2CO3 aqueous solution, and then concentrated under reduced pressure to obtain 151 g of crude 26.1 as yellow oil.

[0212] Step 9 : Dissolve 43.6 g of 26.1 in 109 ml of toluene, then add a solution of 9 g of glycidol in 109 ml of toluene. Heat the reaction mixture to 100°C and stir for 8 hours. Run three batches in parallel, then combine them, evaporate, and dry. Add the residue to MTBE, adjust the pH to 5 with HCl, then separate the phases. Adjust the pH of the aqueous phase to 8 with saturated NaHCO3 solution, extract with ethyl acetate, and concentrate the organic phase under reduced pressure to obtain 161 g of crude 35.1 as yellow oil.

[0213] Step 10 : 161 g of 35.1 was dissolved in 800 ml of DCM, and then 62.2 g of triethylamine, 11.2 g of DMAP, and 169 g of palmitoyl chloride were added sequentially. The resulting reaction mixture was stirred at 20°C for 1 hour, and then poured into water. After separation, the organic phase was concentrated under reduced pressure and then purified by silica column chromatography (n-heptane / siRNA) to obtain 146 g of pure 36.1 as yellow oil.

[0214] Step 11 : 6 g of Pd / C was suspended in 300 ml of AcOH, then 67 g of 36.1 was added, and the reactor was flushed with 30 psi of H2. The resulting reaction mixture was stirred at 30°C for 24 hours. Two batches were run in parallel and worked up together. After filtration through Celite, the filtrate was poured into water and the pH was adjusted to 7 with saturated NaHCO3 solution. After extraction with ethyl acetate, the organic phase was concentrated under reduced pressure. The residue was recrystallized from ACN and subsequently purified by silica column chromatography (n-heptane / siRNA / MeOH) to obtain 38.6 g of pure NLG2 as a white solid.

[0215] Step 12 : 20 g of NLG2 is suspended in 100 ml of ACN, and then 7.8 g of methyl iodide is added. The resulting reaction mixture is stirred at 40°C for 16 hours. After filtration and concentration under reduced pressure, 21 g of crude 38.1 is obtained as a white solid.

[0216] Step 13 : 21 g of 38.1 is added to a 1:1:1 mixture (315 ml) of toluene, 2-propanol, and 10% aqueous NaCl solution. The resulting reaction mixture is stirred at 35°C for 48 hours. After isolation, the crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol under RT for 1 hour to obtain 10 g of pure CLG2 as a white solid. Characterization by ICP-MS confirms that CLG2 is a chloride salt.

[0217] Example 2b: Ether variant The following describes the synthetic pathways for oligoglycerol-containing lipids of formula (II) with ether links, 84.1 and 85.1, starting from 2.1 and 1,2-isopropylideneglycerol. The synthesis proceeds stereospecifically, and therefore, different stereoisomers can also be produced, if necessary, by selecting enantiomerically pure starting materials for the synthesis of 2.1 and step 6 (R- or S-glycidol and R- or S-1,2-isopropylideneglycerol). This synthetic pathway can be applied to other oligoglycerol-containing lipids of formula (II) having ether-linked fatty acid chains.

[0218] [ka]

[0219] Step 1: Dihydropyran is added to the solution of 2.1 and PPTS in DCM, and the resulting reaction mixture is stirred at RT until the reaction is complete. The mixture is diluted with diethyl ether, washed with brine, then dried over Na2SO4, and concentrated under reduced pressure to obtain crude 3.4.

[0220] Step 2: Dissolve 3.4 in 10:1 (vol / vol) DCM / H2O and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. After work-up, purify the crude residue by silica gel chromatography (n-heptane / ethyl acetate) to obtain 4.2.

[0221] Step 3 : Dissolve 4.2 in DCM, then add triethylamine, followed by dropwise addition of MsCl. Stir the reaction mixture at 20°C until complete, then pour into water and extract with DCM. Dry the organic phase over Na2SO4, filter, and concentrate under reduced pressure to obtain crude 27.2.

[0222] Step 4 : Dissolve 27.2 in EtOH, and add the resulting solution to a MeNH2 solution (33% wt) in EtOH. Heat the reaction mixture to 55°C, stir until complete, and then dry by evaporation. Add the residue to MeOH / toluene, wash with a 10% Na2CO3 aqueous solution, and concentrate under reduced pressure to obtain crude 28.2.

[0223] Step 5 : Trifluoromethanesulfonic acid anhydride is added to DCM at 0°C, followed by pyridine anhydride to form a white precipitate. The cooling bath is removed, and a solution of palmityl alcohol 70.1 in DCM is added dropwise over 10 minutes with stirring. The solution is stirred at RT until the reaction is complete. Water is then added, and the mixture is extracted with DCM. The combined organic layer is 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 71.1 as a waxy white solid.

[0224] Step 6 : 1,2-Isopropylidene-rac-glycerol is dissolved in NMP, then PMBCl is added, and the solution is cooled to 0°C. NaH is carefully added, and the resulting reaction mixture is stirred at 25°C until the reaction is complete. The mixture is poured into ice water and extracted with ethyl acetate. 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.

[0225] Step 7 : Dissolve 31.2 in MeOH and cool the solution to 0°C. Carefully add concentrated HCl and stir the resulting reaction mixture at 20°C until the reaction is complete. Quench the mixture with saturated K2CO3 aqueous solution to adjust the pH to 7-8, and then extract with siRNA. Dry the organic phase over Na2SO4 and concentrate under reduced pressure to obtain crude 32.2.

[0226] Step 8 : 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 the reaction is complete. DCM is removed under reduced pressure. Hexane is added, the mixture is sonicated, and then filtered through Celite. The filtrate is evaporated under reduced pressure, and the residue is purified by silica gel chromatography (n-heptane / ethyl acetate) to obtain 72.1.

[0227] Step 9 : Dissolve 72.1 in 10:1 (vol / vol) DCM / H2O and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. After work-up, purify the crude residue by silica gel chromatography (n-heptane / ethyl acetate) to obtain 73.1.

[0228] Step 10 : 73.1, triphenylphosphine, tetrabutylammonium iodide, and anhydrous 1,2-dibromoethane were combined in a flask and heated to 60°C. The reaction mixture was stirred at 60°C until the reaction was 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 obtain 74.1.

[0229] Step 11 : Dissolve 28.1, N,N-diisopropylethylamine, and 74.1 in acetonitrile and stir at room temperature under a nitrogen atmosphere until the reaction is complete. Then remove the solvent under reduced pressure. Dissolve the residue in DCM and wash with water. Dry the organic fraction over MgSO4 and remove the solvent under reduced pressure to obtain 82.2.

