Novel lipid nanoparticle formulations for nucleic acid delivery

Abstract

The present invention provides novel lipid nanoparticles comprising novel ionizable lipids and messenger RNA (mRNA) useful for nucleic acid delivery, as well as related pharmaceutical compositions or vaccines as defined herein, which are intended for use in human or veterinary medicine, and in particular for use in the treatment and / or prevention of cancer.

Description

[Technical Field]

[0001] The present invention provides novel lipid nanoparticles comprising novel ionizable lipids and messenger RNA (mRNA) useful for nucleic acid delivery, as well as related pharmaceutical compositions or vaccines as defined herein, which are intended for use in human or veterinary medicine, and in particular for use in the treatment and / or prevention of cancer. [Background technology]

[0002] Cancer is a major global health problem and one of the leading causes of death worldwide. Traditional cancer treatments, such as surgery, chemotherapy, and radiation therapy, have limited effectiveness and can cause serious side effects. Immunotherapy, which utilizes the power of the immune system to target cancer cells, is emerging as a promising new approach to cancer treatment. Despite significant progress in the development of cancer therapies, there is still a need for new and more effective treatments, especially for cancers that are resistant to existing therapies or have a poor prognosis.

[0003] In recent years, mRNA has emerged as a promising therapeutic tool for treating cancer and other diseases. This is because it can be used to direct the production of proteins that can inhibit or otherwise reduce the proliferation or survival of cancer cells. Advantages of using mRNA include its transient expression and non-transformation properties. That is, mRNA does not need to enter the nucleus to be expressed, and furthermore, it is not integrated into the host genome, thus eliminating the risk of carcinogenesis.

[0004] The use of mRNA in therapeutic settings currently faces two challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to gain access to intracellular compartments where the relevant translation mechanisms reside. Therefore, to overcome these challenges, various delivery strategies are being developed, including the use of lipid nanoparticle formulations that can protect mRNA to prevent degradation and promote its uptake into target cells.

[0005] Despite all prior art, there is a need for alternative polymer-binding lipids, alternative ionizable lipids, and alternative lipid nanoparticles containing these alternative lipids that provide one or more properties such as improved in vivo efficacy, improved transfection process, improved toxicity, improved cost and design simplicity, reduced cytotoxicity, better targeting ability, enhanced short-term and / or long-term immunity, or enhanced endosomal escape of molecules such as nucleic acids. Furthermore, there remains a need for improved PEG-free (PEG-less) lipid nanoparticles for mRNA delivery. Preferably, these PEG-less lipid nanoparticles provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or topical delivery, and provide intracellular delivery of nucleic acids. In addition, it is desirable that these PEG-less lipid nanoparticles containing RNA or mRNA be well-tolerated, provide appropriate therapeutic indicators, and therefore patient treatment with effective amounts of nucleic acids does not involve unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages.

[0006] Human type I interferons (IFNs) are a large subgroup of interferon proteins that help regulate the activity of the immune system. Mammalian types are called IFN-a (alpha), IFN-b (beta), IFN-k (kappa), IFN-d (delta), IFN-e (epsilon), IFN-x (tau), IFN-w (omega), and IFN-z (zeta, also known as limitin). Type I interferons have shown efficacy against the replication of various viruses, including Zika virus, Chikungunya virus, Flavivirus, and Hepatitis C virus. In some aspects and embodiments, the inventors have surprisingly found that the lipid nanoparticles of the present invention, namely phosphatidylserine (preferably DPhyPS) and novel polyoxazoline polymer-bound lipids (preferably PMOZ lipids), more preferably low in PMOZ lipids (i.e., about 1 mol%), in LNP formulations, increase the levels of different cytokines, preferably IFNa and / or IFNb, after prime and / or boost vaccination, and through this increase, a higher immune response, i.e., after prime vaccination and / or after first and / or second boost vaccination. • High CD4 T cell response, • High CD8 T cell response, • High humoral response as measured by IgG1 and / or IgG2a / IgG2a[b] titers. • Multifunctional and activated cytotoxic CD8 + A high percentage of T cells, ·Multifunctional and activated cytotoxic CD8+T EM A high percentage of cells, • A high percentage of multifunctional and activated CD4 T cells, and / or • Multifunctional and activated CD4T EM High percentage of cells We found that this can be induced. Therefore, the use of lipid nanoparticles in the present invention offers remarkable advantages in the context of cancer treatment. Furthermore, surprisingly, it was found that the use of a low percentage of PMOZ lipids correlated with an increase in transfection rate.

[0007] Accordingly, the inventors have remarkably discovered a method for inducing interferon (IFN) production. This method comprises administering the lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention to a subject of interest, thereby increasing IFN production after administration of the aforementioned lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention. Furthermore, the inventors have remarkably discovered a method comprising administering the lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention to a subject of interest in an amount sufficient to induce an immune response in said subject. Preferably, the immune response involves cytokine production, and more preferably, the immune response involves the regulation of type I IFN, type II IFN, and / or type III IFN. More specifically, the inventors have remarkably discovered a method comprising administering the lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention to a subject of interest in an amount sufficient to induce an immune response in said subject. Preferably, the immune response involves the production of type I IFN, and more preferably, the immune response involves the regulation of IFNa, IFNb, IFNe, IFNk, or IFN, most preferably IFNa and / or IFNb.

[0008] In particular, polyoxazoline (POZ) or poly(2-methyl-2-oxazoline) (PMOZ) lipid conjugates have been demonstrated to be suitable components for the assembly of RNA nanoparticles. Poly(2-oxazoline) is a type of polymer formed by cation ring opening and was first identified and synthesized more than 50 years ago (Non-Patent Literature 1). These polymers are nonionic, biostable, soluble in water and some polar organic solvents, and can be synthesized from readily available, non-toxic, and non-explosive starting materials. The N-carbonyl side chains on the polymer chain give it the appearance of a "pseudopolypeptide." POZs with shorter side chains are generally more water-soluble than POZs with longer side chains. For example, PMOZ is composed of repeating units of 2-methyl-2-oxazoline (CAS registry number: 161358-46-9) and is rapidly excreted by the kidney without significant accumulation in tissues (Non-Patent Literature 2). POZ / PMOZ-lipid conjugates enable the production of RNA nanoparticles using different techniques, resulting in particles with specified surface properties and a controlled size range. Production can be carried out using robust methods compliant with pharmaceutical manufacturing requirements. The particles can be end-functionalized using different moieties to modulate charge or introduce specific molecular moieties, such as ligands.

[0009] Furthermore, the inventors surprisingly found that LNPs of the present invention containing phosphatidylserine (preferably DPhyPS) and novel polyoxazoline polymer-bound lipids, preferably PMOZ lipids, and more preferably LNP formulations with a PMOZ lipid content reduced to about 1 mol%, possess favorable physiological and chemical properties when measured by PDI and size measurement after freezing and thawing, or after lyophilization and reconstitution. The inventors surprisingly found that the PMOZ-LNPs were superior in terms of size (smaller size) and PDI after the lipid nanoparticles of the present invention were subjected to thermal stress, i.e., after freeze / thaw cycles or lyophilization and reconstitution. Furthermore, an unfavorable increase in size and PDI was observed in PEG-LNPs upon dilution. Specifically, PEG-LNPs showed increased size and PDI upon dilution. Such increases in size and PDI were not observed or were not significant in LNPs containing PMOZ as a binding lipid. Even when PMOZ-LNPs showed a size increase comparable to PEG-LNPs after dilution or freeze / thaw, PMOZ-LNPs remained smaller than PEG-LNPs. This is advantageous because, according to knowledge in the field, smaller particles are associated with higher immunogenicity. (Non-Patent Documents 3, 4, and 5)

[0010] Furthermore, there is a need in the art to provide alternative and novel lipid nanoparticle formulations suitable for various therapeutic interventions in patients, such as tumor treatment methods aimed at inducing a T-cell response to tumors based on tumor antigen expression by encoding mRNA in antigen-presenting cells (APCs). Target cells for such interventions are, for example, dendritic cells (DCs) present in lymph nodes (LNs) or the spleen. Therefore, mRNA encoding polypeptides containing one or more epitopes can be used to deliver epitopes to patients induced from tumor-associated antigens encoded by overly upregulated RNA transcripts. Dendritic cells (DCs) present in the spleen represent antigen-presenting cells that are particularly important for the mRNA expression of epitopes. Here, the inventors have found that LNPs containing phosphatidylserine, preferably DPhyPS, are highly suitable for cancer treatment, and surprisingly, after administration of these LNPs, high mRNA expression is produced in immune cells, the spleen, and / or dendritic cells, respectively, and a high immune response is induced as described below.

[0011] In summary, there is a constant need for improved lipid nanoparticle compositions used in molecular therapy techniques for the treatment and / or prevention of cancerous diseases, infectious diseases, or for providing therapeutic proteins. Given the circumstances described above, the object of the present invention is to provide improved lipid nanoparticle compositions for introducing mRNA into primate cells, including human cells.

[0012] Another object of the present invention can be seen with respect to the use of the novel ionizable lipid and phosphatidylserine to produce (i) novel ionizable lipids, (ii) novel lipid nanoparticles comprising a combination of the ionizable lipid and phosphatidylserine, and (iii) improved lipid nanoparticles. Furthermore, an object of the present invention can also be seen with respect to the use of the novel polymerizable lipid, novel ionizable lipid and phosphatidylserine to produce (i) novel polymer-bound lipids and novel ionizable lipids, (ii) novel lipid nanoparticles comprising a combination of the novel polymer-bound lipid and ionizable lipid and phosphatidylserine, and (iii) improved with respect to the production of anti-PEG antibodies (i.e., the novel lipid nanoparticles do not produce anti-PEG antibodies), as well as improved with respect to the enhanced physiological and chemical properties of the lipid nanoparticles during (i) freezing and thawing or (ii) lyophilization and reconstitution for storage or shipment, for example.

[0013] Therefore, the present invention achieves all its objectives by providing lipid nanoparticles containing novel ionizable lipids and novel lipid compositions suitable for mRNA delivery. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Kagiya et al., J Polym Sci B Polym Lett 1966;4441-5 [Non-Patent Document 2] Gaertner et al., Journal of Controlled Release 119 (2007) 291-300 [Non-Patent Document 3] Li et al., 2014, Journal of controlled release, 173, 148-157 [Non-Patent Document 4] Ott et al., Vaccine, 1995 13(16), 1557-1562

Non-Patent Document 5

Summary of the Invention

[0015] In a first aspect, the present invention relates to · from about 45 mol% to about 65 mol% of an ionizable lipid, preferably of formula (II): R a -A-R b Formula (II) an ionizable lipid represented thereby, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, and (R a is

[0016]

Chemical formula

[0017] selected from R b is

[0018]

Chemical formula

[0020] In one embodiment of the first aspect, the present invention is a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof, b) A carrier composition comprising the phospholipid phosphatidylserine, preferably about 2.5 mol% or 5 mol% phosphatidylserine, more preferably about 2.5 mol% or 5 mol% DPhyPS, a polymer-bound lipid represented by formula (I), and an ionizable lipid represented by formula (II) described below. This relates to a lipid nanoparticle composition containing [the specified substance].

[0021] In another embodiment, the lipid nanoparticles preferably comprise an ionizable lipid

[0022] [Chemical formula]

[0023] and contain it. In another embodiment, the lipid nanoparticles preferably comprise an ionizable lipid

[0024] [Chemical formula]

[0025] and contain it. In another embodiment, the lipid nanoparticles preferably comprise an ionizable lipid

[0026] [Chemical formula]

[0027] and contain it. [[ID=4l]]In another embodiment, the lipid nanoparticles preferably are · from about 45 mol% to about 65 mol% of an ionizable lipid, preferably about 49 mol% or about 59 mol% of an ionizable lipid, and · from about 4 mol% to about 15 mol%, preferably from about 4 mol% to about 9 mol% of a phospholipid, preferably about 5 mol% or about 7.5 mol% of a phospholipid, more preferably about 7.5 mol% of DPhyPE, and · about 2.5 mol% of phosphatidylserine, preferably DPhyPS, • Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of PMOZ lipids, preferably PMOZ lipids that do not contain sulfur groups (-S-), more preferably PMOZ lipids containing a linker group [linker] of (C(O)CH2CH2C(O)NH), and even more preferably (i)n=50, i.e., "PMOZ4" having 50 monomer repeats (abbreviated as "N-methyl-2-(N4',N4'-di(tetradecyl)succinamide)-poly[(N-acetyl)ethylamine" or "PMOz-DM-amide").

[0028] [ka]

[0029] Or (ii) n=115, i.e., "PMOZ4" having 115 monomer repeats.

[0030] [ka]

[0031] PMOZ lipids, • Approximately 29 mol% to approximately 41 mol% of sterols, preferably cholesterol, • One or more nucleic acids, preferably mRNA and Includes.

[0032] In another embodiment, lipid nanoparticles are preferably • Approximately 49 mol% or approximately 59 mol% of ionizable lipids, • Approximately 5 mol% or approximately 7.5 mol% of DPhyPE, • Approximately 2.5 mol% or approximately 5 mol% of phosphatidylserine, preferably DPhyPS, · About 0.5 mol% to about 1 mol%, preferably about 1 mol% of PMOZ lipid, preferably a PMOZ lipid that does not contain a sulfur group (-S-), more preferably a PMOZ lipid containing a linker group [linker] that is (C(O)CH2CH2C(O)NH), even more preferably (i) n = 50, that is, "PMOZ4" having 50 monomer repetitions

[0033]

Chemical formula

[0034] or (ii) n = 115, that is, "PMOZ4" having 115 monomer repetitions

[0035]

Chemical formula

[0036] and a PMOZ lipid that is · About 29 mol% to about 41 mol% of cholesterol, and · One or more nucleic acids, preferably mRNA are included.

[0037] In another embodiment, the lipid nanoparticle preferably contains one or more nucleic acids, preferably mRNA, and the lipid composition of the lipid nanoparticle is (i) About 49 mol% of C24, about 40 mol% of cholesterol, about 7.5 mol% of DPhyPE, about 2.5 mol% of DPhyPS, and about 1 mol% of PMOZ4 having n = 50, that is, 50 monomer repetitions (ii) About 59 mol% of C24, about 30 mol% of cholesterol, about 7.5 mol% of DPhyPE, about 2.5 mol% of DPhyPS, and about 1 mol% of PMOZ4 having n = 50, that is, 50 monomer repetitions (iii) Approximately 49 mol% C24, approximately 40.5 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 0.5 mol% PMOZ4 having n=115, i.e., 115 monomer repeats. (iv) PMOZ4 having approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=115, i.e., 115 monomer repeats. It is selected from the group consisting of the following.

[0038] In another embodiment, the lipid nanoparticles preferably comprise one or more nucleic acids, preferably mRNA, and the lipid composition of the lipid nanoparticles is (i) Approximately 59 mol% C28, approximately 30 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats. (ii) PMOZ4 having approximately 49 mol% C28, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., 50 monomer repeats. It is selected from the group consisting of the following.

[0039] In another embodiment, the lipid nanoparticles preferably comprise one or more nucleic acids, preferably mRNA, and about 49 mol% C29, about 40 mol% cholesterol, about 7.5 mol% DPhyPE, about 2.5 mol% DPhyPS, and about 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats.

[0040] In another embodiment, lipid nanoparticles are preferably • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids represented by formula (II) shown below and above, more preferably ionizable lipid C24, ionizable lipid C28, or ionizable lipid C29, • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, • Approximately 2.5 mol% to approximately 5 mol% of phosphatidylserine, preferably DPhyPS, • Approximately 25 mol% to approximately 45 mol% of sterols, preferably cholesterol, • Approximately 1 mol% to approximately 2 mol% of PEG lipids, preferably DMG-PEG2000, • One or more nucleic acids, preferably mRNA and Includes.

[0041] In another embodiment of the first aspect, the antigen is derived from a tumor antigen, a pathogenic antigen, an allergen antigen, or an autoimmune autoantigen, preferably from a tumor antigen. In another embodiment of the first aspect, the amount of phosphatidylserine is 9 mol% or less, preferably 5 mol% or less, of the total molar amount of all lipid excipients in the composition.

[0042] In another embodiment of the first aspect, the carrier composition is a lipid nanoparticle composition. In yet another embodiment, the lipid nanoparticle composition is (i) Preferably an ionizable lipid represented by formula (II), more preferably C24, C28 or C29, preferably C24, (ii) Steroids and, (iii) A further phospholipid (preferably the further phospholipid is DPhyPE) added to phosphatidylserine, which is preferably DPhyPS, (iv) Preferably a polymer-bound lipid represented by formula (I), more preferably "PMOZ4" It also includes.

[0043] In another embodiment, the present invention relates to a method for delivering a vaccine composition comprising at least one nucleic acid encoding at least one antigen or a fragment or variant thereof to the spleen or lymph nodes, wherein the carrier composition comprises the phospholipid phosphatidylserine compared to vaccine compositions that do not contain phosphatidylserine. Generally, in certain embodiments, the disclosure relates to directing LNPs containing mRNA to the lymphoid system, specifically focusing on secondary lymphoid organs, particularly the spleen. In various embodiments, the targeted cells are those present in lymph nodes or the splenic cells themselves. Furthermore, the targets may be antigen-presenting cells or dendritic cells, such as professional antigen-presenting cells within the spleen. As a result, the RNA compositions or formulations disclosed herein may be used to deliver RNA to these specific target cells. The lymphoid system is an integral part of both the circulatory and immune systems and includes a network of vessels that transport lymph. This encompasses lymphoid organs, vascular networks, and circulating lymph. Primary lymphoid organs, such as the thymus and bone marrow, produce lymphocytes from immature progenitor cells, while secondary lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and induce adaptive immune responses. Lipid-based RNA delivery systems tend to accumulate naturally in the liver due to the discontinuous nature of the hepatic vascular system or lipid metabolism. In specific embodiments, the site where RNA expression is intended is the liver and its corresponding tissues. In a more specific preferred embodiment, the site where RNA expression is intended is the spleen. In a more specific preferred embodiment, the site where RNA expression is intended is the lymph node.

[0044] In a second embodiment, the present invention relates to a pharmaceutical composition comprising a vaccine composition according to the first embodiment and a pharmaceutically acceptable carrier, diluent or excipient, preferably the pharmaceutical composition is a sterile solid composition for reconstitution with a sterile liquid carrier, the composition further comprising one or more inactive components selected from pH modifiers, bulking agents, stabilizers, nonionic surfactants and antioxidants, and the sterile liquid carrier is an aqueous carrier.

[0045] In a third embodiment, the present invention relates to a vaccine composition according to a first embodiment or a pharmaceutical composition according to a second embodiment for use in the treatment or prevention of infectious diseases; cancer or neoplastic diseases, disorders or conditions; liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis and liver cancer; allergies; or autoimmune diseases, disorders or conditions in a subject. In a very preferred embodiment of the third embodiment of the present invention, the present invention relates to a vaccine composition for use in the treatment or prevention of cancer or neoplastic diseases.

[0046] In a fourth embodiment, the present invention relates to a kit or parts kit comprising a vaccine composition according to the first embodiment or a pharmaceutical composition according to the second embodiment, wherein the kit or parts kit may optionally include a liquid medium for dissolution and may also include technical instructions providing information on the use and dosage of the components.

[0047] In a fifth embodiment, the present invention relates to a method for treating or preventing cancer or neoplastic diseases, disorders or conditions; infectious diseases; liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis and liver cancer; allergies; or autoimmune diseases, disorders or conditions in a subject, the method being: a) A step of preparing a vaccine composition according to the first embodiment, a pharmaceutical composition according to the second embodiment, or a kit or parts kit according to the fourth embodiment. b) The step of applying or administering a vaccine composition or pharmaceutical composition or kit or parts kit to a target tissue or living organism. Includes.

[0048] In a sixth embodiment, the present invention relates to a method for inducing an immune response in a subject, the method comprising administering to the subject an amount effective in producing an antigen-specific immune response in the subject of a vaccine composition of the first embodiment or a pharmaceutical composition of the second embodiment.

[0049] In a seventh embodiment, the present invention relates to the use of a vaccine composition according to the first embodiment, a pharmaceutical composition according to the second embodiment, or a kit or parts kit according to the fourth embodiment, the use of which is for the purpose of (i) inducing an immune response, (ii) inducing an antigen-specific T cell response, or preferably (iii) inducing a CD8+ T cell response in a subject.

[0050] In another embodiment, the present invention relates to a method for inducing interferon (IFN) production, comprising administering the lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention to a target subject, wherein IFN production increases after administration of the lipid nanoparticles, pharmaceutical composition, or kit or parts kit of the present invention.

[0051] In other embodiments, the present invention relates to a method comprising administering to a subject of interest an amount sufficient to induce an immune response in the subject, preferably an immune response involving cytokine production, and more preferably an immune response involving the regulation of type I, type II, and / or type III IFNs.

[0052] In other embodiments, the present invention relates to a method comprising administering to a subject of interest an amount sufficient to induce an immune response in the subject, preferably the immune response involving the production of type I IFN, more preferably the immune response involving the regulation of IFNa, IFNb, IFNe, IFNk, or IFN, most preferably IFNa and / or IFNb.

[0053] In another embodiment, the present invention relates to a novel polymer-bound lipid useful for delivering nucleic acids to living cells. In a specific embodiment, the polymer-bound lipid is given by formula (I): [P]-[Linker]-[L] Formula (I) The compound represented by, or its pharmaceutically acceptable salt, prodrug, tautomer or stereoisomer, [P] represents at least one polyoxazoline (POZ) monomer unit.

[0054] [ka]

[0055] It is a homopolymer portion containing, Here, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, and n has an average value in the range of about 45 to about 55, preferably about 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. [linker] is any linker group, [L] represents the lipid portion.

[0056] In another embodiment, the polymer-bound lipid is [P], Poly(2-methyl-2-oxazoline) (PMOZ)

[0057] [ka]

[0058] Poly(2-ethyl-2-oxazoline) (PEOZ)

[0059] [ka]

[0060] Poly(2-propyl-2-oxazoline) (PPOZ)

[0061] [ka]

[0062] Poly(2-butyl-2-oxazoline) (PBOZ)

[0063] [ka]

[0064] Poly(2-isopropyl-2-oxazoline) (PIPOZ)

[0065] [ka]

[0066] Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), and Poly(2-dimethylamino-2-oxazoline)(PDMAOx) It comprises a heteropolymer or homopolymer moiety containing multiple monomer units selected from the group consisting of, Preferably, [P] is a homopolymer moiety containing multiple PMOZ or PEOZ monomer units, and more preferably, [P] contains or consists solely of multiple PMOZ monomer units. (i)n has an average value in the range of about 45 to about 55, preferably about 50, or (ii)n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa.

[0067] In a very preferred embodiment, the homopolymer moiety [P] is selected from the group consisting of PMeOz50 (polymethyloxazoline or poly(2-methyl-2-oxazoline) having 50 repeats), PEtOz50 (polyethyloxazoline having 50 repeats), PMeOz25 (polymethyloxazoline having 25 repeats), and PEtOz25 (polyethyloxazoline having 25 repeats), preferably PMeOz50 (polymethyloxazoline or poly(2-methyl-2-oxazoline) having 50 repeats).

[0068] In another embodiment, the polymer-bound lipid is selected from the group consisting of POZ-monoacylglycerol bonds, POZ-diacylglycerol bonds, POZ-dialkyloxypropyl bonds, POZ-steroid or POZ-sterol bonds, POZ-phospholipid bonds, POZ-ceramide bonds, and mixtures thereof. In a further embodiment, the lipid moiety [L] comprises at least one linear or branched saturated or unsaturated alkyl chain containing 6 to 30 carbon atoms, preferably, the lipid moiety [L] comprises at least one linear or branched saturated alkyl chain, the alkyl chain may be optionally interrupted by one or more biodegradable groups, and may further / or optionally contain one terminal biodegradable group, the biodegradable group being, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 Alkenyl), zwitterionic linker, ester-free linker portion and ester-containing linker portion (-C(O)O- or -OC(O)-), amide (-C(O)NH-), disulfide (-SS-), carbonyl (-C(O)-), ether (-O-), thioether (-S-), oxime (e.g., -C(H)=NO- or -ON=C(H)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), (-NHC(O)CH2CH2C(O)-), -C(R5)=N-, -N=C(R 5 )-,-C(R 5 )=NO-, -ON=C(R 5 )-, -OC(O)O-, -C(O)N(R 5 ), -N(R 5 )C(O)-, -C(S)(NR 5 )-, (NR 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R 5)2O-, -C(O)(CR 3 R 4 )C(O)O-, or -OC(O)(CR 3 R 4 )C(O)-, carbonate (-OC(O)O-), nitrogen (N), succinoyl, succinate, phosphate ester (-O-(O)POH-O-), cyclic compounds, heterocyclic compounds, piperidine, pyrazine, pyridine, piperazine, and sulfonic acid esters, and combinations thereof, selected from the group, where R 3 , R 4 and R 5 These are independently H or alkyl (e.g., C1-C4 alkyl).

[0069] In further embodiments, the lipid moiety [L] comprises two linear unsaturated alkyl chains containing 6 to 30 carbon atoms, preferably the lipid moiety [L] comprises at least one linear or branched saturated alkyl chain, the alkyl chain may be optionally interrupted by one or more biodegradable groups, and may further / or optionally contain one terminal biodegradable group, the biodegradable group being, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 Alkenyl), zwitterionic linker, ester-free linker moiety, and ester-containing linker moiety (-C(O)O- or -OC(O)-), amide (-C(O)NH-), disulfide (-SS-), carbonyl (-C(O)-), ether (-O-), thioether (-S-), oxime (e.g., -C(H)=NO- or -ON=C(H)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), (-NHC(O)CH2CH2C(O)-), -C(R5)=N-, -N=C(R 5 )-,-C(R 5 )=NO-, -ON=C(R 5 )-, -OC(O)O-, -C(O)N(R 5 ), -N(R 5)C(O)-, -C(S)(NR 5 )-, (NR 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, or -OC(O)(CR 3 R 4 )C(O)-, carbonate (-OC(O)O-), nitrogen (N), succinoyl, succinate, phosphate ester (-O-(O)POH-O-), cyclic compounds, heterocyclic compounds, piperidine, pyrazine, pyridine, piperazine, and sulfonic acid esters, and combinations thereof, selected from the group, where R 3 , R 4 and R 5 These are independently H or alkyl (e.g., C1-C4 alkyl).

[0070] In a further embodiment, the lipid moiety [L] comprises two linear unsaturated alkyl chains, each having 14 carbon atoms. In a more preferred embodiment, the polymer-bound lipid comprises a lipid moiety [L] containing ditetradecylamine and a linker group [linker], preferably the linker group [linker] is (-NHC(O)CH2CH2C(O)-). In a more preferred embodiment, the lipid moiety [L] contains ditetradecylamine, and the linker moiety [linker], preferably (-NHC(O)CH2CH2C(O)-), forms an amide bond by bonding to the N atom of ditetradecylamine. In a more preferred embodiment, the polymer-bound lipid comprises a linker (-NHC(O)CH2CH2C(O)-), and the linker is oriented such that a carboxamide bond is formed through bonding to the N atom of ditetradecylamine.

[0071] In a more preferred embodiment, the polymer-bound lipid comprises a lipid moiety [L] containing ditetradecylamine and a linker group [linker], preferably the linker group [linker] is (C(O)CH2CH2C(O)NH). In a more preferred embodiment, the lipid moiety [L] contains ditetradecylamine, and the linker moiety [linker], preferably (-NHC(O)CH2CH2C(O)-), forms an amide bond by bonding to the N atom of ditetradecylamine.

[0072] In a very preferred embodiment, the lipid portion [L] is the lipid portion used in "PMOZ4". In a very preferred embodiment, the linker portion [linker] is the linker portion used in "PMOZ4".

[0073] In a more preferred embodiment, the present invention relates to a polymer-bound lipid having a lipid moiety [L] which is a lipid moiety used in "PMOZ4". In another most preferred embodiment, the present invention relates to a polymer-bound lipid having a linker moiety [linker] which is a linker used in "PMOZ4".

[0074] [ka]

[0075] In another embodiment, the present invention relates to at least one polyoxazoline (POZ) monomer unit

[0076] [ka]

[0077] We provide novel lipid nanoparticles containing a homopolymer moiety, Here, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, and n has an average value in the range of about 45 to about 55, preferably about 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. Preferably, the homopolymer portion containing multiple monomer units includes poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly(2-propyl-2-oxazoline) (PPOZ), poly(2-butyl-2-oxazoline) (PBOZ), poly(2-isopropyl-2-oxazoline) (PIPOZ), poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), or poly(2-dimethylamino-2-oxazoline) (PDMAOx).

[0078] Most preferably, R is C1 (i.e., CH3 or methyl), which yields polymethyloxazoline or poly(2-methyl-2-oxazoline), i.e., "PMOZ".

