Novel lipid nanoparticles for delivery of nucleic acids

Abstract

The present invention relates to novel polymer-conjugated lipids and novel compositions comprising said novel polymer-conjugated lipids that are useful for delivering nucleic acids to living cells.

Description

[Technical Field]

[0001] The present invention relates to lipid nanoparticles containing polyoxazoline (POZ) or poly(2-methyl-2-oxazoline) (PMOZ)-lipids and nucleic acids, preferably mRNA, useful as mRNA-based vaccines or mRNA-based therapeutics. The present invention also relates to compositions containing lipid nanoparticles (LNPs) containing POZ- or PMOZ-moiety-containing lipids (POZ-lipids, PMOZ-lipids) and mRNA, and to the use of said LNPs or compositions containing POZ- or PMOZ-lipids for preparing pharmaceutical compositions, particularly vaccine compositions, for use in the prevention or treatment of, for example, infectious diseases, tumor or cancer diseases, allergies, or autoimmune diseases. The present invention also relates to mRNA-based therapeutic LNP compositions for protein replacement therapy encoded by mRNA, wherein the LNP compositions contain POZ- or PMOZ-lipids. An example of this is an indication or disease that can be cured by liver transplantation (e.g., OTC deficiency). The present invention further describes therapies using the above-mentioned LNPs or compositions containing POZ- or PMOZ-moiety-containing lipids for protein replacement therapy. The present invention further describes methods for treating or preventing the above diseases. The mRNA may also encode an antibody, an antibody fragment, an antibody variant, adduct or derivative, such as a single chain variable fragment, a diabody or a triabody. [Background technology]

[0002] Generally, vaccines can be subdivided into "first," "second," and "third" generation vaccines. "First generation" vaccines are typically whole organism vaccines. They are based on either live attenuated or killed pathogens, such as viruses, bacteria, etc. The main drawback of live attenuated vaccines is the risk of reversion to life-threatening mutations. Thus, although such pathogens are attenuated, they may still inherently pose unpredictable risks. Killed pathogens may not be as effective as desired in generating specific immune responses. To minimize these risks, "second generation" vaccines have been developed. These are typically subunit vaccines consisting of defined antigens or recombinant protein components derived from pathogens.

[0003] Genetic vaccines, i.e., vaccines for genetic vaccination, are usually understood as "third generation" vaccines. They are typically composed of genetically engineered nucleic acid molecules that allow the expression of peptides or protein (antigen) fragments characteristic of pathogens or tumor antigens in vivo. When administered to a patient, genetic vaccines are expressed after uptake by target cells. Expression of the administered nucleic acid molecule results in the production of the encoded protein. When these proteins are recognized as foreign by the patient's immune system, an immune response is elicited.

[0004] Both DNA and RNA can be used as nucleic acid molecules for administration in the context of genetic vaccination. DNA is known to be relatively stable and easy to handle. However, the use of DNA carries the risk of unwanted insertion of the administered DNA fragment into the patient's genome, potentially resulting in mutagenic events such as loss of function of the impaired gene. An additional risk is the unwanted production of anti-DNA antibodies. Another drawback is that DNA administration limits the level of expression of the encoded peptide or protein achievable because DNA must enter the nucleus to be transcribed before the resulting mRNA can be translated. Among other reasons, the level of expression of the administered DNA depends on the presence of specific transcription factors that regulate DNA transcription. In the absence of such factors, DNA transcription does not produce satisfactory amounts of RNA. As a result, the level of translated peptide or protein obtained is limited.

[0005] The use of messenger RNA (mRNA) to deliver genetic information to target cells offers an attractive alternative to DNA. Advantages of using mRNA include transient expression and non-transforming properties. mRNA does not need to enter the nucleus to be expressed, and it cannot integrate into the host genome, thereby eliminating the risk of oncogenesis. Thus, using RNA instead of DNA for genetic vaccination minimizes or avoids the risk of unwanted genomic integration and the production of anti-DNA antibodies. However, RNA is considered to be a fairly unstable molecular species that can be easily degraded by ubiquitous RNases. mRNA vaccines containing antigen-encoding mRNA complexed with protamine have already been described in the prior art (e.g., Non-Patent Document 1, or Non-Patent Document 2, Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, and Patent Document 5).

[0006] The use of RNA or mRNA in therapeutic settings currently faces two challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to gain access to intracellular compartments where the relevant translation machinery resides. Lipid nanoparticles formed from cationic and neutral lipids, cholesterol, PEGylated lipids, and other lipid components, such as RNA or mRNA, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. Patent documents 6 and 7 describe lipid nanoparticle compositions containing unmodified and nucleotide-modified RNA encoding different antigens in this regard; both references are incorporated herein by reference in their entirety. In addition to molecular composition, parameters such as particle size, charge, or grafting with molecular components such as polyethylene glycol (PEG) or ligands play a role in delivery efficiency. FDA-approved PEG grafting is believed to reduce particle-serum interactions, increase serum stability, and extend circulation time, which is beneficial for certain targeting approaches. Furthermore, PEGylation can be used for particle engineering. For example, when preparing lipid nanoparticles (LNPs) by mixing an aqueous phase of RNA with an oil phase of lipids, a certain amount of PEG-conjugated lipid is required in the lipid mixture; otherwise, the particles will aggregate during the mixing process. It has been shown that particle size can be tuned by varying the molar fraction of PEG-lipids containing PEG at various molar masses. Similarly, particle size can be tuned by varying the molar mass of the PEG fraction of the PEGylated lipid. Typical accessible diameters range between 30 and 200 nm (Non-Patent Document 3). Particles formed in this way have the added advantage of interacting less with serum components due to the PEG fraction and having a longer circulatory half-life, which is desirable in many drug delivery approaches. In summary, without PEG-lipids, particles of discrete diameters cannot be formed; the particles will eventually form large aggregates and precipitate.

[0007] Thus, in the technique of forming LNPs from ethanol and aqueous phases, one of the primary functions of the PEG-lipids is to promote particle self-assembly by forming a steric barrier on the surface of the resulting particles that form when nucleic acids are rapidly mixed in an ethanol solution containing RNA-binding lipids. The steric PEG barrier prevents particle-particle fusion and promotes the formation of a uniform population of LNPs with diameters less than 100 nm.

[0008] PEG is the most widely used "stealth" polymer for drug delivery and is considered the gold standard. To generate uniform, colloidally stable nanoparticle populations, PEG lipids are typically incorporated into the system due to their hydrophilic steric hindrance properties (the PEG shell prevents electrostatic or van der Waals attractions that lead to aggregation). PEGylation allows for the attraction of a water envelope around the polymer, which protects the RNA complex from opsonization by serum proteins, increases serum half-life, reduces rapid renal clearance, and improves pharmacokinetic behavior. Variation in the lipid acyl chain length (C 18 , C 16 or C 14 ) modifies the stability of PEG-lipid incorporation into particles, i.e., PEG shedding, leading to modulation of in vivo bioperformance and pharmacokinetics. Short acyl chains (C) dissociate in vivo from LNPs with a half-life of less than 30 minutes. 14 The use of PEG-lipids with a PEG-MW of 1000 or more results in optimal hepatocyte gene silencing capacity (Non-Patent Document 4; Non-Patent Document 5). Furthermore, by varying the PEG-lipid parameters, strict control of particle size can be achieved; i.e., a higher PEG-MW or a higher molar fraction of PEG-lipid in the particles results in smaller particles.

[0009] Despite these advantages, PEGylation of nanoparticles can also result in various adverse effects that may be detrimental to their intended use for drug delivery. PEGylation of liposomes and LNPs is known to reduce cellular uptake and endosomal escape, ultimately lowering overall transfection efficiency. Indeed, the PEG shell creates a steric barrier to efficient particle binding to cells and also prevents endosomal release by preventing membrane fusion between liposomes and endosomal membranes. For this reason, the type and amount of PEG-lipid used must always be carefully adjusted. On the one hand, the PEG-lipid should provide sufficient stealth effect for in vivo and stabilization, but on the other hand, it should not interfere with transfection. This phenomenon is known as the "PEG dilemma."

[0010] In addition to reducing transfection efficiency, PEGylation has been associated with accelerated blood clearance (ABC) and storage disease induced by anti-PEG antibodies and / or complement activation (Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes into rats can significantly alter the pharmacokinetic behavior of the second dose when administered several days later (Non-Patent Document 9; Non-Patent Document 10). The phenomenon of "accelerated blood clearance" (ABC) appears to be inversely proportional to the PEG content of liposomes. The presence of anti-PEG antibodies in the plasma induces higher particle clearance by the mononuclear phagocyte system (MPS), ultimately reducing drug efficacy. This phenomenon further affects even patients who have never been treated with drugs containing PEG, due to the ubiquity of PEG in, for example, the cosmetic industry (ointments, creams, shampoos, toiletries, lotions) and pesticides.

[0011] PEG is also suspected to induce complement activation, which can lead to hypersensitivity reactions, also known as Complement-Activation Related Pseudo-Allergy (CARPA). It is not yet clear from the literature whether complement activation is due to nanoparticles in general or the presence of PEG in particular.