[0230] Step 12 : Dissolve 82.2 in MeOH and heat to 40°C, then add solutions of formic acid and oxalic acid in water. Stir the resulting reaction mixture at 40°C until the reaction is complete. Dilute the mixture with brine and extract with MeTHF. Separate the organic phase, dry it over Na2SO4, and concentrate under reduced pressure. Dissolve the crude residue in THF and cool to 0°C. Add a mixture of TBAF and acetic acid at 0°C, and stir the reaction mixture at 30°C until the reaction is complete. Pour the solution into a mixture of siRNA and saturated NaHCO3 solution and separate the organic phase. Wash the organic phase with saturated NaHCO3 solution and brine, dry it over Na2SO4, and concentrate under reduced pressure. Purify the residue by silica column chromatography (n-heptane / siRNA / MeOH) to obtain pure 83.1.

[0231] Step 13 : 83.1 is suspended in ACN, and then methyl iodide is added. The resulting reaction mixture is stirred at 40°C until the reaction is complete. After filtration and concentration under reduced pressure, crude 84.1 is obtained.

[0232] Step 14 : 84.1 is added to a 1:1:1 mixture of toluene, 2-propanol, and a 10% aqueous NaCl solution. The resulting reaction mixture is stirred at 35°C until the reaction is complete. After isolation, the crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol under RT for 1 hour to obtain pure 85.1. Characterization by ICP-MS confirms that 85.1 is a chloride salt.

[0233] 4.3 Example 3: Synthesis route of anionic lipid of formula (II) Example 3a: Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phospho-diglycerol and 1,2-dipalmitoyl-sn-glycero-3-phospho-triglycerol The synthetic route for producing DPPG2 and DPPG3 is described in patent application WO 97 / 30058 A1 as phosphatidyl oligoglycerol (PG). x This method was described as part of the synthesis of molecular class glycerophospholipids of formula (II). This synthesis method can be applied to other anionic oligo-containing lipids of formula (II) that have esterified saturated fatty acid chains.

[0234] Example 3b: Synthesis of anionic lipid of formula (II) linked by unsaturated esters The synthetic pathway shown below is suitable for anionic lipids of formula (II) linked by monovalent and polyunsaturated esters. In particular, the following examples demonstrate the synthesis of DOPG2 having an oleyl lipid chain. The synthesis proceeds via the intermediate of formula 4.2. DOPG2 is obtained in a total of 13 synthetic steps, as well as in 4 steps from 4.2 and 34.1. The synthesis proceeds stereospecifically, and therefore different stereoisomers can also be produced by selecting enantiomerically pure starting materials in steps 1, 2, and 5 (R- or S-glycidol and R- or S-1,2-isopropylideneglycerol) as needed.

[0235] [ka]

[0236] Step 1 : To a suspension of NaH in DMF, glycidol is added at 0°C, followed by the addition of BnBr. The resulting reaction mixture is heated to RT and stirred until the reaction is complete. Water is then added, and the mixture is extracted with DCM. 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 1.2.

[0237] Step 2 : Dissolve 1.2 in DMF, then add sn-1,2-IPG, followed by tBuOK at 0-5°C, and stir the resulting reaction mixture under RT until the reaction is complete. Then pour the reaction mixture into ice water and extract with ELISA. Wash the organic phase with brine, dry over Na2SO4, and concentrate under reduced pressure to obtain crude 2.2.

[0238] Step 3 : Dihydropyran is added to a solution of 2.2 and PPTS in DCM, and the resulting reaction mixture is stirred at RT until the reaction is complete. The mixture is diluted with diethyl ether, washed with brine, then dried over Na2SO4, and concentrated under reduced pressure to obtain crude 3.2.

[0239] Step 4 : Add 3.2 to the suspension of Pd / C in EtOH / DCM, and then degas. Stir the resulting mixture under H2 (0.5 MPa) RT until the reaction is complete. Filter the mixture and concentrate the filtrate under reduced pressure. Purify the crude 4.2 by silica gel chromatography (n-heptane / ethyl acetate).

[0240] Step 5 : Dissolve 1,2-isopropylideneglycerol in DMF, then add BnBr and cool the solution to 0°C. Carefully add tBuONa and stir the reaction mixture at 25°C until the reaction is complete. Pour the mixture into ice water, adjust the pH to 6-7 with aqueous HCl, and then extract with siRNA. Wash the organic phase with brine, dry over Na2SO4, and concentrate under reduced pressure to obtain crude 31.1.

[0241] Step 6 : Dissolve 31.1 in MeOH and cool the solution to 0°C. Carefully add concentrated HCl and stir the resulting reaction mixture at 20°C until the reaction is complete. Quench the mixture with saturated K2CO3 aqueous solution to adjust the pH to 7-8, and then extract with ELISA. Dry the organic matter over Na2SO4 and concentrate under reduced pressure to obtain crude 32.1.

[0242] Step 7 : Pyridine and DMAP are added to the DCM solution of 32.1, and then cooled to 5°C. Acetyl chloride is carefully added, and the resulting reaction mixture is stirred at 20°C until the reaction is complete. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 33.1.

[0243] Step 8 : Pd / C is suspended in EtOH, then 33.1 and DCM are added, and the reactor is flushed with H2. The resulting reaction mixture is stirred at 25°C under H2 (5 bar) until the reaction is complete. 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.

[0244] Step 9 : DIPEA is added to a suspension of 34.1 in MeTHF and DCM, and the mixture is then cooled to -15°C. 2-Cyanoethyl-N,N-diisopropyl chlorophosphoramidite (PAMCl) is dissolved in DCM, the solution is cooled to -20°C, and then added to the reaction mixture. The resulting mixture is stirred at -15°C for 1 hour, followed by 1.5 hours at 0°C and 1 hour at 20°C. 4,5-Dicyanoimidazole (DCI) and 4.2 are then dissolved in MeTHF, and the solution is slowly added to the reaction mixture at 20°C. If necessary, after stirring for 1 hour, an additional amount of DCI is added as a solution in DCM, and the entire mixture is stirred overnight at 20°C. A 35% w / w aqueous H2O2 solution is then added over 1 hour at 0°C, followed by stirring at 20°C for another hour. The reaction mixture is diluted with a 5 wt% aqueous Na2S2O5 solution and extracted with MeTHF. The organic phase is dried with Na2SO4, and the solvent is removed under reduced pressure. The crude intermediate 55.1 is purified by silica chromatography (DCM / acetone).