[0079] In a further embodiment, the present invention provides vaccine compositions comprising lipid nanoparticles of the present invention, or kits or parts kits comprising ionizable lipids and polymer-bound lipids of the present invention, which are used as pharmaceuticals and / or for the prevention, suppression, treatment and / or improvement of infectious diseases including viral, bacterial or protozoal infections, cancer or neoplastic diseases.

[0080] In a further embodiment, the present invention provides a method for treating or preventing infectious diseases; cancer or neoplastic diseases, disorders or conditions; liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis and liver cancer; allergies; or autoimmune diseases, disorders or conditions, the method being a) A step of preparing a homopolymer moiety containing at least one polyoxazoline (POZ) monomer, preferably a lipid nanoparticle containing a polymer-bound lipid of the present disclosure, a vaccine composition, or a kit or parts kit of the present disclosure, b) A step of applying or administering mRNA, lipid nanoparticles, vaccine composition, or kit or parts kit to tissue or a living organism. Includes.

[0081] In another specific embodiment, the present invention relates to lipid nanoparticles comprising a novel polymer-bound lipid represented by formula (I), a novel ionizable lipid represented by formula (II), phosphatidylserine, and DPhyPE, wherein the lipid nanoparticles are useful for delivering nucleic acids to living cells.

[0082] In one embodiment, the present invention relates to a novel cationic lipid useful for the delivery of nucleic acids to living cells. The cationic lipid is defined by formula (II): R a -AR b Formula (II) It is an ionizable lipid represented by Here, R a teeth,

[0083] [ka]

[0084] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0085] [ka]

[0086] , or -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1Selected from -N(CH3)2, A is -S-, -SS-, -SC(O)-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, or -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon or nitrogen atom, These choices are all independent of one another, However, optionally, R 1 , R 2 and R 5 All are ethane, A is SS-, and R a and R b If they are the same, R 4 teeth

[0087] [ka]

[0088] isn't it. In this respect, Arcaneil is (-C n H 2n- is a term meaning a radical, and thus, for example, "alkanediyl having 2 to 8 carbon atoms" means -C2H4-, -C3H6-, -C4H8-, -C5H 10 -, -C6H 12 -, -C7H 14 -, or -C8H 16 - and is equivalent to an alkanediyl group having the formula. In other words, alkanediyl is a series of divalent radicals of general formula C n H 2n derived from aliphatic hydrocarbons. Unless otherwise specified, these alkanediyls include substituted alkanediyls.

[0089] In another embodiment, when R 1 , R 2 and R 5 are all ethanediyl and A is S-S-, and R a and R b are the same, then R 4 is

[0090]

Chemical formula

[0091] not, or, in one embodiment, the lipid represented by formula (II) is not lipid C23 disclosed in Table 1 of the present specification, nor lipid SS-EC described below (to avoid doubt, in some selected embodiments, the cationic lipid COASTSOME® SS-EC is excluded from embodiments relating to the cationic lipid represented by formula (II)).

[0092] In another aspect, the present invention provides a novel composition incorporating a cationic lipid such as the novel cationic lipid defined above. These cationic lipids and compositions have been found to be particularly effective for the introduction of nucleic acids into living cells. For example, they enable the improvement of RNA (e.g., mRNA) vaccines, i.e., mRNA-based vaccines, against specific infectious diseases or tumors.

[0093] In a further embodiment, the present invention provides for the pharmaceutical use of compositions incorporating cationic lipids and nucleic acid compounds, particularly for use as vaccines, and for methods of vaccination based on these vaccines.

[0094] In another aspect of the present invention, the present invention also provides kits, particularly parts kits, comprising an mRNA compound comprising an mRNA sequence as defined herein and at least one lipid represented by formula (I) or formula (II) as defined herein.

[0095] In another aspect of the present invention, the present invention also provides a pharmaceutical composition comprising lipid nanoparticles of the present disclosure, a kit or parts kit of the present disclosure, or a vaccine composition of the present disclosure, the pharmaceutical composition comprising an effective amount of a cancer antigen or viral antigen, preferably mRNA encoding a cancer antigen, and used for vaccination and / or treatment in a subject.

[0096] In yet another aspect of the present invention, the present invention provides improved lyophilizable lipid nanoparticles having advantageous physiological and chemical properties after lyophilization and reconstitution.

[0097] In yet another embodiment of the present invention, the present invention provides improved lipid nanoparticles having advantageous physiological and chemical properties after freezing and thawing. definition For clarity and readability, the following scientific background information and definitions are provided. Any technical feature referred to or disclosed herein may be part of, or be read in, any, each embodiment and all embodiments of the present invention. Additional definitions and explanations may be provided in the context of this disclosure.

[0098] Unless otherwise defined or required by the specific context, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art in the relevant field.

[0099] Unless the context indicates or requires otherwise, the words “comprise,” “comprises,” and “comprising,” as well as similar expressions, should be interpreted in this specification and in the claims as “including, but not limited to.” For the purposes of the present invention, it should also be understood that the term “consisting of” is considered a preferred embodiment of the term “comprising.” Hereinafter, where a group is defined as containing at least a certain number of embodiments, this also means that it preferably includes a group consisting only of these embodiments.

[0100] Expressions such as "one embodiment," "embodiment," and "specific embodiment" mean that a particular feature, characteristic, or a specific group or combination of features, characteristics, or features, as referred to in combination with each expression, is present in at least one embodiment of the present invention. The appearance of these expressions in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, specific features, characteristics, or features can be combined in any suitable manner in one or more embodiments.

[0101] The singular forms "a," "an," and "the" should be understood as referring to multiple things unless the context explicitly indicates otherwise. In the context of numbers, percentages should be understood as a percentage of the total number of each item. In other cases, unless the context indicates otherwise, percentages should be understood as weight percentages (weight %).

[0102] As used herein, “compound” means a chemical substance, which is a material consisting of molecules having essentially the same chemical structure and properties. For low molecular weight compounds, the molecules are typically identical in terms of their atomic composition and structural arrangement. For high molecular weight or polymer compounds, the molecules of the compound are very similar, but not all of them are necessarily identical. For example, a polymer segment specified to consist of 50 monomer units may also contain individual molecules having, for example, 48 or 53 monomer units.

[0103] The term "molecule" can be used as a synonym for "compound" or an individual (i.e., single) molecule. Any reference to a compound or part having a functional group that can be ionized under physiological conditions should be understood to include the ionized form of the respective compound or part. Conversely, any reference to a compound or part having an ionized functional group that can also exist in a non-ionized form under physiological conditions should be understood to include the non-ionized form of the respective compound or part. For example, a disclosure of a compound having a carboxyl group should be interpreted as referring to each compound having either a non-ionized carboxyl group or an ionized carboxylate group.

[0104] As used herein, “physiological conditions” refers to an aqueous environment having a pH within the pH range known from human physiology, including both extracellular and intracellular conditions. This pH range is approximately pH 1 to pH 9. Depending on the context, physiological conditions may also refer to near-neutral conditions such as approximately pH 5 to pH 8.5, or approximately 5.5 to pH 8.

[0105] Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, that is, amphiphilic compounds that possess lipid-like physical properties. In the context of this invention, the term lipid is considered to encompass lipids.

[0106] In the context of the present invention, the phrase "selected from the group consisting of..." followed by a particular group of elements (e.g., "A, B, and C") is not intended to be limited to that group within the context of the present invention. In other words, such a phrase does not indicate that the disclosure is closed to elements not listed; that is, alternative meanings are also included in the group following this term. Therefore, in the context of the present invention, the phrase "selected from the group consisting of..." followed by a particular group of elements (i.e., "A, B, and C") should be understood as "selected from A, B, and C" or "A, B, or C," encompassing other structurally and functionally related and unrelated elements that are not mentioned.

[0107] The term "approximately" is used when a parameter or value does not necessarily have to be identical, i.e., 100% the same. Thus, "approximately" means that a parameter or value may deviate by 0.1% to 20%, preferably 0% (i.e., preferably identical to the value given after "approximately") and also preferably 0.1% to 10%; in particular, by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Those skilled in the art will understand, for example, that a particular parameter or value may vary slightly depending on the method used to determine the parameter. For example, if a particular parameter or value is defined herein as having a length of, for example, "about 1000 nucleotides," then its length may deviate by 0.1% to 20%, preferably 0.1% to 10%, in particular by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Thus, those skilled in the art will see that in specific examples, its length may deviate by 1 to 200 nucleotides, preferably 1 to 100 nucleotides, in particular by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nucleotides. In other words, the term "approximately" preferably refers to the exact value or parameter given after the term "approximately," but also preferably refers to a value or parameter that deviates as described above (i.e., approximately 1 mol% may mean 1%, but this value may also deviate as described above).

[0108] The term "cationic" means that each structure has a positive charge either permanently or not permanently, depending on certain conditions such as pH, unless the different meanings are clear from the specific context. Thus, the term "cationic" encompasses both "permanently cationic" and "cationizable" or "ionizable", and vice versa. As used herein, the term "cationizable" means that a compound, or a group or atom, is positively charged in a low pH environment and uncharged in a high pH environment. Even in a non-aqueous environment where the pH value cannot be determined, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or active hydrogen ion. This depends on the individual properties of the cationizable or polycationizable compound, particularly the pK a of each cationizable group or atom that is charged or uncharged at its respective pH or hydrogen ion concentration. In a dilute aqueous environment, the fraction of cationizable compounds, groups or atoms having a positive charge can be estimated using the well-known Henderson-Hasselbalch equation by those skilled in the art. For example, if a compound or moiety is cationizable, it is preferably positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably a pH value of 9 or less, 8 or less, 7 or less, most preferably a physiological pH value, such as about 7.3 to 7.4, i.e., under physiological conditions, particularly under the physiological salt conditions of cells in vivo. In a preferred embodiment, the cationizable compound or moiety is mainly neutral at a physiological pH value, such as about 7.0 to 7.4, but is preferably positively charged at a lower pH value. In some embodiments, the pK a of the cationizable compound or moiety preferably ranges from about 5 to about 7. In some embodiments, the protonatable lipid has a pK a of the protonatable group in the range of about 4 to about 11, such as a pK a of about 5 to about 7.

[0109] Unless a different meaning is evident from the specific context, the term "cationic" means that each structure has a positive charge, either permanently or not permanently, depending on certain conditions such as pH. Therefore, the term "cationic" encompasses both "permanently cationic" and "cationizable." For example, a compound or moiety having a primary, secondary, or tertiary amino group can exist in a predominantly positively charged state under physiological conditions, and is therefore cationic, and more specifically, cationizable.

[0110] As used herein, “permanently cationic” means that each compound, or group or atom, is positively charged at any pH value or hydrogen ion activity in its environment. Often, the positive charge arises from the presence of a quaternary nitrogen atom. If a compound has multiple such positive charges, it may be called permanently polycationic, a subcategory of permanently cationic compounds.

[0111] Similarly, the terms “anionic,” “anionizable,” and “persistently anionic” are used to have similar meanings to “cationic,” “cationizable,” and “persistently cationic,” except that the charge of the respective compound, group, or atom is negative rather than positive.

[0112] The term "neutral," when applied to compounds such as lipids or steroids, or to a group or part thereof, means that it is neither cationic nor anionic, for example, a hydrocarbon, that does not have functional groups that can be ionized under physiological conditions; or it means that it is both cationic and anionic under typical physiological conditions, i.e., zwitterionic, such as a typical natural phosphatidylcholine.

[0113] As used herein, “lipids” refer to a group of organic compounds characterized by being derivatives of fatty acids (e.g., esters) and generally insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids” including fats, oils, and waxes; (2) “compound lipids” including phospholipids and glycolipids; and (3) “derived lipids” such as steroids. With respect to glycolipids, in certain embodiments, the LNP comprises a glycolipid (e.g., monosialoganglioside GM1).

[0114] In this context, the prefix "poly-" refers to multiple atoms or groups in a compound that possess their respective properties. When enclosed in parentheses, the presence of multiple atoms or groups is not mandatory. For example, (poly)cationicity means cationic and / or polycationic. However, the absence of the prefix should not be interpreted as excluding multiple atoms or groups. For instance, a polycationic compound is also a cationic compound and can be referred to as such.

[0115] The term “nucleic acid” means any compound comprising DNA or RNA, or any compound comprising them. The term may also be used for polynucleotides and / or oligonucleotides. Whenever a nucleic acid or nucleic acid sequence encoding a particular protein and / or peptide is referred to herein, such nucleic acid or nucleic acid sequence preferably also includes a regulatory sequence that enables its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the particular protein or peptide, in a suitable host, e.g., human.

[0116] In a particularly preferred embodiment, the artificial nucleic acid, nucleic acid, or RNA is mRNA, more preferably isolated mRNA. mRNA technology is particularly preferred in the context of the present invention because it allows for controlled dosage, transient and controlled expression, complete degradation of mRNA after protein synthesis, and does not pose a risk of insertional mutations compared to viral systems.

[0117] In the context of the present invention, the term "nucleoside modification" refers to nucleic acids such as mRNA compounds or molecules that contain nucleosides not normally present in natural mRNA, preferably non-natural nucleosides. In particular, this term preferably refers to mRNA nucleosides other than adenine, guanine, cytosine, uracil, and thymine.

[0118] The term "nucleoside" generally refers to a compound consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base. The term "nucleotide" generally refers to a nucleoside that contains a phosphate group attached to a sugar.

[0119] A "peptide" refers to an oligomer or polymer of at least two amino acid monomers linked by peptide bonds. This term does not limit the length of the amino acid polymer chain. A peptide may contain, for example, fewer than 50 monomer units. Longer peptides, typically having 50 to 600 monomer units, or more specifically 50 to 300 monomer units, are also called polypeptides.

[0120] A "protein" is defined as comprising or consisting of one or more polypeptides folded into a three-dimensional form that facilitate biological functions. Immune System: The immune system can protect an organism from infection. When a pathogen breaches the organism's physical barriers and invades, the innate immune system provides an immediate but nonspecific response. If the pathogen evades this innate response, the vertebrate develops an adaptive immune system, which is a second layer of defense. Here, the immune system adapts its response to improve its recognition of the pathogen during infection. Thus, even after the pathogen has been eliminated, this improved response is retained in the form of immunological memory, allowing the adaptive immune system to launch a faster and more powerful attack each time it encounters the pathogen. According to this, the immune system consists of the innate immune system and the adaptive immune system. Each of these two parts contains so-called humoral and cellular components.

[0121] Immune Response: An immune response can typically be either a specific response of the adaptive immune system to a particular antigen (a so-called specific or adaptive immune response) or a nonspecific response of the innate immune system (a so-called nonspecific or innate immune response). This invention relates to the core of the specific response of the adaptive immune system (adaptive immune response). In particular, this invention relates to an adaptive immune response to infection by a virus, such as the influenza virus. Preferably, this invention also relates to an immune response following the administration of a cancer vaccine to a cancer patient. The specific response may be supported by an additional nonspecific response (innate immune response). Therefore, this invention also relates to compounds for the simultaneous stimulation of the innate and adaptive immune systems to induce an efficient adaptive immune response.

[0122] Adaptive Immune System: The adaptive immune system consists of highly specialized systemic cells and processes that eliminate or prevent pathogenic proliferation. Adaptive immune responses provide the vertebrate immune system with the ability to recognize and remember (generate immunity to) specific pathogens and initiate a stronger attack each time it encounters them. This system is highly adaptable due to somatic hypermutation (processes with increased frequency of somatic mutations) and V(D)J gene rearrangements (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Since gene rearrangements result in irreversible changes in the DNA of each cell, all of that cell's progeny (offspring) inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are key to longevity-specific immunity. Immune network theory is a theory of how the adaptive immune system works, based on the interaction between the variable regions of T cells, B cells, and molecules produced by T cells and B cells that possess these variable regions.

[0123] Adaptive Immune Response: Adaptive immune responses are typically understood to be antigen-specific. Antigen specificity allows for the generation of a response adapted to a specific antigen, pathogen, or pathogen-infected cell. The ability to initiate these adapted responses is maintained in the body by "memory cells." If a pathogen infects the body more than once, these specific memory cells are used to rapidly eliminate this pathogen. In this context, the first step of an adaptive immune response is the activation of different immune cells that can induce an antigen-specific immune response by naive antigen-specific T cells or antigen-presenting cells. This occurs in lymphoid tissues and organs through which naive T cells constantly pass. Cell types that can act as antigen-presenting cells include, among others, dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in inducing an immune response. Dendritic cells take up antigens by phagocytosis and macropinocytosis, and are stimulated, for example, by contact with a foreign antigen, to migrate to local lymphoid tissues where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced to express MHC molecules by infectious agents or other appropriate stimuli. The unique ability of B cells to bind soluble protein antigens via their receptors and internalize them can also be important in inducing T cells. Antigen presentation on MHC molecules leads to T cell activation, thereby inducing their proliferation and differentiation into armed effector T cells. The most important functions of effector T cells are the killing of infected cells by CD8+ cytotoxic T cells, the activation of macrophages by Th1 cells which together constitute cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibodies and thus drive the humoral immune response. T cells do not directly recognize and bind to antigens, but instead recognize antigens by T cell receptors which recognize short peptide fragments of pathogen-inducing protein antigens that are bound to MHC molecules on the surface of other cells, for example.

[0124] Cellular immunity / cellular immune response: Cellular immunity typically relates to the activation of macrophages, natural killer cells (NKs), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. More generally, cellular immunity relates to the activation of cells in the immune system, without the involvement of antibodies. Cellular immune responses are characterized by the activation of antigen-specific cytotoxic T lymphocytes that can induce apoptosis in somatic cells that exhibit antigen epitopes on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells exhibiting tumor antigens; the activation of macrophages and natural killer cells so that they can destroy pathogens; and the stimulation of cells to secrete various cytokines that affect the function of other cells involved in adaptive and innate immune responses.

[0125] Humoral immunity / humoral immune response: Humoral immunity typically refers to antibody production and any associated auxiliary processes. Humoral immune responses typically may be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. Humoral immunity may also typically refer to the effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation, and phagocytic opsonin enhancement and pathogen elimination.

[0126] Innate immune system: Also known as the nonspecific immune system, the innate immune system includes cells and mechanisms that nonspecifically protect the host from infection by other organisms. This means that cells in the innate system recognize and respond to pathogens in a general way, but unlike the adaptive immune system, they do not confer persistent immunity or protective immunity to the host. The innate immune system responds to various factors, including pathogen-associated molecular pattern (PAMP) receptors, ligands for Toll-like receptors (TLRs), lipopolysaccharides, TNF-alpha, CD40 ligands, cytokines, monokines, lymphokines, interleukins, or chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and IFN-alpha. It can be activated by other adjuncts such as IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, ligands for human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, ligands for mouse Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13, ligands for NOD-like receptors, ligands for RIG-I-like receptors, immunostimulant nucleic acids, immunostimulant RNA (isRNA), CpG-DNA, antibacterial agents, or antiviral agents. Typically, the innate immune response includes the recruitment of immune cells to the site of infection through the production of chemical factors, including specialized chemical mediators called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, blood, and lymph by specialized leukocytes; activation of the adaptive immune system through a process known as antigen presentation; and / or acting as a physical and chemical barrier to infectious agents.

[0127] Adjuvants / Adjuvant Components: An adjuvant or adjuvant component is, in a broad sense, a drug or composition that can modify, for example, enhance the effectiveness of other agents, such as drugs or vaccines (e.g., pharmacologically or immunologically). Conventionally, in the context of the present invention, this term refers to a compound or composition that acts as an immunostimulant carrier or auxiliary and / or other pharmaceutically active compound. This term should be interpreted broadly and refers to a wide range of substances that can increase the immunogenicity of an antigen incorporated with or co-administered with the adjuvant in question. In the context of the present invention, adjuvants preferably enhance the specific immunogenic effect of the activator of the present invention. Typically, "adjuvant" and "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be divided into, for example, immunostimulants, antigenic delivery systems, or even combinations thereof.

[0128] The term "adjuvant" is typically understood to refer to a drug that does not confer immunity itself. Adjuvants nonspecifically support the immune system to enhance antigen-specific immune responses, for example, by promoting antigen presentation to the immune system or induction of nonspecific innate immune responses. Furthermore, adjuvants can modulate antigen-specific immune responses, preferably by shifting a dominant Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, adjuvants can favorably modulate cytokine expression / secretion, antigen presentation, type of immune response, etc.

[0129] Immunostimulating RNA: In the context of this invention, immunostimulating RNA (isRNA) is typically RNA capable of inducing the innate immune response itself. It usually does not possess an open reading frame and therefore does not provide peptide antigens or immunogens, but it induces an innate immune response by binding, for example, to a specific type of Toll-like receptor (TLR) or other suitable receptor. However, mRNA that does possess an open reading frame and encodes peptides / proteins (e.g., antigenic function) can also induce an innate immune response.

[0130] As used herein, the term “antibody” includes both intact antibodies and antibody fragments. Typically, an intact “antibody” is an immunoglobulin that specifically binds to a particular antigen. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgE, IgA, and IgD. Typically, an intact antibody is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a “light” chain and a “heavy” chain. An “antibody fragment” includes a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; tribes; tetras; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, an antibody fragment includes an isolated fragment, an "Fv" fragment consisting of a heavy chain variable region and a light chain variable region, a recombinant single-chain polypeptide molecule ("ScFv protein") in which the light chain variable region and the heavy chain variable region are linked by a peptide linker, and a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region. Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs). Suitable antibodies that can be encoded by the therapeutic RNA of the present invention include monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, Fab fragments, or bispecific antibodies. In the context of the present invention, an antibody may be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0131] In the context of this invention, the term “antigen” typically refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and can induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or include, a peptide or protein that can be presented to T cells by MHC. In the sense of this invention, an antigen may be a product of translation of a provided nucleic acid molecule, preferably mRNA as defined herein. In this context, peptide and protein fragments, variants, and derivatives containing at least one epitope are also understood as antigens. Thus, the term “antigen” as used herein is intended to refer to a substance that is recognized and understood by those skilled in the art, and can be recognized, for example, by the immune system, preferably the adaptive immune system, and can induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or include, a peptide or protein that can be presented to T cells by MHC. Peptide or protein fragments, variants, and derivatives derived from, for example, cancer antigens, containing at least one epitope can also be understood as antigens. In the context of the present invention, the antigen may be the product of translation of a provided therapeutic RNA (e.g., coding RNA, replicon RNA, mRNA). The term “antigenic peptide or protein” is intended to be recognized and understood by those skilled in the art and to refer to a peptide or protein derived from an (antigenic) protein that can stimulate the body’s adaptive immune system to produce an adaptive immune response, for example. Thus, “antigenic peptide or protein” includes at least one epitope or antigen (e.g., tumor antigen, viral antigen, bacterial antigen, protozoan antigen) of the protein from which it is derived. In the context of the present invention, the antigen may be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0132] In the context of nucleic acids, that is, with respect to nucleic acids "derived from" another nucleic acid, the term "derived from" as used herein means that a nucleic acid derived from another nucleic acid shares at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity with the nucleic acid from which it is derived. Those skilled in the art will notice that sequence identity is typically calculated for nucleic acids of the same kind, i.e., DNA sequences or RNA sequences. Thus, when DNA is "derived from" RNA, or RNA is "derived from" DNA, it is understood that in the first step, the RNA sequence is converted to the corresponding DNA sequence (in particular by substituting U with T throughout the sequence), or vice versa, a DNA sequence is converted to the corresponding RNA sequence (in particular by substituting T with U throughout the sequence). The sequence identity of the DNA sequence or the RNA sequence is then determined. Preferably, a nucleic acid “derived from” also refers to a nucleic acid that is modified compared to the nucleic acid from which it is derived, for example, to further increase RNA stability and / or to prolong and / or increase protein production. In the context of amino acid sequences, the term “derived from” means that an amino acid sequence derived from (another) amino acid sequence shares at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity with the amino acid sequence from which it is derived.

[0133] Epitope (also called "antigen determinant"): In the context of the present invention, a T cell epitope or portion of a protein may include a fragment having a length of about 6 to about 20 or more amino acids, for example, preferably about 8 to about 10, for example 8, 9 or 10 (or even 11 or 12 amino acids), which is processed and presented by an MHC class I molecule, or a fragment having a length of about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more, which is processed and presented by an MHC class II molecule, and these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule.

[0134] A B cell epitope is typically a fragment located on the outer surface of a (natural) protein or peptide antigen, as defined herein, which is recognizable by an antibody, i.e., in its natural form, and preferably has 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids.

[0135] Such epitopes of proteins or peptides may be further selected from any of the variants of such proteins or peptides referred herein. In this context, the antigenic determinant may be a three-dimensional structure or discontinuous epitope composed of segments of the protein or peptide as defined herein, which are discontinuous in the amino acid sequence of the protein or peptide as defined herein but can be assembled into a three-dimensional structure consisting of a single polypeptide chain or into a continuous or linear epitope.

[0136] An "immunotogenic composition" is a composition that promotes immune tolerance to an antigen in cells or cell lines, and the antigen may be a self-antigen or a non-self-antigen. In other words, there is no immune response to the antigen, or the immune response is reduced. Conversely, the vaccine composition according to the present invention induces an immune response to a specific antigen, i.e., an antigen encoded by at least one nucleic acid. The antigen may also be a self-antigen or a non-self-antigen, and the overall objective of the vaccine composition of the present invention is to produce a (strong) immune response to this antigen, while the overall objective of the immunotolerogenic composition is to suppress the immune response to this antigen, at least partially, and in the best case, completely.

[0137] An "immunotogenic nucleic acid" is a nucleic acid that promotes immune tolerance to an antigen in a cell or cell line, and the nucleic acid may be a chemically modified mRNA and / or may encode an immunotolerogenic polypeptide. Conversely, at least one nucleic acid according to the present invention encodes at least one antigen or fragment thereof to which a (potent) immune response is desired and induced upon administration.

[0138] An "immunotomogenic polypeptide" is a polypeptide that promotes immune tolerance in a cell or cell line by acting on an underlying pathway, particularly by inhibiting mediators underlying such pathways, typically by reducing the immune response. Therefore, an immunotolerogenic polypeptide may be an inhibitor of mTOR, IL-2, IL-10, or an antibody reactive to CD3 or CD40. Conversely, at least one antigen or fragment thereof according to the present invention does not promote immune tolerance in a cell or cell line, but rather induces a (potent) immune response to itself.

[0139] The immunotolerogenic composition may, in particular, contain immunotolerogenic nucleic acids, which promote immune tolerance as described above. The immunotolerogenic composition may further contain a specific antigen, resulting in the absence or reduction of an immune response to this specific antigen due to the presence of the immunotolerogenic nucleic acid. Conversely, since the overall objective of the vaccine composition of the present invention is to elucidate a (strong) immune response to at least one antigen or fragment thereof that is encoded (and not to block or reduce an immune response to a co-administered antigen, as is the objective of the immunotolerogenic composition), the vaccine composition according to the present invention, in a preferred embodiment, does not contain an antigen but naturally contains at least one nucleic acid encoding at least one antigen or fragment thereof. In yet another preferred embodiment, the vaccine composition according to the present invention contains at least one nucleic acid encoding at least one antigen or fragment thereof as the sole payload and therefore cannot contain an antigen (the immunotolerogenic composition discussed in this paragraph contains an antigen in addition to an immunotolerogenic nucleic acid).

[0140] According to the present invention, the term “vaccine” refers to a pharmaceutical preparation (pharmaceutical composition) or product that, when administered, induces an immune response, particularly a cellular immune response and / or a humoral immune response, that is suitable for recognizing and attacking diseased cells, such as pathogens or cancer cells. Vaccines may be used for the prevention or treatment of disease. The terms “preventive vaccine” or “preventive agent”: Both terms refer to any agent that, when administered to a subject, has a preventive effect and / or induces a desired biological and / or pharmacological effect. In connection therewith, this definition also applies to the term “preventive vaccination.” The terms “therapeutic vaccine” or “therapeutic agent”: Both terms refer to any agent that, when administered to a subject, has a therapeutic and / or preventive effect and / or induces a desired biological and / or pharmacological effect. In connection therewith, this definition also applies to the term “therapeutic vaccination.” In this regard, the use of a pharmaceutical composition, a preventive or therapeutic vaccine or a preventive or therapeutic agent is a pharmaceutical intended to therapeutically or preventively induce an immune response in a subject that requires it. In this regard, the term "vaccine" is typically understood as a preventive or therapeutic material that provides at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to produce an adaptive immune response.

[0141] In the context of this invention, the term “antigen-donating mRNA” is typically an mRNA having at least one open reading frame that can be translated by the cell or organism to which the mRNA is donated. The product of this translation is a peptide or protein that can act as an antigen, preferably an immunogen. The product may also be a fusion protein composed of two or more immunogens, for example, a fusion protein consisting of two or more epitopes, peptides, or proteins derived from the same or different viral proteins, where the epitopes, peptides, or proteins may be linked by linker sequences.