[0012] The presence of PEG in lipid nanoparticles can also induce specific immune responses. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotides or plasmid DNA induced a strong immune response in mice, resulting in rapid blood clearance of subsequent administration. The magnitude of this response was sufficient to induce significant morbidity and, in some cases, mortality. The use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG-lipids abolished the response, suggesting that the rapid clearance of liposome-encapsulated ODN from the blood depended on the presence of PEG-lipids in the membrane. The production of anti-PEG antibodies and putative complement activation were plausible explanations for the rapid clearance of vesicles from the blood (Non-Patent Document 11).

[0013] PEG can induce an immune response and should be avoided in certain applications where multiple injections are required, such as in mRNA-based treatments, e.g., for protein replacement therapy, where the risk may be particularly high due to the potential inherent immunogenicity of RNA.

[0014] Furthermore, for storage and transportation purposes, LNPs are often stored at low temperatures, e.g., frozen or lyophilized (freeze-dried). Prior to administration to patients, frozen LNPs must be thawed to room temperature, and lyophilized LNPs must be reconstituted into solution. During thawing or reconstitution, respectively, a frequently observed problem is changes in physicochemical properties, such as increases in LNP diameter and PDI. Standard LNP formulations containing PEG-lipids have been found to be highly sensitive to dilution and freeze / thaw cycles. This is reflected by significant effects on either or both LNP diameter and mRNA encapsulation efficiency (EE). Addressing this issue is important because freezing at low concentrations allows for the preparation of single-dose vials, as opposed to the current presentation as concentrated multi-dose vials that require a dilution step before administration. More specifically, at concentrations below 1 g / L, a significant effect on LNP diameter was observed after a single freeze / thaw cycle, with particle size nearly doubling for some dilutions.

[0015] Thus, there is a need in the art for efficient methods and compositions for introducing RNA into cells that avoid the drawbacks associated with the use of PEG. Accordingly, an object of the present invention is to provide efficient methods and compositions for introducing RNA into cells that avoid the described drawbacks associated with the use of PEG. The present invention solves this objective and addresses these and other needs. There is also a need for improved PEG-free lipid nanoparticles for delivering RNA. Preferably, these PEG-free lipid nanoparticles will provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. Furthermore, these PEG-free lipid nanoparticles containing RNA or mRNA should be well tolerated and provide a sufficient therapeutic index so that treatment of a patient with an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risks to the patient. The present invention provides these and related advantages. Therefore, despite all prior art, there is still a need for alternative polymer-conjugated lipids, alternative cationic lipids, and consequently alternative lipid nanoparticles comprising said alternative lipids, which provide one or more of the following properties: reduced cytotoxicity of molecules, such as nucleic acids, better targeting ability, enhanced short-term and / or long-term immunity, or facilitated endosomal escape.Therefore, despite the vast amount of research carried out to date in the field of polymer-conjugated lipids, lipid nanoparticles and cationic lipids, it is desired to develop further polymer-conjugated lipids, lipid nanoparticles and cationic lipids that can improve or eliminate one or more of the above-mentioned problems, or the in vivo efficacy, toxicity, cost and design simplicity of transfection process.

[0016] Specifically, the inventors have surprisingly found that the RNA particle formulations described herein meet the above requirements. 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)s are a class of polymers formed by cationic ring-opening that were first identified and synthesized over 50 years ago (Non-Patent Document 12). These polymers are nonionic, biostable, soluble in water and some polar organic solvents, and can be synthesized from readily available, non-toxic, non-explosive starting materials. N-carbonyl side chains on the polymer chain give them a "pseudo-polypeptide" appearance. POZs with shorter side chains are generally more water-soluble than POZs with longer side chains. PMOZ is composed of repeating units of, for example, 2-methyl-2-oxazoline (CAS Registry Number: 161358-46-9) and is rapidly excreted by the kidney without significant accumulation in tissues (Non-Patent Document 13). POZ / PMOZ-lipid conjugates enable the production of RNA nanoparticles by various techniques, resulting in defined surface properties and a controlled diameter range. Production can be carried out by robust methods that comply with pharmaceutical manufacturing requirements. The particles can be end-group functionalized with various moieties to adjust the charge or introduce specific molecular moieties, such as ligands.

[0017] Here, the present inventors have surprisingly found that LNPs of the present invention containing a novel polyoxazoline polymer-conjugated lipid, preferably PMOZ-lipid, have advantageous physicochemical properties as measured by PDI and diameter measurements after freezing and thawing, or lyophilization and reconstitution. The present inventors have surprisingly found that PMOZ-LNPs are superior in terms of diameter (smaller diameter) and PDI after subjecting the lipid nanoparticles of the present invention to thermal stress, i.e., freeze / thaw cycles or lyophilization and reconstitution, respectively. Furthermore, an unfavorable increase in diameter and PDI was observed for PEG-LNPs upon dilution, i.e., PEG-LNPs had increased diameter and PDI upon dilution. For LNPs containing PMOZ as a conjugated lipid, no increase in diameter or PDI, or no significant increase, was observed. Even though PMOZ-LNPs show an increase in diameter upon dilution or freeze / thaw within a similar range to PEG-LNPs, they are still smaller than PEG-LNPs, which may be preferable since knowledge in the field indicates that smaller particles are more immunogenic (Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16).

[0018] Thus, the objects of the present invention can also be seen in terms of (i) novel polymer-conjugated lipids, (ii) novel lipid nanoparticles comprising said novel polymer-conjugated lipids, and (iii) the use of said novel polymer-conjugated lipids to produce lipid nanoparticles that are improved with respect to the generation of anti-PEG antibodies (i.e., the novel lipid nanoparticles do not generate anti-PEG antibodies) and with respect to enhanced physicochemical properties upon (i) freezing and thawing or (ii) lyophilization and reconstitution of said lipid nanoparticles, for example, for storage and transport. These objects, as well as further objects described in the "Background of the Invention," are solved by the subject matter of the present invention. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] European Patent No. 1083232 [Patent Document 2] International Publication No. 2010037539 [Patent Document 3] International Publication No. 2012116811 [Patent Document 4] International Publication No. 2012116810 [Patent Document 5] International Publication No. 2015024665 [Patent Document 6] International Publication No. 2018078053 [Patent Document 7] International Publication No. 2016176330 [Non-patent literature]

[0020] [Non-Patent Document 1] PMID27336830 [Non-patent document 2] PMID23159882 [Non-patent document 3] Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1(8):e37, PMID:23344179 [Non-patent document 4] Chen et al., 2014, J Control Release 196:106-12 [Non-Patent Document 5] Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163 [Non-patent document 6] Bendele A et al., 1998, Toxicological Sciences 42, 152-157 [Non-Patent Document 7] Young MA et al., 2007, Translational Research 149(6), 333-342 [Non-patent document 8] S.M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478 [Non-Patent Document 9] Laverman P et al., 2001, J Pharmacol Exp Ther. 298(2), 607-12 [Non-Patent Document 10] Ishida et al., 2006, J Control Release 115(3), 251-8 [Non-Patent Document 11] Semple et al., 2005, J Pharmacol Exp Ther. 312(3), 1020-6 [Non-Patent Document 12] Kagiya et al., J Polym Sci B Polym Lett 1966;4:441-5 [Non-Patent Document 13] Gaertner et al., Journal of Controlled Release 119 (2007) 291-300 [Non-Patent Document 14] Li et al., 2014, Journal of controlled release, 173, 148-157 [Non-Patent Document 15] Ott et al., Vaccine, 1995, 13(16), 1557-1562 [Non-Patent Document 16] Shah et al., 2014. Nanomedicine, 9(17), 2671-2681 Summary of the Invention

[0021] In one aspect, the present invention relates to novel polymer-conjugated lipids useful for the delivery of nucleic acids into living cells. In a specific embodiment, the polymer-conjugated lipid is a compound according to formula (I): [P]-[linker]-[L] Formula (I) or a pharmaceutically acceptable salt, prodrug, tautomer or stereoisomer thereof (In the formula, [P] is at least one polyoxazoline (POZ) monomer unit

[0022] [ka]

[0023] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value ranging from about 45 to about 55, preferably n is about 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. is a homopolymer moiety comprising [linker] is an optional linker group; [L] is the lipid moiety) is.

[0024] In another embodiment, the polymer-conjugated lipid has as [P]: Poly(2-methyl-2-oxazoline) (PMOZ)

[0025] [ka]

[0026] Poly(2-ethyl-2-oxazoline) (PEOZ)

[0027] [ka]

[0028] Poly(2-propyl-2-oxazoline) (PPOZ)

[0029] [ka]

[0030] Poly(2-butyl-2-oxazoline) (PBOZ)

[0031] [ka]

[0032] Poly(2-isopropyl-2-oxazoline) (PIPOZ)

[0033] [ka]

[0034] Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), and Poly(2-dimethylamino-2-oxazoline) (PDMAOx) and a heteropolymer portion or a homopolymer portion comprising a plurality of monomer units selected from the group consisting of: Preferably, [P] is a homopolymer moiety comprising a plurality of PMOZ or PEOZ monomer units, more preferably, [P] comprises a plurality of PMOZ monomer units, or preferably consists of a plurality of PMOZ monomer units; (i) n has an average value in the range of about 45 to about 55, preferably n is about 50; or (ii) n is selected so that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0035] In a highly 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).