[0245] Step 10 : DBU is added to a solution of 55.1 dissolved in MeTHF, and the reaction mixture is stirred in RT for 2 hours. Then Pd / C is added, and the resulting reaction suspension is stirred under an H2 atmosphere (1.2 bar) for 1 hour. The reaction mixture is filtered through Celite, washed with 1 M aqueous citric acid solution, and the organic phase is separated. The organic phase is washed with 5 wt% aqueous NaHCO3 solution, dried over Na2SO4, and concentrated under reduced pressure to obtain 58.1.

[0246] Step 11 : 58.1, K2CO3, and a 2:1 mixture of MeOH / H2O were stirred at RT until the reaction was complete. The MeOH was removed under reduced pressure, and the aqueous phase was extracted with MeTHF. The organic phase was separated, dried over Na2SO4, and concentrated under reduced pressure to obtain crude 61.1.

[0247] Step 12 : Pyridine and DMAP are added to a solution of 61.1 in DCM, and the mixture is then cooled to 5°C. Oleyl chloride is carefully added, and the resulting reaction mixture is stirred at 20°C until the reaction is complete. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 64.1.

[0248] Step 13 : Dissolve 64.1 in MeOH, heat to 40°C, and then add solutions of formic acid and oxalic acid in water. Stir the resulting reaction mixture at 40°C until the reaction is complete. Dilute the mixture with brine and extract with MeTHF. Separate the organic phase, dry over Na2SO4, and concentrate under reduced pressure. Purify the crude residue by silica column chromatography (n-heptane / siRNA / MeOH) to obtain pure 67.1.

[0249] Example 3c: Synthesis of ether-bonded anionic lipid of formula (II) The following synthetic pathway is suitable for anionic lipids of formula (II) linked by saturated, monounsaturated, and polyunsaturated ethers. In particular, the following examples show the synthesis of 104.ω1 having an oleyl lipid chain. The synthesis proceeds via the intermediate of formula 4.2. 104.ω1 is obtained in a total of eight synthetic steps, as well as in four steps from 4.2 and 73.ω1. The synthesis proceeds stereospecifically, and therefore different stereoisomers can also be produced as needed by selecting enantiomerically pure starting materials for the synthesis of 4.2 and step 2 (R- or S-1,2-isopropylideneglycerol).

[0250] [ka]

[0251] Step 1: Trifluoromethanesulfonic acid anhydride is added to DCM at 0°C, followed by pyridine anhydride to form a white precipitate. The cooling bath is removed, and a solution of oleyl alcohol 70ω1 in DCM is added dropwise over 10 minutes with stirring. The solution is stirred at RT until the reaction is complete. Water is then added, and the mixture is extracted with DCM. The combined organic layer is 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 71ω1 as a waxy white solid.

[0252] Step 2: 1,2-Isopropylideneglycerol is dissolved in NMP, then PMBCl is added and the solution is cooled to 0°C. NaH is carefully added, and the resulting reaction mixture is stirred at 25°C until the reaction is complete. The mixture is poured into ice water and extracted with ethyl acetate. 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.

[0253] Step 3: Dissolve 31.2 in MeOH and cool the solution to 0°C. Carefully add concentrated HCl and stir the resulting reaction mixture at 20°C until the reaction is complete. Quench the mixture with saturated K2CO3 aqueous solution to adjust the pH to 7-8, and then extract with siRNA. Dry the organic phase over Na2SO4 and concentrate under reduced pressure to obtain crude 32.2.

[0254] Step 4: DCM is added to a mixture of 71.ω1, 32.2, and 1,8-bis(dimethylamino)naphthalene. The reaction mixture is stirred under reflux until the reaction is complete. DCM is removed under reduced pressure. Hexane is added, the mixture is sonicated, and then filtered through Celite. The filtrate is evaporated under reduced pressure, and the residue is purified by silica gel chromatography (n-heptane / ethyl acetate) to obtain 72.ω1.

[0255] Step 5 : Dissolve 72.ω1 in 10:1 (vol / vol) DCM / H2O and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. After work-up, purify the crude residue by silica gel chromatography (n-heptane / ethyl acetate) to obtain 73.ω1.

[0256] Step 6: DIPEA is added to a suspension of 73.ω1 in MeTHF and DCM, and the mixture is then cooled to -15°C. 2-Cyanoethyl-N,N-diisopropyl chlorophosphoramidite (PAMCl) is dissolved in DCM, the solution is cooled to -20°C, and then added to the reaction mixture. The resulting mixture is stirred at -15°C for 1 hour, followed by 1.5 hours at 0°C and 1 hour at 20°C. 4,5-Dicyanoimidazole (DCI) and 4.2 are then dissolved in MeTHF, and the solution is slowly added to the reaction mixture at 20°C. If necessary, after stirring for 1 hour, an additional amount of DCI is added as a solution in DCM, and the entire mixture is then stirred overnight at 20°C. A 35% w / w aqueous H2O2 solution is then added over 1 hour at 0°C, followed by stirring at 20°C for another 1 hour. The reaction mixture is diluted with a 5 wt% aqueous Na2S2O5 solution and extracted with MeTHF. The organic phase is dried with Na2SO4, and the solvent is removed under reduced pressure. The crude intermediate 98.ω1 is purified by silica chromatography (DCM / acetone).

[0257] Step 7: DBU is added to a solution of 98.ω1 dissolved in MeTHF, and the reaction mixture is stirred in RT for 2 hours. The reaction mixture is filtered through Celite, washed with 1M citric acid aqueous solution, and the organic phase is separated. The organic phase is washed with 5 wt% NaHCO3 aqueous solution, dried over Na2SO4, and concentrated under reduced pressure to obtain 101.ω1.

[0258] Step 8: Dissolve 101.ω1 in MeOH and heat to 40°C, then add solutions of formic acid and oxalic acid in water. Stir the resulting reaction mixture at 40°C until the reaction is complete. Dilute the mixture with brine and extract with MeTHF. Separate the organic phase, dry over Na2SO4, and concentrate under reduced pressure. Purify the crude residue by silica column chromatography (n-heptane / siRNA / MeOH) to obtain pure 104.ω1.