[0142] The term “artificial mRNA” (sequence) can typically be understood as an mRNA molecule that does not exist in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to their individual sequences (which do not exist in nature) and / or other modifications, such as structural modifications of nucleotides that do not exist in nature. Typically, artificial mRNA molecules can be designed and / or produced by genetic engineering methods that correspond to a desired artificial sequence (heterogeneous sequence) of nucleotides. In this context, the artificial sequence is usually a sequence that cannot exist in nature; that is, the artificial sequence differs from the wild-type sequence by at least one nucleotide. The term “wild-type” can be understood as a sequence that exists in nature. Furthermore, the term “artificial nucleic acid molecule” is not limited to meaning “a single molecule,” but is typically understood to include an ensemble of identical molecules. Thus, the term can refer to multiple identical molecules contained in an aliquot.

[0143] In a very preferred embodiment, the nucleic acid of the present invention is "isolated" mRNA. "Isolated": As used herein, the term "isolated" means, with respect to a nucleic acid molecule, preferably isolated mRNA, or polypeptide, that the nucleic acid molecule, preferably isolated mRNA, or polypeptide is in a state other than its natural environment, e.g., away from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule, preferably isolated mRNA, or polypeptide is substantially free from other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule, preferably isolated mRNA, or polypeptide may be in a highly purified form, i.e., ultrapure 95% or ultrapure 99%. As used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in dimers or alternative physical forms, such as phosphorylated or derivatized forms. Isolated substances may also have varying levels of purity with respect to the substance they associate with. Isolated substances and / or entities may also be isolated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In the context of this invention, the specification and the claims, the term “mRNA” preferably means “isolated mRNA” and vice versa.

[0144] As used herein in the context of nucleic acid sequences or amino acid sequences, the terms “heterogeneous” or “heterogeneous sequence” refer to sequences (e.g., DNA, RNA, amino acids) as recognized and understood by those skilled in the art, and are intended to refer to sequences derived from a different gene, a different allele, or a different species. If two sequences cannot be derived from the same gene or the same allele, these sequences are typically understood to be “heterogeneous.” That is, heterogeneous sequences may be derived from the same organism, but they do not naturally exist in the same nucleic acid molecule, such as the same RNA or protein.

[0145] Bi / Multicistronic mRNA: mRNA that typically has two (biscistronic) or more (multicistronic) open reading frames (ORFs) (coding regions or coding sequences). In this context, an open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such mRNA yields two (biscistronic) or more (multicistronic) distinct translation products (if the ORFs are not identical). For expression in eukaryotes, such mRNA may contain, for example, an internal ribosome entry site (IRES) sequence.

[0146] Monocistronic mRNA: Monocistronic mRNA can be mRNA that typically contains only one open reading frame (coding sequence or coding region). In this context, the open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

[0147] 3' Untranslated Region (3'-UTR): The 3'-UTR is typically the portion of mRNA located between the protein-coding region (i.e., the open reading frame) and the poly(A) sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by a gene that is transcribed into its respective mRNA during the gene expression process. The genomic sequence is first transcribed into an mRNA precursor, which optionally contains introns. The mRNA precursor is then further processed into mature mRNA in the maturation process. This maturation process includes steps such as 5' capping, splicing of the mRNA precursor to remove any introns, and 3' end modifications, including polyadenylation of the 3' end of the mRNA precursor and any endo- or exonuclease cleavage. In the context of the present invention, the 3'-UTR corresponds to a sequence of mature mRNA located 3' to the stop codon of the protein-coding region, preferably immediately 3' to the stop codon of the protein-coding region, and extending to the nucleotide immediately 5' to the poly(A) sequence, preferably immediately 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence may be an RNA sequence in the mRNA used to define the 3'-UTR sequence, for example, or a DNA sequence corresponding to such an RNA sequence. In the context of the present invention, the term "3'-UTR of a gene," such as "3'-UTR of the albumin gene," is the sequence corresponding to the 3'-UTR of the mature mRNA derived from that gene, i.e., the mRNA obtained by gene transcription and maturation of the mRNA precursor. The term "3'-UTR of a gene" encompasses both DNA and RNA sequences of the 3'-UTR.

[0148] 5' Untranslated Region (5'-UTR): The 5'-UTR is typically understood to be a specific section of messenger RNA (mRNA). It is located at 5' of the open reading frame of the mRNA. Typically, the 5'-UTR begins at the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'-UTR may be post-transcriptionally modified, for example, by the addition of a 5' cap. In the context of this invention, the 5'-UTR corresponds to a sequence of mature mRNA located between the 5' cap and the start codon. Preferably, the 5'-UTR corresponds to a sequence extending from a nucleotide located 3' relative to the 5' cap, preferably a nucleotide immediately 3' relative to the 5' cap, to a nucleotide located 5' relative to the start codon of the protein-coding region, preferably a nucleotide immediately 5' relative to the start codon of the protein-coding region. The nucleotide located immediately 3' to the 5' cap of mature mRNA typically corresponds to the transcription start site. The term “corresponding” means that the 5'-UTR sequence may be an RNA sequence in the mRNA used to define the 5'-UTR sequence, for example, or a DNA sequence corresponding to such an RNA sequence. In the context of this invention, the term “5'-UTR of a gene,” such as “5'-UTR of a TOP gene,” refers to the sequence corresponding to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by gene transcription and maturation of the mRNA precursor. The term “5'-UTR of a gene” encompasses both the DNA and RNA sequences of the 5'-UTR.

[0149] 5'-terminal oligopyrimidine tract (TOP): A 5'-terminal oligopyrimidine tract (TOP) is an elongation of a pyrimidine nucleotide located in the 5' terminal region of a nucleic acid molecule, typically such as the 5' terminal region of a particular mRNA molecule or the 5' terminal region of a functional entity of a particular gene, e.g., the 5' terminal region of a transcribed area. This sequence usually begins with a cytidine, corresponding to the transcription start site, followed by an elongation of approximately 3 to 30 pyrimidine nucleotides. For example, a TOP may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or even more nucleotides. Due to the pyrimidine elongation, the 5'-TOP ends at 5' of the first purine nucleotide located downstream of the TOP. Messenger RNAs containing a 5'-terminal oligopyrimidine tract are often called TOP mRNAs. Therefore, genes providing such messenger RNAs are called TOP genes. TOP sequences are found, for example, in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.

[0150] TOP motif: In the context of the present invention, the TOP motif is a nucleic acid sequence corresponding to the 5'-TOP as defined above. Thus, the TOP motif in the context of the present invention is preferably an extension of a pyrimidine nucleotide having a length of 3 to 30 nucleotides. Preferably, the TOP motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, and most preferably at least 8 pyrimidine nucleotides, wherein the pyrimidine nucleotide extension preferably begins with a cytosine nucleotide at its 5' end. In TOP genes and TOP mRNA, the TOP motif preferably begins with a transcription start site at its 5' end and ends with 1 nucleotide 5' relative to the first purine residue in the gene or mRNA. The TOP motif in the sense of the present invention is preferably located at the 5' end of a sequence representing the 5'-UTR, or at the 5' end of a sequence encoding the 5'-UTR. Therefore, preferably, an extension of three or more pyrimidine nucleotides is referred to as a "TOP sequence" in the sense of the present invention if it is located at the 5' end of the mRNA of the present invention, the 5'-UTR element of the mRNA of the present invention, or a nucleic acid sequence derived from the 5'-UTR of the TOP gene described herein. In other words, an extension of three or more pyrimidine nucleotides that is not located at the 5' end of the 5'-UTR or 5'-UTR element, but is located somewhere within the 5'-UTR or 5'-UTR element, is preferably not referred to as a "TOP motif".

[0151] TOP genes: TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. As defined above, the 5'-UTR of a TOP gene corresponds to the sequence of the 5'-UTR of mature mRNA induced from the TOP gene, preferably extending from a nucleotide located 3' relative to the 5' cap to a nucleotide located 5' relative to the start codon. The 5'-UTR of a TOP gene typically does not contain any start codon, preferably neither an upstream AUG (uAUG) nor an upstream open reading frame (uORF). In this context, the upstream AUG and upstream open reading frame are understood to be the AUG and open reading frame located 5' of the start codon (AUG) of the open reading frame to be translated. The 5'-UTR of a TOP gene is generally quite short. The length of the 5'-UTR of the TOP gene can vary between 20 and up to 500 nucleotides, typically less than about 200 nucleotides, preferably less than about 150 nucleotides, and more preferably less than about 100 nucleotides. An exemplary 5'-UTR of the TOP gene in the sense of the present invention is a nucleic acid sequence extending from the nucleotide at position 5 to the nucleotide immediately 5' relative to the start codon (e.g., ATG) in the sequence or its homolog or variant as described in SEQ ID NOs. 1-1363, SEQ ID NOs. 1395, SEQ ID NOs. 1421 and SEQ ID NOs. 1422 of International Publication No. 2013143700, the disclosure of which is incorporated herein by reference. In this context, a particularly preferred fragment of the 5'-UTR of the TOP gene is the 5'-UTR of the TOP gene lacking the 5'-TOP motif. The term “5'-UTR of the TOP gene” preferably refers to the naturally occurring 5'-UTR of the TOP gene.

[0152] Nucleic acid sequences, particularly fragments of mRNA: A fragment of a nucleic acid sequence consists of sequential extensions of nucleotides corresponding to sequential extensions of nucleotides in the full-length nucleic acid sequence that forms the basis of the fragment's nucleic acid sequence, representing at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length nucleic acid sequence. In the sense of the present invention, such a fragment is preferably a functional fragment of a full-length nucleic acid sequence.

[0153] In the context of the present invention, a "fragment" or "variant" of a protein or peptide is defined as having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of the elongation of at least 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide. %, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity. More preferably, a "fragment" or "variant" of a protein or peptide used herein is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the protein or peptide from which the variant is derived.

[0154] Nucleic acid sequence variants, particularly mRNA variants: A nucleic acid sequence variant refers to a variant of a nucleic acid sequence that forms the basis of a nucleic acid sequence. For example, a variant nucleic acid sequence may exhibit deletions, insertions, additions, and / or substitutions of one or more nucleotides compared to the nucleic acid sequence from which the variant is induced. Preferably, a nucleic acid sequence variant is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the nucleic acid sequence from which the variant is induced. Preferably, the variant is a functional variant. A nucleic acid sequence "variant" may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% nucleotide identity over an extension of 10, 20, 30, 50, 75, or 100 nucleotides of such a nucleic acid sequence.

[0155] Stabilized nucleic acids, preferably mRNA: Stabilized nucleic acids, preferably mRNA, typically exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and / or exovivo degradation (e.g., by manufacturing processes prior to vaccine administration, e.g., during the preparation of the vaccine solution to be administered). RNA stabilization can be achieved, for example, by providing a 5' cap structure, a poly-A tail, or any other UTR modification. This can also be achieved by chemical modification or modification of the G / C content of the nucleic acid. Various other methods are known in the art and can be considered in the context of the present invention.

[0156] RNA In Vitro Transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to the process by which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template for producing RNA transcripts. According to the present invention, RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, preferably a linear plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases include T7, T3, and SP6 RNA polymerases. The DNA template for in vitro RNA transcription can be obtained by cloning nucleic acids, particularly cDNA corresponding to each RNA to be transcribed in vitro, and introducing this into a suitable vector for in vitro transcription, such as plasmid DNA. In a preferred embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme before being transcribed in vitro. cDNA can be obtained by reverse transcription or chemosynthesis of mRNA. Furthermore, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0157] Methods for in vitro transcription are known in the art (see, for example, Geall et al. (2013) Semin.Immunol.25(2):152-159; Brunelle et al. (2013) Methods Enzymol.530:101-14). Reagents used in the above methods typically include the following: 1) A linearized DNA template having a promoter sequence that has high binding affinity to each RNA polymerase, such as RNA polymerase encoded by a bacteriophage. 2) Ribonucleoside triphosphates (NTPs) for four bases (adenine, cytosine, guanine, and uracil), 3) Depending on the case, the capped analog defined above (e.g., m7G(5')ppp(5')G(m7G)), 4) DNA-dependent RNA polymerases (e.g., T7, T3, or SP6 RNA polymerases) that can bind to promoter sequences within a linearized DNA template. 5) Ribonuclease (RNase) inhibitors to inactivate any contaminated RNase, 6) In some cases, pyrophosphatases that break down pyrophosphates, which can inhibit transcription, 7) Mg as a cofactor for polymerase 2+ MgCl2, which supplies ions, 8) A buffer for maintaining an appropriate pH value, which may contain an optimal concentration of an antioxidant (e.g., DTT) and / or a polyamine such as spermidine.

[0158] Full-length protein: As used herein, the term “full-length protein” typically refers to a protein that substantially contains the entire amino acid sequence of a naturally occurring protein. Nevertheless, amino acid substitutions in a protein, such as those resulting from mutation, are also included in the term “full-length protein.”

[0159] Protein Fragments: In the context of the present invention, a “fragment” of a protein or peptide may include a sequence of a protein or peptide as defined herein that is typically N-terminal and / or C-terminally cleaved with respect to its amino acid sequence (or its coding nucleic acid molecule) compared to the amino acid sequence (or its coding nucleic acid molecule) of the original (natural) protein. Thus, such cleavage may exist at the amino acid level or, correspondingly, at the nucleic acid level. Therefore, sequence identity with respect to such a fragment as defined herein may preferably refer to the entire protein or peptide as defined herein, or the entire (coding) nucleic acid molecule of such a protein or peptide.

[0160] In the context of a gene's nucleic acid sequence, the term “variant” refers to a nucleic acid sequence variant, i.e., a nucleic acid sequence or gene that contains at least one nucleic acid that differs from the reference (or “parent”) nucleic acid or the reference (or “parent”) nucleic acid sequence of the gene. Thus, a variant nucleic acid or gene may preferably contain at least one mutation, substitution, insertion, or deletion in its nucleic acid sequence compared to its respective reference sequence. Preferably, the term “variant” as used herein includes naturally occurring variants of a nucleic acid sequence or gene, and engineered variants. Thus, “variant” as defined herein may be derived from a reference nucleic acid sequence, isolated from a reference nucleic acid sequence, related to a reference nucleic acid sequence, based on a reference nucleic acid sequence, or homologous to a reference nucleic acid sequence. The "variant" may preferably have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence of the respective naturally occurring (wild-type) nucleic acid sequence or gene, or its homolog, fragment, or derivative.

[0161] Furthermore, the term “variant” as used herein in the context of proteins or peptides is intended to be recognized and understood by those skilled in the art and to refer to a protein or peptide variant having an amino acid sequence different from the original sequence in one or more mutations, such as one or more substitutions, insertions, and / or deletions of amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, e.g., its specific antigenic properties, compared to the full-length natural protein. A “variant” of a protein or peptide as defined herein may include conserved amino acid substitutions compared to its natural, i.e., unmutated physiological sequence. These amino acid sequences and their coding nucleotide sequences fall under the term “variant” as defined herein. Substitutions in which amino acids of the same class are exchanged with each other are called conserved substitutions. In particular, these are amino acids having aliphatic side chains, amino acids having positively or negatively charged side chains, amino acids having aromatic groups in their side chains or amino acids, and amino acids having side chains that can enter hydrogen bridges, e.g., hydroxyl functional groups. This means, for example, that an amino acid with a polar side chain may be substituted by another amino acid with a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain may be substituted by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine), or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at sequence positions that do not cause modification to the three-dimensional structure and do not affect the binding domain. Modification to the three-dimensional structure by insertion or deletion can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy). A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over an elongation of at least 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide.Preferably, the protein variant includes a functional variant of the protein, meaning that the variant exhibits the same effect or functionality as the protein from which it is induced, or at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the effect or functionality.

[0162] Furthermore, in the context of nucleic acid sequences or genes, the term “fragment” refers to a continuous subsequence of a full-length reference (or “parent”) nucleic acid sequence or gene. In other words, a “fragment” can typically be a shorter portion of a full-length nucleic acid sequence or gene. Thus, a fragment typically consists of a sequence that is identical to the corresponding extension within a full-length nucleic acid sequence or gene. This term includes naturally occurring fragments as well as manipulated fragments. In the context of the present invention, a preferred fragment of a sequence consists of a continuous extension of nucleic acid corresponding to the continuous extension of the entity within the nucleic acid or gene from which the fragment is induced, representing at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the entire (i.e., full-length) nucleic acid sequence or gene from which the fragment is induced. The sequence identity expressed with respect to such a fragment preferably refers to the entire nucleic acid sequence or gene. Preferably, the “fragment” may comprise a nucleic acid sequence having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, or even more preferably 97% sequence identity with the reference nucleic acid sequence or gene from which it is derived.

[0163] In this context, protein fragments may typically contain amino acid sequences having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, or even more preferably at least 97% sequence identity with the amino acid sequence of the respective naturally occurring full-length protein.

[0164] The term “identity” as used herein in the context of nucleic acid sequences or amino acid sequences is intended to be recognized and understood by those skilled in the art and to refer, for example, to the percentage of two sequences that are identical. To determine the percentage of two sequences that are identical, for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, preferably aa sequences encoded by nucleic acid sequences as defined herein, or aa sequences themselves, the sequences can be aligned and then compared with each other. Thus, for example, a position in a first sequence can be compared to a corresponding position in a second sequence. If the position in the first sequence is occupied by the same residue as the position in the second sequence, the two sequences are identical at this position. Otherwise, the sequences are different at this position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted into the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted into the second sequence to allow for further alignment. Therefore, the percentage of identical arrays is a function obtained by dividing the number of identical positions by the total number of positions that are occupied only in one array. The percentage of identical arrays can be determined using an algorithm, such as the one built into the BLAST program.

[0165] In the context of the present invention, a protein or peptide fragment may further include a sequence of a protein or peptide as defined herein, having, for example, at least 5 amino acid lengths, preferably at least 6 amino acid lengths, preferably at least 7 amino acid lengths, more preferably at least 8 amino acid lengths, even more preferably at least 9 amino acid lengths; even more preferably at least 10 amino acid lengths; even more preferably at least 11 amino acid lengths; even more preferably at least 12 amino acid lengths; even more preferably at least 13 amino acid lengths; even more preferably at least 14 amino acid lengths; even more preferably at least 15 amino acid lengths; even more preferably at least 16 amino acid lengths; even more preferably at least 17 amino acid lengths; even more preferably at least 18 amino acid lengths; even more preferably at least 19 amino acid lengths; even more preferably at least 20 amino acid lengths; even more preferably at least 25 amino acid lengths; even more preferably at least 30 amino acid lengths; even more preferably at least 35 amino acid lengths; even more preferably at least 50 amino acid lengths; and most preferably at least 100 amino acid lengths. For example, such fragments may have a length of about 6 to about 20 or more amino acids, for example preferably about 8 to about 10, for example 8, 9 or 10 amino acids (or even 6, 7, 11 or 12 amino acids), and may be fragments processed and presented by MHC class I molecules, or preferably about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid lengths, and may be fragments processed and presented by MHC class II molecules, and these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule, i.e., the fragments are typically not recognized in their native form. Protein or peptide fragments may contain at least one epitope of these proteins or peptides. Furthermore, it can also be understood that protein domains, such as the extracellular domain, intracellular domain or transmembrane domain of a protein and shortened or cleaved versions of the protein constitute protein fragments.

[0166] Protein Variants: “Variants” of proteins or peptides, as defined in the context of this invention, can be made having an amino acid sequence different from the original sequence due to one or more mutations, such as substitutions, insertions, and / or deletions of amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, e.g., its specific antigenic properties, compared to the full-length natural protein. “Variants” of proteins or peptides, as defined herein, may include conserved amino acid substitutions compared to their natural, i.e., unmutated physiological sequence. These amino acid sequences and their coding nucleotide sequences fall under the term “variant” as defined herein. Substitutions in which amino acids of the same class are exchanged with each other are called conserved substitutions. In particular, these are amino acids having aliphatic side chains, amino acids having positively or negatively charged side chains, amino acids having aromatic groups in their side chains or amino acids, and amino acids having side chains that can enter hydrogen bridges, e.g., hydroxyl functional groups. This means, for example, that an amino acid with a polar side chain may be substituted by another amino acid with a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain may be substituted by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine), or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at sequence positions that do not cause modification to the three-dimensional structure and do not affect the binding domain. Modification to the three-dimensional structure by insertion or deletion can be easily determined, for example, using CD spectroscopy (circular dichroism spectrum) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, Modern Physical Methods in Biochemistry, Neuberger et al. (eds.), Elsevier, Amsterdam).

[0167] A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over an extension of 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide.

[0168] Furthermore, variants of proteins or peptides as defined herein, which can be encoded by nucleic acid molecules, may also include sequences in which the nucleotides of the encoding nucleic acid sequence are exchanged by degeneracy of the genetic code without resulting in a change in the respective amino acid sequence of the protein or peptide; that is, the amino acid sequence or at least a portion of it is not different from the original sequence in one or more mutations within the sense described above.

[0169] Sequence Identity: To determine the percentage of identical sequences between two sequences, for example, nucleic acid sequences or amino acid sequences as defined herein, preferably amino acid sequences encoded by the nucleic acid sequence of a polymer carrier as defined herein, or the amino acid sequences themselves, the sequences can be aligned and then compared to each other. For example, a position in a first sequence can be compared to a corresponding position in a second sequence. If a position in the first sequence is occupied by the same component (residue) as a position in the second sequence, the two sequences are identical at that position. Otherwise, the sequences differ at that position. If an insertion occurs in the second sequence compared to the first, a gap can be inserted into the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first, a gap can be inserted into the second sequence to allow for further alignment. Therefore, the percentage of identical sequences is a function of the number of identical positions divided by the total number of positions that include positions occupied only in one sequence. The percentage of identical sequences can be determined using a mathematical algorithm. Preferred but not limited examples of mathematical algorithms that can be used are those of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such algorithms are incorporated into the BLAST program. Sequences that are identical to the sequences of the present invention to a certain extent can be identified by this program.

[0170] Derivatives of proteins or peptides: Derivatives of peptides or proteins are typically understood to be molecules derived from another molecule, such as the aforementioned peptide or protein. “Derivatives” of peptides or proteins also include fusions containing the peptide or protein used in this invention. For example, a fusion may include a label, such as an epitope, e.g., a FLAG epitope or a V5 epitope. For example, the epitope is a FLAG epitope. Such tags are useful, for example, for purifying fusion proteins.

[0171] Pharmacologically effective dose: In the context of this invention, the pharmaceutically effective dose is typically understood to be an amount sufficient to induce an immune response. Carrier: In the context of the present invention, a carrier is typically a compound that facilitates the transport and / or complexation of another compound. The carrier may form a complex with the other compound. A polymer carrier is a carrier formed of a polymer.

[0172] Medium: Typically, a drug, such as a carrier, that may be used within a pharmaceutical composition or vaccine to facilitate the administration of the components of the pharmaceutical composition or vaccine to an individual. [Brief explanation of the drawing]

[0173] The figures shown below are for illustrative purposes only and further illustrate the present invention. These figures should not be construed as limiting the present invention to them. [Figure 1] Figure 1 (tumor antigen Trp2 im injection; complete details can be seen in Example 8) shows that vaccination with LNP1 containing Trp2 mRNA showed a favorable immune response. Figure 1A shows IFNγ+TNFα+CD107a+ CD8+ T cells (multifunctional CD107a+CD8+ T cells), and Figure 1B shows IFNγ+TNFα+CD4+ T cells (multifunctional CD4+ T cells) (LNP1 = black circle, buffer = black square). [Figure 2]Figure 2 (tumor antigen Trp2 im injection; complete details can be seen in Example 9) shows that vaccination with LNP1-LNP7 containing Trp2 mRNA showed a very good immune response. Figure 2A shows IFNγ+TNFα+CD107a+ CD8+ T cells (pluripotent and activated CD107a+CD8+ T cells), and Figure 2B shows IFNγ+TNFα+CD4+ T cells (pluripotent and activated CD4+ T cells) (LNP1=black circle, LNP2=black square, LNP3=black inverted triangle, LNP4=black triangle, LNP5=white circle, LNP6=white square, LNP7=cross, buffer is on the x-axis level). [Figure 3] Tumor antigen Trp2 im injection; complete details can be seen in Example 10. Figure 3A shows that LNPs containing DPhyPS (black squares) showed a higher CD8 T cell response compared to LNPs without DPhyPS (white circles) after vaccination and peptide restimulation with a Trp2 immunodominant epitope. Buffer control = black triangle. Furthermore, Figure 3B shows that LNPs containing DPhyPS (black squares) had a higher IgG2a endpoint titer compared to LNPs without DPhyPS (white circles). Buffer control = black triangle. [Figure 4] Figure 4 (tumor antigen Trp2 im injection; full details can be seen in Example 12) shows that vaccination with different LNPs containing Trp2-encoding mRNA induced a very good immune response, as measured by IFNγ+TNFα+CD8+ T cells (pluripotent and activated CD107a+CD8+ T cells) (LNP1=black circle, LNP2=white circle, LNP3=white square, LNP4=white diamond, buffer=black square on x-axis level). [Figure 5] Figure 5 (tumor antigen Trp2 im injection; full details can be seen in Example 12) shows that vaccination with different LNPs containing Trp2-encoding mRNA induced a very good immune response, as measured by IFNγ+TNFα+CD107a+CD8+ T cells (pluripotent and activated CD107a+CD8+ T cells) (LNP1=black circle, LNP2=white circle, LNP3=white square, LNP4=white diamond, buffer=black square on x-axis level). [Figure 6] Figure 6 (tumor antigen Trp2 im injection; full details can be seen in Example 12) shows that vaccination with different LNPs containing Trp2-encoding mRNA induced a very good immune response, as measured in multifunctional CD8+ TEM cells (IFNγ+TNFα+CD107a+CD8+TEM cells) (LNP1=black circle, LNP2=white circle, LNP3=white square, LNP4=white diamond, buffer=black square on x-axis level). [Figure 7] Figure 7 (tumor antigen Trp2 im injection; complete details can be seen in Example 12) shows that vaccination with different LNPs containing Trp2-encoding mRNA induced a very good immune response, as measured by IFNγ+TNFα+CD4+ T cells (pluripotent and activated CD4+ T cells) (LNP1=black circle, LNP2=white circle, LNP3=white square, LNP4=white diamond, buffer=black square on x-axis level). [Modes for carrying out the invention]

[0174] This disclosure is described in detail below, but it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, and that these are subject to change. The terminology used herein is intended solely to describe specific embodiments and is not intended to limit the scope of this disclosure, and it should be understood that the scope of this disclosure is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0175] Polymer-bound lipids Under storage conditions or during formulation, lipid-based carriers may undergo charge-induced aggregation, a state that may be undesirable for the stability of the lipid-based carrier. Therefore, it may be desirable to include lipid compounds that can reduce aggregation, for example, by sterically stabilizing the lipid-based carrier. Such steric stabilization may occur when the compound has a sterically bulky but uncharged portion that shields or blocks the charged portion of the lipid-based carrier from close proximity to other lipid-based carriers in the composition. In the context of the present invention, stabilization of the lipid-based carrier is achieved by including lipids that may include lipids having sterically bulky groups located outside the lipid-based carrier after its formation.

[0176] The terms “aggregation-reducing lipid” or “polymer-conjugated lipid” refer to molecules having a lipid portion and a portion suitable for reducing or preventing aggregation of a lipid-based carrier containing cargo, preferably mRNA. Thus, “aggregation-reducing lipid,” also referred to herein as “polymer-conjugated lipid,” is a lipid containing a polymer as an aggregation-reducing group. A polymer should be understood as a substance or material consisting of a very large molecule or macromolecule composed of many repeating subunits, as is evident from the context of this invention. A suitable polymer in the context of this invention may be a hydrophilic polymer. Therefore, in a preferred embodiment, the lipid-based carrier of the pharmaceutical composition contains a polymer-conjugated lipid.

[0177] In a preferred embodiment of the present invention, the LNP comprises a lipid conjugate, preferably a polymer-bound lipid as described above and below herein, preferably "PMOZ4", an ionizable lipid as described above and below herein, preferably an ionizable lipid represented by formula (II), more preferably C24, C28 or C29, most preferably C24, a steroid and a neutral lipid, and preferably an additional phosphatidylserine as a fifth excipient, preferably DPhyPS.

[0178] In the eighth embodiment, the present invention relates to formula (I): [P]-[Linker]-[L] Formula (I) We provide polymer-bound lipids defined as compounds represented by the following: [P] represents at least one polyoxazoline (POZ) monomer unit.

[0179] [ka]

[0180] A heteropolymer or homopolymer portion, preferably a homopolymer portion, containing the following: Here, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, and n has an average value in the range of about 45 to about 55, preferably about 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. [linker] is any linker group, [L] represents the lipid portion.