[0036] In another embodiment, the polymer-conjugated lipid is selected from the group consisting of a POZ-monoacylglycerol conjugate, a POZ-diacylglycerol conjugate, a POZ-dialkyloxypropyl conjugate, a POZ-steroid or POZ-sterol conjugate, a POZ-phospholipid conjugate, a POZ-ceramide conjugate, 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 optionally being interrupted by one or more biodegradable groups and / or optionally comprising a terminal biodegradable group, including, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9alkenyl), zwitterionic linkers, non-ester and ester-containing linker moieties (-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(R 5 )=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 sulfonate ester, and combinations thereof; R 3 , R 4 and R 5 are independently H or alkyl (e.g., C1-C4 alkyl).

[0037] In a further embodiment, 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 optionally being interrupted by one or more biodegradable groups and / or optionally comprising one terminal biodegradable group, the biodegradable group being selected from the group consisting of, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 alkenyl), zwitterionic linkers, non-ester and ester-containing linker moieties (-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(R 5 )=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 sulfonate ester, and combinations thereof; R 3 , R 4 and R 5 are independently H or alkyl (e.g., C1-C4 alkyl).

[0038] In a further embodiment, the lipid moiety [L] comprises two linear unsaturated alkyl chains each containing 14 carbon atoms. In a further most preferred embodiment, the polymer-conjugated lipid comprises a lipid moiety [L] and a linker group [Linker] comprising ditetradecylamine, preferably the linker group [Linker] is (-NHC(O)CH2CH2C(O)-). In a further preferred embodiment, the lipid moiety [L] comprises ditetradecylamine, and the linker moiety [Linker], preferably (-NHC(O)CH2CH2C(O)-), forms an amide bond through its connection to the N atom of the ditetradecylamine. In a most preferred embodiment, the polymer-conjugated lipid comprises a linker (-NHC(O)CH2CH2C(O)-), and the linker is oriented such that a carboxamide bond is formed through its connection to the N atom of the ditetradecylamine.

[0039] In a further most preferred embodiment, the polymer-conjugated lipid comprises a lipid moiety [L] and a linker group [Linker] comprising ditetradecylamine, preferably the linker group [Linker] is (C(O)CH2CH2C(O)NH). In a further preferred embodiment, the lipid moiety [L] comprises ditetradecylamine, and the linker moiety [Linker], preferably (-NHC(O)CH2CH2C(O)-), forms an amide bond via its connection to the N atom of the ditetradecylamine.

[0040] In a further preferred embodiment, the lipid moiety [L] is the lipid moiety used in "PMOZ2". In a further preferred embodiment, the linker moiety [linker] is the linker moiety used in "PMOZ2".

[0041] In a further most preferred embodiment, the present invention relates to a polymer-conjugated lipid having a lipid moiety [L] that is a lipid moiety used in "PMOZ4." In another most preferred embodiment, the present invention relates to a polymer-conjugated lipid having a linker moiety [Linker] that is a linker used in "PMOZ4."

[0042] In a preferred embodiment, the lipid moiety [L] comprises ditetradecylamine, and the linker group [Linker] is (-NHC(O)CH2CH2C(O)-). In a preferred embodiment, the lipid moiety [L] comprises ditetradecylamine, and the linker moiety [Linker] is connected to the N atom of the ditetradecylamine.

[0043] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: at least one polyoxazoline (POZ) monomer unit

[0044] [ka]

[0045] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value ranging from about 45 to about 55, preferably n is about 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. a homopolymer portion comprising Preferably, the homopolymer moiety comprising multiple monomer units comprises 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), to provide novel lipid nanoparticles.

[0046] Most preferably, R is C1 (i.e., CH3 or methyl), resulting in polymethyloxazoline or poly(2-methyl-2-oxazoline), i.e., "PMOZ."

[0047] In a further aspect, the present invention provides a vaccine composition comprising the lipid nanoparticles of the present invention or a kit or kit of parts comprising the polymer-conjugated lipids of the present invention for use as a medicament and / or for preventing, prophylactically treating and / or ameliorating a disease selected from an infectious disease, including a viral, bacterial or protozoan infectious disease, a cancer or a tumor disease.

[0048] In a further aspect, the present invention provides a method of treating or preventing an infectious disease; a cancer or neoplastic disease, disorder or condition; a liver disease selected from the group consisting of liver fibrosis, cirrhosis and liver cancer; an allergy; or an autoimmune disease, disorder or condition, comprising: a) providing a lipid nanoparticle, a vaccine composition, or a kit or kit-of-parts of the present disclosure, comprising a homopolymer moiety comprising at least one polyoxazoline (POZ) monomer, preferably a polymer-conjugated lipid of the present disclosure; b) applying or administering the mRNA, lipid nanoparticles, vaccine composition, or kit or kit-of-parts to a tissue or organism; The present invention provides a method comprising:

[0049] In another aspect of the present invention, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present disclosure or the kit or kit-of-parts of the present disclosure or the vaccine composition of the present disclosure, for use in vaccinating a subject, the pharmaceutical composition comprising an effective dose of mRNA encoding a viral antigen.

[0050] In yet another aspect of the present invention, the present invention provides improved lyophilizable lipid nanoparticles that have advantageous physicochemical properties after lyophilization and reconstitution. In yet another aspect of the present invention, the present invention provides improved lipid nanoparticles that have advantageous physicochemical properties after being frozen and thawed.

[0051] definition For clarity and readability, the following scientific background information and definitions are provided. Any technical feature mentioned in or disclosed herein can be part of or can be read in each and every embodiment of the present invention. Additional definitions and explanations can be provided in the context of this disclosure.

[0052] Unless otherwise defined or unless specific context requires, all technical terms used herein have the same meaning as commonly understood by those skilled in the relevant art.

[0053] Unless the context indicates or requires otherwise, the words "comprise," "comprises," and "comprising," and similar expressions, are to be interpreted in the present specification and claims in an open and inclusive sense, as meaning "including, but not limited to." It should also be understood that for purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising." Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this is also intended to encompass groups that preferably consist only of these embodiments.

[0054] The phrases "one embodiment," "embodiment," "specific embodiment," and the like mean that the particular feature, property, or characteristic, or particular group or combination of features, properties, or characteristics, recited in connection with the respective phrase, is present in at least one embodiment of the invention. The appearances of these phrases in various places throughout this specification do not necessarily refer to the same embodiment. Furthermore, the particular features, properties, or characteristics may be combined in any suitable manner in one or more embodiments.

[0055] The singular forms "a," "an," and "the" are to be understood as including plural references unless the context clearly dictates otherwise. Percentages in numerical contexts should be understood as being relative to the total number of the respective item. In other cases, unless the context dictates otherwise, percentages should be understood as percentages by weight (wt %).

[0056] As used herein, " compound " refers to a chemical substance, which is a material that consists of molecules that have 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 polymeric compounds, the molecules of the compound are very similar, but not all of them are necessarily identical.For example, a polymer segment that is specified as consisting of 50 monomer units may also contain individual molecules that have, for example, 48 or 53 monomer units.

[0057] The term "molecule" may be used synonymously with "compound" or an individual (ie, single) molecule. Any reference to a compound or moiety having a functional group that is ionizable under physiological conditions should be understood to include the ionized form of the respective compound or moiety. Conversely, any reference to a compound or moiety having an ionizable functional group that may also exist in a non-ionized form under physiological conditions should be understood to include the non-ionized form of the respective compound or moiety. For example, a disclosure of a compound having a carboxyl group should be interpreted as referring to the respective compound having a non-ionized carboxyl group or an ionized carboxylate group.

[0058] 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. An approximation of this pH range is from about pH 1 to about pH 9. Depending on the context, physiological conditions can also refer to near-neutral conditions, such as from about pH 5 to about pH 8.5, or from about pH 5.5 to about pH 8.

[0059] Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, i.e., amphiphilic compounds with lipid-like physical properties. In the context of the present invention, the term lipid is considered to encompass lipidoids.

[0060] In the context of the present invention, the term "selected from the group consisting of" followed by a particular group of elements (e.g., "A, B, and C") is intended to be non-limiting within the context of the present invention. In other words, such terms do not indicate that the disclosure is closed to unlisted elements, i.e., alternative meanings are included within the group following the term. Thus, in the context of the present invention, the term "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 "is A, B, or C," encompassing other structurally and functionally related as well as unrelated, unrecited elements.

[0061] The term "about" is used when a parameter or value does not necessarily have to be identical, i.e., 100% identical. Thus, "about" means that the parameter or value may deviate by 0.1% to 20%, preferably 0.1% to 10%; particularly, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Those skilled in the art will recognize that, for example, a particular parameter or value may vary slightly based on the method by which the parameter is determined. For example, if a particular parameter or value is defined herein as having a length of, for example, "about 1000 nucleotides," that length can deviate by 0.1% to 20%, preferably 0.1% to 10%, particularly 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Thus, those skilled in the art will understand that in certain specific examples, the length can deviate by 1 to 200 nucleotides, preferably 1 to 100 nucleotides, particularly 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.