[0259] 4.4 Example 4: Efficacy of NLG2 used as an ionizable lipid LNPs were prepared using the lipids described herein by the IJM method (Garcia, et al. 2022) according to Example 1. The IJM NanoScaler (Knauer) consists of five impingement jet mixers (IJMs), three pumps, and two valves. The IJM NanoScaler (Knauer) was operated at a total flow rate (TFR) of 4.5 ml / min and configured for LNP production in an appropriate configuration that maintained a 3:1 aqueous phase to organic phase ratio. The LNPs obtained after formulation were diluted with PBS (pH 7.4). Vivaspin was used in the subsequent purification step to remove excess organic phase. TM I used 500 (Sigma).

[0260] The cationizable lipid NLG2 can be used in RNA-LNP formulations to form particles with desirable particle size and PDI, but it showed low mRNA encapsulation efficiency (between 15–25%) (measured by Ribogreen) (Carrasco, et al. 2021) (Table 2). This was tested by replacing the cationizable lipid with NLG2 in formulation B-based LNP formulations (46.3% ALC-0315) and formulation A formulations (50% SM-102). This was not an unexpected result because NLG2 consists of an unbranched linear alkyl chain in the lipid tail, which is described as necessary for successfully complex mRNA or siRNA. x By using branched fatty acid chains, the inclusion efficiency can likely be significantly improved. NLG2-containing RNA-LNPs also have a pKa value of approximately 5.2, which is lower than that observed in known RNA-LNPs (6.2-6.6) (Table 2).

[0261] [Table 2]

[0262] As a result, NLG2-containing LNPs showed negligible in vitro transfection efficiency (≤1%) compared to LNP formulation B (Figure 1). This was the case regardless of the type of helper lipid present in the composition (DSPC or DOPE).

[0263] Furthermore, increasing the amount of DOPE helper lipids from 20% to 30% or varying the N / P ratio within the range of 3.0 to 10.0 resulted in an inclusion efficiency of less than 25% (data not shown). In addition, compared to the control LNP formulation B, these NLG2-containing LNPs had an in vitro transfection efficiency of <1%.

[0264] 4.5 Example 5: Efficacy of DPPG2 lipid as a shielding lipid in mRNA-LNP preparations Next, we investigated the effects of three different concentrations (2%, 5%, and 10%) of DPPG2 in RNA-LNP by replacing PEG-DMG as the shielding lipid in formulation B.

[0265] Surprisingly, using DPPG2 as the sole shielding lipid resulted in dispersion-stable particles with a size of 130–160 nm, exhibiting a narrow size distribution indicated by a PDI ≤ 0.20 (Table 2). In particular, formulations containing 5% and 10% anionic DPPG2 showed excellent mRNA encapsulation efficiency (≥ 80%) (Table 3). This data is particularly noteworthy, as the literature suggests that PEG lipids are essential for producing LNPs within an acceptable size range.

[0266] [Table 3]

[0267] As the DPPG2 content in PEG-free RNA-LNPs increased, the particle zeta potential also decreased significantly from -12 mV (2 mol% DPPG2) to -23 mV (10 mol% DPPG2) (Table 3). RNA-LNPs of formulation B had a zeta potential of -6.2 ± 0.6 mV. This data suggests that DPPG2, a negatively charged phospholipid, significantly affects the surface charge, and that DPPG2 and DPPG3 preferentially reside in the monolayer on the LNP surface.

[0268] When the stability of mRNA encapsulated in all DPPG2-containing RNA-LNPs was tested, the mRNA remained intact after 48 hours, with the 10% DPPG2 ratio showing the most superior performance (Figure 2). This data demonstrates the dispersion stability of DPPG2-based PEG-free RNA-LNPs.

[0269] Interestingly, the presence of serum-containing medium significantly positively affected the in vitro transfection efficiency of DPPG2-containing LNPs (based on formulation B), resulting in a considerable increase compared to PEGylated formulation B (Figure 3). The difference in transfection can be explained by improved interaction between DPPG2-based RNA-LNPs and the target cell surface, and / or improved cellular uptake, and / or enhanced release from endosomes. This effect may be due to the significantly smaller glycerol head groups of G2 and G3 lipids compared to PEG lipids, which are often known to sterically inhibit such interactions.

[0270] The adverse effects of large PEG head groups on nucleic acid uptake by target cells in vivo and in vitro have been documented (Song, et al. 2001). Interestingly, when we investigated liposome-cell interactions of classical drug-filled anionic liposomes containing 5% DPPG or 5% DSPE-PEG, respectively, anionic DPPG2 liposomes showed higher liposome-cell interactions (twice as measured by FACS analysis) compared to their PEGylated counterparts, but importantly, this was only in serum-free medium, i.e., in the absence of proteins and other serum components. As expected, both anionic liposome formulations containing either 5% DPPG2 or 5% DSPE-PEG2000 (with equivalent zeta potentials) showed almost undetectable cell targeting after protein adsorption (Petrini, et al. 2021).

[0271] Importantly, all DPPG2-containing LNPs showed a clear dose-response relationship in the test range of 50 ng to 500 ng. This is a crucial prerequisite for the development of therapeutic RNA-LNPs for patient treatment. This is in contrast to the PEGylated Formulation B formulation, which showed consistently low transfection efficiencies (Figures 4 and 5).

[0272] To investigate whether the addition of G2 or G3 lipids to LNP preparations can prevent agglutination with blood components such as red blood cells after systemic injection, PEGylated RNA-LNPs containing G2 or G3 lipids were added to a red blood cell suspension. All RNA-LNPs with G2 or G3 lipid denaturation prevented agglutination with red blood cells.

[0273] The effects of DPPG2 and PEG lipids as shielding components also play a significant role in LNP size. This was demonstrated by experiments using selected RNA-LNPs (Table 4). As expected, the size of PEG-free formulation B was larger (134 nm) compared to each PEG-containing formulation (77 nm) (Table 4). Importantly, the sizes of all three LNPs were in the range of approximately 77–130 nm, which is suitable for systemic administration to patients, while the DPPG2 formulations had a particle size of 128 nm and a good PDI of 0.15. It is worth mentioning that they had transfection efficiencies exceeding 70%.

[0274] PEG-free formulation A increased in particle size from 116 nm to 145 nm after 24 hours at 4°C (see Table 4).