[0181] In formula (I), R in [P] is preferably C1 (methyl), which yields the PMOZ unit. The present invention relates to the use of novel polymer-bound lipids comprising phosphatidylserine (preferably DPhyPS) and polyoxazoline (POZ) represented by formula (I), wherein [P] is preferably Poly(2-methyl-2-oxazoline) (PMOZ), Poly(2-ethyl-2-oxazoline) (PEOZ), Poly(2-propyl-2-oxazoline) (PPOZ), Poly(2-butyl-2-oxazoline) (PBOZ), Poly(2-isopropyl-2-oxazoline) (PIPOZ), Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), or Poly(2-dimethylamino-2-oxazoline)(PDMAOx) Includes, Furthermore / or based on our remarkable discovery that the use of lipid nanoparticles (LNPs) containing phosphatidylserine (preferably DPhyPS) and these novel polymer-binding lipids is extremely effective for delivering nucleic acids such as mRNA to living organisms such as humans. This has made it possible for us to create improved vaccines that deliver, for example, mRNA compounds encoding antigenic peptides or proteins and induce antigen-specific immune responses very efficiently at very low doses, thereby avoiding the drawbacks associated with the use of PEG. This disclosure addresses these and other needs. A further advantage achieved by the present invention is, quite remarkably, that we have discovered a class of formulations for delivering mRNA vaccines in vivo that, according to aspects and embodiments of the present invention, result in a significantly enhanced, and in many ways synergistic, immune response, including functional antibody production with enhanced antigen-generating and neutralizing capabilities. These results can be achieved even when administering significantly lower doses of mRNA compared to the mRNA doses used in other classes of lipid-based formulations. The formulations of the present invention demonstrated a significant and unpredictable in vivo immune response sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents. In summary, the inventors of the present invention can surprisingly demonstrate that LNPs with equivalent or even enhanced performance can be obtained by using several different polymer-bound lipids represented by formula (I), e.g., PMOZ lipids, in combination with phosphatidylserine (preferably DPhyPS), instead of standard PEG lipids. This unexpected finding could be verified by using several different LNP compositions, namely, the inventors found that polymer-bound lipids represented by formula (I), in combination with phosphatidylserine (preferably DPhyPS), can clearly enhance prior art LNP compositions. Even more surprisingly, it was found that reducing the PMOZ density of the formulation to about 1 mol% enhanced the antigen-specific T cell response and / or neutralization titer in vivo.

[0182] Accordingly, the present invention is directed toward compositions comprising polymer-bound lipids according to formula (I), preferably POZ lipids represented by formula (I), more preferably PMOZ lipids as described above and below herein, and ionizable lipids represented by formula (II), preferably C24, C28, or C29 as described above and below herein, more preferably C24, in combination with phosphatidylserine (preferably DPhyPS) as described below herein. Thus, all the options and preferences disclosed for polymer-bound lipids represented by formula (I), preferably POZ lipids, more preferably PMOZ lipids, are also applicable to compositions in this aspect of the present invention. In other words, the specifically disclosed embodiments of polymer-bound lipids, preferably POZ lipids, more preferably PMOZ lipids, and particularly preferred PMOZ lipids DMG-PMOZ should be understood to also define specific preferred embodiments of compositions according to the present invention, i.e., compositions characterized by comprising a PMOZ lipid according to one of the specific selections described herein. In other words, the term “polymer-bound lipid” refers to a molecule that includes both a lipid portion and a polymer portion. Preferably, the polymer-bound lipid represented by formula (I) is a POZ lipid, more preferably a PMOZ lipid. Therefore, the terms “POZ lipid” or “PMOZ lipid” refer to a molecule containing both a lipid moiety and a POZ moiety, or each a PMOZ moiety. Thus, “PMOZ lipid” should be understood as a lipid containing at least one polyoxazoline (POZ) unit, i.e., at least one homopolymer moiety containing a PMOZ unit.

[0183] The composition may further include active and / or inactive excipients as described below. In one specific embodiment, the composition comprises a polymer-bound lipid represented by formula (I), preferably a PMOZ lipid, in addition to one or more lipids selected from the group consisting of (a) a steroid, (b) a neutral lipid, (c) an ionizable lipid preferably represented by formula (II), more preferably C24, C28 or C29, most preferably C24, and (d) phosphatidylserine, preferably DPhyPS.

[0184] In another embodiment, [P] is Poly(2-methyl-2-oxazoline) (PMOZ)

[0185] [ka]

[0186] Poly(2-ethyl-2-oxazoline) (PEOZ)

[0187] [ka]

[0188] Poly(2-propyl-2-oxazoline) (PPOZ)

[0189] [ka]

[0190] Poly(2-butyl-2-oxazoline) (PBOZ)

[0191] [ka]

[0192] Poly(2-isopropyl-2-oxazoline) (PIPOZ)

[0193] [ka]

[0194] Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), and Poly(2-dimethylamino-2-oxazoline)(PDMAOx) A heteropolymer or homopolymer moiety comprising multiple monomer units selected from the group consisting of, Preferably, [P] is a homopolymer moiety containing multiple PMOZ or PEOZ monomer units, and more preferably, [P] contains or consists solely of multiple PMOZ monomer units. (i)n has an average value in the range of about 45 to about 55, preferably about 50, or (ii)n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa.

[0195] In another embodiment, [P] is

[0196] [ka]

[0197] It is a heteropolymer or homopolymer moiety containing multiple monomer units selected from the group consisting of the following: In yet another embodiment, [P] of the polymer-bound lipid represented by formula (I) is selected from the group consisting of poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) and poly(2-dimethylamino-2-oxazoline) (PDMAOx).

[0198] In further embodiments, the polymer-bound lipid represented by formula (I) is selected from the group consisting of POZ-monoacylglycerol bonds, POZ-diacylglycerol bonds, POZ-dialkyloxypropyl bonds, POZ-steroid or POZ-sterol bonds, POZ-phospholipid bonds, POZ-ceramide bonds, and mixtures thereof.

[0199] In a preferred embodiment, the polymer-bound lipid includes a moiety based on 1,2-dimyristoyl-rac-glycerol (DMG).

[0200] [ka]

[0201] A more preferred embodiment of the [P] portion (polymethyloxazoline) of PMOZ: For PMOZ, the preferred average molecular mass of the [P] portion is approximately 3.8 kDa to 4.8 kDa, approximately 3.9 kDa to 4.7 kDa, approximately 4 kDa to 4.6 kDa, approximately 4.1 kDa to 4.5 kDa, approximately 4.2 kDa to 4.4 kDa, or most preferably approximately 4.3 kDa. Other preferred average molecular masses of the [P] portion are (i) approximately 3.9 kDa to 4.4 kDa, approximately 3.9 kDa to 4.1 kDa, or approximately 4.2 kDa to 4.4 kDa.

[0202] In a more preferred embodiment, the average molecular mass of the [P] portion is greater than 4.3 kDa. In other preferred embodiments, preferred average molecular masses of the [P] portion are about 4.25 kDa to about 4.675 kDa, about 4.675 kDa to about 5.1 kDa, about 5.1 kDa to about 5.525 kDa, about 5.525 kDa to about 5.95 kDa, about 5.95 kDa to about 6.375 kDa, about 6.375 kDa to about 6.8 kDa, or greater than 6.8 kDa.

[0203] In other preferred embodiments,

[0204] [ka]

[0205] For PMOZ represented by , n has an average value in the range of approximately 40 to approximately 80, preferably approximately 45 to approximately 70, more preferably approximately 50 to approximately 60, or most preferably, n has an average value of approximately 50.

[0206] In a more preferred embodiment for PMOZ, n has an average value greater than 50. In other preferred embodiments, n has an average value of about 55, about 60, about 65, about 70, about 75, or about 80.

[0207] Therefore, the PMOZ portion is preferably a PMOZ portion having a molecular mass of about 4.3 kDa, but shorter and longer portions can also be used. In other preferred embodiments, the "n" in the [P] portion for the novel polymer-bound lipid is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4 The values ​​are 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 100, preferably 25, and even more preferably 50. In a more preferred embodiment, the "n" of the monomer compound of [P] is selected such that the [P] portion has an average molecular weight of 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, or 8 kDa, preferably 2.5 kDa, and more preferably 5 kDa.

[0208] In a further particular preferred embodiment, n is a novel polymer-bound lipid, where the [P] portion is approximately 2kDa, 2.1kDa, 2.2kDa, 2.3kDa, 2.4kDa, 2.5kDa, 2.6kDa, 2.7kDa, 2.8kDa, 2.9kDa, 3kDa, 3.1kDa, 3.2kDa, 3.3kDa, 3.4kDa, 3.5kDa, 3.6kDa, 3.7kDa, 3.8kDa, 3.9kDa, 4kDa, 4.1kDa, 4.2kDa, 4.3kDa, 4.4kDa, 4.5kDa, 4.6kDa, 4.7kDa, 4.8kDa, 4.9kDa, 5kDa, 5.1kDa, 5.2kDa, 5.3kDa, 5.4kDa, 5.5kDa, 5.6kDa, 5.7kDa , 5.8kDa, 5.9kDa, 6kDa, 6.1kDa, 6.2kDa, 6.3kDa, 6.4kDa, 6.5kDa, 6.6kDa, 6.7kDa, 6.8kDa, 6.9kDa, 7kDa, 7.1kDa, 7.2kDa, 7.3kDa, 7.4kDa, 7.5kDa, 7.6kDa, 7.7kDa, 7.8kDa, 7.9kDa, 8kDa, 8.1kDa, 8.2kDa, 8.3kDa, 8.4kDa, 8.5kDa, 8.6kDa, 8.7kD a, 8.8kDa, 8.9kDa, 9kDa, 9.1kDa, 9.2kDa, 9.3kDa, 9.4kDa, 9.5kDa, 9.6kDa, 9.7kDa, 9.8kDa, 9.9kDa, 10kDa, 10.1kDa, 10.2 Selected to have an average molecular weight of kDa, 10.3kDa, 10.4kDa, 10.5kDa, 10.6kDa, 10.7kDa, 10.8kDa, 10.9kDa, 11kDa, 11.1kDa, 11.2kDa, 11.3kDa, 11.4kDa, 11.5kDa, 11.6kDa, 11.7kDa, 11.8kDa, 11.9kDa, 12kDa, or greater than 12kDa (all the values ​​mentioned above in this paragraph shall be considered "approximate" values).

[0209] In a more preferred embodiment, the polymer-bound lipid is [P], where n has a [P] portion of approximately 2kDa, 2.1kDa, 2.2kDa, 2.3kDa, 2.4kDa, 2.5kDa, 2.6kDa, 2.7kDa, 2.8kDa, 2.9kDa, 3kDa, 3.1kDa, 3.2kDa, 3.3kDa, 3.4kDa, 3.5kDa, 3.6kDa, 3.7kDa, 3.8kDa, 3.9kDa, 4kDa, 4.1kDa, 4.2kDa, 4.3kDa, 4.4 kDa, 4.5kDa, 4.6kDa, 4.7kDa, 4.8kDa, 4.9kDa, 5kDa, 5.1kDa, 5.2kDa, 5.3kDa, 5.4kDa, 5.5kDa, 5.6kDa, 5.7kDa, 5.8kDa, 5.9kD a, 6kDa, 6.1kDa, 6.2kDa, 6.3kDa, 6.4kDa, 6.5kDa, 6.6kDa, 6.7kDa, 6.8kDa, 6.9kDa, 7kDa, 7.1kDa, 7.2kDa, 7.3kDa, 7.4kDa, 7. 5kDa, 7.6kDa, 7.7kDa, 7.8kDa, 7.9kDa, 8kDa, 8.1kDa, 8.2kDa, 8.3kDa, 8.4kDa, 8.5kDa, 8.6kDa, 8.7kDa, 8.8kDa, 8.9kDa, 9kDa , 9.1kDa, 9.2kDa, 9.3kDa, 9.4kDa, 9.5kDa, 9.6kDa, 9.7kDa, 9.8kDa, 9.9kDa, 10kDa, 10.1kDa, 10.2kDa, 10.3kDa, 10.4kDa, 10. It contains a poly(2-methyl-2-oxazoline) (PMOZ) moiety selected to have an average molecular weight of 5kDa, 10.6kDa, 10.7kDa, 10.8kDa, 10.9kDa, 11kDa, 11.1kDa, 11.2kDa, 11.3kDa, 11.4kDa, 11.5kDa, 11.6kDa, 11.7kDa, 11.8kDa, 11.9kDa, 12kDa, or greater than 12kDa (all the aforementioned values ​​in this paragraph shall be considered "approximate" values).

[0210] In a more preferred embodiment, the polymer-bound lipid is [P], where n is in increasing order of preference. n has an average value in the range of approximately 40 to approximately 60. n has an average value in the range of approximately 45 to approximately 55. n has an average value in the range of approximately 46 to approximately 54. n has an average value in the range of approximately 47 to approximately 53. n has an average value in the range of approximately 48 to approximately 52. n has an average value in the range of approximately 49 to approximately 51. n has an average value of approximately 50, and n=50 It contains a polyethyl oxazoline (PEOZ) moiety selected from the group consisting of the following.

[0211] Furthermore, in the most preferred embodiment, the polymer-bound lipid is [P], where n is in increasing order of preference. n has an average value in the range of approximately 40 to approximately 60. n has an average value in the range of approximately 45 to approximately 55. n has an average value in the range of approximately 46 to approximately 54. n has an average value in the range of approximately 47 to approximately 53. n has an average value in the range of approximately 48 to approximately 52. n has an average value in the range of approximately 49 to approximately 51. n has an average value of approximately 50, and n=50 It contains a poly(2-methyl-2-oxazoline) (PMOZ) moiety selected from the group consisting of the following.

[0212] For "PMOZ1" to "PMOZ5", preferably "PMOZ4", n has an average value in the range of preferably 2 to 200, preferably 20 to 100, more preferably 24 to 26, even more preferably about 100, or even more preferably 45 to 50, most preferably 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa.

[0213] For "PMOZ4", n is more preferably an average value in the range of about 100, or n is selected such that the [P] portion has an average molecular weight of about 7kDa to about 11kDa, about 8kDa to about 10kDa, about 8kDa to about 9kDa, and about 8kDa.

[0214] A very preferred embodiment of the [P] portion (polyethyloxazoline) of PEOZ: For PEOZ, the preferred average molecular mass of the [P] portion is approximately 4.5 kDa to 5.5 kDa, approximately 4.6 kDa to 5.4 kDa, approximately 4.7 kDa to 5.3 kDa, approximately 4.8 kDa to 5.2 kDa, approximately 4.9 kDa to 5.1 kDa, and most preferably approximately 5 kDa.

[0215] In a more preferred embodiment, the average molecular mass of the [P] portion is greater than 5 kDa. In other preferred embodiments, the preferred average molecular mass of the [P] portion is about 4.95 kDa to about 5.445 kDa, about 5.445 kDa to about 5.94 kDa, about 5.94 kDa to about 6.435 kDa, about 6.435 kDa to about 6.93 kDa, about 6.93 kDa to about 7.425 kDa, about 7.425 kDa to about 7.92 kDa, or greater than 7.92 kDa.

[0216] In other preferred embodiments,

[0217] [ka]

[0218] For the PEOZ represented by , n has an average value in the range of approximately 40 to approximately 80, preferably approximately 45 to approximately 70, more preferably approximately 50 to approximately 60, or most preferably an average value of approximately 50.

[0219] In a more preferred embodiment for PEOZ, n has an average value greater than 50. In other preferred embodiments, n has an average value of about 55, about 60, about 65, about 70, about 75, or about 80.

[0220] Therefore, the PEOZ portion is preferably a PEOZ portion having a molecular mass of about 5 kDa, but shorter and longer portions can also be used. A more very preferred embodiment for the [P] portion of PPOZ (polypropyl oxazoline) or the [P] portion of PIPOZ (poly-2-isopropyl-2-oxazoline): For PPOZ or equivalent PIPOZ, the preferred average molecular mass of the [P] portion is about 5.2 kDa to about 6.2 kDa, about 5.3 kDa to about 6.1 kDa, about 5.4 kDa to about 6 kDa, about 5.5 kDa to about 5.9 kDa, about 5.6 kDa to about 5.8 kDa, most preferably about 5.7 kDa.

[0221] In a more preferred embodiment, the average molecular mass of the [P] portion is greater than 5.7 kDa. In other preferred embodiments, preferred average molecular masses of the [P] portion are about 5.65 kDa to about 6.215 kDa, about 6.215 kDa to about 6.78 kDa, about 6.78 kDa to about 7.345 kDa, about 7.345 kDa to about 7.91 kDa, about 7.91 kDa to about 8.475 kDa, about 8.475 kDa to about 9.04 kDa, or greater than 9.04 kDa.

[0222] In other preferred embodiments,

[0223] [ka]

[0224] For PPOZ represented by or equivalently PIPOZ (poly(2-isopropyl-2-oxazoline)), n has an average value in the range of about 40 to about 80, preferably about 45 to about 70, more preferably about 50 to about 60, and most preferably an average value of about 50.

[0225] In a more preferred embodiment for PPOZ or equally for PIPOZ, n has an average value greater than 50. In other preferred embodiments, n has an average value of about 55, about 60, about 65, about 70, about 75, or about 80.

[0226] Therefore, the PPOZ or equivalent PIPOZ portion is preferably a PPOZ or equivalent PIPOZ portion having a molecular mass of about 5.7 kDa, but shorter and longer portions can also be used.

[0227] In one embodiment, the lipid moiety [L] represented by formula (I)([P]-[linker]-[L]) comprises at least one linear or branched saturated or unsaturated alkyl chain containing 6 to 30 carbon atoms, preferably the lipid moiety [L] comprises at least one linear or branched saturated alkyl chain, the alkyl chain may be optionally interrupted by one or more biodegradable groups, and may further / or optionally contain one terminal biodegradable group, the biodegradable group being, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 Alkenyl), zwitterionic linker, ester-free linker portion and ester-containing linker portion (-C(O)O- or -OC(O)-), amide (-C(O)NH-), disulfide (-SS-), carbonyl (-C(O)-), ether (-O-), thioether (-S-), oxime (e.g., -C(H)=NO- or -ON=C(H)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), (-NHC(O)CH2CH2C(O)-), -C(R5)=N-, -N=C(R 5 )-,-C(R 5 )=NO-, -ON=C(R 5 )-, -OC(O)O-, -C(O)N(R 5 ), -N(R 5 )C(O)-, -C(S)(NR 5 )-, (NR5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, or -OC(O)(CR 3 R 4 )C(O)-, carbonate (-OC(O)O-), nitrogen (N), succinoyl, succinate, phosphate ester (-O-(O)POH-O-), cyclic compounds, heterocyclic compounds, piperidine, pyrazine, pyridine, piperazine, and sulfonic acid esters, and combinations thereof, selected from the group, where R 3 , R 4 and R 5 These are independently H or alkyl (e.g., C1-C4 alkyl).

[0228] In another embodiment, the lipid moiety [L] comprises at least one linear or branched saturated or unsaturated alkyl chain containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, preferably in the range of 10 to 20 carbon atoms, more preferably in the range of 12 to 18 carbon atoms, even more preferably 14, 16, or 18 carbon atoms, even more preferably 16 or 18 carbon atoms, and most preferably 14 carbon atoms, all selections being independent of each other.

[0229] In another embodiment, the linker group [linker] includes an amide linker moiety, preferably an ester linker moiety, or the linker group [linker] has the following structure:

[0230] [ka]

[0231] In further embodiments, polymer-bound lipids have the following structure:

[0232] [ka]

[0233] Here, the linker group is selected from any one of the linker groups disclosed herein, preferably a linker group containing an ester moiety. n has an average value in the range of 2 to 200, preferably 20 to 100, more preferably 24 to 26, even more preferably about 100, still even more preferably 45 to 50, most preferably 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. Most preferably, the polymer-bound lipid is DMG-PMOZ, and n has an average value of 45 to 50, most preferably 50.

[0234] In another preferred embodiment, the polymer-bound lipid is

[0235] [ka]

[0236] It has the structure ["PMOZ1"], and more preferably n=50, i.e., 50 monomer repeats. In another preferred embodiment, the polymer-bound lipid is

[0237] [ka]

[0238] It has the structure ["PMOZ3"], and more preferably n=50, i.e., 50 monomer repeats. In another preferred embodiment, the polymer-bound lipid is

[0239] [ka]

[0240] It has the structure ["PMOZ5"], and more preferably n=50, i.e., 50 monomer repeats. In another preferred embodiment, the polymer-bound lipid is

[0241] [ka]

[0242] It has the structure ["PMOZ2"], and more preferably n=50, i.e., 50 monomer repeats. In the most preferred embodiment, the polymer-bound lipid is

[0243] [ka]

[0244] It has the structure ["PMOZ4"], more preferably n=50, i.e., 50 monomer repeats, i.e.,

[0245] [ka]

[0246] That is the case. Surprisingly, the inventors have identified the polymer-bound lipids mentioned above and below, preferably "PMOZ1", "PMOZ2", "PMOZ3", "PMOZ4", or "PMOZ5", most preferably "PMOZ4", or each as a linker group [linker]

[0247] [ka]

[0248] We have found it advantageous that the polymer-bound lipids comprise succinate, a peptide bond (-CO-NH-), an amine, or a secondary amine, and more preferably the linker group [linker] comprises succinamidyl (-NHC(O)CH2CH2C(O)-) or (-NHC(O)CH2CH2C(O)-), which have particular advantages with respect to manufacturing efficiency or general synthesis, preferably GMP manufacturing efficiency. In other words, the production of these polymer-bound lipids can be carried out in a way that is easier to implement, more practical, simpler, and / or more cost-effective. In other words, the general synthesis of these compounds containing the preferred linkers mentioned above is easier and more practical. Finally, polymer-bound lipids having the aforementioned [linker] group are more stable with respect to chemical stability. In other words, the polymer-bound lipids and [linkers] disclosed above have highly advantageous and unexpected behavior with respect to synthesis and production.

[0249] In another very preferred embodiment, the linker group [linker] preferably includes an amide linker moiety. In a more very preferred embodiment, the linker group [linker] preferably includes an ester linker moiety.

[0250] In a more very preferred embodiment, the linker group [linker] preferably includes a succinate linker moiety. In another very preferred embodiment, the linker group [linker] comprises both an ester linker moiety and an amide linker moiety. In another preferred embodiment, the linker group [linker] comprises both an ester linker moiety, an amine linker moiety and an amide linker moiety.

[0251] In another very preferred embodiment, the linker group [linker] is preferably

[0252] [ka]

[0253] The structure is such that the linker group [linker] is preferably an amine, and preferably a secondary amine linker moiety. In further embodiments, the lipid nanoparticles include polymer-bound lipids of the present disclosure.

[0254] In a more preferred embodiment, the polymer-bound lipid of the present invention does not contain a polyethylene glycol (PEG) moiety or residue, and / or does not contain a sulfur group (-S-), and / or does not contain a terminal nucleophile.

[0255] In a more preferred embodiment, the polymer-bound lipid of the present invention does not contain a polyethylene glycol (PEG) moiety or residue. In a more preferred embodiment, the polymer-bound lipid of the present invention does not contain a sulfur group (-S-).

[0256] In a more preferred embodiment, the polymer-bound lipid of the present invention does not contain terminal nucleophiles. In a more preferred embodiment, the polymer-bound lipid of the present invention does not contain sulfur groups (-S-) and terminal nucleophiles.

[0257] In a more preferred embodiment, the polymer-bound lipid, preferably a POZ lipid or PMOZ lipid, is not covalently bound to the bioactive component, and the bioactive component is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA.

[0258] In further embodiments, the lipid nanoparticles do not contain polyethylene glycol (PEG) lipid binding or binding of PEG and lipid-like material, preferably PEG-free, and / or (ii) the polymer-bound lipids of the present invention do not contain sulfur groups (-S-), terminal nucleophiles, and / or are not covalently bound to a biologically active component which is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA or any combination thereof.

[0259] In another, highly preferred embodiment, the polymer-bound lipid of the present invention contains neither sulfur (S) nor sulfur groups (-S-). In further embodiments, the lipid nanoparticles of the present invention further comprise a sterol or steroid selected from the group consisting of cholesterol, cholesteryl hemisuccinate (CHEMS), and derivatives thereof, and preferably the lipid nanoparticles further comprise cholesterol.

[0260] In yet another embodiment, the lipid nanoparticles of the present invention are based on the molar percentage with the total lipid component or excipient composition set to 100%, (i) an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, or about 10 mol% of the polymer-bound lipids of the present invention disclosed herein, or (ii) More preferably, polymer-bound lipids as described herein in amounts of about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, or about 1 mol% Includes.

[0261] In further embodiments, the bioactive component contained within the lipid nanoparticles is preferably a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or modified messenger RNA (mRNA) containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA or any combination thereof, preferably the bioactive component is chemically modified mRNA or unmodified mRNA, and more preferably the bioactive component is unmodified mRNA.

[0262] In a very preferred embodiment, the nucleic acid compound is artificial mRNA or isolated mRNA. In other preferred embodiments, the novel polymer-bound lipids may be derived from polymer-bound lipids disclosed in International Publication No. 2018078053 (i.e., N,N-ditetradecylacetamide compounds or lipids derived from those described in claim 5 of International Publication No. 2018078053), the entire disclosure of International Publication No. 2018078053 being incorporated herein by reference.

[0263] In this specification, all chemical compounds referred to throughout the specification can be prepared by methods known to those skilled in the art, and the starting materials and / or reagents used in these processes can be obtained through the ordinary knowledge of those skilled in the art, based on common sense (e.g., textbooks, or patent applications such as International Publication Nos. 2022173667, 2009043027, 2013067199, 2010006282, 2009089542, 2016019340, 2008106186, 2020264505, and 2020023947, etc. (the full disclosures of these patent applications are incorporated herein by reference)).

[0264] In other aspects and embodiments of the present invention, commercially available DMG-PEG2000 (DMG-PEG2K or PEG2000-DMG, or "1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000") is a preferred polymer-binding lipid for the LNP of the present invention.

[0265] [ka]

[0266] Regarding the amounts of each excipient, it is preferable that the ionizable lipid is incorporated in a relatively high molar amount compared to the molar amount of the polymer-bound lipid of formula (I) present in the composition or lipid nanoparticles of the present invention. Furthermore, it is preferable that the molar amount of the ionizable lipid is higher than the molar amount of the neutral lipid in the composition or nanoparticles. In addition, the molar amount of the steroid may optionally be higher than the molar amount of the polymer-bound lipid of formula (I).

[0267] In certain embodiments, the LNP comprises one or more additional lipids that stabilize particle formation during particle formation. Suitable stabilizing lipids include neutral lipids and anionic lipids. In various embodiments, the molar ratio of ionizable lipids (e.g., lipids of formula (I)) to neutral lipids is in the range of about 2:1 to about 8:1, about 3:1 to about 7:1, or about 4:1 to about 6:1.

[0268] In a particular embodiment, the polymer-bound lipid of formula (I) is present in the LNP in an amount of about 1 mol% to about 10 mol% relative to the total lipid content of the nanoparticles. In one embodiment, the polymer-bound lipid of formula (I) is present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, the polymer-bound lipid of formula (I) is present in the LNP in an amount of about 1 mol% or about 1.5 mol%. In a preferred embodiment, the polymer-bound lipid of formula (I) is present in the LNP in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, or about 10 mol%, based on the molar percentage with the total lipid component or excipient composition as 100%, preferably about 5 mol%, more preferably about 2.5 mol%, or similarly preferably about 1.7 mol%.

[0269] In a particular very preferred embodiment, the polymer-bound lipid according to formula (I) is present in the LNP in an amount of about 0.1 mol% to about 1 mol% relative to the total lipid content of the nanoparticles. In one embodiment, the polymer-bound lipid according to formula (I) is present in the LNP in an amount of about 0.5 mol% to about 1 mol%. In one embodiment, the polymer-bound lipid according to formula (I) is present in the LNP in an amount of about 0.7 mol% to about 1.3 mol%. In a preferred embodiment, the polymer-bound lipid according to formula (I) is present in the LNP in amounts of about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, most preferably about 1 mol%, preferably 0.5 mol%, more preferably 0.7 mol%, or similarly preferably 0.8 or 0.9 mol%, most preferably about 1 mol%, based on the molar percentage with the total lipid component or excipient composition as 100%.

[0270] In some embodiments, the lipid-based carrier contains polymer-bound lipids in amounts of about 3 mol%, 2 mol%, or less than 1 mol%, based on the total moles of lipids in the lipid-based carrier. In further embodiments, the lipid-based carrier contains polymer-bound lipids in amounts of about 0.1% to about 10% on a molar basis, for example, about 0.5% to about 10%, about 0.5% to about 5%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100% of the total moles of lipids in the lipid-based carrier). In other preferred embodiments, the lipid-based carrier contains about 1.0% to about 2.0% on a molar basis (based on 100% total moles of lipids in the lipid-based carrier), for example, about 1.2% to about 1.9%, about 1.2% to about 1.8%, about 1.3% to about 1.8%, about 1.4% to about 1.8%, about 1.5% to about 1.8%, about 1.6% to about 1.8%, particularly about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most preferably 1.7% of polymer-bound lipids. In other preferred embodiments, the lipid-based carrier contains about 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, preferably 2.5%, of polymer-bound lipids on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).