[0062] The term "cationic" means that the respective structure has a positive charge, either permanently or not, depending on certain conditions, such as pH, unless a different meaning is clear from the specific context. Thus, the term "cationic" encompasses both "permanently cationic" and "cationisable." 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 or active hydrogen ion concentration. This depends on the individual properties of the cationizable or polycationizable compound, particularly the pK of each cationizable group or atom, which is charged or uncharged at that pH or hydrogen ion concentration. a In a dilute aqueous environment, the fraction of cationizable compounds, groups, or atoms that are positively charged can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, if a compound or moiety is cationizable, it is preferred that it be positively charged at pH values ​​of about 1-9, preferably 4-9, 5-8, or even 6-8, more preferably 9 or less, 8 or less, or 7 or less, and most preferably at physiological pH values, e.g., about 7.3-7.4, i.e., under physiological conditions, particularly the physiological salt conditions of cells in vivo. In embodiments, it is preferred that the cationizable compound or moiety is primarily neutral at physiological pH values, e.g., about 7.0-7.4, but positively charged at lower pH values. In some embodiments, the pK of the cationizable compound or moiety is a A preferred range of is about 5 to about 7. In some embodiments, the protonatable lipid has a pK of the protonatable group in the range of about 4 to about 11. a , for example, a pK of about 5 to about 7 a It has.

[0063] Unless a different meaning is clear from the specific context, the term "cationic" means that the respective structure has a positive charge, either permanently or not, depending on certain conditions, such as pH. Thus, the term "cationic" encompasses both "permanently cationic" and "cationizable." For example, compounds or moieties having primary, secondary, or tertiary amino groups can exist in a predominantly positively charged state under physiological conditions and are therefore cationic, or more specifically, cationizable.

[0064] As used herein, "permanently cationic" means that each compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment.In most cases, the positive charge is caused by the presence of quaternary nitrogen atoms.If a compound has multiple such positive charges, it can be called permanently polycationic, which is a subcategory of permanently cationic.

[0065] Similarly, the terms "anionic," "anionizable," and "permanently anionic" are used to mean similar things to "cationic," "cationizable," and "permanently cationic," except that the charge on the respective compound, group, or atom is negative rather than positive.

[0066] The term "neutral" when applied to a compound, such as a lipid or steroid, or to a group or moiety, means that it is neither cationic nor anionic, i.e., a compound that does not have functional groups that are ionizable under physiological conditions, such as a hydrocarbon; or that it is both cationic and anionic, i.e., zwitterionic, under typical physiological conditions, such as a typical naturally occurring phosphatidylcholine.

[0067] As used herein, "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids. With respect to glycolipids, in certain embodiments, the LNPs comprise a glycolipid (e.g., monosialoganglioside GM1).

[0068] In this context, the prefix "poly-" refers to multiple atoms or groups with respective properties in a compound. When placed within parentheses, the presence of the plural is optional. For example, (poly)cationic means cationic and / or polycationic. However, the absence of the prefix should not be construed as excluding the plural. For example, polycationic compounds are also cationic compounds and may be referred to as such.

[0069] The term "nucleic acid" refers to any compound comprising or consisting of DNA or RNA. This term can be used for polynucleotides and / or oligonucleotides. Whenever a nucleic acid or nucleic acid sequence encoding a specific protein and / or peptide is referred to herein, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises control sequences that allow its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the specific protein or peptide, in a suitable host, e.g., a human.

[0070] In a particularly preferred embodiment, artificial nucleic acid, nucleic acid or RNA is mRNA, more preferably isolated mRNA.Compared with viral systems, mRNA allows for controlled dosage, transient and controlled expression, complete degradation of mRNA after protein synthesis, and does not bring about the risk of insertion mutation, so mRNA technology is particularly preferred in the context of the present invention.

[0071] In the context of the present invention, the term "nucleoside modification" refers to a nucleic acid, such as an mRNA compound or molecule, that contains a nucleoside that is not normally present in natural mRNA, preferably a non-natural nucleoside. In particular, the term preferably refers to an mRNA nucleoside other than adenine, guanine, cytosine, uracil, and thymine.

[0072] 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 includes a phosphate group attached to the sugar.

[0073] "Peptide" refers to an oligomer or polymer of at least two amino acid monomers linked by a peptide bond. The 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 50-600 monomer units, more specifically 50-300 monomer units, are also called polypeptides.

[0074] A "protein" comprises or consists of one or more polypeptides folded into a three-dimensional form that facilitates a biological function. An "influenza pandemic" or "pandemic flu" can occur when a non-human (novel) influenza virus acquires the ability for efficient and sustained human-to-human transmission and then spreads worldwide. Influenza viruses that have the potential to cause a pandemic are called "influenza viruses with pandemic potential" or "pandemic influenza viruses."

[0075] Examples of influenza viruses with pandemic potential include two different "bird flu" viruses, avian influenza A (H5N1) and avian influenza A (H7N9). Because these are non-human viruses (i.e., they are novel in humans and circulate in birds in some parts of the world), there is little to no immunity to these viruses among people. Although human infections with these viruses have occurred rarely, if any of these viruses were to change so that they could readily infect humans and spread easily from person to person, an influenza pandemic could occur.

[0076] Pandemic influenza / flu vaccine or pandemic influenza / flu vaccine: A vaccine directed against pandemic influenza virus is referred to herein as a pandemic influenza / flu vaccine or pandemic influenza / flu vaccine.

[0077] Flu / Influenza Season: Flu season is a recurring annual period characterized by epidemics of influenza (flu) outbreaks. Seasons occur during the cold seasons in each hemisphere. Influenza activity can sometimes be predicted and even tracked geographically. The onset of major influenza activity in each season varies by location, but in any specific location, these minor outbreaks typically take about three weeks to peak and another three weeks to significantly decline. Influenza vaccination is used to reduce the effects of influenza seasons; pneumonia vaccination further reduces the effects and complications of influenza seasons. Because winter occurs at different times of the year in the Northern and Southern Hemispheres, there are actually two influenza seasons each year.

[0078] Seasonal influenza / flu vaccine or seasonal influenza / flu vaccine: A vaccine directed against seasonal influenza viruses during influenza season is referred to herein as a "seasonal influenza / flu vaccine or seasonal influenza / flu vaccine."

[0079] Immune system: The immune system can protect an organism from infection. When a pathogen breaches the organism's physical barriers and invades the organism, the innate immune system provides an immediate but non-specific response. If a pathogen evades this innate response, vertebrates are equipped with a second layer of defense, the adaptive immune system. Here, the immune system adapts its response to improve pathogen recognition during infection. Therefore, even after the pathogen is 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 the pathogen is encountered. Accordingly, the immune system includes the innate immune system and the adaptive immune system. Each of these two parts contains what are called humoral and cellular components.

[0080] Immune response: An immune response can typically be either a specific reaction of the adaptive immune system against a specific antigen (the so-called specific or adaptive immune response) or a non-specific reaction of the innate immune system (the so-called non-specific or innate immune response). The present invention relates to the core of the specific reaction of the adaptive immune system (adaptive immune response). In particular, the present invention relates to an adaptive immune response against infection by a virus, such as influenza virus. However, this specific response can be supported by an additional non-specific reaction (innate immune response). Therefore, the present invention also relates to compounds for simultaneous stimulation of the innate and adaptive immune systems to elicit an efficient adaptive immune response.

[0081] Adaptive immune system: The adaptive immune system is composed of highly specialized cells and processes throughout the body that eliminate or prevent pathogenic proliferation. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (generating immunity) and launch a more powerful attack each time the pathogen is encountered. This system is highly adaptable due to somatic hypermutation (a process in which the frequency of somatic mutations increases) and V(D)J gene rearrangement (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. Because gene rearrangement results in irreversible changes in each cell's DNA, all of that cell's progeny (descendants) inherit genes encoding the same receptor specificity, including memory B and T cells, which are key to long-lived specific immunity. Immune network theory is a theory of how the adaptive immune system works, based on the interactions between the variable regions of T and B cell receptors and molecules produced by T and B cells that contain the variable regions.

[0082] Adaptive immune response: Adaptive immune responses are typically understood to be antigen-specific. Antigen specificity allows for the generation of responses tailored to specific antigens, pathogens, or pathogen-infected cells. The ability to mount these tailored 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 the pathogen. In this context, the first step in the adaptive immune response is the activation of different immune cells capable of inducing antigen-specific immune responses 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 eliciting an immune response. Dendritic cells take up antigens by phagocytosis and macropinocytosis and, stimulated, for example, by contact with foreign antigens, 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 and internalize soluble protein antigens via their receptors may also be important in inducing T cells. Presentation of antigens 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 and 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 antigens but instead recognize them via T cell receptors that recognize short peptide fragments of pathogen-derived protein antigens, for example, bound to MHC molecules on the surface of other cells.