[0275] [Table 4]

[0276] To further evaluate the storage stability of different RNA-LNPs, we compared two formulations containing either DPPG2 or DMG-PEG as a shielding lipid (Table 5). The DPPG2-containing RNA-LNP had a lipid composition of ALC-0315 / DSPC / Chol / DPPG2 46.3:9.4:34.3:10 (mol / mol), while the PEGylated RNA-LNP consisted of ALC-0315 / DSPC / Chol / DMG-PEG 46.3:9.4:42.7:1.6 (mol / mol). 25 mM acetate (pH 4.0), with or without 10% sucrose as a cryoprotective agent, was used as a buffer for both formulations. The formulations were stored at 2–8°C or -20°C for 48 hours and 2 weeks. Notably, both formulations showed sustained stability, maintaining a constant particle size even after 1 week of storage at 2–8°C. However, when stored at -20°C, both formulations showed a similar increase in particle size. Clearly, the presence of sucrose as a model cryoprotectant did not affect the size increase. In all formulations analyzed by agarose gel electrophoresis, the encapsulation efficiency of GFP mRNA remained good (data not shown).

[0277] The transfection efficiencies of different formulations stored under different conditions were measured according to Example 4. Surprisingly, as shown in Figure 11, the DPPG2-containing formulation showed the highest transfection efficiency when stored at 4°C without cryoprotection.

[0278] [Table 5]

[0279] 4.6 Example 6: Efficacy of DPPG3 lipid as a shielding lipid in mRNA-LNP preparations / DPPG3-containing preparations in which PEG lipids are completely replaced In the next step, we investigated the ability of DPPG3 to replace PEG-DMG as a shielding lipid in RNA-LNPs. The lipid composition and characterization results of the formulations are shown in Table 6.

[0280] DPPG3 (5 or 10 mol%) was successfully used to replace the shielding lipid DMG-PEG in RNA-LNPs in both formulation A and formulation B-like LNPs. RNA-LNPs with clearly defined size, narrow size distribution (PDI < 0.2), and good mRNA encapsulation efficiency (70–83%) were produced.

[0281] [Table 6]

[0282] Furthermore, the in vitro transfection efficiency of DPPG3-containing RNA-LNPs (in serum-containing culture medium) was improved compared to that of PEGylated formulation B's RNA-LNPs (Figure 6). A particularly significant increase in transfection efficiency was observed for PEG-free formulation A's RNA-LNPs, which were treated with SM-102 and DPPG3, respectively, as cationizable barrier lipids (Figure 6).

[0283] [Table 7]

[0284] To evaluate the transfection efficiency of different RNA-LNPs stored for up to three weeks, a comparison was made between two formulations shown in Table 14, each containing either DPPG3 or DMG-PEG as a shielding lipid, as disclosed above. The DPPG3-containing RNA-LNP (DPPG3-containing A-like LNPA) had a lipid composition of SM-102 / DSPC / Chol / DPPG3 50:10:30:10 (mol / mol), while the formulation A-like PEGylated RNA-LNP consisted of SM-102 / DSPC / Chol / DMG-PEG 50:10:38.5:1.5 (mol / mol).

[0285] JPEG2026534185000038.jpg46147

[0286] The transfection efficiencies of different LNPs containing 50, 150, and 300 ng of RNA in HEK-293 cells were measured immediately after production (week 0), and after storage at 4°C for 1 and 3 weeks. As shown in Figure 13, notably, all RNA-LNP formulations containing DPPG3 showed superior transfection efficiencies even after 3 weeks compared to the control (B-like LNP).

[0287] 4.7 Example 7: Effect of cationic CLG2 lipids in mRNA-LNP Next, we investigated the possibility of increasing the zeta potential of formulations A and B-based PEGylated RNA-LNPs while achieving well-controlled particle size and maintaining satisfactory transfection efficiency. For this purpose, we used a novel lipid, CLG2, a cationic member of the G2 lipid family that carries a permanent positive charge.

[0288] The initial experiments aimed to test whether cationic CLG2 could replace the neutrally charged helper lipid DSPC in formulations A and B. The final lipid composition and characterization results are shown in Table 8. The PDI of CLG2-containing PEGylated RNA-LNPs (Table 8) was comparable to that of control formulation B (Table 6). However, the particle size was larger in the former. The encapsulation efficiency of CLG2-containing PEGylated RNA-LNPs was >80%. The pKa values ​​(measured by TNS assay) ranged from 6.2 to 6.3 for all three formulations. All formulations still had a negative zeta potential, which is advantageous for in vivo administration, but it was important to note that it was slightly higher than that of formulation B (Table 6). However, since PEG is known to effectively shield the charge of membrane-forming lipids, the effect of CLG2 on surface charge may be underestimated by the zeta potential results.

[0289] Surprisingly, cationic CLG2 can replace neutral DSPC as a helper lipid in PEGylated RNA-LNP formulations, thereby modulating the zeta potential and potentially affecting the protein corona of LNPs obtained in vivo after systemic administration.

[0290] Importantly, the particle size of PEGylated CLG2-containing RNA-LNPs was influenced by the structure of the cationizable lipid and the amount of CLG2 used (Table 8). The particle sizes were 170 nm (PDI 0.19) and 218 nm (PDI 0.19) for RNA-LNPs using SM-102 (formulation A-like) and ALC-0315 (formulation B-like), and for RNA-LNPs using approximately 10 mol% CLG2 as a helper lipid, respectively. When the CLG2 content in RNA-LNPs using 50 mol% SM-102 (formulation A-like) was increased from 10 mol% to 20 mol%, the particle size decreased from 170 nm (PDI 0.19) to 148 nm (PDI 0.17). Therefore, it can be considered that smaller LNPs can be generated by incorporating higher concentrations of CLG2, even in the absence of any PEG lipids. mRNA in the CLG2 formulation was not degraded even after 48 hours (data not shown).

[0291] [Table 8]

[0292] In the presence of serum-containing culture medium, the in vitro transfection efficiency of PEGylated RNA-LNPs containing CLG2 as a helper lipid showed a significant improvement compared to PEGylated LNPs found in formulation B (Figure 7).

[0293] [Table 9]

[0294] As expected, when CLG2-containing formulations A and B used CLG2 with linear acyl chains to replace each ionizable lipid with branched alkyl chains in the LNP formulation, they were only able to form microparticles with a wide size distribution (>0.25) (Table 9).

[0295] Interestingly, in the absence of PEG-DMG or DPPG2, CLG2 also failed to stabilize RNA-LNPs (see Table 10), resulting in undesirable particle size and PDI even after buffer exchange.