[0271] In a very preferred embodiment, the lipid-based carrier contains about 2.5% of polymer-bound lipids on a molar basis (based on 100% of the total moles of lipids in the lipid-based carrier). In this regard, the polymer-bound lipids may be polymer-bound lipids containing P(M)OZ, preferably "PMOZ4".

[0272] In some embodiments, the lipid nanoparticles contain polymer-bound lipids of the present disclosure in molar ratios of about 0.5 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol% (based on 100% total moles of lipids in the lipid-based carrier).

[0273] In other preferred embodiments, the lipid-based carrier comprises about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, or about 1.5 mol% of the polymer-bound lipid of the present invention (based on 100% total moles of lipids in the lipid-based carrier), or less than about 1.5 mol% of the polymer-bound lipid of the present invention. In other preferred embodiments, the lipid-based carrier comprises about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, or about 1.5 mol% of the PMOZ lipid of the present invention, preferably "PMOZ4", or less than about 1.5 mol% of the PMOZ lipid of the present invention, preferably "PMOZ4". In other embodiments, the lipid-based carrier comprises about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, or about 1.5 mol% of PEG lipids, preferably DMG-PEG2000, or less than about 1.5 mol% of PEG lipids, preferably DMG-PEG2000 (based on 100% total moles of lipids in the lipid-based carrier).

[0274] In a preferred embodiment, the content of the polymer-bound lipid according to formula (I) of the present invention is about 1 to 5 mol% of the total lipid content of the formulation, preferably 1.7 mol% or 2.5 mol% (based on 100% total moles of lipid in the lipid-based carrier). In another very preferred embodiment, the lipid-based carrier contains about 1% or less than 1% of the polymer-bound lipid on a molar basis (based on 100% total moles of lipid in the lipid-based carrier). In another very preferred embodiment, the lipid-based carrier contains less than 1% on a molar basis (based on 100% total moles of lipid in the lipid-based carrier), i.e., preferably an amount of polymer-bound lipid selected from the group consisting of about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, and about 0.9 mol% (based on 100% total moles of lipid in the lipid-based carrier). In other preferred embodiments, the lipid-based carrier comprises about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, or about 10 mol% of the polymer-bound lipids of the present invention (based on 100% total moles of lipids in the lipid-based carrier), or more preferably about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, or about 1 mol% of the polymer-bound lipids of the present invention, preferably "PMOZ4".

[0275] In various embodiments, the molar ratio of ionizable lipids (e.g., lipids of formula (II)) to polymer-bound lipids according to formula (II) is in the range of about 100:1 to about 25:1, about 50:1 to about 25:1, or about 40:1 to about 25:1.

[0276] In various embodiments, the molar ratio of ionizable lipids to polymer-bound lipids is in the range of approximately 100:1 to approximately 25:1. Cationic ionizable lipids or cationizable lipids Ionizable lipids of LNPs protonate when the pH drops below the pK of the lipid's ionizable group, but become increasingly neutral at higher pH values. Therefore, at pH values ​​below the pK, the lipids can associate with negatively charged nucleic acids. In certain embodiments, the ionizable lipids include zwitterionic lipids that become positively charged as the pH decreases.

[0277] In the novel ionizable lipid shown below by formula (II), the degradable / biodegradable portion A comprises two structures R which may be the same or different. a and R b Connect R a and R b Each of these contains at least one basic, i.e., cationic moiety, which contains a tertiary nitrogen atom. a and R b At least one of the components has a substantially lipophilic tail structure and at least one ester group. The degradable / biodegradable portion A may be selected from the following functional groups: -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-. In one preferred embodiment, A is a portion or group containing one or more sulfur atoms, such as -S-, -SS-, or -SC(O)-N(H)-. In a particularly preferred embodiment of embodiment A shown herein, A is -S-, and the ionizable lipid is R a -SR b It can be expressed as, where R a and R b The following can be selected according to the above definition.

[0278] In a very preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -AR b Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, Ra teeth,

[0279] [ka]

[0280] Selected from, R b teeth,

[0281] [ka]

[0282] Selected from, A is -S-, R 1 These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. These choices are all independent of each other.

[0283] In another highly preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): Ra -AR b Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth

[0284] [ka]

[0285] And, R b teeth

[0286] [ka]

[0287] And, A is -S-; R 1 These are ethanediyl or linear or unbranched alkanediyl having two carbon atoms. R 2 It is an alkanediyl having two carbon atoms, R 3 ha-R 5 -C(O)-O-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 3 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. These choices are all independent of each other.

[0288] In another highly preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -ARb Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth

[0289] [ka]

[0290] And, R b teeth

[0291] [ka]

[0292] And, A is -S-, R 1 These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R 2 It is an alkanediyl having two carbon atoms, R 3 ha-R 5 -C(O)-O-, -R 5 -OC(O)-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 6 carbon atoms, These choices are all independent of each other.

[0293] In another highly preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -AR b Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth,

[0294] [ka]

[0295] Selected from, R b teeth,

[0296] [ka]

[0297] Selected from, A is -S-, R 1 These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R 2 It is an alkanediyl having two carbon atoms, R 3 ha-R 5 -C(O)-O-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 3 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. These choices are all independent of each other.

[0298] In a more very preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -AR b Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth,

[0299] [ka]

[0300] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0301] [ka]

[0302] , -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 Selected from -N(CH3)2, A is -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, R 1 These are ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms, where each substituted carbon atom is either unsubstituted or substituted with one or more C1-C4 alkyl, C1-C4 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5-OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms, and the lipophilic substituent having 12 to 36 carbon atoms is (i) a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or (ii) derived from tocopherol or tocotrienol. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon (CH) or nitrogen atom bonded to a hydrogen atom. These choices are all independent of each other.

[0303] In a more very preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -AR b Formula (II) A lipid represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth,

[0304] [ka]

[0305] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0306] [ka]

[0307] , or -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1Selected from -N(CH3)2, A is -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, preferably A is -S-. R 1 These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. These choices are all independent of each other.

[0308] In a more very preferred embodiment, the ionizable lipid contained in the lipid nanoparticles of the present invention is of formula (II): R a -AR b Formula (II) It is a lipid represented by, Here, R a teeth,

[0309] [ka]

[0310] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0311] [ka]

[0312] , -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 Selected from -N(CH3)2, A is -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, preferably A is -S-. R 1 is an optionally substituted ethanediyl, propanediyl, butanediyl, or a linear or unbranched alkanediyl having 2 to 8 carbon atoms, preferably R 1 is ethane, R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom or a nitrogen atom. These choices are all independent of one another. However, optionally, R 1, R 2 and R 5 All are linear unsubstituted ethanediyls, A is -SS-, and R a and R b If they are the same, R 4 teeth

[0313] [ka]

[0314] isn't it. In another embodiment, the present invention relates to a novel ionizable lipid useful for the delivery of nucleic acids to living cells. The ionizable lipid is given by formula (II): R a -AR b Formula (II) It is a compound represented by the following: Here, R a teeth,

[0315] [ka]

[0316] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0317] [ka]

[0318] , -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 Selected from -N(CH3)2, A is -S-, -SS-, -SC(O)-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, preferably A is -S-. R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, or -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom or a nitrogen atom. These choices are all independent of one another, However, optionally, R 1 , R 2 and R 5 All are ethanes, A is -SS-, and R a and R b If they are the same, R 4 teeth

[0319] [ka]

[0320] isn't it. In yet another embodiment, embodiment A, the present invention is defined by formula (II): R a -AR b Formula (II) This provides a novel ionizable lipid defined as a compound represented by, or a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof, Here, R a teeth,

[0321] [ka]

[0322] or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0323] [ka]

[0324] , -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 Selected from -N(CH3)2, A is -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-, R 1 These are ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms, where each substituted carbon atom is either unsubstituted or substituted with one or more C1-C4 alkyl, C1-C4 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5-C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms, and this lipophilic substituent having 12 to 36 carbon atoms is either a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or is derived from alpha-tocopherol. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon (CH) or nitrogen atom bonded to a hydrogen atom. These choices are all independent of one another, However, optionally, (i)R 3 ga-R 5 (ii)R 1 and R 2 (iii)R 5 (iv)A is -SS- and (v)R a and R b If they are the same, R 4 teeth

[0325] [ka]

[0326] Instead, Furthermore, (i)R 3 (ii)R 1 and R 2 (iii) A is a linear unsubstituted ethanediyl, and (iv) R a and R b If they are the same, R 4 teeth

[0327] [ka]

[0328] but

[0329] [ka]

[0330] Not either, Alternatively, as an alternative to the above conditions, ionizable lipids may be:

[0331] [ka]

[0332] It is not a lipid selected from the group consisting of the following. In other embodiments, R of formula (II) 4 teeth

[0333] [ka]

[0334] That is the case. In one preferred embodiment, A is -S- and R a and R b They are identical, R 4 teeth

[0335] [ka]

[0336] That is the case. In another preferred embodiment, A is -S- and R 4 teeth

[0337] [ka]

[0338] That is the case. In a preferred embodiment, Ra teeth,

[0339] [ka]

[0340] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from, R b teeth,

[0341] [ka]

[0342] , or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from. R a and R b Since they may be different from each other, they can be selected independently. As mentioned above, R a teeth,

[0343] [ka]

[0344] or -R 1 -N(H)-C(O)-R 3 -R 4 It may be selected from, preferably X is CH and R b teeth,

[0345] [ka]

[0346] , -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1-N(CH3)2 may be selected, and preferably X is CH. Furthermore, as mentioned above, R a teeth,

[0347] [ka]

[0348] It may be selected from, preferably X is CH and R b teeth,

[0349] [ka]

[0350] X may be selected from the following, and preferably X is CH. In one preferred embodiment, R a and R b At least one of them is R 2 and A or R 1 It contains a piperidine or piperazine-derived 6-membered ring structure between and . This has at least one tertiary nitrogen atom located in the vicinity of part A and a lipophilic tail structure R 4 From at least one spacer (R 2 ), means that it is separated by any ester group. 3 The potential advantage of having multiple ester groups (if present) lies in the fact that the hydrolytically unstable ester bonds further enhance the degradation of lipids in physiological environments, such as the intracellular environment.

[0351] In a further embodiment, R a and R b Both contain a piperidine or piperazine-derived 6-membered ring structure. Also, R a and R b Both

[0352] [ka]

[0353] And preferably, ionizable lipids in which X is CH are also preferred, R a and R b These may be selected independently or they may be the same. In a further embodiment, R a teeth

[0354] [ka]

[0355] And preferably X is CH and / or R b teeth

[0356] [ka]

[0357] Preferably, X is CH, and R 2 It acts as a linker or spacer between the respective basic piperidine or piperazine derivative ring structure and the ester group. As mentioned above, R 2 It is defined as an alkanediyl having 2 to 8 carbon atoms. 2 The chain may be linear or branched, and otherwise (i.e., except for the branched portion) is preferably unsubstituted. In one embodiment, R 2 is a linear unsubstituted alkanediyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In another embodiment, R 2 R is a linear unsubstituted alkanediyl having 2 to 6 carbon atoms. In a further preferred embodiment, R 2 R is a linear unsubstituted ethanediyl or propanediyl. For example, R a and R b Both are

[0358] [ka]

[0359] It may be, preferably, X is N or CH, and R 2 This is selected from straight-chain unsubstituted alkanediyls having 2 to 6 carbon atoms, such as ethanediyl or propanediyl.

[0360] R in equation (II) 4 R is defined as a lipophilic substituent having 12 to 36 carbon atoms. a The "tail" end, and in some cases R b (However, R b ga-R 1 The "tail" end (if not N(CH3)2) is thought to confer the lipophilicity typically required for molecules to cross biological membranes. Therefore, R 4 In principle, R may be any structure that is substantially lipophilic. For example, hydrocarbon structures are lipophilic. In one embodiment, R 4 It may consist only of carbon and hydrogen atoms at at least one occurrence site. In one preferred embodiment, R 4 R represents a linear or branched alkyl or alkenyl, preferably having 12 to 25 carbon atoms. The branched alkyl or alkenyl may optionally have multiple side chains, for example, two, three, four or more methyl side chains. In another embodiment, R 4 This may be an alkyl or alkenyl containing a single alkyl or alkenyl side chain having, for example, 2 to 10 carbon atoms. For example, R 4 The molecule may be 1-n-hexyl-n-nonyl (or 7-n-pentadecyl) or 2-n-hexyl-n-decyl. In other embodiments, the lipophilic substituent may optionally contain one or more heteroatoms such as O, S, or N. In other embodiments, the lipophilic substituent may optionally contain one or more saturated, unsaturated, or aromatic ring structures that may contain one or more heteroatoms such as O, S, or N.

[0361] R 4It may also contain a small number of heteroatoms, such as oxygen atoms, as long as its primarily lipophilic properties are maintained. In one embodiment, R 4 It contains one or more oxygen atoms and does not contain any other heteroatoms. 4 It may also include ring structures such as aromatic rings or aliphatic ring structures, which may optionally contain one or more oxygen atoms. If heteroatoms and / or ring structures are present, they are not at the terminal end of the “tail” but at any R 3 It is preferable that it be located on the structural side. In one embodiment, R 4 R is a lipophilic group derived from tocopherol or tocotrienol. In one embodiment, R 4 It is lipophilic, derived from alpha-tocopherol, and in particular,

[0362] [ka]

[0363] And in particular, R 1 , R 2 and R 5 Not all of them are linear unsubstituted ethanediyls, A is -SS-, and R a and R b They are identical. The “lipophilic groups derived from tocopherols or tocotrienols” as used herein include derivatives of tocopherols and tocotrienols, and in particular derivatives having the structures shown in Scheme 1 below, namely derivatives derived from alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol.

[0364] [ka]

[0365] Scheme 1: Tocopherol derivatives have saturated phytyl chains, and tocotrienol derivatives have polyunsaturated phytyl chains. For both tocopherol and tocotrienol derivatives, isoforms are defined by R1 and R2 selected from CH3 and H. Thus, as shown, for example, when R1 is CH3 and R2 is CH3, the resulting derivatives are alpha-isoforms of tocopherol and tocotrienol, respectively (called alpha-tocopherol derivatives and alpha-tocotrienol derivatives, respectively). The OH group is a bonding site, as shown in the two structures on the left side of the figure, and therefore is naturally absent in the derivatives.

[0366] In particular, in the preferred embodiment of the above-described aspect A, R 4 The group is either a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or a lipophilic group selected from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and derivatives of delta-tocotrienol as shown herein in Scheme 1.

[0367] In particular, in yet another preferred embodiment of the above embodiment A, R 4 is a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or

[0368] [ka]

[0369] That is the case. Preferably, all chiral centers of the vitamin E or tocopherol moiety have the (R) configuration. It should be understood herein that other embodiments in which the chiral centers of the tocopherol may vary, i.e., not all of them have the (R) configuration, are also included in the scope of the present invention.

[0370] In a very preferred embodiment, the ionizable lipid is given by formula (II): R a -AR b Formula (II) Defined as a compound represented by, Here, R a teeth

[0371] [ka]

[0372] And, R b teeth,

[0373] [ka]

[0374] Selected from, A is -S-, X is a carbon atom, R 1 is an optionally substituted ethanediyl, propanediyl, butanediyl, or a linear or unbranched alkanediyl having 2 to 8 carbon atoms, preferably R 1 is ethane, R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 This is a lipophilic substituent having 12 to 36 carbon atoms, preferably

[0375] [ka]

[0376] And, R 5 It is an alkanediyl having 1 to 6 carbon atoms, These choices are all independent of each other.

[0377] In other preferred embodiments, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, in the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP being selected from structures C1-C23 in Table 1, or C1-C27 respectively, or a lipid derived from formula (I) of PCT patent application PCT / EP2019 / 086825 or a subsequent patent application claiming priority to said application, namely International Publication No. 2021123332. In other embodiments, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, in the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP being derived from structures C1-C23, or C1-C27 respectively, in Table 1 of the PCT patent application PCT / EP2019 / 086825 or a subsequent patent application claiming priority to the said application, i.e., International Publication No. 2021123332, where element "A" of formula (I) of PCT / EP2019 / 086825 is -S-. Accordingly, formulas C1-C23, or C1-C27 respectively, of the PCT patent application PCT / EP2019 / 086825 or a subsequent patent application claiming priority to the said application, i.e., International Publication No. 2021123332, and specific disclosures therein are incorporated herein by reference.

[0378] In a very preferred embodiment, the ionizable lipid of the present invention is selected from the group consisting of C24, C28, and C29. In a more very preferred embodiment, the ionizable lipid of the present invention is C24.

[0379] In a more very preferred embodiment, the ionizable lipid of the present invention is C28. In a more very preferred embodiment, the ionizable lipid of the present invention is C29. In further embodiments, the ionizable lipid is preferably selected from the ionizable lipids listed in Table 1 herein.

[0380] [Table 1]

[0381] JPEG2026509258000088.jpg216170

[0382] Accordingly, the present invention is directed toward a composition comprising one ionizable lipid selected from the group consisting of compounds C24, C28, and C29, preferably as described above and below in this specification, in combination with a polymer-bound lipid represented by formula (I) as described herein, preferably "PMOZ4". Even more preferably, the present invention is directed toward a composition comprising one ionizable lipid selected from the group consisting of compounds C24, C28, and C29, preferably as described above and below in this specification, in combination with a polymer-bound lipid represented by formula (I) as described herein, preferably "PMOZ4", and further in combination with lipids DPhyPE and DPhyPS.

[0383] In a very preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, of the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP having a structure "C24", which is the most preferred structure for the ionizable lipids contained in the lipid nanoparticle composition of the present invention.

[0384] [ka]

[0385] In another very preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, of the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP having a structure "C28", which is another very preferred structure for the ionizable lipids contained in the lipid nanoparticle composition of the present invention.

[0386] [ka]

[0387] In another very preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, of the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP having a structure "C29", which is another very preferred structure for the ionizable lipids contained in the lipid nanoparticle composition of the present invention.

[0388] [ka]

[0389] In another embodiment of the present invention, a method for synthesizing the ionizable lipids of the present invention, preferably a method for synthesizing ionizable lipid C28 or ionizable lipid C29 according to the synthesis route described in the examples of the present invention, i.e., · "Synthesis of lipid C28 (bis((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)O,O'-(((thiobis(ethane-2,1-diyl))bis(piperidine-1,3-diyl))bis(ethane-2,1-diyl))disuccinate (VitE-C4DE-meta-Pip-S))" or each • "Synthesis of lipid C29 (bis((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)O,O'-(((thiobis(ethane-2,1-diyl))bis(piperidine-4,1-diyl))bis(ethane-2,1-diyl))disuccinate (VitE-C4DE-reversePip-S))" It will be provided.

[0390] Cationic, ionizable, or cationizable lipids include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N- (1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5,1,2-dilinoleylcarbamoyl Oxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3 - Trimethylaminopropane chloride salt (DLin-TMA.Cl), ICE (imidazole-based), HGT5000, HGT5001, DMDMA, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), HGT4003, 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-di Methyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1, 1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazandiyl)didodecane-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl )-N1,N16-Diundecyl-4,7,10,13-Tetraazahexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-Tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-Tetraen-19-yloxy)-N,N-Dimethylpropane-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-Tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE), manufactured by GIBCO / BRL [Grand Island, New York]); LIPOFECTAMINE® (commercially available cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), manufactured by GIBCO / BRL); and TRANSFECTAM® (commercially available ionizable lipids containing dioctadecylamideglycylcarboxyspermine (DOGS) in ethanol, manufactured by Promega Corporation) This includes (manufactured by) Corp. [Madison, Wisconsin] or any combination of the foregoing. Further suitable ionizable lipids used in the compositions and methods of the present invention include those described in International Publication No. 2010053572 (and in particular CI2-200 as described in paragraph

[0225] ) and International Publication No. 2012170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see U.S. Patent Application Publication No. 20150140070).

[0391] In some embodiments, the ionizable lipid may be an amino lipid. Representative aminolipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), and 1,2-dilinoleyloxy-3- This includes (N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and MC3 (U.S. Patent Application Publication No. 20100324120).

[0392] In preferred embodiments, the ionizable lipid may be an amino alcohol lipidoid. The amino alcohol lipidoids that can be used in the present invention may be prepared by the method described in U.S. Patent No. 8,450,298, which is incorporated in whole into this specification. Suitable (ionizable) lipids may also be compounds disclosed in Tables 1, 2, and 3 of International Publication No. 2017075531, which is incorporated into this specification, and as defined in claims 1 to 24 thereof.

[0393] In another embodiment, suitable lipids may also be compounds disclosed in International Publication No. 2015074085 (i.e., compounds designated in ATX-001 to ATX-032 or claims 1 to 26), U.S. Patent Applications Publications 61 / 905,724 and 15 / 614,499, or U.S. Patents 9,593,077 and 9,567,296, which are incorporated herein by reference in whole.

[0394] In other embodiments, suitable ionizable lipids may also be compounds disclosed in International Publication No. 2017117530, which is incorporated herein by reference in whole (i.e., lipids 13, 14, 15, 16, 17, 18, 19, 20, or compounds specified in the claims).

[0395] In a preferred embodiment, the ionizable or ionizable lipid may also be selected from the lipids disclosed in International Publication No. 2018078053 (i.e., lipids derived from formulas I, II, and III of International Publication No. 2018078053, or lipids specified in claims 1 to 12 of International Publication No. 2018078053), the entire disclosure of International Publication No. 2018078053 is incorporated herein by reference. In that context, the lipids disclosed in Table 7 of International Publication No. 2018078053 (e.g., lipids derived from formulas I-1 to I-41) and the lipids disclosed in Table 8 of International Publication No. 2018078053 (e.g., lipids derived from formulas II-1 to II-36) may be suitably used in the context of the present invention. Accordingly, formulas I-1 to I-41 and II-1 to II-36 of International Publication No. 2018078053, and specific disclosures related thereto, are incorporated herein by reference.

[0396] In a preferred embodiment, the ionizable lipid may be derived from Formula III of the published PCT patent application, International Publication No. 2018078053. Accordingly, Formula III of International Publication No. 2018078053 and the specific disclosures relating thereto are incorporated herein by reference.

[0397] In further embodiments, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, of the composition is complexed with one or more lipids to form an LNP, the ionizable lipid of the LNP being selected from structures III-1 to III-36 in Table 9 of the published PCT patent application, International Publication No. 2018078053. Accordingly, formulas III-1 to III-36 of International Publication No. 2018078053 and the specific disclosures thereof are incorporated herein by reference. In certain embodiments, the ionizable lipid as defined herein, more preferably the ionizable lipid compound III-3 ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) is present in the LNP in an amount of about 30 mol% to about 80 mol%, preferably about 30 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, and more preferably about 47.4 mol%, relative to the total lipid content of the LNP. When two or more ionizable lipids are incorporated into an LNP, these percentages are applied to the combined ionizable lipids.

[0398] In a preferred embodiment, the ionizable lipid is present in the LNP in an amount of about 30 mol% to about 70 mol%. In one embodiment, the ionizable lipid is present in the LNP in an amount of about 40 mol% to about 60 mol%, for example, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, or about 65 mol%. In a preferred embodiment, the ionizable lipid is present in the LNP in amounts of about 47 mol% to about 48 mol%, for example, about 47.0 mol%, about 47.1 mol%, about 47.2 mol%, about 47.3 mol%, about 47.4 mol%, about 47.5 mol%, about 47.6 mol%, about 47.7 mol%, about 47.8 mol%, about 47.9 mol%, and about 50.0 mol%, with about 47.4 mol% being particularly preferred.

[0399] In other embodiments, cationic or ionizable lipids are

[0400] [ka]

[0401] JPEG2026509258000093.jpg255170

[0402] That is the case. In preferred embodiments, the amino or ionizable lipids as defined herein have at least one protonable or deprotonable group such that they are positively charged at a pH below physiological pH (e.g., pH 7.4) and preferably neutral at a second pH above physiological pH. It will be understood, of course, that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of the dominant species and do not require that all lipids exist in either a charged or neutral form. Lipids having two or more protonable or deprotonable groups, or being zwitterionic, are not excluded and may be equally appropriate in the context of the present invention. In some embodiments, the protonizable lipids have a pKa of protonable groups in the range of about 4 to about 11, e.g., about 5 to about 7.

[0403] An LNP may contain two or more (different) ionizable lipids as defined herein. The ionizable lipids may be selected to contribute to different advantageous properties. For example, ionizable lipids with different properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in an LNP. In particular, ionizable lipids may be selected such that the properties of the mixed LNP are more desirable than the properties of the individual lipids in a single LNP.

[0404] In other embodiments, the ionizable lipids of this disclosure are compounds of formula (Cat-II):

[0405] [ka]

[0406] Alternatively, these may be N-oxides, or salts or isomers thereof. R1 is C5-30 alkyl, C5-20 alkenyl, -R * Selected from the group consisting of YR'', -YR'', and -R''M'R', R2 and R3 are H, C 1~14 Alkyl, C 2~14 Alkenil, -R * YR”, -YR”, and -R * R2 and R3 are independently selected from the group consisting of OR, or together with the atoms to which they are bonded, they form a heterocycle or a carbon ring. R4 is hydrogen, C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, CHQR, -CQ(R)2, and unsubstituted C 1~6 Selected from the group consisting of alkyl groups, where Q is a carbocyclic, heterocyclic, -OR, or -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R , -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2RS, -O(CH2) n Selected from OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5. Each R5 is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyl and H, Each R6 is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyl and H, M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl groups, and heteroaryl groups, where M'' is bonded, C 1~13 Alkyl or C 2~13 It is alkenyl, R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyl and H, R8 is C 3~6 Selected from the group consisting of carbon rings and heterocycles, R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6 Selected from the group consisting of carbon rings and heterocycles, Each R is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyl and H, Each R' is C 1~18 Alkyl, C 2~18 Alkenil, -R * Independently selected from the group consisting of YR'', -YR'', and H, Each R is C 3~15 Alkyl and C 3~15 Independently selected from the group consisting of alkenils, Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils, Each Y is independent of C 3~6 It is a carbon ring, Each X is independently selected from the group consisting of F, Cl, Br, and I, m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, and R4 is -(CH2) n Q, -(CH2) nIf it is CHQR, -CHQR, or -CQ(R)2, then (i) if n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) if n is 1 or 2, Q is not a 5-membered, 6-membered, or 7-membered heterocycloalkyl.

[0407] As used herein, the term “ionizable lipid” has its usual meaning in the art and may refer to a lipid containing one or more charged moieties. In some embodiments, the ionizable lipid may be positively charged or negatively charged. The ionizable lipid may be positively charged and in that case may be called an “ionizable lipid.” Thus, the terms “cationic lipid” and “ionizable lipid” may be used interchangeably throughout this specification. In certain embodiments, the ionizable lipid molecule may contain an amine group and may be called an ionizable aminolipid. As used herein, “charged moiety” refers to a chemical moiety having a formal electronic charge, e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In certain embodiments, the charged moieties include amine groups. Examples of negatively charged groups or their precursors include carboxylic acid groups, sulfonic acid groups, sulfate groups, phosphonic acid groups, phosphate groups, and hydroxyl groups. The charge of a charged moiety may, in some cases, change depending on environmental conditions; for example, pH changes can alter the charge of a moiety and / or make it charged or uncharged. In general, the charge density of a molecule can be chosen as desired. It should be understood that the terms “charged” or “charged moiety” do not refer to “partially negative charge” or “partially positive charge” on the molecule. The terms “partially negative charge” and “partially positive charge” are given their usual meanings in the art. A "partial negative charge" can arise when a functional group contains a bond that is polarized such that the electron density is pulled toward one atom of the bond, creating a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this way.In some embodiments, the ionizable lipid is an ionizable aminolipid, sometimes referred to in the art as an "ionizable cationic lipid" or "cationic ionizable lipid." In one embodiment, the ionizable aminolipid may have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0408] Furthermore, regarding preferred compositions, (i) The ionizable lipid can be selected from the compounds in Table 1, and / or (ii) Neutral lipids or neutral phospholipids include 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also called 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also called dioleoylphosphatidylcholine), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (D PPC (also called dipalmitoylphosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), 1,2-dipalmitoyl-s n-glycero-3-phosphoethanolamine (DPPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoyl-phosphatidylethanolamine 1, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-Monomethylphosphoethanolamine, 16-O-Dimethylphosphatidylethanolamine, 1,2-Dielucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-Transphosphatidylethanolamine, 1-Stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dieridoyl-sn-glycero-3-phosphoethanolamine (TransDOPE), 1-Stearoyl-2-Linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE), 1-Tridecanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-Oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt), 1-Palmitoi 2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS), 1-1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-O- Hexadecanyl-2-O-(9Z-octadecenyl)-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesterylhemis A zwitterionic compound selected from the group consisting of succinoyl-sn-glycero-3-phosphocholine (DChemsPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethylhydrogen phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethylethyl phosphate (DOCPe), and 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (Edelfosine), and / or, (iii) Polymer-bound lipids are given by formula (I): [P]-[Linker]-[L] Formula (I) It is a polymer-bound lipid represented by [P] represents at least one polyoxazoline (POZ) monomer unit:

[0409] [ka]

[0410] It is a homopolymer portion containing, In the formula, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, n has an average value in the range of about 45 to about 55, preferably about 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. [linker] is any linker group, [L] represents the lipid portion.