[0083] Cellular immunity / cell-mediated immune response: Cellular immunity typically involves the activation of macrophages, natural killer cells (NK), and antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. In a more general way, cellular immunity is not related to antibodies but rather to the activation of cells of the immune system. The cellular immune response is characterized by activating antigen-specific cytotoxic T lymphocytes, which can induce apoptosis in somatic cells that display antigen epitopes on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells that display tumor antigens; activating macrophages and natural killer cells so that they can destroy pathogens; and stimulating cells to secrete various cytokines that affect the function of other cells involved in adaptive and innate immune responses.

[0084] Humoral immunity / humoral immune response: Humoral immunity typically refers to antibody production and the ancillary processes that may accompany it. Humoral immune responses can typically be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. Humoral immunity can also typically refer to the effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation, and opsonization promotion of phagocytosis and pathogen elimination.

[0085] Innate immune system: Also known as the nonspecific immune system, the innate immune system comprises cells and mechanisms that nonspecifically defend the host against infection by other organisms. This means that the cells of the innate system recognize and respond to pathogens in a general way, but unlike the adaptive immune system, they do not confer long-lasting or protective immunity on the host. The innate immune system may, for example, react with pathogen-associated molecular pattern (PAMP) receptors, such as ligands for Toll-like receptors (TLRs), or with lipopolysaccharide, TNF-alpha, CD40 ligand, or with 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, IFN-alpha, , IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and other auxiliary substances such as 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, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibacterial agents, or antiviral agents. Typically, the response of the innate immune system involves recruitment of immune cells to the site of infection through the production of chemical factors, including specialized chemical messengers called cytokines; activation of the complement cascade; recognition and removal of foreign substances present in organs, tissues, blood, and lymph by specialized white blood cells; 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.

[0086] Adjuvant / Adjuvant Component: An adjuvant or adjuvant component, in a broad sense, is an agent or composition (e.g., pharmacological or immunological) that can modify, e.g., enhance, the effectiveness of other agents, such as drugs or vaccines. Conventionally, in the context of the present invention, this term refers to a compound or composition that serves as a carrier or auxiliary substance for an immunogen 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 or co-administered with the adjuvant in question. In the context of the present invention, an adjuvant preferably enhances the specific immunogenic effect of an active agent of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be divided, for example, into immunostimulants, antigenic delivery systems, or even combinations thereof.

[0087] The term "adjuvant" is typically understood not to include agents that confer immunity themselves. Adjuvants nonspecifically support the immune system to enhance antigen-specific immune responses, for example, by promoting antigen presentation to the immune system or inducing nonspecific innate immune responses. Furthermore, adjuvants can preferably modulate antigen-specific immune responses, for example, by shifting predominantly Th2-based antigen-specific responses to more Th1-based antigen-specific responses, or vice versa. Thus, adjuvants can advantageously modulate cytokine expression / secretion, antigen presentation, type of immune response, etc.

[0088] Immunostimulatory RNA: In the context of the present invention, immunostimulatory RNA (isRNA) can typically be RNA that can induce an innate immune response itself. It usually does not have an open reading frame, so it does not provide a peptide antigen or immunogen, but it induces an innate immune response, for example, by binding to a specific type of Toll-like receptor (TLR) or other appropriate receptor. However, of course, mRNA that has an open reading frame and encodes a peptide / protein (e.g., an antigenic function) can also induce an innate immune response.

[0089] 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 can 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" 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; tetramers; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments consisting of heavy and light chain variable regions, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of amino acid residues mimicking a hypervariable region. Examples of antigen-binding fragments of antibodies 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, antibodies can be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0090] The term "antigen" in the context of the present invention 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 the adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC. In the sense of the present invention, an antigen can be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, fragments, variants, and derivatives of peptides and proteins 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, 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 the adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC. Fragments, variants, and derivatives of peptides or proteins containing at least one epitope, for example, derived from a cancer antigen, are also understood as antigens. In the context of the present invention, an antigen may be a product of translation of a provided therapeutic RNA (e.g., coding RNA, replicon RNA, mRNA). The term "antigenic peptide or protein" is recognized and understood by those skilled in the art and is intended to refer to, for example, a peptide or protein derived from an (antigenic) protein that can stimulate the body's adaptive immune system to result in an adaptive immune response. Thus, an "antigenic peptide or protein" includes at least one epitope or antigen of the protein from which it is derived (e.g., a tumor antigen, a viral antigen, a bacterial antigen, a protozoan antigen). 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.

[0091] In the context of nucleic acids, i.e., with respect to a nucleic acid "derived from" (another) nucleic acid, the term "derived from" as used throughout this specification means that the nucleic acid derived from (another) nucleic acid shares, for example, 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 be aware that sequence identity is typically calculated for nucleic acids of the same type, i.e., DNA or RNA sequences. Thus, when DNA is "derived from" RNA, or when RNA is "derived from" DNA, it will be understood that in the first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by substituting U for T throughout the sequence), or vice versa, the DNA sequence is converted to the corresponding RNA sequence (particularly by substituting T for U throughout the sequence). The sequence identity of the DNA sequence or the sequence identity of the RNA sequence is then determined. Preferably, a nucleic acid "derived from" a nucleic acid also refers to a nucleic acid that has been modified compared to the nucleic acid from which it is derived, e.g., to even 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, e.g., 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.

[0092] Epitope (also called "antigenic determinant"): A T cell epitope or portion of a protein in the context of the present invention may include a fragment having a length of preferably about 6 to about 20 or more amino acids, e.g., a fragment having a length of preferably about 8 to about 10, e.g., 8, 9, or 10 (or even 11 or 12) amino acids, that is processed and presented by MHC class I molecules, or a fragment having a length of preferably about 13 or more amino acids, e.g., 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, that is processed and presented by MHC class II molecules, and these fragments may be selected from any portion 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.

[0093] A B-cell epitope is typically a fragment located on the outer surface of a (natural) protein or peptide antigen as defined herein, which can be recognized by an antibody, i.e. in its native form, and which preferably has 5 to 15 amino acids, more preferably has 5 to 12 amino acids, even more preferably has 6 to 9 amino acids.

[0094] Such epitopes of proteins or peptides may further be selected from any of the variants of such proteins or peptides mentioned herein. In this context, an antigenic determinant may be a conformational or discontinuous epitope made up of segments of a protein or peptide as defined herein that are discontinuous in the amino acid sequence of the protein or peptide as defined herein, but that are grouped together in a three-dimensional structure made up of a single polypeptide chain, or a continuous or linear epitope.

[0095] A "tolerogenic composition" is a composition that promotes immune tolerance in a cell or cell line to an antigen, which may be a self or non-self antigen. In other words, there is no or a reduced immune response to the antigen. In contrast, a 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 or non-self antigen, and the overall goal of a vaccine composition of the present invention is to generate a (strong) immune response to this antigen, while the overall goal of a tolerogenic composition is to at least partially, and at best completely, suppress the immune response to this antigen.

[0096] A "tolerogenic nucleic acid" is a nucleic acid that promotes immune tolerance in a cell or cell line to an antigen; the nucleic acid may be a chemically modified mRNA and / or may encode a tolerogenic polypeptide. Conversely, at least one nucleic acid according to the invention encodes at least one antigen or a fragment thereof against which a (strong) immune response is desired and / or induced upon administration.

[0097] A "tolerogenic polypeptide" is a polypeptide that promotes immune tolerance in a cell or cell line, typically by reducing the immune response through acting on an underlying pathway, in particular by inhibiting an underlying mediator in such a pathway. Thus, a tolerogenic polypeptide can be an inhibitor of mTOR, IL-2, IL-10, or an antibody reactive with CD3 or CD40. Conversely, at least one antigen or a fragment thereof according to the present invention does not promote immune tolerance in a cell or cell line, but induces a (strong) immune response against itself.

[0098] The tolerogenic composition may contain, in particular, a tolerogenic nucleic acid that promotes the aforementioned immune tolerance. The tolerogenic composition may further contain a specific antigen, so that the presence of the tolerogenic nucleic acid results in the absence or reduction of an immune response to this specific antigen. Conversely, since the overall purpose of the vaccine composition of the present invention is to elicit a (strong) immune response directed to the encoded at least one antigen or fragment thereof (and not, as is the purpose of the tolerogenic composition, to block or reduce an immune response directed to a co-administered antigen), the vaccine composition according to the present invention, in a preferred embodiment, does not contain an antigen, but of course still 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 only payload and therefore cannot contain an antigen (the same applies to the tolerogenic composition discussed in this paragraph in addition to the tolerogenic nucleic acid).

[0099] The term "vaccine" is understood to be a prophylactic or therapeutic material that typically provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to produce an adaptive immune response.

[0100] The term "antigen-providing mRNA" in the context of the present invention typically refers to an mRNA having at least one open reading frame that can be translated by the cell or organism that provided the mRNA. The product of this translation is an antigen, preferably a peptide or protein that can act as an immunogen. The product may also be a fusion protein composed of two or more immunogens, such as a fusion protein consisting of two or more epitopes, peptides, or proteins derived from the same or different viral proteins, and the epitopes, peptides, or proteins may be linked by a linker sequence.