[0296] [Table 10]

[0297] 4.8 Example 8: Efficacy of CLG2 (cationic lipid) and DPPG2 as helper lipids in mRNA-LNPs In the next step, we combined two members of the G2 lipid family, anionic DPPG2 and cationic CLG2, in RNA-LNPs, replacing the helper lipid DSPC and PEGylation stabilizer lipid, respectively. Table 11 shows the lipid compositions used and the results of characterization of the prepared formulations. Both RNA-LNP formulations had good pKa values ​​and good inclusion efficiency. Furthermore, the potential of CLG2 to modulate the zeta potential of RNA-LNPs was again demonstrated. This is clearly shown by comparing RNA-LNPs containing 9.4% CLG2 and 10% DPPG2 (zeta potential: -6.5±0.4mV; Table 11) or RNA-LNPs containing 9.4% CLG2 and 1.6% DMG-PEG (-1.0±0.5mV; Table 8) with RNA-LNPs containing 0% CLG2, 9.4% DSPC, and 10% DPPG2 (-23±1.8mV; Table 3).

[0298] An LNP formulation containing nearly equimolar amounts of negatively charged DPPG2 (10%) and positively charged CLG2 (9.4%) had a similar zeta potential (-6.5±0.4mV) compared to a "standard" LNP formulation B (zeta potential -6.2±0.4V) containing 9.4% zwitterionic DSPC.

[0299] Notably, RNA-LNPs composed of CLG2 and DPPG2 showed up to 10 times higher in vitro transfection efficiency than PEGylated RNA-LNPs (see Figure 8).

[0300] Therefore, by combining the cationic and anionic members of the G2 and G3 lipid families, Onpattro, the LNP of approved COVID-19 vaccines (formulations B and A) and approved siRNAs, is used. (登録商標) This allows for the design and fabrication of PEG-free mRNA LNPs that share several characteristics (including nearly identical zeta potentials) (Urits, et al. 2020).

[0301] CLG2 may also promote the regulation of the charge (zeta potential) of LNP formulations in the presence of shielding agents, such as PEGylated lipids or anionic members of the G2 or G3 lipid family.

[0302] [Table 11]

[0303] Therefore, the presence of G2 or G3 lipids in RNA LNP formulations may have the potential to ensure systemic stability without loss of transfection activity due to PEGylation.

[0304] Using DPPG2 or DPPG3 as a substitute for PEG lipids in LNPs may offer a promising solution for in vivo application of RNA-LNPs. Furthermore, cationic G2 or G3 lipids, which also appear to be LNP-compatible, may function as cationic components that modulate the zeta potential as needed.

[0305] 4.9 Example 9: Compatibility of NLG2 and CLG2 with classical DODMA / DOPE lipids The miscibility of the oligoglycerol lipids NLG2 and CLG2 with DODMA (1,2-dioleyloxy-3-dimethylaminopropane), a representative member of classical ionizable lipids, was tested. In contrast to ALC-0315 and SM-102, the hydrophobic moiety in DODMA consists of two unbranched, unsaturated carbon atoms linked to the propane backbone by ether bonds.18 These are fatty acids. Furthermore, the change in the identity of the zwitterionic phospholipid was tested by replacing DSPC in the LNP with DOPE according to the composition shown in Table 12. The LNP was prepared using the formulation method utilizing impact jet technology (IJM) as described above.

[0306] As shown in Table 12, LNPs with a size range of 100–190 nm, a narrow polydispersity index (PDI), and an encapsulation efficiency of at least 95% were successfully generated. The possibility of generating LNPs with a size of approximately 100 nm was demonstrated (containing both NLG2 and CLG2-containing LNPs).

[0307] [Table 12]

[0308] Transfection efficiency was measured in the HEK293 cell line. Three total formulations containing NLG2, CLG2, or both CLG2 and NLG2 in addition to DODMA as an ionizable lipid showed superior transfection efficiency compared to formulation B-based formulations (containing ALC-0315 as an ionizable lipid) (with varying mRNA amounts of 50 ng, 150 ng, and 300 ng). Furthermore, compared to a four-component formulation consisting of DODMA (50%):DSPC (10%):cholesterol (38.5%):DMG-PEG (1.5%), the addition of CLG2 or (especially) NLG2 resulted in at least equivalent five-component formulations with lower mRNA amounts (50 ng) and significantly improved transfection efficiency (Figure 9). Formulations containing both DOPE phospholipids and NLG2 were most efficient in in vitro mRNA delivery compared to DSPC-containing LNPs, likely due to improved endosomal escape. In conclusion, the miscibility of NLG2 and CLG2 with DOPE and DODMA as alternatives to DSPC and branched-chain ionizable lipids enables the generation of LNPs with high in vitro potency, respectively.

[0309] Furthermore, the compatibility of NLG2 and CLG2 with DODMA indicates that oligoglycerol lipids containing fatty acids attached to the glycerol backbone via ester bonds can synergistically interact with lipids in which fatty acid chains are attached to the propane backbone via ether bonds. These results clearly demonstrate that a novel class of oligoglycerol lipids is broadly and universally applicable for the rational design of LNPs.

[0310] 4.10 Example 10: NLG2 in 3-component LNP We investigated whether NLG2 could completely replace zwitterionic DSPC as a PEG-blocking lipid and helper lipid during LNP formation. This approach enabled the generation of a three-component LNP formulation with a diameter of approximately 100 nm and a narrow size distribution. The LNPs were generated and analyzed as described above.

[0311] As shown in Table 13, formulations in which PEG lipids and DSPC helper lipids were completely replaced with NLG2 (and a portion of ALC-0315) in molar ratios of 21% and 25% were found to form LNPs with a vesicle size of approximately 100 nm and monodisperse distribution (PDI 0.12-0.17), and an mRNA encapsulation efficiency of approximately 80% (Table 13). Successful mRNA encapsulation in the three-component formulation containing NLG2 based on Formulation B was further confirmed using agarose gel electrophoresis (data not shown).

[0312] [Table 13]

[0313] Both NLG2-containing formulations showed superior in vitro transfection efficiency compared to the Formulation B-like control formulation (Figure 10).

[0314] These results clearly demonstrate the diverse potential of oligoglycerol lipids in LNP formation. These experiments show that their dual function as both shielding and helper lipids reduces the complexity of LNP formulations by eliminating the need for a fourth component, which is a significant advantage in the manufacture and storage of approved vaccines for human use.