[0411] In other preferred embodiments, the lipid-based carrier comprises cationic or ionizable lipids. Cationic or ionizable lipids in lipid-based carriers can be cationizable or ionizable; that is, they protonate when the pH drops below the pK of the lipid's ionizable group, but become increasingly neutral at higher pH values. Therefore, at pH values ​​below the pK, the lipid can associate with negatively charged nucleic acids. In certain embodiments, the ionizable lipids include zwitterionic lipids that become positively charged as the pH decreases.

[0412] In preferred embodiments, the lipid-based carrier preferably comprises a cationic or ionizable lipid having a net positive charge at physiological pH, and more preferably the cationic or ionizable lipid comprises a tertiary or quaternary nitrogen group. Therefore, in preferred embodiments, the lipid-based carrier comprises a cationic or ionizable lipid selected from aminolipids.

[0413] In further embodiments, the lipid formulation comprises a cationic or ionizable lipid defined by formula I in paragraph

[0251] of International Publication No. 2021222801 or a lipid selected from the disclosures in paragraphs

[0260] or

[0261] of International Publication No. 2021222801. In other embodiments, the lipid formulation comprises a cationic or ionizable lipid selected from the group consisting of ATX-001 to ATX-132, preferably ATX-0126, as disclosed in claim 90 of International Publication No. 2021183563. The disclosures of International Publication No. 2021222801 and International Publication No. 2021183563, in particular the aforementioned lipids, are incorporated herein by reference.

[0414] Furthermore, suitable ionizable lipids may be selected or derived from the ionizable lipids according to each of the PCT claims 1 to 14 of the published patent application, International Publication No. 2021123332, or Table 1 of International Publication No. 2021123332, and the disclosures relating to each of the claims 1 to 14 or Table 1 of International Publication No. 2021123332 are incorporated herein by reference. Accordingly, suitable ionizable lipids may be selected or derived from the ionizable lipids according to compounds 1 to 27 (C1 to C27) of Table 1 of International Publication No. 2021123332.

[0415] In other embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises an ionizable lipid selected from or derived from (COATSOME® SS-EC) SS-33 / 4PE-15 (see C23 in Table 1 of International Publication No. 2021123332).

[0416] In other embodiments, the lipid-based carrier of the pharmaceutical composition (e.g., LNP) comprises an ionizable lipid selected from or derived from 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, also known as SM-102 (Jackson LA, Anderson EJ, Rouphael NG, Roberts PC, Makhene M, Coler RN et al. An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med. 2020 Nov. 12;383(20):1920-31 or Hassett KJ, Higgins Higgins J, Woods A, Levy B, Xia Y, Hsiao CJ et al., Impact of lipid nanoparticle size on mRNA vaccine immunogenicity. J Control Release. July 10, 2021; 335:237-46 (see Lipid H). Other preferred lipid-based carriers (e.g., LNPs) for pharmaceutical compositions are squalamide ionizable aminolipids, more preferably formulas (M1) and (M2):

[0417] [ka]

[0418] It contains an ionizable lipid selected from the group consisting of, Substituents (e.g., R1, R2, R3, R5, R6, R7, R 10 M, M1, m, n, o, l) are defined in claims 1 to 13 of U.S. Patent No. 1,0392,341, which is incorporated herein by reference in its entirety.

[0419] Therefore, in a preferred embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises an ionizable lipid selected from or derived from ALC-0315, SM-102, SS-33 / 4PE-15, HEXA-C5DE-PipSS, or compounds C24, C28, or C29 mentioned above.

[0420] In a particularly preferred embodiment, the lipid-based carrier of the pharmaceutical composition, preferably LNP, comprises an ionizable lipid selected from or derived from ALC-0315 or SM-102.

[0421] In some embodiments, the lipid-based carrier of the present invention comprises two or more (different) ionizable lipids as defined herein. Generally, the amount of ionizable lipids in a composition (and therefore in lipid nanoparticles) is typically at least about 20 mol% of the total molar amount of all lipid excipients in the composition (or nanoparticles). In another embodiment, the amount of ionizable lipids is at least about 25 mol%, or at least 30 mol%, respectively. In other preferred embodiments, the amount of ionizable lipids in the composition is about 30 mol% to about 70 mol%, or about 40 mol% to about 70 mol%, or about 45 mol% to about 65 mol%, respectively; for example, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, Approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, approximately 65 mol%, or 70 mol%, or approximately 40 mol% to approximately 60 mol%; for example, approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, or approximately 60 mol%.

[0422] In a preferred embodiment, the ionizable lipid is present in the lipid-based carrier in an amount of about 30 mol% to about 70 mol%. In one embodiment, the ionizable lipid is present in the lipid-based carrier in an amount of about 40 mol% to about 60 mol%, for example, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol%. In preferred embodiments, the ionizable lipid is present in the lipid-based carrier in amounts of about 47 mol% to about 48 mol%, for example, about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, and 50.0 mol%, respectively, with 47.4 mol% being particularly preferred. In other preferred embodiments, the ionizable lipid is present in the lipid-based carrier in amounts of about 55 mol% to about 65 mol%, for example, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, or about 65 mol%, respectively, with 59 mol% being particularly preferred.

[0423] In some embodiments, ionizable lipids are present in the LNP in a ratio of about 20 mol% to about 70 mol% or 75 mol%, or about 45 mol% to about 65 mol%, or about 35 mol% to about 45 mol%, or about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol% of the total lipids present in the LNP. In further embodiments, the LNP contains ionizable lipids in a molar basis of about 25% to about 75%, for example, about 20% to about 70%, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 57.1%, about 50%, or about 40% in a molar basis (based on 100% of the total moles of lipids in the lipid nanoparticles). In some embodiments, the ratio of ionizable lipids to nucleic acids (e.g., coding RNA or DNA) is about 3 to about 15, for example, about 5 to about 13 or about 7 to about 11.

[0424] In some embodiments, the ratio of ionizable lipids to RNA is about 3 to about 15, for example, about 5 to about 13 or about 7 to about 11. References to other suitable cationic or ionizable, neutral, steroid / sterol, or polymer-bound lipids: Other suitable cationic or ionizable, neutral, steroid / sterol or polymer-bound lipids are described in International Publication Nos. 2010053572, 2011068810, 2012170889, 2012170930, 2013052523, 2013090648, 2013149140, 2013149141, 2013151663, 2013151664, 2013151665, and 2013151666. 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Publication No. 20130178541, U.S. Patent Application Publication No. 20130225836, and U.S. Patent Application Publication No. 20140039032, which specifically relate to cationic or ionizable, neutral, sterol, or polymer-bound lipids suitable for lipid-based carriers, are incorporated herein by reference.

[0425] steroid A "steroid" is an organic compound that has four rings arranged in a specific molecular configuration. Steroids contain the following carbon skeletons:

[0426] [ka]

[0427] Steroids and neutral steroids both include naturally occurring steroids and their analogues (e.g., cholesteryl hemisuccinate (CHEMS), an amphiphilic lipid consisting of succinic acid esterified to the beta-hydroxyl group of cholesterol as a cholesterol derivative). Using the definition of “neutral” as provided herein, a neutral steroid may be a steroid that has neither ionizable atoms nor groups under physiological conditions, or it may be a zwitterionic steroid. In one preferred embodiment, the neutral steroid contains neither ionizable atoms nor groups under physiological conditions. In some preferred embodiments, the steroid or steroid analogue is cholesterol. The terms “steroid” and “neutral steroid” are used interchangeably herein.In other embodiments, sterols are phytosterols, such as β-sitosterol, campesterol, stigmasterol, fucosterol, stigmathanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestanone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5a-cholesta-7-en-3β-ol, 3,6,9-triol Xaoctan-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, citcalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, dimosterol, diosgenin, f Cholesterol, fecosterol, or fecosterol, or their salts or esters; cholesterol, cholesterol succinate, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, cholesteryl phosphorylcholine; and sodium cholate may be selected from the group.

[0428] In further embodiments, the steroid is an imidazole cholesterol ester or "ICE" as disclosed in paragraphs

[0320] and

[0339] -

[0340] of International Publication No. 2019226925, which is incorporated herein by reference in whole.

[0429] In other preferred embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, a steroid analog, or a sterol. Preferably, the steroid, steroid analogue, or sterol may be derived from or selected from cholesterol, cholesteryl hemisuccinate (CHEMS), and their derivatives. In other embodiments, the lipid-based carrier of the pharmaceutical composition includes a steroid, steroid analogue, or sterol derived from a phytosterol (e.g., a sitosterol such as beta-sitosterol), preferably a compound having the structure of Formula I as disclosed in claim 1 of International Publication No. 2020061332; the disclosures of International Publication No. 2020061332, particularly the disclosures of Formula I and phytosterols, are incorporated herein by reference. In further embodiments, the steroid is an imidazole cholesterol ester or "ICE" as disclosed in paragraphs

[0320] and

[0339] -

[0340] of International Publication No. 2019226925; the entirety of International Publication No. 2019226925 is incorporated herein by reference.

[0430] In a particularly preferred embodiment, the lipid-based carrier of the pharmaceutical composition contains cholesterol. The molar ratio of ionizable lipids to cholesterol in lipid-based carriers can range from approximately 2:1 to approximately 1:1.

[0431] In some embodiments, the lipid-based carrier contains about 10 mol% to about 60 mol% or about 25 mol% to about 40 mol% of sterols (based on 100% of the total moles of lipids in the lipid-based carrier). In one embodiment, the sterols constitute about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipids present in the lipid-based carrier. In another embodiment, the lipid-based carrier contains about 5% to about 50% on a molar basis, for example, about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 48 mol%, about 40 mol%, about 38.5 mol%, about 35 mol%, about 34.4 mol%, about 31.5 mol%, or about 30 mol% sterols (based on 100% total moles of lipids in the lipid-based carrier). In a preferred embodiment, the lipid-based carrier contains about 28 mol%, about 29 mol%, or about 30 mol% sterols (based on 100% total moles of lipids in the lipid-based carrier). In the most preferred embodiment, the lipid-based carrier contains about 40.9 mol% sterols (based on 100% total moles of lipids in the lipid-based carrier).

[0432] The amount of steroids in the composition may be at least about 10 mol%, or in the range of about 10 mol% to about 60 mol%, or about 20 mol% to about 50 mol%, or about 25 mol% to about 45 mol%, respectively; for example, it may be about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol%, respectively. Also, to avoid any doubt, the molar percentages are relative to the total molar amount of all lipid excipients in the composition.

[0433] neutral lipids, neutral phospholipids The “neutral lipids,” also called “helper lipids,” according to the present invention are preferably phospholipids or neutral phospholipids. As used herein, “neutral phospholipids” are typically amphiphilic compounds comprising molecules having two hydrophobic fatty acid “tails” and a hydrophilic “head” containing a phosphate group. The phosphate group may be modified with simple organic molecules such as choline, ethanolamine, or serine. Phospholipids are abundant in nature. For example, phospholipids make up a significant proportion of additives in biological membranes. As used herein, the terms “phospholipids” or “neutral phospholipids” encompass both natural and synthetic phospholipids.

[0434] The terms “neutral lipid,” “neutral phospholipid,” or “zwitterionic compound,” as used interchangeably herein, refer to any one of several lipid species that exist at physiological pH uncharged or in a neutral zwitterionic form. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, which are further described below herein.

[0435] According to one preferred embodiment, the composition comprises a zwitterionic neutral lipid such as phosphatidylcholine or phosphatidylethanolamine. Suitable examples of phosphatidylcholine include natural or purified mixtures, sometimes called "lecithin" or "phosphatidylcholine," typically derived from egg yolk or soy; or highly purified or semi-synthetic compounds such as phosphatidylcholine having two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc.

[0436] In another preferred embodiment, the neutral lipid or neutral phospholipid is, but is not limited to, 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also known as 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmi Toyl-sn-glycero-3-phosphocholine (also called DPPC, dipalmitoylphosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine ( POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Phosphoethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dielucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-disquareoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dierydoyl-sn-glycero-3-phosphoethanolamine (Trans-DOPE), 1-Stearoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine (SLPE), 1-Tridecanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-Oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine ( (Sodium salt) (POPS), 1-1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-difytanol-sn-glycero-3-phospho-L-serine (sodium salt), 1-O-hexadecanyl-2-O-(9Z-oc Tadenyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesterylhemisuccinoyl It is a zwitterionic compound selected from the group consisting of 2-sn-glycero-3-phosphocholine (DChemsPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelhosine).

[0437] In another preferred embodiment, the neutral lipid according to the present invention is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In a more preferred embodiment, the neutral lipid according to the present invention is 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC). In an even more preferred, particularly preferred embodiment, the neutral lipid according to the present invention is 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE). An advantage of the present invention related to the use of DPhyPE is its high membrane-fusing ability due to its bulky tail, which enables it to fuse with endosomal lipids at a high level. Therefore, in another embodiment, the present invention relates to the use of lipids having high membrane-fusing ability in lipid-based carriers or nucleic acid-lipid particles, preferably DPhyPE as shown herein.

[0438] [ka]

[0439] Specifically, the advantageous use of 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), preferably in combination with the lipids of the present invention disclosed herein, for delivering mRNA vaccines in vivo, resulting in a significantly enhanced immune response, is a remarkable discovery made by the inventors, similar to specific aspects and embodiments of the present invention. In other words, the inventors have surprisingly found that the use of DPhyPE offers a clear advantage over DSPC, which has been used in the art to date as a standard neutral lipid in virtually all prior art LNP compositions for vaccine settings, specifically, but not limited to mRNA and also siRNA. In other words, the compositions of the present invention have highly advantageous and unpredictable behavior in vivo, resulting in a highly enhanced immune response.

[0440] Furthermore, the data presented in the examples demonstrate that the significantly enhanced immune response using the compositions of the present invention, i.e., all RNA vaccines of the present invention, is more useful than the present invention. Surprisingly, in contrast to prior art knowledge indicating that DSPC is the most common and undoubtedly neutral lipid for lipid nanoparticles, the inventors found that it is preferable to use DPhyPE in the mRNA formulation of the composition for producing the vaccine.

[0441] DSPC, DOPC, or DOPE, which are routinely used in the art as phospholipids in LNPs, each have two C molecules, as is evident from the structures shown below in this specification. 18 It has side chain arms.

[0442] [ka]

[0443] Surprisingly, in a further embodiment of the present invention, the inventors have found that the addition of phospholipids having shorter alkyl chains than, for example, prior art DSPC or DOPE is highly beneficial to the efficacy of the lipid nanoparticles of the present invention, including the ionizable lipids and polymer-bound lipids of the present invention according to formula (I), compared to lipid nanoparticles that do not contain the phospholipids having shorter alkyl chains.

[0444] In other preferred embodiments, the lipid-based carrier (e.g., LNP) comprises a neutral lipid or a phospholipid. The term "neutral lipid" refers to one of several lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Suitable neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids used in the particles described herein is generally guided by considering, for example, lipid particle size and the stability of the lipid particles in the bloodstream. Preferably, the neutral lipids are lipids having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipid contains saturated fatty acids having carbon chain lengths in the range of C10 to C20. In another embodiment, neutral lipids having monounsaturated fatty acids having carbon chain lengths in the range of C10 to C20 are used. Furthermore, neutral lipids having a mixture of saturated and unsaturated fatty acid chains can be used.

[0445] In some embodiments, the lipid-based carrier comprises one or more neutral lipids, the neutral lipids being distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)- Selected from the group comprising cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), or mixtures thereof.

[0446] In other preferred embodiments, the neutral lipid of the lipid-based carrier (e.g., LNP) of the pharmaceutical composition is selected from or derived from 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE).

[0447] Therefore, in a preferred embodiment, the lipid-based carrier of the pharmaceutical composition (e.g., LNP) comprises a neutral lipid selected from or derived from DSPC or DPhyPE, and further comprises phosphatidylserine, preferably DPhyPS.

[0448] In various embodiments, the molar ratio of ionizable lipids to neutral lipids in the lipid-based carrier is in the range of approximately 2:1 to approximately 8:1. In a preferred embodiment, the lipid nanoparticles of the present invention contain about 5 mol% to about 15 mol% of phospholipids, preferably DPhyPE, calculated from the total lipids present in the lipid-based carrier. In one embodiment, the lipid-based carrier contains about 3 mol% to about 12 mol% or about 5 mol% to about 10 mol% of phospholipids, preferably DPhyPE, calculated from the total lipids present in the lipid-based carrier. In some embodiments, the lipid nanoparticles of the present invention contain about 5 mol% to about 25 mol%, preferably about 5 mol% to about 15 mol%, or about 8 mol% to about 12 mol%, more preferably about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%, calculated from the total lipids present in the lipid-based carrier. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 1 mol% to about 6 mol%, preferably about 2.5 mol% to about 5 mol%, more preferably about 2.5 mol%, of phospholipids, preferably DPhyPE. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol%, of phospholipids, preferably DPhyPE. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 0.25 mol%, about 0.5 mol%, about 0.75 mol%, about 1 mol%, about 1.25 mol%, about 1.5 mol%, about 1.75 mol%, about 2 mol%, about 2.25 mol%, about 2.5 mol%, about 2.75 mol%, about 3 mol%, about 3.25 mol%, about 3.5 mol%, about 3.75 mol%, about 4 mol%, about 4.25 mol%, about 4.5 mol%, about 4.75 mol%, or about 5 mol% of phospholipids, preferably DPhyPE. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 1.5 mol%, about 2 mol%, or about 2.5 mol% of phospholipids, preferably DPhyPE. In a still more preferred embodiment, the lipid nanoparticles of the present invention contain about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol% of phospholipids, preferably DPhyPE.In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, or about 7.5 mol% of phospholipids, preferably DPhyPE. Similarly, in a more preferred embodiment, the lipid nanoparticles of the present invention contain about 5 mol% DPhyPE or about 7.5 mol% DPhyPE.

[0449] Phospholipid phosphatidylserine The inventors have also found, to their surprise, that the immune response can be enhanced by adding at least one further neutral lipid, particularly a second neutral lipid, to the above neutral lipid (see corresponding examples). As described above, it is preferable for the (first) neutral lipid of the present invention to have two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc. (this means in particular that the fatty acyl moieties are fairly long, starting with a moiety having 14 carbon atoms). The inventors have found that the addition of phosphatidylserine, particularly DPhyPS, provides a beneficial effect.

[0450] As used herein, the term "phosphatidylserine" refers to a compound comprising a serine head group and one or more tail groups bonded to a carbon atom of glycerol via a phosphodiester. Preferably, the tail groups are fatty acids bonded to another carbon atom of glycerol via an ester. Preferably, as used herein, the term "phosphatidylserine" refers to a compound comprising a serine head group and one or more tail groups bonded to a carbon atom of glycerol via a phosphodiester, wherein the tail groups are fatty acids bonded to another carbon atom of glycerol via an ester. The fatty acids may preferably be saturated fatty acids selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid. Fatty acids may also be unsaturated fatty acids selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoleidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. Fatty acids may also be branched-chain fatty acids, particularly phytanic acid.

[0451] Examples are shown below, for example, in the case of DPhyPS, WT-PS (i.e., 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine or 18:0-18:1 PS, WT-PS with two different fatty acid / alkyl chains, which is widely distributed among animals, plants and microorganisms), 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS and 18:1-PS DOPS, serine is bonded to the first carbon atom of glycerol via a phosphodiester, while the second and third carbon atoms of glycerol are bonded to the fatty acids, respectively, via esters. In this configuration, the two fatty acids may be identical (see, e.g., DPhyPS, 16:0 PS, 14:0-PS, 10:0-PS, 6:0-PS and 18:1-PS DOPS) or different (see, e.g., WT-PS or 18:0-18:1 PS). In other examples, for instance, in 18:1-Lyso PS and 18:0-Lyso PS, serine is similarly bonded to the first carbon atom of glycerin via a phosphodiester, while a single additional carbon atom of glycerin is bonded to a fatty acid via an ester, leaving a single OH group on the remaining carbon atoms of glycerin. Such a configuration is typically called “lysophosphatidylserine,” which, in consideration of the above definition, is included in the term “phosphatidylserine” as used herein. Preferred embodiments relating to DPhyPS are described herein in the section “A Fourth Set of Embodiments.”

[0452] In a preferred embodiment, phosphatidylserine is selected from the group consisting of DPhyPS, WT-PS, 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS, 18:1-PS DOPS, 18:1-Lyso PS, and 18:0-Lyso PS. The phosphatidylserine is DPhyPS or WT-PS (18:0-18:1 PS), most preferably DPhyPS, and most preferably in a molar ratio of 2.5 mol% in lipid nanoparticles.

[0453] In other preferred embodiments, the lipid nanoparticles of the present invention further include "DPhyPS" (1,2-difitanoyl-sn-glycero-3-phospho-L-serine; 4ME 16:0 PS) or "WT-PS" (i.e., 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine or 18:0-18:1 PS, due to two different fatty acid / alkyl chains of WT-PS which are widely distributed among animals, plants and microorganisms), 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS and 18:1-PS DOPS, where serine is bonded to the first carbon atom of glycerin via a phosphodiester, while the second and third carbon atoms of glycerin are bonded to the fatty acids, respectively, via esters. The structures of the phosphatidylserine mentioned above are as follows (it should be noted that all of these lipids are commercially available, for example, from Avanti Polar Lipids):

[0454] [ka]

[0455] JPEG2026509258000101.jpg255170

[0456] JPEG2026509258000102.jpg252170

[0457] JPEG2026509258000103.jpg171170

[0458] Further examples of saturated phosphatidylserine include 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimiristoyl-sn-glycero-3-phosphoserine (dimiristoylphosphatidylserine; DMPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (distearoylphosphatidylserine; DSPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (dipalmitoylphosphatidylserine; DPPS), and 1-myristoyl-2-palmitoyl-s Examples include n-glycero-3-phosphoserine (MPPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphoserine (PMPS), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphoserine (MSPS), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoserine (PSPS), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphoserine (SPPS), and 1-stearoyl-2-myristoyl-sn-glycero-3-phosphoserine (SMPS). In some embodiments, phosphatidylserine comprises a stearoyl (18:0) moiety, an oleoyl (18:1) moiety, an eicosatetraenoyl (20:4) moiety, a docosahexaenoyl (22:06) moiety, or a combination thereof. In other embodiments, PS is La-phosphatidylserine (brain, pig; CAS registry number 383907-32-2).

[0459] Therefore, in the most preferred embodiments and aspects of the present invention, the lipid nanoparticles of the present invention comprise "DPhyPS" (1,2-difitanoyl-sn-glycero-3-phospho-L-serine; 4ME 16:0 PS). In yet more preferred embodiments and aspects, the lipid nanoparticles of the present invention comprise DPhyPS, a cationic lipid according to formula (II), preferably C24, C28, or C29, more preferably C24, and a polymer-bound lipid according to formula (I), preferably "PMOZ4". In other embodiments and aspects, the lipid nanoparticles of the present invention comprise DPhyPS, a cationic lipid according to formula (II), preferably C24, C28, or C29, most preferably C24, and DMG-PEG2000.

[0460] In a preferred embodiment, the lipid nanoparticles of the present invention contain about 5 mol% to about 15 mol% of phosphatidylserine, preferably DPhyPS, calculated from the total lipids present in the lipid-based carrier. In one embodiment, the lipid-based carrier contains about 3 mol% to about 12 mol% or about 5 mol% to about 10 mol% of phosphatidylserine, preferably DPhyPS, calculated from the total lipids present in the lipid-based carrier. In some embodiments, the lipid nanoparticles of the present invention contain about 5 mol% to about 25 mol%, preferably about 5 mol% to about 15 mol%, or about 8 mol% to about 12 mol%, more preferably about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%, calculated from the total lipids present in the lipid-based carrier. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 1 mol% to about 6 mol%, preferably about 2.5 mol% to about 5 mol%, more preferably about 2.5 mol%, of phosphatidylserine, preferably DPhyPS. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol%, of phosphatidylserine, preferably DPhyPS. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 0.25 mol%, about 0.5 mol%, about 0.75 mol%, about 1 mol%, about 1.25 mol%, about 1.5 mol%, about 1.75 mol%, about 2 mol%, about 2.25 mol%, about 2.5 mol%, about 2.75 mol%, about 3 mol%, about 3.25 mol%, about 3.5 mol%, about 3.75 mol%, about 4 mol%, about 4.25 mol%, about 4.5 mol%, about 4.75 mol%, or about 5 mol% of phosphatidylserine, preferably DPhyPS. In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 1.5 mol%, about 2 mol%, or about 2.5 mol% of phosphatidylserine, preferably DPhyPS.In a more preferred embodiment, the lipid nanoparticles of the present invention contain about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol% of phosphatidylserine, preferably DPhyPS. Similarly, in a more preferred embodiment, the lipid nanoparticles of the present invention contain about 2.5 mol% of DPhyPS or about 5 mol% of DPhyPS.

[0461] Lipid nanoparticle composition The terms “lipid nanoparticle composition” and “composition” are used interchangeably herein. In the context of the present invention, lipid nanoparticles are not limited to any particular form and should be interpreted as including any form produced by combining ionizable lipids and optionally one or more further lipids, for example, in an aqueous environment and / or in the presence of nucleic acid compounds. For example, liposomes, lipid complexes, lipoplexes, etc., fall within the range of lipid nanoparticles.

[0462] In the context of the present invention, “composition” means any type of composition in which a specified component may be incorporated with any further excipients, and usually with at least one pharmaceutically acceptable carrier or excipient. Thus, a composition may be a dry composition such as a powder or granules, or a solid unit such as a lyophilized form or a tablet. Alternatively, a composition may be in liquid form, and each excipient may be incorporated independently in dissolved or dispersed (e.g., suspension or emulsified) form. In one preferred embodiment, the composition is formulated as a sterile solid composition, such as a powder or lyophilized form for reconstitution with an aqueous liquid carrier. Such formulations are also preferred for versions of the composition comprising nucleic acid cargo, which are described in more detail below. In this regard, similarly, the term “lipid composition” is used in relation to lipid nanoparticles and each lipid contained in the lipid nanoparticles. That is, the lipid nanoparticles preferably have a lipid composition comprising different lipids, namely ionizable lipids, phospholipids, sterols, polymer-bound lipids, and further phospholipids, preferably phosphatidylserine, as disclosed throughout the context of this application.

[0463] In some embodiments, the lipid nanoparticles disclosed herein, which encapsulate nucleic acids, are lyophilized lipid nanoparticles. Lyophilized lipid nanoparticles are lipid nanoparticles from which the liquid (e.g., water) has been removed by freeze-drying, wherein a liquid product is frozen and then placed under vacuum to remove the solvent (e.g., water) by sublimation, leaving a composition substantially free of the solvent (e.g., water). In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain polymer-bound lipids of the present invention, preferably lipids containing polyoxazolines, more preferably PMOZ lipids. In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain nucleic acids. In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain nucleic acids encapsulated within the lipid nanoparticles. In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain compounds of formula I. In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain PMOZ. In some embodiments, the lyophilized lipid nanoparticles disclosed herein contain lipids, nucleic acids, compounds of formula I, or any mixture thereof.

[0464] In the composition of the present invention, ionizable lipids may be present within or as part of lipid nanoparticles (LNPs). In other words, such a composition contains lipid nanoparticles, and the ionizable lipids are present within the lipid nanoparticles.

[0465] As used herein, “nanoparticles” are submicron particles having any structure or form. Submicron particles may also be called colloids or colloidal. With respect to the material on which nanoparticles are based, and their structure or form, nanoparticles can be classified, to name only a few of the possible names for certain types of nanoparticles, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polyplexes, mixtures or hybrid complexes.

[0466] As defined above, lipid nanoparticles include any type of nanoparticles formed or co-formed by lipids. In particular, lipid nanoparticles can be co-formed by a combination of lipids, including at least one amphiphilic, vesicle-forming lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0467] In some embodiments, such lipid nanoparticles comprise an ionizable lipid (e.g., a lipid of formula (II)) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-binding lipids (e.g., polymer-binding lipids such as the polymer-binding lipid having formula (I) described above). Compositions comprising the ionizable lipids, steroids, neutral lipids, and polymer-binding lipids according to formula (I) as defined herein are now considered by the inventors to exist, at least in an aqueous environment, as compositions comprising lipid nanoparticles typically formed by these excipients.

[0468] LNPs may contain any lipids capable of forming particles to which one or more nucleic acid molecules are bound or to which one or more nucleic acid molecules are encapsulated. In some embodiments, mRNA, or a portion thereof, is encapsulated in an aqueous space surrounded by the lipid portion of the lipid nanoparticle, or some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses. In some embodiments, mRNA, or a portion thereof, is associated with lipid nanoparticles.