[0101] The term "artificial mRNA" (sequence) can be understood to mean an mRNA molecule that typically does not occur in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such an mRNA molecule can be non-natural due to its individual sequence (not occurring in nature) and / or other modifications, such as structural modifications of non-naturally occurring nucleotides. Typically, an artificial mRNA molecule can be designed and / or produced by genetic engineering methods corresponding to a desired artificial sequence of nucleotides (heterologous sequence). In this context, an artificial sequence is usually a sequence that cannot occur in nature, i.e., the artificial sequence differs from the wild-type sequence by at least one nucleotide. The term "wild-type" can be understood as a naturally occurring sequence. Furthermore, the term "artificial nucleic acid molecule" is not limited to meaning "one single molecule" but is typically understood to include an ensemble of identical molecules. Thus, this term can refer to multiple identical molecules contained in an aliquot.

[0102] In a highly preferred embodiment, the nucleic acid of the invention is an "isolated" mRNA. "Isolated": As used herein, the term "isolated" with respect to a nucleic acid molecule, preferably an isolated mRNA or polypeptide, means that the nucleic acid molecule, preferably an isolated mRNA or polypeptide, is in a state other than its natural environment, such as away from blood and / or animal tissue. In some embodiments, the isolated nucleic acid molecule, preferably an isolated mRNA or polypeptide, is substantially free of 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 an isolated mRNA or polypeptide, may be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms. Isolated materials may also have various levels of purity with respect to the materials with which they are associated. Isolated substances and / or entities may also be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of other components with which they were initially associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, more than about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In the context of the present invention, specification, and claims, the term "mRNA" preferably means "isolated mRNA," and vice versa.

[0103] The term "heterologous" or "heterologous sequence," as used throughout this specification in the context of a nucleic acid sequence or amino acid sequence, refers to a sequence (e.g., DNA, RNA, amino acid) that is recognized and understood by those skilled in the art and is intended to refer to a sequence derived from another gene, another allele, or another species. Two sequences are typically understood to be "heterologous" when they cannot be derived from the same gene or the same allele. That is, a heterologous sequence may be derived from the same organism, but does not naturally occur in the same nucleic acid molecule, e.g., the same RNA or protein.

[0104] Bi- / multicistronic mRNA: An mRNA that typically has two (bicistronic) or more (multicistronic) open reading frames (ORFs) (coding regions or sequences). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA results in two (bicistronic) or more (multicistronic) separate translation products (if the ORFs are not identical). For expression in eukaryotes, such an mRNA may contain, for example, an internal ribosome entry site (IRES) sequence.

[0105] Monocistronic mRNA: A monocistronic mRNA can be an mRNA that typically contains only one open reading frame (coding sequence or region). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

[0106] 3'-untranslated region (3'-UTR): The 3'-UTR is typically a portion of an mRNA located between the protein-coding region (i.e., open reading frame) and the poly(A) sequence of the mRNA. The 3'-UTR of an mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by a gene, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into a pre-mRNA, which optionally contains an intron. The pre-mRNA is then further processed into a mature mRNA in the maturation process. This maturation process includes the steps of 5'-capping, splicing the pre-mRNA to remove the optional intron, and polyadenylation of the 3' end of the pre-mRNA, and optional endo- or exonuclease cleavage, and other 3'-end modifications. In the context of the present invention, the 3'-UTR is located 3' to the stop codon of the protein-coding region, preferably immediately 3' to the stop codon of the protein-coding region, and corresponds to the sequence of a mature mRNA extending to the nucleotide 5' to the poly(A) sequence, preferably immediately 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence can be an RNA sequence, for example, in the mRNA used to define the 3'-UTR sequence, 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," refers to a sequence corresponding to the 3'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the mRNA precursor. The term "3'-UTR of a gene" encompasses both the DNA and RNA sequences of the 3'-UTR.

[0107] 5'-untranslated region (5'-UTR): 5'-UTR is typically understood to be a specific section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'-UTR begins at the transcription initiation 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 the present invention, the 5'-UTR corresponds to the sequence of the mature mRNA located between the 5'-cap and the start codon. Preferably, the 5'-UTR corresponds to the sequence extending from the nucleotide located 3' to the 5'-cap, preferably the nucleotide located immediately 3' to the 5'-cap, to the nucleotide located 5' to the start codon of the protein-coding region, preferably the nucleotide located immediately 5' to the start codon of the protein-coding region. The nucleotide located immediately 3' to the 5' cap of a mature mRNA typically corresponds to the transcription start site. The term "corresponding" means that the 5'-UTR sequence can be an RNA sequence, for example, in the mRNA used to define the 5'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the context of the present invention, the term "5'-UTR of a gene," such as "5'-UTR of a TOP gene," refers to a sequence corresponding to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the mRNA precursor. The term "5'-UTR of a gene" encompasses the DNA sequence and RNA sequence of the 5'-UTR.

[0108] 5'-Terminal Oligopyrimidine Tract (TOP): A 5'-Terminal Oligopyrimidine Tract (TOP) is a stretch of pyrimidine nucleotides typically located in the 5'-terminal region of a nucleic acid molecule, such as the 5'-terminal region of a particular mRNA molecule or the functional entity of a particular gene, e.g., the 5'-terminal region of a transcribed region. This sequence begins with a cytidine, usually corresponding to the transcription start site, followed by a stretch of pyrimidine nucleotides, usually about 3 to 30. For example, a TOP can 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. The pyrimidine stretch, and thus the 5'-TOP, ends one nucleotide 5' from the first purine nucleotide located downstream of the TOP. Messenger RNAs containing 5'-terminal oligopyrimidine tracts are often called TOP mRNAs. Therefore, genes that provide such messenger RNAs are called TOP genes. TOP sequences are found in genes and mRNAs that code for peptide elongation factors and ribosomal proteins, for example.

[0109] TOP motif: In the context of the present invention, a TOP motif is a nucleic acid sequence corresponding to the 5'-TOP as defined above. Thus, a TOP motif in the context of the present invention is a stretch of pyrimidine nucleotides, preferably having a length of 3 to 30 nucleotides. Preferably, a 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, and the stretch of pyrimidine nucleotides preferably starts with a cytosine nucleotide at its 5'-end. In TOP genes and TOP mRNAs, the TOP motif preferably starts at its 5'-end with the transcription start site and ends one nucleotide 5' to the first purine residue in the gene or mRNA. A TOP motif in the sense of the present invention is preferably located at the 5'-end of a sequence representing a 5'-UTR or the 5'-end of a sequence encoding a 5'-UTR. Thus, preferably, when a stretch of three or more pyrimidine nucleotides is located at the 5'-end of the respective sequence, such as the mRNA of the present invention, the 5'-UTR element of the mRNA of the present invention, or the nucleic acid sequence derived from the 5'-UTR of a TOP gene described herein, it is referred to as a "TOP motif" in the sense of the present invention. In other words, a stretch 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 anywhere within the 5'-UTR or 5'-UTR element, is preferably not referred to as a "TOP motif".

[0110] TOP gene: 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 a mature mRNA derived from the TOP gene, preferably extending from the nucleotide located 3' to the 5' cap to the nucleotide located 5' to the start codon. The 5'-UTR of a TOP gene typically does not contain any start codon, preferably an upstream AUG (uAUG) or an upstream open reading frame (uORF). In this context, the upstream AUG and upstream open reading frame are typically understood to be the AUG and open reading frame located 5' to 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 a TOP gene can vary between 20 nucleotides and up to 500 nucleotides, and is typically less than about 200 nucleotides, preferably less than about 150 nucleotides, and more preferably less than about 100 nucleotides. Exemplary 5'-UTRs of TOP genes within the meaning of the present invention are nucleic acid sequences extending from the 5th nucleotide to the nucleotide immediately 5' to the start codon (e.g., ATG) in the sequences according to SEQ ID NOs: 1-1363, 1395, 1421, and 1422 of WO2013143700, or homologs or variants thereof, the disclosures of which are incorporated herein by reference. In this context, a particularly preferred fragment of the 5'-UTR of a TOP gene is a 5'-UTR of a TOP gene lacking the 5'-TOP motif. The term "5'-UTR of a TOP gene" preferably refers to the 5'-UTR of a naturally occurring TOP gene.

[0111] Fragments of nucleic acid sequences, in particular mRNA: A fragment of a nucleic acid sequence consists of a consecutive stretch of nucleotides corresponding to a consecutive stretch of nucleotides in the full-length nucleic acid sequence that is the basis of the nucleic acid sequence of the fragment, 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. Such fragments in the sense of the present invention are preferably functional fragments of the full-length nucleic acid sequence.

[0112] In the context of the present invention, a "fragment" or "variant" of a protein or peptide is at least 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 16 %, 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 as 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 it is derived.

[0113] Variants of nucleic acid sequences, particularly mRNA: A variant of a nucleic acid sequence refers to a variant of the nucleic acid sequence underlying the nucleic acid sequence. For example, a variant nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the nucleic acid sequence from which the variant is derived. Preferably, a variant of a nucleic acid sequence 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 derived. Preferably, the variant is a functional variant. A "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75, or 100 nucleotides of such a nucleic acid sequence.