[0315] 4.11 Example 11: Efficacy of (10%) DPPG2-containing LNP: In vivo in mice To understand the in vivo distribution of LNPs containing DPPG2 as a shielding lipid, BALB / c mice (male, 6 weeks old, approximately 20g) were intravenously administered LNPs containing Luc-mRNA (see Table 14 for details of the formulations). The groups were treated with B-like LNPs (containing DPPG2), LNP formulation B (containing PEG), and saline as a control (n=5 per group). Each experimental group received a single dose of 20 μg (100 μL) of luciferase-mRNA (Luc-mRNA) encapsulated in LNPs. Four hours after administration, ex vivo imaging for luminescence was performed using an in vivo imaging system (IVIS) (Wang, et al. 2023). Ex vivo analysis was performed on various organs, including the liver, lungs, heart, kidneys, spleen, lymph nodes, and blood samples. In particular, as shown in Figure 12, the accumulation of LNPs containing DPPG2 was significantly reduced in the liver compared to conventional LNPs containing PEG. These results clearly demonstrate the liver bypass effect of DPPG2-containing LNPs and the potential for oligoglycerol lipids to alter the in vivo distribution of mRNA-carrying LNPs.

[0316] [Table 14]

[0317] item This application also discloses the following items:

[0318] Item 1. a) At least one formula (I)

[0319] [ka]

[0320] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (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 at least one additional lipid selected from A composition containing, preferably lipid nanoparticles (LNPs).

[0321] Item 2. a) At least one formula (IA)

[0322] [ka]

[0323] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (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 at least one additional lipid selected from A composition of item 1, including the following:

[0324] Item 3. a) At least one formula (I)

[0325] [ka]

[0326] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid A composition of item 1, including the following:

[0327] Item 4. a) At least one formula (IA)

[0328] [ka]

[0329] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b)(ii) at least one structural lipid A composition containing any one of items 1 to 3.

[0330] Item 5. a) At least one formula (I)

[0331] [ka]

[0332] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition of item 1, including the following:

[0333] Item 6. a) At least one formula (IA)

[0334] [ka]

[0335] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition containing any one of items 1, 2, or 5.

[0336] Item 7. a) At least one formula (I)

[0337] [ka]

[0338] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition of item 1, including the following:

[0339] Item 8. a) At least one formula (IA)

[0340] [ka]

[0341] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition containing any one of items 1, 2, or 7.

[0342] Item 9. a) At least one formula (I)

[0343] [ka]

[0344] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y=0 or 1; and z=0 or 1; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b)(iii) at least one helper lipid A composition of item 1, including the following:

[0345] Item 10. a) At least one formula (IA)

[0346] [ka]

[0347] (In the formula, A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = a linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b)(iii) at least one helper lipid A composition containing any one of items 1, 2, or 9.

[0348] Item 11. At least one oligoglycerol-containing lipid of formula (I) or (IA) is A = OP(-O - A composition containing any one of items 1 to 10, which is phosphatidyl oligoglycerol that is (=O)-O.

[0349] Item 12. At least one oligoglycerol-containing lipid of formula (I) or (IA) is A = N + R 4 R 5 A composition comprising any one of items 1 to 11, which is a cationic oligoglycerol-containing lipid.

[0350] Item 13. At least one oligoglycerol-containing lipid of formula (I) or (IA) is present, A = NR 3 A composition comprising any one of items 1 to 12, which is a cationizable oligoglycerol-containing lipid.

[0351] Item 14. At least one oligoglycerol-containing lipid of formula (I) or (IA) is A = OP(-O - Phosphatidyl oligoglycerol is (=O)-O, and A = N + R 4 R 5 A composition comprising any one of items 1 to 13, comprising a cationic oligoglycerol-containing lipid of formula (I) or (IA).

[0352] Item 15. Any one of items 1 to 14 comprising at least one oligoglycerol-containing lipid of formula (I) or (IA) comprising 2 to 4 glycerol units, preferably 2 or 3 glycerol units, and more preferably 2 glycerol units.

[0353] Item 16.R 1 and R 2 However, a composition comprising any one of items 1 to 15, which is independently a linear or branched hydrocarbon group.

[0354] Item 17.R 1 and R2 A composition comprising any one of items 1 to 16, which independently comprises saturated, monounsaturated, or polyunsaturated hydrocarbon groups.

[0355] Item 18.R 1 and R 2 However, any one of items 1 to 17 is a hydrocarbon group having independently 13 to 19 carbon atoms, and in particular 15 or 17 carbon atoms.

[0356] Item 19.R 1 and R 2 However, the composition is one of items 1 to 18, which is a linear saturated C13-C19 alkyl group.

[0357] Item 20.R 3 = C1-C5 alkyl groups, especially R 3 = CH3, and / or R 4 and R 5 However, each is independently a C1-C5 alkyl group, and especially R 4 = R 5 = One of the compositions from items 1 to 19 that is CH3.

[0358] Item 21. At least one oligoglycerol-containing lipid is present in formula (IIA)

[0359] [ka]

[0360] (wherein the formula n=2 to 20; and in particular n=2 or 3; and A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R5 Each of these is a hydrocarbon group having either hydrogen or 1 to 5 carbon atoms independently; (y=0 or 1; and z=0 or 1) One of the compositions from items 1 to 20 that is one of the following.

[0361] Item 22. At least one oligoglycerol-containing lipid is formed by formula (II)

[0362] [ka]

[0363] (wherein the formula n=2 to 20; and in particular n=2 or 3; and A = OP(-O - )(=O)-O; or NR 3 ; or N + R 4 R 5 ; R 1 and R 2 These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R 3 , R 4 , and R 5 Each of these is a hydrocarbon group having either hydrogen or 1 to 5 carbon atoms independently; (y=0 or 1; and z=0 or 1) One of the compositions from items 1 to 21 that is one of the following.

[0364] Item 23. Expression

[0365] [ka]

[0366] A composition comprising any one of items 1 to 22, which contains an oligoglycerol-containing lipid having [a certain property].

[0367] Item 24. Expression

[0368] [ka]

[0369] A composition comprising any one of items 1 to 22, which contains an oligoglycerol-containing lipid having [a certain property].

[0370] Item 25. Expression

[0371] [ka]

[0372] A composition comprising any one of items 1 to 22, which contains an oligoglycerol-containing lipid having [a certain property].

[0373] Item 26. Expression

[0374] [ka]

[0375] A composition comprising any one of items 1 to 22, which contains an oligoglycerol-containing lipid having [a certain property].

[0376] Item 27. Expression

[0377] [ka]

[0378] A composition containing an oligoglycerol-containing lipid, one of items 1 to 22.

[0379] Item 28. Expression

[0380] [ka]

[0381] A composition containing an oligoglycerol-containing lipid, one of items 1 to 22.

[0382] Item 29. (a) At least one oligoglycerol-containing lipid of formula (I) or (IA), optionally (i) A = OP(-O - Phosphatidyl oligoglycerol of formula (I) or (IA) such that )(=O)-O; and (ii) A = N + R 4 R 5 A cationic oligoglycerol-containing lipid of formula (I) or (IA) that is at least one such lipid; and (b)(i) at least one cationizable lipid; and (b)(ii) at least one structural lipid A composition containing any one of items 1 to 28.