[0469] As noted herein, compositions comprising lipid excipients typically form lipid nanoparticles, at least in an aqueous environment. As defined herein, the nanoparticles are primarily submicron in size. In certain embodiments, mRNA, when present in lipid nanoparticles, is resistant to degradation by nucleases in aqueous solution. As used herein, the average diameter may be represented by the z-mean determined by dynamic light scattering. In one embodiment, the composition is a sterile liquid composition comprising lipid nanoparticles having an average hydrodynamic diameter (or average particle size) determined by dynamic laser scattering from about 30 nm to about 800 nm. In various embodiments, lipid nanoparticles are found in the following ranges: approximately 30nm to 150nm, approximately 50nm to 200nm, approximately 60nm to 200nm, approximately 70nm to 200nm, approximately 80nm to 200nm, approximately 90nm to 200nm, approximately 90nm to 200nm, approximately 90nm to 190nm, approximately 90nm to 180nm, approximately 90nm to 170nm, approximately 90nm to 160nm, approximately 90nm to 150nm, approximately 90nm to 140nm, approximately 90nm to 130nm, approximately 90nm to 120nm, approximately 90nm to 100nm, and approximately 70nm to 9nm. They have an average diameter of 0 nm, approximately 80 nm to 90 nm, approximately 70 nm to 80 nm, or approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, and are substantially non-toxic. In another preferred embodiment of the present invention, the lipid nanoparticles have a hydrodynamic diameter in the range of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, about 60 nm to about 150 nm, or about 60 nm to about 120 nm, or about 80 nm to about 160 nm, or about 90 nm to about 140 nm, 50 nm to about 300 nm, or about 60 nm to about 250 nm, or about 60 nm to about 200 nm, or about 70 nm to 200 nm, or about 75 nm to about 160 nm, or about 100 nm to about 140 nm, or about 90 nm to about 140 nm. The range of about 50 nm to about 60 nm or about 60 nm to about 80 nm is also preferred.

[0470] Compositions comprising the lipid excipients described herein that yield the lipid nanoparticles of the present invention may be relatively homogeneous. The polydispersity index (PDI) can be used to indicate the homogeneity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle compositions of the present invention may have polydispersity indices ranging from about 0 to about 0.35, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, or 0.35. In some embodiments, the polydispersity index (PDI) of the nanoparticle composition may be about 0.1 to about 0.2.

[0471] Various optional features, choices, and preferences relating to the compositions of the present invention are generally described herein and will be clearly understood by those skilled in the art, and all of these also apply to lipid nanoparticles. Similarly, the choices and preferences also apply to compositions comprising such lipid nanoparticles.

[0472] For example, lipid nanoparticles according to one preferred embodiment include an ionizable lipid as defined above, a neutral lipid which may be DPhyPE, a steroid which may be cholesterol, and formula (I): [P]-[Linker]-[L] Formula (I) It includes polymer-bound lipids which may be polymer-bound lipids represented as, [P] represents at least one polyoxazoline (POZ) monomer unit.

[0473] [ka]

[0474] It is a homopolymer portion containing, In the formula, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, n has an average value in the range of about 45 to about 55, preferably about 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, most preferably about 4.3 kDa. [linker] is any linker group, [L] is the lipid portion, and the ionizable lipid may be selected from compounds C24 to C29 listed in Table 1, or preferably the ionizable lipid is preferably ionizable lipid structure C24, C28 or C29, most preferably C24.

[0475] Therefore, in the context of the present invention, mRNA is preferably contained in a liquid or semiliquid composition, and the mRNA is complexed or associated with lipid nanoparticles according to one of the preferred embodiments. That is, in a preferred embodiment, the liquid or semiliquid composition contains a complex comprising mRNA, the complex preferably existing as lipid nanoparticles as defined herein.

[0476] As used herein, lipid nanoparticles are typically formed by their respective excipients and reflect the same quantitative ratio of excipients as the entire composition containing the nanoparticles; therefore, references to the molar amount of lipid excipients in the composition of the present invention should also be understood as describing the molar amount of each excipient in the lipid nanoparticles contained in the composition.

[0477] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C24 and polymer-bound lipid, n=50, i.e., "PMOZ 4", having 50 monomer repeats.

[0478] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C28 and polymer-bound lipid, n=50, i.e., "PMOZ 4", having 50 monomer repeats.

[0479] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C29 and polymer-bound lipid, n=50, i.e., "PMOZ 4", having 50 monomer repeats.

[0480] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C24 and polymer-bound lipid, n=115, i.e., "PMOZ 4", having 115 monomer repeats.

[0481] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C28 and polymer-bound lipid, n=115, i.e., "PMOZ 4", having 115 monomer repeats.

[0482] In a more very preferred embodiment, the lipid nanoparticles of the present invention include a C29 and polymer-bound lipid, "PMOZ4" having n=115, i.e., 115 monomer repeats.

[0483] In further embodiments, the lipid nanoparticles of the present invention include C24 and polymer-bound lipid DMG-PEG2000. In further embodiments, the lipid nanoparticles of the present invention include C28 and polymer-bound lipid DMG-PEG2000.

[0484] In further embodiments, the lipid nanoparticles of the present invention include C29 and polymer-bound lipid DMG-PEG2000. In one embodiment, the composition is (a) 30 to 70 mol% of preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24, (b) A steroid in an amount of approximately 20-50 mol%, (c) Approximately 5-25 mol% of neutral lipids, (d) A polymer-bound lipid preferably according to formula (I) in an amount of about 1 to 10 mol%, more preferably about 1 mol%, or less than about 1 mol%, and (e) Phosphatidylserine in an amount of about 1-6 mol%, preferably DPhyPS Contains lipid nanoparticles, Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0485] In another embodiment, the composition is (a) 40-70 mol% of preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24, (b) Steroids in amounts of 20-50 mol%, (c) 5-15 mol% of neutral lipids, (d) A polymer-bound lipid according to formula (I) in an amount of 1 to 10 mol%, more preferably about 1 mol% or less than about 1 mol%, and (e) Phosphatidylserine in an amount of about 1-6 mol%, preferably DPhyPS Contains lipid nanoparticles, Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0486] In one embodiment, the composition is (a) 20 to 60 mol% of preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24, (b) Steroids in amounts of 25-55 mol%, (c) 5-25 mol% of neutral lipids, (d) A polymer-bound lipid according to formula (I) in an amount of 1 to 5 mol%, more preferably about 1 mol% or less than about 1 mol%, and (e) Phosphatidylserine in an amount of about 1-6 mol%, preferably DPhyPS Contains lipid nanoparticles, Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0487] In a further embodiment, the composition, (a) 45-65 mol% of preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24, (b) Steroids in amounts of 25-45 mol%, (c) 8-12 mol% of neutral lipids, (d) A polymer-bound lipid according to formula (I) in an amount of 1 to 2 mol%, preferably 1.7 mol%, more preferably about 1 mol%, or less than about 1 mol%, and (e) Phosphatidylserine in an amount of about 1-6 mol%, preferably DPhyPS Contains lipid nanoparticles, Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0488] In a more preferred embodiment, the composition is (a) 45-65 mol% of preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24, (b) Steroids in amounts of 25-45 mol%, (c) 8-12 mol% of neutral lipids, (d) A polymer-bound lipid according to formula (I) in an amount of 1 to 2 mol%, preferably 1.7 mol%, more preferably about 1 mol%, or less than about 1 mol%, and (e) Phosphatidylserine in an amount of about 1-6 mol%, preferably DPhyPS Contains lipid nanoparticles, Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0489] In these embodiments, the ionizable lipid is preferably a compound selected according to any one of the preferences disclosed herein. For example, the ionizable lipid may be selected from the compounds listed in Table 1. Furthermore, these embodiments may also include steroids, neutral lipids, and / or polymer-bound lipids selected according to any one of the preferences disclosed herein. In all embodiments in which compositions or lipid nanoparticles described herein are listed and mol% values ​​are given for each excipient, each amount should be considered to be relative to the total molar amount of all lipid excipients in the lipid nanoparticles.

[0490] In a more preferred embodiment, the composition or lipid nanoparticles described herein comprise 59 mol% of an ionizable lipid according to formula (I), 10 mol% of a neutral lipid, 29.3 mol% of a steroid, and 1.7 mol% of a polymer-bound lipid.

[0491] In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% ionizable lipid, 10 mol% DPhyPE, 29.3 mol% cholesterol, and 1.7 mol% polymer-bound lipid according to formula (I). In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% ionizable lipid, 10 mol% DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% polymer-bound lipid according to formula (I). In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% ionizable lipid, 10 mol% DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% "DMG-PMOZ".

[0492] [ka]

[0493] In summary, the selection of preferred lipid compositions according to more specific embodiments of the present invention includes at least five lipid excipients disclosed herein in Table E.

[0494] [Table 2]

[0495] Furthermore, Table F shows preferred lipid formulations of the present invention, indicating distinct molar percentages of at least four lipid excipients in the composition of the present invention. For example, a preferred lipid composition includes the molar percentages of lipids disclosed in row "F1", i.e., 59 mol% ionizable lipids, 29.3 mol% sterols, 10 mol% neutral lipids, and 1.7 mol% polymer-bound lipids. Another example of a preferred lipid composition includes the molar percentages of lipids disclosed in row "F31", i.e., 45 mol% ionizable lipids, 43.5 mol% sterols, 10 mol% neutral lipids, and 1.5 mol% polymer-bound lipids.

[0496] [Table 3]

[0497] Therefore, in a more preferred implementation of the present invention, the compositions of the present invention are named F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15, F16, F17, F18, F19, F20, F21, F22, F23, F24, F25, F26, F27, F28, F29, F3 0, F31, F32, F33, F34, F35, F36, F37, F38, F39, F40, F41, F42, F43, F44, F45, F46, F47, F48, F49, F50, F51, F52, F53, F54, F55, F56, F57, F58, F59, F60, F61, F62, F63, F64, F65, F66, F6 The mixture comprises excipients disclosed in Table E, selected from the group consisting of excipient combination designations E1, E2, E3, E4, E5, E6, E7, and E8

[0498] In preferred embodiments, the compositions or lipid nanoparticles described herein are (i) about 40 to about 60 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), preferably C24, C28 or C29, most preferably about 49 mol% of C24, (ii) Approximately 20 mol% to approximately 50 mol% cholesterol, more preferably approximately 40 mol% cholesterol, (iii) about 2 mol% to about 15 mol% of phospholipids, preferably DPhyPE, more preferably about 7.5 mol% of DPhyPE. (iv) about 1 mol% to about 6 mol% phosphatidylserine, preferably DPhyPS, more preferably about 2.5 mol% DPhyPS, and (v) Approximately 0.5 mol% to approximately 2 mol% of polymer-bound lipids, preferably polymer-bound lipids according to formula (I), preferably PMOZ4 having n=50, i.e., 50 monomer repeats, or n=115, i.e., 115 monomer repeats, more preferably approximately 1 mol% of PMOZ4 The mol% (ratio) of each lipid is selected so that the total of all five excipients equals 100%.

[0499] In a very preferred embodiment, the compositions or lipid nanoparticles described herein are (i) Approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, or approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), preferably C24, C28 or C29, more preferably C24, (ii) Approximately 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, or 45 mol% cholesterol, (iii) Approximately 0.5 mol%, approximately 1 mol%, approximately 1.5 mol%, approximately 2 mol%, approximately 2.5 mol%, approximately 3 mol%, approximately 3.5 mol%, approximately 4 mol%, approximately 4.5 mol%, approximately 5 mol%, approximately 5.5 mol%, approximately 6 mol%, approximately 6.5 mol%, approximately 7 mol%, approximately 7.5 mol%, approximately 8 mol%, approximately 8.5 mol%, approximately 9 mol%, approximately 9.5 mol%, or approximately 10 mol% of phospholipids, preferably DPhyPE. (iv) about 0.25 mol%, about 0.5 mol%, about 0.75 mol%, about 1 mol%, about 1.25 mol%, about 1.5 mol%, about 1.75 mol%, about 2 mol%, about 2.25 mol%, about 2.5 mol%, about 2.75 mol%, about 3 mol%, about 3.25 mol%, about 3.5 mol%, about 3.75 mol%, about 4 mol%, about 4.25 mol%, about 4.5 mol%, about 4.75 mol%, or about 5 mol% of phosphatidylserine, preferably DPhyPS, and (v) polymer-bound lipids in amounts of approximately 0.1 mol%, approximately 0.2 mol%, approximately 0.3 mol%, approximately 0.4 mol%, approximately 0.5 mol%, approximately 0.6 mol%, approximately 0.7 mol%, approximately 0.8 mol%, approximately 0.9 mol%, approximately 1 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, or approximately 2 mol%, preferably polymer-bound lipids according to formula (I), more preferably PMOZ4 The mol% (ratio) of each lipid is selected so that the total of all five excipients equals 100%.

[0500] In other embodiments, the compositions or lipid nanoparticles described herein are, (i) Approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, or approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), preferably C24, C28 or C29, most preferably C24. (ii) Approximately 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, or 45 mol% cholesterol, (iii) Approximately 0.5 mol%, approximately 1 mol%, approximately 1.5 mol%, approximately 2 mol%, approximately 2.5 mol%, approximately 3 mol%, approximately 3.5 mol%, approximately 4 mol%, approximately 4.5 mol%, approximately 5 mol%, approximately 5.5 mol%, approximately 6 mol%, approximately 6.5 mol%, approximately 7 mol%, approximately 7.5 mol%, approximately 8 mol%, approximately 8.5 mol%, approximately 9 mol%, approximately 9.5 mol%, or approximately 10 mol% of phospholipids, preferably DPhyPE. (iv) about 0.25 mol%, about 0.5 mol%, about 0.75 mol%, about 1 mol%, about 1.25 mol%, about 1.5 mol%, about 1.75 mol%, about 2 mol%, about 2.25 mol%, about 2.5 mol%, about 2.75 mol%, about 3 mol%, about 3.25 mol%, about 3.5 mol%, about 3.75 mol%, about 4 mol%, about 4.25 mol%, about 4.5 mol%, about 4.75 mol%, or about 5 mol% of phosphatidylserine, preferably DPhyPS, and (v) Approximately 0.1 mol%, approximately 0.2 mol%, approximately 0.3 mol%, approximately 0.4 mol%, approximately 0.5 mol%, approximately 0.6 mol%, approximately 0.7 mol%, approximately 0.8 mol%, approximately 0.9 mol%, approximately 1 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, or approximately 2 mol% of polymer-bound lipids, preferably PEG polymer-bound lipids, more preferably DMG-PEG2000. The mol% (ratio) of each lipid is selected so that the total of all five excipients equals 100%.

[0501] The zeta potential of a nanoparticle composition can be used to indicate the interfacial dynamic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Lipid nanoparticles according to the present invention may exhibit a relatively neutral zeta potential due to the presence of both negatively and positively charged compounds. The zeta potential (sometimes abbreviated as "charge") can be determined along with the particle size by dynamic light scattering and laser Doppler microelectrophoresis, for example, using a Malvern Zetasizer Nano (Malvern Instruments Ltd.; located in Malvern, UK). Those skilled in the art will know of the many suitable methods available for measuring the zeta potential, for example, by diluting LNP to 0.01 mg / mL mRNA in 0.1 × PBS and measuring with a Malvern Zetasizer (Nano ZS), or generally by measuring in 0.1 N PBS at pH 7.5. Depending on the amount and nature of the charged compounds in the lipid nanoparticles, the nanoparticles can be characterized by their zeta potential. In a preferred embodiment, the zeta potential is in the range of approximately -50mV to approximately +50mV. In another preferred embodiment, the zeta potential is in the range of approximately -25mV to approximately +25mV. In some embodiments, the zeta potential of the lipid nanoparticles of the present invention may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV. Preferably, the zeta potential of the lipid nanoparticles of the present invention is in the range of -50mV to +50mV, preferably in the range of -25mV to +25mV, more preferably in the range of -10mV to +10mV, and most preferably in the range of -5mV to +5mV.

[0502] In certain embodiments, the LNP comprises one or more targeting moieties that can target the LNP to a cell or a population of cells. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

[0503] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells and induce the internalization of the LNP. For example, in one embodiment, one or more internalization domains bind to receptors found on the cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP can bind to a biomolecule in vivo, thereby allowing the LNP-binding biomolecule to be recognized by a cell surface receptor and induce internalization. For example, in one embodiment, the LNP binds to systemic ApoE, which leads to the uptake of the LNP and associated cargo. In certain embodiments of the present invention, ApoE can be supplemented in the medium or pharmaceutical composition used.

[0504] Preferably, in one embodiment, the composition of the present invention comprises a biologically active component, preferably nucleic acid, more preferably mRNA, and even more preferably, (a) mRNA comprising at least one coding sequence encoding a peptide or protein or a fragment or variant thereof, wherein the peptide or protein is an antigen, and the antigen is preferably derived from a pathogenic antigen, tumor antigen, allergen antigen or autoimmune autoantigen or a fragment or variant thereof, or (b) mRNA containing at least one coding sequence that encodes a therapeutic protein or a fragment or variant thereof. Therefore, therapeutic proteins are, (i) Therapeutic proteins used to treat cancer or neoplastic diseases, (ii) Enzyme replacement therapy for the treatment of metabolic, endocrine, or amino acid disorders, or therapeutic proteins used to replace deleted, deficient, or mutated proteins. (iii) Therapeutic proteins used to treat blood disorders, circulatory disorders, respiratory disorders, infectious diseases or immunodeficiency. (iv) Therapeutic proteins used in hormone replacement therapy, (v) Therapeutic proteins used to reprogram somatic cells into pluripotent or totipotent stem cells, (vi) Therapeutic proteins used as adjuvants or immunostimulants, (vii) Therapeutic antibodies, therapeutic proteins, (viii) Therapeutic proteins that are gene editing agents, and (ix) Therapeutic proteins used to treat or prevent liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer. Selected from the group consisting of, Preferably, the therapeutic protein is a therapeutic protein used to treat cancer or neoplastic diseases.

[0505] In a preferred embodiment of the present invention, the lipid nanoparticles are at least one polyoxazoline (POZ) monomer unit.

[0506] [ka]

[0507] It contains polymer-bound lipids, R is a C1-9 alkyl or C2-9 alkenyl, preferably a C1 or C2 alkyl, and n has an average value in the range of 2-200, preferably 20-100, more preferably 24-26 or 45-50, or n is selected to have an average molecular weight of 1.5-22 kDa for the [P] portion, more preferably 2-19 kDa, even more preferably about 7.5 kDa or about 15 kDa, preferably 1-15 kDa, more preferably 2-12.5 kDa, more preferably about 5 kDa or about 10 kDa, even more preferably about 2 kDa-2.5 kDa or about 4 kDa-5 kDa. Preferably, the homopolymer portion containing multiple monomer units includes poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), poly(2-propyl-2-oxazoline) (PPOZ), poly(2-butyl-2-oxazoline) (PBOZ), poly(2-isopropyl-2-oxazoline) (PIPOZ), poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), or poly(2-dimethylamino-2-oxazoline) (PDMAOx). More preferably, the polymer-bound lipids described above or below in this specification, most preferably "PMOZ4", are included.

[0508] As described in the section "Polymer-bound lipids," in other specific aspects and embodiments of the present invention, commercially available DMG-PEG2000 (DMG-PEG2K or PEG2000-DMG), which uses the PEG lipid DMG-PEG2000 as the sole polymer-bound lipid instead of polyoxazoline (POZ) polymer-bound lipids in certain LNPs of the present invention, is also a preferred polymer-bound lipid for certain LNPs of the present invention.

[0509] [ka]

[0510] In a preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably formula (II): R a -AR b Formula (II) An ionizable lipid represented by, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer thereof, (R a teeth,

[0511] [ka]

[0512] Selected from, R b teeth,

[0513] [ka]

[0514] Selected from, A is -S-, R 1 These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R 2 It is an alkanediyl having 2 to 8 carbon atoms, R 3 This is not required; if present, use -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 It is -NH-C(O)O-, R 4 These are lipophilic substituents having 12 to 36 carbon atoms, and lipophilic substituents having 12 to 36 carbon atoms are derived from tocopherols or tocotrienols. R 5 It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. (All of these choices are independent of one another.) • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, of phospholipids, preferably selected from DSPC and DPhyPE (more preferably the phospholipid is DPhyPE), • Approximately 1 mol% to approximately 6 mol%, preferably approximately 2.5 mol% to approximately 5 mol%, more preferably approximately 2.5 mol%, of phosphatidylserine, preferably DPhyPS. • Approximately 25 mol% to approximately 45 mol% of sterols, preferably cholesterol. • Less than approximately 1.5 mol%, preferably about 1 mol%, of polymer-bound lipids, preferably PMOZ lipids, more preferably PMOZ lipids that do not contain sulfur groups (-S-), and • One or more nucleic acids, preferably mRNA Includes, More preferably, ionizable lipids

[0515] [ka]

[0516] It is characterized by being such. In another preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, of phospholipids, preferably selected from DSPC and DPhyPE (more preferably the phospholipid is DPhyPE), • Approximately 1 mol% to approximately 6 mol%, preferably approximately 2.5 mol% to approximately 5 mol%, more preferably approximately 2.5 mol%, of phosphatidylserine, preferably DPhyPS. • Approximately 25 mol% to approximately 45 mol% of sterols, preferably cholesterol. • Less than approximately 1.5 mol%, preferably about 1 mol%, of polymer-bound lipids, preferably PMOZ lipids, more preferably PMOZ lipids that do not contain sulfur groups (-S-), and • One or more nucleic acids, preferably mRNA Includes, Preferably, the ionizable lipid is

[0517] [ka]

[0518] It is characterized by being such. In another preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, of phospholipids, preferably selected from DSPC and DPhyPE (more preferably the phospholipid is DPhyPE), • Approximately 1 mol% to approximately 6 mol%, preferably approximately 2.5 mol% to approximately 5 mol%, more preferably approximately 2.5 mol%, of phosphatidylserine, preferably DPhyPS. • Approximately 25 mol% to 45 mol% of sterols, preferably cholesterol; • Less than approximately 1.5 mol%, preferably about 1 mol%, of polymer-bound lipids, preferably PMOZ lipids, more preferably PMOZ lipids that do not contain sulfur groups (-S-), and • One or more nucleic acids, preferably mRNA Includes, Preferably, the ionizable lipid is

[0519] [ka]

[0520] It is characterized by being such. In another preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), more preferably CVL1(C24), preferably approximately 49 mol% or approximately 59 mol% of CVL1(C24), • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, preferably approximately 5 mol% or approximately 7.5 mol%, more preferably approximately 7.5 mol% DPhyPE. • Approximately 2.5 mol% phosphatidylserine, preferably DPhyPS, • Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of PMOZ lipids that do not contain sulfur groups (-S-), more preferably (i)n=50, i.e., "PMOZ4" having 50 monomer repeats.

[0521] [ka]

[0522] Or (ii) n=115, i.e., "PMOZ4" having 115 monomer repeats.

[0523] [ka]

[0524] • Approximately 29 mol% to approximately 41 mol% of sterols, preferably cholesterol, and • One or more nucleic acids, preferably mRNA Includes.

[0525] In another preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), more preferably CVL1-meth(C28), preferably approximately 49 mol% or approximately 59 mol% of CVL1-meth(C28), • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, more preferably approximately 5 mol% DPhyPE or approximately 7.5 mol% DPhyPE. • Approximately 2.5 mol% or approximately 5 mol% phosphatidylserine, preferably DPhyPS, - Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of PMOZ lipids that do not contain sulfur groups (-S-), more preferably "PMOZ4" having (i)n=50, i.e., 50 monomer repeats.

[0526] [ka]

[0527] Or (ii) n=115, i.e., "PMOZ4" having 115 monomer repeats.

[0528] [ka]

[0529] • Approximately 29 mol% to approximately 41 mol% of sterols, preferably cholesterol, and • One or more nucleic acids, preferably mRNA Includes.

[0530] In another preferred embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), more preferably CVL1-para(C29), preferably approximately 49 mol% or approximately 59 mol% of CVL1-para(C29), more preferably approximately 49 mol% of CVL1-para(C29), • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, more preferably approximately 5 mol% DPhyPE or approximately 7.5 mol% DPhyPE, even more preferably approximately 7.5 mol% DPhyPE. • Approximately 2.5 mol% phosphatidylserine, preferably DPhyPS, • Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of PMOZ lipids that do not contain sulfur groups (-S-), more preferably (i)n=50, i.e., "PMOZ4" having 50 monomer repeats.

[0531] [ka]

[0532] Or (ii) n=115, i.e., "PMOZ4" having 115 monomer repeats.

[0533] [ka]

[0534] • Approximately 29 mol% to approximately 41 mol% of sterols, preferably cholesterol, and • One or more nucleic acids, preferably mRNA Includes.

[0535] In another preferred embodiment, the lipid nanoparticles of the present invention contain one or more nucleic acids, preferably mRNA, and (i) Approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats. (ii) Approximately 59 mol% C24, approximately 30 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats. (iii) Approximately 49 mol% C24, approximately 40.5 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 0.5 mol% PMOZ4 having n=115, i.e., 115 monomer repeats, (iv) PMOZ4 having approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=115, i.e., 115 monomer repeats. It comprises a lipid nanoparticle composition selected from the group consisting of the following.

[0536] In another preferred embodiment, the lipid nanoparticles of the present invention contain one or more nucleic acids, preferably mRNA, and (i) Approximately 59 mol% C28, approximately 30 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats, (ii) PMOZ4 having approximately 49 mol% C28, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=50, i.e., 50 monomer repeats. It comprises a lipid nanoparticle composition selected from the group consisting of the following.

[0537] In another preferred embodiment, the lipid nanoparticles of the present invention comprise a lipid nanoparticle composition comprising one or more nucleic acids, preferably mRNA, and about 49 mol% C29, about 40 mol% cholesterol, about 7.5 mol% DPhyPE, about 2.5 mol% DPhyPS, and about 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats.

[0538] In another embodiment, the lipid nanoparticles of the present invention are • Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably ionizable lipids according to formula (II), more preferably C24, C28 or C29, most preferably C24. • Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol% of DPhyPE. • Approximately 2.5 mol% to approximately 5 mol% of phosphatidylserine, preferably DPhyPS. • Approximately 25 mol% to approximately 45 mol% of sterols, preferably cholesterol. • Approximately 1 mol% to approximately 2 mol% of PEG lipids, preferably DMG-PEG2000, and • One or more nucleic acids, preferably mRNA Includes.

[0539] In another embodiment, the lipid nanoparticles of the present invention contain one or more nucleic acids, preferably mRNA, and (i) Approximately 49 mol% C24, approximately 39.3 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1.7 mol% DMG-PEG2000, (ii) Approximately 59 mol% C28, approximately 29.3 mol% cholesterol, approximately 5 mol% DPhyPE, approximately 5 mol% DPhyPS and approximately 1.7 mol% DMG-PEG2000, (iii) Approximately 49 mol% C29, approximately 39.3 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1.7 mol% DMG-PEG2000 It comprises a lipid nanoparticle composition selected from the group consisting of the following.

[0540] In some embodiments, the lipid nanoparticles comprise 20-60% ionizable lipids, 5-25% non-ionizable lipids, 25-55% sterols, and 0.5-15% polymer-bound lipids of the present disclosure.

[0541] In some embodiments, the lipid nanoparticles contain ionizable lipids in a molar ratio of 20-60%. For example, the lipid nanoparticles may contain ionizable lipids in molar ratios of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60%. In some embodiments, the lipid nanoparticles contain ionizable lipids in molar ratios of 20%, 30%, 40%, 50%, or 60%.

[0542] In other embodiments, the lipid nanoparticles contain non-ionizable lipids in a molar ratio of 5-25%. For example, the lipid nanoparticles may contain non-ionizable lipids in molar ratios of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25%. In some embodiments, the lipid nanoparticles contain non-ionizable lipids in molar ratios of 5%, 10%, 15%, 20%, or 25%.

[0543] In other embodiments, the lipid nanoparticles contain sterols in a molar ratio of 25-55%. For example, the lipid nanoparticles may contain sterols in molar ratios of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55%. In some embodiments, the lipid nanoparticles contain sterols in molar ratios of 25%, 30%, 35%, 40%, 45%, 50%, or 55%.

[0544] Encapsulation / complexation into LNPs: In a preferred embodiment of the second aspect, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, and optionally at least one further nucleic acid, is complexed with one or more lipids (e.g., ionizable lipids and / or neutral lipids), thereby encapsulated, partially encapsulated, or associated with them to form liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes.