[0114] 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 ex vivo degradation (e.g., by manufacturing steps 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 polyA 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. A variety of other methods are known in the art and are contemplated in the context of the present invention.

[0115] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to a process in which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template to generate RNA transcripts. RNA can be obtained, according to the present invention, 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. DNA templates for in vitro RNA transcription can be obtained by cloning nucleic acids, particularly cDNAs corresponding to the respective RNAs to be in vitro transcribed, and introducing them 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 an appropriate restriction enzyme before being transcribed in vitro. cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. Furthermore, DNA templates for in vitro RNA synthesis can also be obtained by gene synthesis.

[0116] Methods for in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14). Reagents used in the methods typically include: 1) a linearized DNA template having a promoter sequence with high binding affinity for a respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); 3) optionally a cap analog as defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase) that can bind to a promoter sequence within a linearized DNA template; 5) optionally, a ribonuclease (RNase) inhibitor to inactivate any contaminating RNases; 6) pyrophosphatase, which degrades pyrophosphate, which can potentially inhibit transcription; 7) Mg as a cofactor for polymerases 2+ MgCl2, which provides ions; 8) A buffer to maintain an appropriate pH value, which may also contain optimal concentrations of antioxidants (e.g., DTT) and / or polyamines such as spermidine.

[0117] Full-length protein: As used herein, the term "full-length protein" refers to a protein that contains substantially the entire amino acid sequence of a protein, typically occurring in nature. Nevertheless, amino acid substitutions in the protein, for example, due to mutations, are also encompassed by the term full-length protein.

[0118] Fragment of a protein: A "fragment" of a protein or peptide in the context of the present invention may comprise a protein or peptide sequence as defined herein that is N-terminally and / or C-terminally truncated, typically with respect to its amino acid sequence (or its encoding nucleic acid molecule), compared to the amino acid sequence of the original (natural) protein (or its encoding nucleic acid molecule). Such truncation may thus occur at the amino acid level or, correspondingly, at the nucleic acid level. Sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein, or to the entire (encoding) nucleic acid molecule of such a protein or peptide.

[0119] The term "variant" in the context of the nucleic acid sequence of a gene refers to a nucleic acid sequence variant, i.e., a nucleic acid sequence or gene comprising a nucleic acid sequence that differs by at least one nucleic acid from the reference (or "parent") nucleic acid sequence of the reference (or "parent") nucleic acid or 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 the respective reference sequence. Preferably, the term "variant" as used herein includes naturally occurring variants of a nucleic acid sequence or gene, as well as engineered variants. Thus, a "variant" as defined herein may be derived from, isolated from, related to, based on, or homologous to a reference nucleic acid sequence. "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%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence identity to the respective naturally occurring (wild-type) nucleic acid sequence or gene, or the nucleic acid sequence of a homologue, fragment or derivative thereof.

[0120] The term "variant" as used throughout this specification in the context of a protein or peptide is also recognized and understood by those skilled in the art and is intended to refer to a protein or peptide variant having an amino acid sequence that differs 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., their specific antigenic properties, as compared to the full-length native protein. A "variant" of a protein or peptide as defined herein may contain conservative amino acid substitutions compared to its native, i.e., non-mutated, physiological sequence. These amino acid sequences, as well as their encoding nucleotide sequences, are particularly included in the term "variant" as defined herein. Substitutions in which amino acids from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chain or amino acid, and amino acids with side chains capable of entering hydrogen bridges, e.g., with hydroxyl functional groups. This means, for example, that an amino acid having a polar side chain is replaced by another amino acid having a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain is replaced by another amino acid having a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine), or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are particularly possible at sequence positions that do not cause modifications to the three-dimensional structure or affect the binding region. Modifications to the three-dimensional structure due to insertions or deletions 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 a stretch of at least 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide.Preferably, a variant of a protein includes a functional variant of a protein, meaning that the variant exhibits the same effect or functionality as the protein from which it is derived, or at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the effect or functionality.

[0121] The term "fragment" in the context of a nucleic acid sequence or gene also refers to a contiguous 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 identical to a corresponding stretch within a full-length nucleic acid sequence or gene. This term includes naturally occurring fragments as well as engineered fragments. A preferred fragment of a sequence in the context of the present invention consists of a contiguous stretch of nucleic acid corresponding to a contiguous stretch of an entity in the nucleic acid or gene from which the fragment is derived, 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 derived. Sequence identity indicated for such fragments preferably refers to the entire nucleic acid sequence or gene. Preferably, a "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%, and most preferably at least 95% or even 97% sequence identity to a reference nucleic acid sequence or gene from which it is derived.

[0122] Also in this context, a fragment of a protein may typically comprise an amino 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%, and most preferably at least 95% or even 97% sequence identity to the amino acid sequence of the respective naturally occurring full-length protein.

[0123] The term "identity" used throughout this specification in the context of nucleic acid or amino acid sequences is recognized and understood by those skilled in the art and is intended to refer, for example, to the percentage of identity between two sequences. To determine the percentage identity between two sequences, such as nucleic acid sequences or amino acid (aa) sequences defined herein, preferably the aa sequences encoded by the nucleic acid sequences defined herein, or the aa sequences themselves, the sequences can be aligned and then compared to each other. Thus, for example, a position in a first sequence can be compared to the corresponding position in a second sequence. If a position in the first sequence is occupied by the same residue as in the second sequence, the two sequences are identical at this position. Otherwise, the sequences differ 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 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 further alignment. Therefore, the percentage that two sequences are identical is a function of the number of identical positions divided by the total number of positions, including positions that are only occupied in one sequence. The percentage that two sequences are identical can be determined using an algorithm, such as the algorithm built into the BLAST program.

[0124] Fragments of proteins or peptides in the context of the present invention may further comprise protein or peptide sequences as defined herein, for example having a length of at least 5 amino acids, preferably at least 6 amino acids, preferably at least 7 amino acids, more preferably at least 8 amino acids, even more preferably at least 9 amino acids; even more preferably at least 10 amino acids; even more preferably at least 11 amino acids; even more preferably at least 12 amino acids; even more preferably at least 13 amino acids; even more preferably at least 14 amino acids; even more preferably at least 15 amino acids; even more preferably at least 16 amino acids; even more preferably at least 17 amino acids; even more preferably at least 18 amino acids; even more preferably at least 19 amino acids; even more preferably at least 20 amino acids; even more preferably at least 25 amino acids; even more preferably at least 30 amino acids; even more preferably at least 35 amino acids; even more preferably at least 50 amino acids; and most preferably at least 100 amino acids. For example, such fragments can be about 6 to about 20 or more amino acids in length, e.g., fragments processed and presented by MHC class I molecules, preferably about 8 to about 10, e.g., 8, 9, or 10 amino acids (or even 6, 7, 11, or 12 amino acids) in length, or fragments processed and presented by MHC class II molecules, preferably about 13 or more amino acids in length, e.g., 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length, and these fragments can be selected from any portion 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. Fragments of proteins or peptides can include at least one epitope of these proteins or peptides. Furthermore, domains of proteins, e.g., extracellular, intracellular, or transmembrane domains of proteins, and truncated or truncated versions, can be understood to constitute fragments of proteins.

[0125] Protein variant: A "variant" of a protein or peptide as defined in the context of the present invention can be created, which has an amino acid sequence that differs from the original sequence by 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, such as its specific antigenic properties, as compared with the full-length natural protein. A "variant" of a protein or peptide as defined in the context of the present specification can contain conservative amino acid substitutions as compared with its natural, i.e., non-mutated, physiological sequence. These amino acid sequences and their encoding nucleotide sequences are particularly included in the term "variant" as defined herein. Substitutions in which amino acids from the same class are exchanged for each other are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chain or amino acid, and amino acids with side chains that can enter into hydrogen bridges, for example, with hydroxyl functional groups. This means, for example, that an amino acid with a polar side chain is replaced by another amino acid with a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain is replaced by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). In particular, insertions and substitutions are possible at sequence positions that do not cause modifications to the three-dimensional structure or affect the binding region. Modifications to the three-dimensional structure due to insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, Modern Physical Methods in Biochemistry, Neuberger et al. (eds.), Elsevier, Amsterdam).

[0126] A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids to such protein or peptide.

[0127] Furthermore, variants of proteins or peptides as defined herein that may be encoded by a nucleic acid molecule may also include sequences in which nucleotides of the encoding nucleic acid sequence have been exchanged due to the degeneracy of the genetic code without resulting in a change in the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least a part of it does not differ from the original sequence in one or more mutations within the above meaning.

[0128] Sequence identity: To determine the percentage of identity between two sequences, such as the nucleic acid sequences or amino acid sequences defined herein, preferably the amino acid sequences encoded by the nucleic acid sequences of the polymeric carriers defined herein or the amino acid sequences themselves, the sequences can be aligned and then compared. Thus, for example, a position in a first sequence can be compared with the corresponding position in a second sequence. If a position in the first sequence is occupied by the same component (residue) as that in the second sequence, the two sequences are identical at this position. Otherwise, the sequences differ 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 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 further alignment. Therefore, the percentage of identity between two sequences is a function of the number of identical positions divided by the total number of positions, including positions that are only occupied in one sequence. The percentage of identity between two sequences can be determined using a mathematical algorithm. A preferred, but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is incorporated into the BLAST program. Sequences that are identical to the sequences of the present invention to a certain degree can be identified by this program.