[0383] Item 30. (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 A composition containing any one of items 1 to 29.

[0384] Item 31. A composition from any one of items 1-30 that is a lipid nanoparticle (LNP).

[0385] Item 32. A composition in which the structural lipid is a sterol, preferably selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomazidine, ursolic acid, and α-tocopherol, from any one of items 1 to 31.

[0386] Item 33. One of the compositions from items 1 to 32 in which the structural lipid is cholesterol.

[0387] Item 34. Helper lipids, Hydrogenated soybean L-α-phosphatidylcholine (HSPC); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-Diolioyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-Dimiristoyl-sn-glycerophosphocholine (DMPC); 1,2-Diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-Palmitoyl-2-olioyl-sn-glycero-3-phosphocholine (POPC) Selected from the group consisting of; and Helper lipids in particular, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) One of the compositions listed in items 1 to 33;

[0388] Item 35. b)(iv) PEG lipids A composition containing any one of items 1-6 and 9-34.

[0389] Item 36. A composition which does not contain PEG lipids, and is one of any one of items 1-6 and 9-34.

[0390] Item 37. One of the compositions from items 1-4, 7-8, 11-28, and 31-36, which does not contain helper lipids.

[0391] Item 38. One of the compositions from items 1-4, 7-8, 11-28, 31-33, and 36-37, which does not contain helper lipids and does not contain PEG lipids.

[0392] Item 39. c) Therapeutic and / or prophylactic agents A composition containing any one of items 1 to 38, further including the above.

[0393] Item 40. A composition of Item 39 in which the therapeutic agent is nucleic acid, particularly ribonucleic acid (RNA).

[0394] Item 41. A composition of Item 40 in which RNA is selected from the group consisting of messenger RNA (mRNA), antisense RNA (aRNA), small interfering RNA (siRNA), CRISPR-RNA (crRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), and mixtures thereof.

[0395] Item 42. A composition of item 39 in which the therapeutic and / or prophylactic agent is a small molecule.

[0396] Item 43. 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) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and at least one of the CLG2s described in item 25 or 26; and e) Optional therapeutic and / or prophylactic agents A composition containing any one of items 1 to 42.

[0397] Item 44. The composition of item 43, which does not contain PEG lipids.

Claims

1. a) At least one equation (I) 【Chemistry 1】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ are each independently hydrogen or a hydrocarbon group having 1 to 5 C atoms; y=0 or 1; and z=0 or 1; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (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 at least one additional lipid selected from A composition comprising, wherein the composition is preferably lipid nanoparticles (LNPs).

2. a) At least one formula (IA) 【Chemistry 2】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (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 at least one additional lipid selected from The composition according to claim 1, comprising:

3. a) At least one equation (I) 【Transformation 3】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y = 0 or 1; and z = 0 or 1; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b) (ii) at least one structural lipid The composition according to claim 1, comprising:

4. a) At least one formula (IA) 【Chemistry 4】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; and b) (i) at least one cationic or cationizable lipid; and b) (ii) at least one structural lipid A composition according to any one of claims 1 to 3, comprising:

5. a) At least one equation (I) 【Transformation 5】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y = 0 or 1; and z = 0 or 1; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 The composition according to claim 1, comprising:

6. a) At least one formula (IA) 【Transformation 6】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition according to any one of claims 1, 2, or 5, comprising:

7. a) At least one equation (I) 【Transformation 7】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; y = 0 or 1; and z = 0 or 1; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 The composition according to claim 1, comprising:

8. a) At least one formula (IA) 【Transformation 8】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. Oligoglycerol-containing lipids; 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 A composition according to any one of claims 1, 2, or 7, comprising:

9. a) At least one equation (I) 【Chemistry 9】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ These are hydrocarbon groups, each independently having 12 to 24 carbon atoms; R ３ , R ４ , and R ５ are each independently hydrogen or a hydrocarbon group having 1 to 5 C atoms; y=0 or 1; and z=0 or 1; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. oligoglycerol-containing lipids; and b) (iii) at least one helper lipid The composition according to claim 1, comprising:

10. a) At least one formula (IA) 【Chemistry 10】 (In the formula, A = OP(-O － ) (=O) - O; or NR ３ ; or N ＋ R ４ R ５ ; R １ and R ２ are each independently a hydrocarbon group having 12 to 24 C atoms; R ３ , R ４ , and R ５ Each is a hydrocarbon group having hydrogen or 1 to 5 carbon atoms independently; and B = A linear or branched oligoglycerol group having 2 to 20 glycerol units. oligoglycerol-containing lipids; and b) (iii) at least one helper lipid A composition according to any one of claims 1, 2, or 9, comprising:

11. formula 【Chemistry 11】 Lipids containing oligoglycerols and / or formula 【Chemistry 12】 Contains oligoglycerol-containing lipids having and / or formula 【Chemistry 13】 A composition according to any one of claims 1 to 10, comprising an oligoglycerol-containing lipid having the properties of

12. (a) At least one oligoglycerol-containing lipid of formula (I) or (IA), optionally (i) A = OP(-O － Phosphatidyl oligoglycerol of formula (I) or (IA) such that ) (= O) - O; and (ii) A = N ＋ R ４ R ５ A cationic oligoglycerol-containing lipid of formula (I) or (IA) that is at least one such lipid; and (b) (i) at least one cationizable lipid; and (b) (ii) at least one structural lipid A composition according to any one of claims 1 to 11, comprising:

13. (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 A composition according to any one of claims 1 to 12, comprising:

14. A composition according to any one of claims 1 to 13, wherein the composition is a lipid nanoparticle (LNP).

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

16. Helper lipids, Hydrogenated soybean L-α-phosphatidylcholine (HSPC); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-Diolioyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-Dimiristoyl-sn-glycerophosphocholine (DMPC); 1,2-Diolioyl-sn-glycero-3-phosphocholine (DOPC); and 1-Palmitoyl-2-Olioyl-sn-Glycerol-3-Phosphocholine (POPC) Selected from the group consisting of; and Helper lipids in particular, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) A composition according to any one of claims 1 to 15.

17. c) Therapeutic and / or prophylactic agents A composition according to any one of claims 1 to 16, further comprising:

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

19. 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) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and CLG ２ at least one of; and e) Optional therapeutic and / or prophylactic agents A composition according to any one of claims 1 to 18, comprising:

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