[0545] Nucleic acids (e.g., DNA or RNA) incorporated into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be located entirely or partially within the internal space of the liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome, within the lipid layer / membrane, or associated with the external surface of the lipid layer / membrane. The incorporation of nucleic acids into liposomes / LNPs is also referred to herein as “encapsulation,” where the nucleic acid, e.g., RNA, is completely contained within the internal space of the liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome. The purpose of incorporating nucleic acids into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes is to protect the nucleic acid, preferably RNA, from environments that may contain enzymes or chemicals or conditions that degrade nucleic acids, and / or systems or receptors that cause rapid efflux of nucleic acids. Furthermore, by incorporating nucleic acids, preferably RNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, nucleic acid uptake can be promoted, thereby enhancing the therapeutic effect of nucleic acids, such as RNA encoding the antigenic SARS-CoV-2 (nCoV-2019) protein. Therefore, incorporating nucleic acids, such as RNA or DNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be particularly suitable for coronavirus vaccines (e.g., SARS-CoV-2 vaccines), for example, intramuscular and / or intradermal administration.

[0546] In this context, the terms “complexed” or “associated” refer to the intrinsically stable binding of nucleic acids and one or more lipids to a larger complex or aggregate that is not via covalent bonds.

[0547] The term “lipid nanoparticles,” also known as “LNPs,” is not limited to any specific form and includes any form produced by combining ionizable lipids and optionally one or more additional lipids, for example, in an aqueous environment and / or in the presence of nucleic acids, such as RNA. For example, liposomes, lipid complexes, SNALPs, lipoplexes, etc., all fall within the scope of lipid nanoparticles (LNPs). Thus, “lipid nanoparticles” (LNPs) are nanoparticles formed of lipids, typically comprising at least one amphiphilic membrane-forming lipid and optionally other lipids, and optionally cargo material such as nucleic acid compounds. As used herein, the expression “lipid nanoparticles” or “LNPs” includes any subtypes and forms of nanoparticles formed or co-formed by lipids, such as the aforementioned liposomes and lipoplexes.

[0548] Liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be of different sizes, including, but are not limited to, multilayer vesicles (MLVs) which may have a diameter of several hundred nanometers and contain a series of concentric bilayers separated by narrow aqueous compartments, small unicellular vesicles (SUVs) which may have a diameter of less than 50 nm, and large monolayer vesicles (LUVs) which may have a diameter between 50 nm and 500 nm.

[0549] The LNPs of the present invention are preferably characterized as microvesicles having an internal water space isolated from an external medium by one or more bilayer membranes. The bilayer membrane of the LNP is typically formed by amphiphilic molecules such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of liposomes can also be formed by amphichromatic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, LNPs typically function to transport at least one nucleic acid, preferably at least one RNA, to a target tissue.

[0550] Therefore, in a preferred embodiment of the second aspect, at least one nucleic acid, preferably at least one RNA, is complexed with one or more lipids to form lipid nanoparticles (LNPs). Preferably, the LNPs are particularly suitable for intramuscular, intradermal, and / or intravenous administration. In a very preferred embodiment, the LNPs are particularly suitable for intramuscular administration.

[0551] Alternatively, the composition may be provided in solid form. In particular, the composition may be provided as a sterile solid composition for reconstitution with a sterile liquid carrier, in which case the solid composition may further contain one or more inert components selected from pH modifiers, fillers, stabilizers, nonionic surfactants, and antioxidants. In this embodiment, the sterile liquid carrier is preferably an aqueous carrier.

[0552] In the context of the present invention, typical “lipid-based carriers” are selected from liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. In the context of the present invention, formulations of lipid nanoparticles relate to the term “lipid-based carrier” encompassing lipid-based delivery systems for RNA containing lipid components. Lipid nanoparticles or lipid-based carriers may further include other components suitable for encapsulating / integrating / complexing RNA, including cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof.

[0553] The RNA of the pharmaceutical composition may be fully or partially incorporated into a lipid-based carrier or encapsulated, and the RNA may be located within the internal space of the lipid-based carrier, within the lipid layer / membrane of the lipid-based carrier, or associated with the external surface of the lipid-based carrier. The incorporation of RNA into a lipid-based carrier may be called “encapsulation.” “Lipid-based carrier” is not limited to any particular form and includes, for example, any form produced by combining polymer-bound lipids and at least one further lipid in an aqueous environment, for example, in the presence of RNA. For example, LNPs, liposomes, lipid complexes, lipoplexes, etc., fall within the scope of the term “lipid-based carrier.” Lipid-based carriers may be of different sizes, including, but are not limited to, multilayer vesicles (MLVs) which may have a diameter of several hundred nanometers and contain a series of concentric bilayers separated by a narrow aqueous compartment, small unicellular vesicles (SUVs) which may have a diameter of less than 50 nm, and large monolayer vesicles (LUVs) which may have a diameter between 50 nm and 500 nm. Liposomes, a specific type of lipid-based carrier, are characterized as microscopic vesicles having an internal aqueous space isolated from other media by one or more bilayer membranes. Within a liposome, at least one RNA is typically located in the internal aqueous space, enclosed within a portion or the entire lipid portion of the liposome. The bilayer membrane of a liposome is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains. Lipid nanoparticles (LNPs), another specific type of lipid-based carrier, are characterized as microscopic lipid particles having a solid or partially solid core. Typically, LNPs do not have an internal aqueous space isolated from the external medium by a bilayer. Within an LNP, at least one RNA can be encapsulated or incorporated into the lipid portion of the LNP, enclosed within a portion or the entire lipid portion of the LNP. LNPs may contain any lipids to which RNA can bind or to form particles in which RNA can be encapsulated. Preferably, the lipid-based carriers are particularly suitable for intramuscular, intradermal, and / or intravenous administration.

[0554] In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition is selected from liposomes, lipid nanoparticles, lipoplexes, and / or nanoliposomes. In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition is lipid nanoparticles (LNPs). In a particularly preferred embodiment, the lipid nanoparticles of the pharmaceutical composition encapsulate at least one RNA of the present invention.

[0555] The terms “encapsulated,” e.g., incorporated, complexed, encapsulated, partially encapsulated, associated, and partially associated refer to the intrinsically stable binding of RNA and one or more lipids to a lipid-based carrier (e.g., a larger complex or aggregate), preferably not by covalent bonds of RNA. RNA encapsulated in a lipid-based carrier may be located entirely or partially within the lipid-based carrier (e.g., the lipid portion and / or internal space) and / or within the lipid layer / membrane of the lipid-based carrier. Encapsulation of RNA in a lipid-based carrier is also referred to herein as “incorporated,” since the RNA is preferably contained within the lipid-based carrier. While not wishing to be bound by theory, the purpose of incorporating or encapsulating RNA in a lipid-based carrier may be to protect the RNA from environments that may contain enzymes, chemicals, or conditions that degrade RNA. Furthermore, incorporating RNA into a lipid-based carrier may facilitate RNA uptake and thus enhance the therapeutic effect of RNA when administered to cells or subjects.

[0556] The terms "membrane-fusing" or "membrane-fusing" are intended to refer to lipids that assist in the fusion of lipid-based carriers or nucleic acid-lipid particles with cell membranes, thereby helping the nucleic acids contained within the lipid-based carriers or nucleic acid-lipid particles to enter cells.

[0557] In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition comprises at least one lipid selected from at least one aggregation-reducing lipid, at least one ionizable lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog.

[0558] In a preferred embodiment, the lipid-bas...

Claims

1. - Approximately 45 mol% to approximately 65 mol% of ionizable lipids, preferably formula (II): R ａ -A-R ｂ Formula (II) An ionizable lipid represented by, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer thereof, (R ａ teeth, 【Chemistry 1】 Selected from, R ｂ teeth, 【Chemistry 2】 Selected from, A is -S-, R １ These are ethanediyl or linear or unbranched alkanediyl having 2-3 carbon atoms. R ２ It is an alkanediyl having 2 to 8 carbon atoms, R ３ is not essential and when present, -R ５ -C(O)-O-, -R ５ -O-C(O)-, -R ５ -C(O)-NH-, -R ５ -OC(O)-NH-, or R ５ -NH-C(O)O- and R ４ This is a lipophilic substituent having 12 to 36 carbon atoms, and the lipophilic substituent having 12 to 36 carbon atoms is derived from tocopherol or tocotrienol. R ５ It is an alkanediyl having 1 to 6 carbon atoms, X is a carbon atom (CH) bonded to a hydrogen atom or a nitrogen atom, preferably a carbon atom (CH) bonded to a hydrogen atom. - A phospholipid in an amount of about 4 mol% to about 15 mol%, preferably about 4 mol% to about 9 mol%, preferably a phospholipid selected from DSPC and DPhyPE (more preferably the phospholipid is DPhyPE), • Approximately 1 mol% to approximately 6 mol%, preferably approximately 2.5 mol% to approximately 5 mol%, more preferably approximately 2.5 mol%, of phosphatidylserine, preferably DPhyPS, • Approximately 25 mol% to approximately 45 mol% of sterols, preferably cholesterol, - A polymer-bound lipid in an amount of less than approximately 1.5 mol%, preferably approximately 1 mol%, preferably a PMOZ lipid, more preferably a PMOZ lipid that does not contain a sulfur group (-S-), - One or more nucleic acids, preferably mRNA and Lipid nanoparticles containing lipids.

2. The ionizable lipids 【Transformation 3】 The lipid nanoparticles according to claim 1.

3. The ionizable lipids 【Chemistry 4】 The lipid nanoparticles according to claim 1.

4. The ionizable lipids 【Transformation 5】 The lipid nanoparticles according to claim 1.

5. - Approximately 45 mol% to approximately 65 mol% of the ionizable lipid, preferably approximately 49 mol% or approximately 59 mol% of the ionizable lipid, - Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, preferably approximately 5 mol% or approximately 7.5 mol%, of the phospholipid, more preferably approximately 7.5 mol% of DPhyPE, - Approximately 2.5 mol% phosphatidylserine, preferably DPhyPS, - Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of the PMOZ lipid, preferably a PMOZ lipid that does not contain a sulfur group (-S-), more preferably (C(O)CH ２ CH ２ PMOZ lipids containing a linker group [linker] which is C(O)NH), more preferably (i)n=50, i.e., "PMOZ4" having 50 monomer repeats. 【Transformation 6】 , or (ii) n = 115, i.e., "PMOZ4" having 115 monomer repeats 【Transformation 7】 PMOZ lipids, • Approximately 29 mol% to approximately 41 mol% of sterols, preferably cholesterol, - One or more nucleic acids, preferably mRNA and Lipid nanoparticles according to any one of claims 1 to 4, comprising:

6. - Approximately 49 mol% or approximately 59 mol% of the ionizable lipid and • Approximately 5 mol% or approximately 7.5 mol% DPhyPE, - Approximately 2.5 mol% or approximately 5 mol% phosphatidylserine, preferably DPhyPS, - Approximately 0.5 mol% to approximately 1 mol%, preferably approximately 1 mol%, of PMOZ lipids, preferably PMOZ lipids that do not contain sulfur groups (-S-), more preferably (C(O)CH ２ CH ２ PMOZ lipids containing a linker group [linker] which is C(O)NH), more preferably (i)n=50, i.e., "PMOZ4" having 50 monomer repeats. 【Transformation 8】 , or (ii) n = 115, i.e., "PMOZ4" having 115 monomer repeats 【Chemistry 9】 PMOZ lipids, • Approximately 29 mol% to approximately 41 mol% cholesterol, - One or more nucleic acids, preferably mRNA and Lipid nanoparticles according to any one of claims 1 to 5, including the lipid nanoparticles described in any one of claims 1 to 5.

7. The lipid nanoparticles comprise one or more nucleic acids, preferably mRNA, and the lipid composition of the lipid nanoparticles is (i) Approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% PMOZ4 having n=50, i.e., 50 monomer repeats. (ii) Approximately 59 mol% C24, approximately 30 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% PMOZ4 having n=50, i.e., 50 monomer repeats. (iii) Approximately 49 mol% C24, approximately 40.5 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 0.5 mol% PMOZ4 having n=115, i.e., 115 monomer repeats. (iv) PMOZ4 having approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% n=115, i.e., 115 monomer repeats. It is selected from the group consisting of, Most preferably, the lipid nanoparticle comprises one or more nucleic acids, preferably mRNA, and the lipid composition of the lipid nanoparticle is about 49 mol% C24, about 40 mol% cholesterol, about 7.5 mol% DPhyPE, about 2.5 mol% DPhyPS, and about 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats, according to claim 2.

8. The lipid nanoparticles comprise one or more nucleic acids, preferably mRNA, and the lipid composition of the lipid nanoparticles is (i) Approximately 59 mol% C24, approximately 30 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% PMOZ4 having n=50, i.e., 50 monomer repeats. (ii) Approximately 49 mol% C24, approximately 40 mol% cholesterol, approximately 7.5 mol% DPhyPE, approximately 2.5 mol% DPhyPS, and approximately 1 mol% PMOZ4 having n=50, i.e., 50 monomer repeats. It is selected from the group consisting of, Most preferably, the lipid nanoparticle comprises one or more nucleic acids, preferably mRNA, and its lipid composition is about 49 mol% C24, about 40 mol% cholesterol, about 7.5 mol% DPhyPE, about 2.5 mol% DPhyPS, and about 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats, according to claim 3.

9. The lipid nanoparticles according to any one of claims 4 to 8, wherein the lipid nanoparticles comprise one or more nucleic acids, preferably mRNA, and about 49 mol% C24, about 40 mol% cholesterol, about 7.5 mol% DPhyPE, about 2.5 mol% DPhyPS, and about 1 mol% n=50, i.e., PMOZ4 having 50 monomer repeats.

10. - Approximately 45 mol% to approximately 55 mol% of ionizable lipids, preferably ionizable lipids of formula (II) as shown in claim 1, more preferably ionizable lipids (C24) as shown in claim 2, - Approximately 4 mol% to approximately 15 mol%, preferably approximately 4 mol% to approximately 9 mol%, DPhyPE - Approximately 1.5 mol% to approximately 3.5 mol% of phosphatidylserine, preferably DPhyPS, • Approximately 35 mol% to approximately 45 mol% of sterols, preferably cholesterol, • "PMOZ4" in amounts of approximately 0.5 mol% to approximately 1.5 mol%, - One or more nucleic acids, preferably mRNA and Lipid nanoparticles containing lipids.

11. - Approximately 48 mol% to approximately 50 mol%, preferably approximately 49 mol%, of ionizable lipid C24, - Approximately 6.5 mol% to approximately 8.5 mol%, preferably approximately 7.5 mol%, of DPhyPE, • Approximately 1.5 mol% to approximately 3.5 mol%, preferably approximately 2.5 mol%, of phosphatidylserine, preferably DPhyPS, • Approximately 35 mol% to approximately 45 mol%, preferably approximately 40 mol%, of sterols, preferably cholesterol, - Approximately 0.5 mol% to approximately 1.5 mol%, preferably approximately 1 mol%, of "PMOZ4", - One or more nucleic acids, preferably mRNA and Lipid nanoparticles according to claim 10, comprising:

12. The lipid nanoparticles according to any one of claims 1 to 11, wherein the lipid nanoparticles contain the mRNA in an amount such that the N / P ratio is in the range of about 5 to about 15, more preferably about 8 to about 12, even more preferably about 9 to about 11, and most preferably about 10.

13. The lipid nanoparticles according to any one of claims 1 to 12, wherein the lipid nanoparticles have a lipid-to-mRNA weight ratio (m / m) in the range of about 20 to about 60, more preferably about 30 to about 50, and even more preferably about 40, about 41, about 42, about 43, about 44 or about 45.

14. The nucleic acid is (a) mRNA comprising at least one coding sequence encoding a peptide or protein or a fragment or variant thereof, wherein the peptide or protein is an antigen, and the antigen is preferably derived from a tumor antigen, pathogenic antigen, allergen antigen or autoimmune autoantigen or a fragment or variant thereof, or (b) mRNA containing at least one coding sequence that encodes a therapeutic protein or a fragment or variant thereof The therapeutic protein is, (i) Therapeutic proteins used to treat cancer or neoplastic diseases, (ii) Enzyme replacement therapy for the treatment of metabolic, endocrine, or amino acid disorders, or therapeutic proteins used to replace deleted, deficient, or mutated proteins. (iii) Therapeutic proteins used to treat blood disorders, circulatory system diseases, respiratory system diseases, infectious diseases or immunodeficiency. (iv) Therapeutic proteins used in hormone replacement therapy, (v) Therapeutic proteins used to reprogram somatic cells into pluripotent or totipotent stem cells, (vi) Therapeutic proteins used as adjuvants or immunostimulants, (vii) Therapeutic antibodies, therapeutic proteins, (viiii) Therapeutic proteins that are gene editing agents, and (ix) Therapeutic proteins used to treat or prevent liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer. Selected from the group consisting of, Preferably, the therapeutic protein is a therapeutic protein used for the treatment of cancer or a neoplastic disease, the lipid nanoparticle according to any one of claims 1 to 13.

15. The nucleic acid is (a) an mRNA comprising at least one coding sequence encoding a peptide or protein or a fragment or variant thereof, wherein the peptide or protein is an antigen, and the antigen is preferably mRNA derived from a tumor antigen or a fragment or variant thereof, or the mRNA comprising at least one coding sequence encoding a therapeutic protein or a fragment or variant thereof, wherein the therapeutic protein is a therapeutic protein used for the treatment of cancer or a neoplastic disease, according to any one of claims 1 to 14.

16. The lipid nanoparticle according to any one of claims 1 to 15, wherein the nucleic acid is mRNA encoding a tumor antigen.

17. The lipid nanoparticles are a sterile solid composition for reconstitution with a sterile liquid carrier, the lipid nanoparticles further comprising one or more inactive components selected from pH modifiers, bulking agents, stabilizers, nonionic surfactants, and antioxidants, and the sterile liquid carrier is an aqueous carrier, according to any one of claims 1 to 16.

18. The lipid nanoparticles are a sterile liquid composition, and the lipid nanoparticles have an average hydrodynamic diameter determined by dynamic laser scattering of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, or 60 nm to about 200 nm, or 70 nm to 200 nm, or about 75 nm to about 160 nm, or about 85 nm to about 140 nm, or 90 nm to about 130 nm, or 50 nm to about 120 nm, according to any one of claims 1 to 17.

19. The lipid nanoparticles according to any one of claims 1 to 18, wherein the lipid nanoparticles exhibit a zeta potential in the range of -50 mV to +50 mV, preferably in the range of -25 mV to +25 mV, more preferably in the range of -10 mV to +10 mV, and most preferably in the range of -5 mV to +5 mV.

20. The lipid nanoparticle according to any one of claims 1 to 19, wherein the mRNA compound is monocistronic, bicistronic, or multicistronic mRNA.

21. The lipid nanoparticle according to any one of claims 1 to 20, wherein the mRNA compound comprises a coding region encoding a peptide or protein, and the coding region has sequence modifications.

22. The sequence modification is selected from G / C content modification, codon modification, codon optimization, or C optimization of the sequence, preferably compared to the coding region of the corresponding wild-type mRNA. - The G / C content in the aforementioned code region is increasing. - The C content in the aforementioned code region is increasing. - The codon usage frequency of the code region is matched to the human codon usage frequency, and / or Lipid nanoparticles according to claim 21, wherein the codon adaptation index (CAI) within the code region is increased or maximized.

23. The aforementioned mRNA compound is a) 5' cap structure, preferably m7GpppN, more preferably cap 1 or m7G(5')ppp(5')(2'OMeA)pG, b) If necessary, preferably, at least one miRNA sequence for a microRNA whose microRNA binding site is selected from the group consisting of miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof. c) at least one 5'-UTR element, d) Code array, e) at least one 3'-UTR element, f) At least one poly(A) array, g) at least one poly(C) sequence, Lipid nanoparticles according to any one of claims 1 to 22, further comprising any combination thereof.

24. The lipid nanoparticle according to any one of claims 1 to 23, wherein the at least one coding RNA comprises a 5' cap structure, preferably an m7G, cap 0, cap 1, cap 2, modified cap 0, or modified cap 1 structure.

25. The at least one coding RNA comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR, preferably the at least one heterologous 5'-UTR comprising a 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a nucleic acid sequence derived from any one homolog, fragment, or variant of these genes, and / or Preferably, the lipid nanoparticle according to any one of claims 1 to 24, wherein the at least one heterologous 3'-UTR comprises a 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a nucleic acid sequence derived from any one homolog, fragment, or variant of any one of these genes.

26. The lipid nanoparticles according to any one of claims 1 to 25, wherein the at least one coding RNA comprises (i) HSD17B4 5'-UTR and PSMB3 3'-UTR, more preferably HSD17B4 5'-UTR (SEQ ID NO: 12, SEQ ID NO: 13) and PSMB3 3'-UTR (SEQ ID NO: 46, SEQ ID NO: 47).

27. The mRNA is free from chemical modifications, preferably from base modifications, more preferably from base modifications selected from the group consisting of pseudouridine (psi or ψ), N1-methylpseudridine (N1MPU, N1Mpsi or N1Mψ), 1-ethylpseudracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, and 5-methoxyuracil, and most preferably from N1-methylpseudridine (N1MPU, N1Mpsi or N1Mψ) modifications, as described in any one of claims 1 to 26.

28. The lipid nanoparticle according to any one of claims 1 to 26, wherein the mRNA compound comprises at least one chemical modification.

29. The lipid nanoparticle according to claim 28, wherein the chemical modification is selected from the group consisting of base modification, sugar modification, skeleton modification, and lipid modification, preferably the chemical modification is base modification, more preferably the base modification is selected from the group consisting of pseudouridine (psi or ψ), N1-methylpseudridine (N1MPU, N1Mpsi or N1Mψ), 1-ethylpseudracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, and 5-methoxyuracil, and most preferably the chemical modification is N1-methylpseudridine (N1MPU, N1Mpsi or N1Mψ).

30. A pharmaceutical composition comprising one or more lipid nanoparticles as defined in any one of claims 1 to 29 and an acceptable pharmaceutical carrier, preferably used in human or veterinary medicine, more preferably used for the prevention or treatment of cancer or infectious disease in a subject, and most preferably used for the prevention or treatment of cancerous disease in a subject.

31. A pharmaceutical composition according to claim 30 or a lipid nanoparticle according to any one of claims 1 to 29, wherein the pharmaceutical is used as a pharmaceutical, and the pharmaceutical is a preventive or therapeutic vaccine.

32. A pharmaceutical composition according to any one of claims 30 to 31, or a lipid nanoparticle according to any one of claims 1 to 29, wherein the target is a vertebrate, preferably a mammal, and is used as a pharmaceutical.

33. A pharmaceutical composition according to any one of claims 30 to 32, or a lipid nanoparticle according to any one of claims 1 to 29, which is used as a pharmaceutical and is useful for treating infectious diseases, cancer, or tumors, and preferably useful for treating cancer or tumors.

34. A kit or parts kit comprising lipid nanoparticles according to any one of claims 1 to 29 or a pharmaceutical composition according to any one of claims 30 to 33, further comprising, if necessary, a liquid medium for dissolution, and, if necessary, technical instructions providing information on the use and dosage of the components.

35. A method for inducing an antigen-specific immune response in a subject, comprising administering to the subject in an amount effective to produce an antigen-specific immune response in the subject a lipid nanoparticle according to any one of claims 1 to 29, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34.

36. A method for preventing, improving, or treating a disease or condition, preferably cancer, in a target subject, comprising administering to the target a lipid nanoparticle according to any one of claims 1 to 29, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34.

37. The method according to any one of claims 35 to 36, wherein administration of the lipid nanoparticles, the pharmaceutical composition, or the kit or parts kit results in the expression of an mRNA-encoded antigen in the target lymphocytes or in the spleen and / or lymph nodes.

38. The method according to any one of claims 35 to 37, wherein the antigen-specific immune response comprises (i) a T cell response, (ii) a B cell response, (iii) a CD4 T cell immune response, (iv) a CD8 T cell immune response, and / or (v) an antigen-specific antibody response, the antigen-specific antibody response being measured by the presence of an antigen-specific antibody in serum, and preferably the antigen-specific immune response comprises a combination of two or more antigen-specific immune responses selected from the group consisting of (i), (ii), (iii), (iv), and (v).

39. The method according to any one of claims 35 to 38, wherein the lipid nanoparticles according to any one of claims 1 to 29, the pharmaceutical composition according to any one of claims 30 to 33, or the kit or parts kit according to claim 34 is administered intravenously, intramuscularly, intradermally, or intratumorally, more preferably intravenously or intramuscularly, most preferably intramuscularly.

40. A method for treating or preventing a disease, the method comprising applying or administering to a target subject of interest the lipid nanoparticles described in any one of claims 1 to 29, the pharmaceutical composition described in any one of claims 30 to 33, or the kit or parts kit described in claim 34, preferably subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial bursa, intranasal cavity, oral, intrasternal, intramedullary, intrahepatic, intrafocal, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, intraocular, intravitreous, subretinal, intranodal, or intratumor, more preferably intramuscular, intradermal, intravenous, or intratumor, more preferably intravenous or intramuscular, most preferably intramuscular.

41. Lipid nanoparticles according to any one of claims 1 to 29 used as a pharmaceutical, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34.

42. Lipid nanoparticles according to any one of claims 1 to 29, used for the prevention or treatment of cancer, autoimmune diseases, infectious diseases, allergies, or protein deficiencies, preferably used for the prevention or treatment of cancer, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34.

43. The following structure 【Chemistry 10】 It can be represented as follows, or the following structure 【Chemistry 11】 An ionizable lipid represented by [formula].

44. A method for synthesizing ionizable lipid C28 or ionizable lipid C29, wherein C28 is used in scheme 1 【Chemistry 12】 According to scheme 2, or C29 【Chemistry 13】 A method of synthesis according to the following.

45. The lipid nanoparticles according to any one of claims 1 to 29, wherein after (i) freezing and thawing or (ii) freeze-drying and reconstitution, the lipid nanoparticles have a lower PDI and / or a smaller size compared to control lipid nanoparticles comprising PEG lipids instead of PMOZ comprising polymer-bound lipids as described in any one of claims 1 to 29.

46. A method for producing lipid nanoparticles according to any one of claims 1 to 29 in a frozen state, wherein the lipid nanoparticles, after thawing, have a lower PDI and / or smaller size compared to control lipid nanoparticles containing PEG lipids instead of polymer-bound lipids as shown in any one of claims 1 to 29.

47. A method for producing lipid nanoparticles according to any one of claims 1 to 29 in a freeze-dried state, wherein the lipid nanoparticles, after reconstitution, have a lower PDI and / or smaller size compared to control lipid nanoparticles containing PEG lipids instead of polymer-bound lipids as shown in any one of claims 1 to 29.

48. An improved freeze-drying process for preparing lipid nanoparticles according to any one of claims 1 to 29, the process comprising using a PMOZ containing a polymer-bound lipid as described in any one of claims 1 to 29 as an excipient instead of a PEG lipid, wherein the lipid nanoparticles, after reconstitution, have a lower PDI and / or smaller size compared to control lipid nanoparticles containing a PEG lipid instead of a PMOZ containing a polymer-bound lipid as described in any one of claims 1 to 29.

49. A method for inducing interferon (IFN) production, comprising administering to a target subject a lipid nanoparticle according to any one of claims 1 to 29, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34, wherein IFN production increases after administration.

50. The method according to claim 49, comprising administering to a target subject in an amount sufficient to induce an immune response in the target subject, preferably the immune response being accompanied by cytokine production, and more preferably the immune response being accompanied by the regulation of type I IFN, type II IFN, and / or type III IFN.

51. The method according to any one of claims 49 to 50, comprising administering to a subject of interest an amount sufficient to induce an immune response in the subject, preferably the immune response being accompanied by the production of type I IFN, more preferably the immune response being accompanied by the regulation of IFNa, IFNb, IFNe, IFNk, or IFN, most preferably IFNa and / or IFNb.

52. The method according to any one of claims 49 to 51, comprising administering to a target subject a lipid nanoparticle according to any one of claims 1 to 29, a pharmaceutical composition according to any one of claims 30 to 33, or a kit or parts kit according to claim 34, wherein, after administration, RNA accumulation and / or RNA expression occur in the spleen and / or lymph nodes.

53. A vaccine composition comprising lipid nanoparticles according to any one of claims 1 to 29, wherein the lipid nanoparticles show an increase of about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after one or more freeze / thaw cycles compared to before the freeze / thaw cycle, preferably an increase of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after the freeze / thaw cycle.

54. A vaccine composition comprising lipid nanoparticles according to any one of claims 1 to 29, wherein the formulation shows an increase of about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after freeze-drying compared to before freeze-drying, preferably an increase of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.

55. A vaccine composition comprising lipid nanoparticles according to any one of claims 1 to 29, wherein the formulation shows an increase of about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after dilution compared to before dilution, preferably an increase of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after dilution.

56. A vaccine composition comprising lipid nanoparticles according to any one of claims 1 to 29, wherein the encapsulation efficiency of the formulation remains substantially the same after storage at about 4°C or below for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer.

57. A vaccine composition comprising lipid nanoparticles according to any one of claims 1 to 29, wherein the average diameter of the LNPs remains substantially the same after storage at about 4°C or below for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer.

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