[0129] Derivative of protein or peptide: Derivative of peptide or protein is typically understood to be a molecule derived from another molecule such as said peptide or protein. "Derivative" of peptide or protein also includes fusions containing the peptide or protein used in the present invention. For example, the fusion contains a tag such as an epitope, for example, 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.

[0130] Pharmaceutically effective amount: A pharmaceutically effective amount in the context of the present invention is typically understood to be an amount sufficient to induce an immune response. Carrier: A carrier in the context of the present invention may typically be a compound that facilitates the transport and / or complexation of another compound. Said carrier may form a complex with said other compound. A polymeric carrier is a carrier made of a polymer.

[0131] Vehicle: Typically, an agent, such as a carrier, that may be used within a pharmaceutical composition or vaccine to facilitate administration of the components of the pharmaceutical composition or vaccine to an individual. The drawings shown below are merely exemplary and further illustrate the invention and should not be construed as limiting the invention thereto. [Brief explanation of the drawings]

[0132] [Figure 1]Organ distribution profiles are shown. When comparing GN01-based PEG-LNPs and PMOZ-LNPs (DMPE-PMOZ-v1), the organ distribution profiles can be considered to be roughly similar. LNPs containing PMOZ-lipids as polymer-conjugated lipids surprisingly have no negative effect on LNP efficacy, and even a positive effect is evident on luciferase levels in the spleen and lymph nodes. As shown herein above, the inventors surprisingly found that PMOZ-LNPs perform significantly better than standard PEG-LNPs. Full details can be found in Example 4. [Figure 2] This shows that a single im immunization with just 1 μg of mRNA formulated in PMOZ-LNP (DMPE-PMOZ-v1) already induced very robust VNT in all animals, well above the protective titer of 0.5 IU / ml. PMOZ-LNP performed significantly better than the standard control; very high levels of rabies VNT were measured for all LNPs. Full details can be found in Example 5. [Figure 3] This shows that a single im immunization with just 1 μg of mRNA formulated into PMOZ-LNP already induced very robust VNT in all animals, well above the protective titer of 0.5 IU / ml. PMOZ-LNP performed significantly better than the standard control, i.e., very high levels of rabies VNT were measured for all LNPs. Full details can be found in Example 7. [Figure 4] These results show that im immunization with just 1 μg of mRNA formulated into PMOZ-LNP induced very high VNT in all animals after booster vaccination (day 28), well above the protective titer of 0.5 IU / ml. PMOZ-LNP performed significantly better than the standard control; very high levels of rabies VNT were measured for all LNPs. Full details can be found in Example 7. [Figure 5A]This shows that iv administration of 2 μg of PpLuc mRNA formulated in PMOZ-LNPs reduced cytokine levels compared to PEG-LNPs. Full details can be found in Example 8. [Figure 5B] This shows that iv administration of 20 μg of PpLuc mRNA formulated in PMOZ-LNPs reduced cytokine levels compared to PEG-LNPs. Full details can be found in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0133] Although the present disclosure will be described in detail below, it should be understood that the present disclosure is not limited to the specific methodology, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0134] Novel polymer-conjugated lipids In a first aspect, the present invention provides a polymer-conjugated lipid defined as a compound according to formula (I): [P]-[linker]-[L] Formula (I) (In the formula, [P] is at least one polyoxazoline (POZ) monomer unit

[0135] [ka]

[0136] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value ranging from about 45 to about 55, preferably n is about 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. a heteropolymer or homopolymer moiety, preferably a homopolymer moiety, comprising [linker] is an optional linker group; [L] is the lipid moiety) to provide.

[0137] R in [P] of formula (I) is preferably C1 (methyl), resulting in a PMOZ unit. In the present invention, preferably, [P] is 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) This invention is based on the surprising discovery by the inventors that the use of novel polymer-conjugated lipids comprising polyoxazolines (POZ) according to Formula (I), including the following, and / or lipid nanoparticles (LNPs) comprising these novel polymer-conjugated lipids, is highly effective for delivering nucleic acids, such as mRNA, to living organisms, such as human individuals. This has enabled the inventors to create improved vaccines that deliver, for example, mRNA compounds encoding antigenic peptides or proteins, and highly efficiently induce antigen-specific immune responses at very low dosages, avoiding the drawbacks associated with the use of PEG. The present disclosure addresses these and other needs. A further advantage achieved by the present invention is that, quite surprisingly, the inventors have discovered a class of formulations for delivering mRNA vaccines in vivo that, in accordance with aspects and embodiments of the present invention, result in significantly enhanced, and in many respects synergistic, immune responses, including enhanced antigen production and functional antibody production with neutralizing capacity. 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 formulation of the present invention has demonstrated significant and unexpected in vivo immune response, sufficient to establish the effectiveness of functional mRNA vaccine as a prophylactic and therapeutic agent.In summary, the present inventors have surprisingly shown that several different polymer-conjugated lipids according to formula (I), such as PMOZ-lipids, can be used to replace standard PEG-lipids to obtain LNPs with comparable or even enhanced performance.This unexpected discovery can be verified by using several different LNP compositions, namely, the present inventors have surprisingly found that the polymer-conjugated lipids according to formula (I) can clearly enhance the LNP compositions of the prior art.

[0138] Therefore, the present invention is directed to a composition comprising a polymer-conjugated lipid according to formula (I), preferably a POZ-lipid according to formula (I), more preferably a PMOZ-lipid, as described herein below.All options and preferences disclosed for the polymer-conjugated lipid according to formula (I), preferably a POZ-lipid, more preferably a PMOZ-lipid itself, are also applicable to the composition in this aspect of the present invention.In other words, the specifically disclosed embodiments of the polymer-conjugated lipid, preferably a POZ-lipid, more preferably a PMOZ-lipid, and the particularly preferred PMOZ-lipid, DMG-PMOZ, should also be understood to define specific preferred embodiments of the composition according to the present invention, that is, the composition characterized by comprising a PMOZ lipid according to one of the specific options described herein.In other words, the term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion.Preferably, the polymer-conjugated lipid according to formula (I) is a POZ-lipid, more preferably a PMOZ-lipid. Thus, the term "POZ-lipid" or "PMOZ-lipid" refers to a molecule containing both a lipid portion and a POZ portion or a PMOZ portion, respectively. Thus, a "PMOZ-lipid" should be understood as a lipid containing at least one homopolymer portion containing at least one polyoxazoline (POZ) unit, preferably a PMOZ unit.

[0139] The composition may contain additional active and / or inactive excipients, as further described below. In one specific embodiment, in addition to the polymer-conjugated lipid according to formula (I), preferably PMOZ-lipid, the composition contains one or more lipids selected from the group consisting of: (a) steroids; (b) neutral lipids; and (c) cationic lipids.

[0140] In another embodiment, [P] is Poly(2-methyl-2-oxazoline) (PMOZ)

[0141] [ka]

[0142] Poly(2-ethyl-2-oxazoline) (PEOZ)

[0143] [ka]

[0144] Poly(2-propyl-2-oxazoline) (PPOZ)

[0145] [ka]

[0146] Poly(2-butyl-2-oxazoline) (PBOZ)

[0147] [ka]

[0148] Poly(2-isopropyl-2-oxazoline) (PIPOZ)

[0149] [ka]

[0150] Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), and Poly(2-dimethylamino-2-oxazoline) (PDMAOx) a heteropolymer or homopolymer moiety comprising a plurality of monomer units selected from the group consisting of: Preferably, [P] is a homopolymer moiety comprising a plurality of PMOZ or PEOZ monomer units, more preferably, [P] comprises a plurality of PMOZ monomer units, or preferably consists of a plurality of PMOZ monomer units; (i) n has an average value in the range of about 45 to about 55, preferably n is about 50; or (ii) n is selected so that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0151] In another embodiment, [P] is

[0152] [ka]

[0153] The polymeric moiety is a heteropolymeric or homopolymeric moiety comprising a plurality of monomeric units selected from the group consisting of: In yet another embodiment, [P] of the polymer-conjugated lipid according to Formula (I) is selected from the group consisting of poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) and poly(2-dimethylamino-2-oxazoline) (PDMAOx).

[0154] In still further embodiments, the polymer-conjugated lipid according to Formula (I) is selected from the group consisting of a POZ-monoacylglycerol conjugate, a POZ-diacylglycerol conjugate, a POZ-dialkyloxypropyl conjugate, a POZ-steroid or POZ-sterol conjugate, a POZ-phospholipid conjugate, a POZ-ceramide conjugate, and mixtures thereof.

[0155] In a preferred embodiment, the polymer-conjugated lipid is 1,2-dimyristoyl-rac-glycerol (DMG).

[0156] [ka]

[0157] Includes parts based on. Preferably, the polymer-conjugated lipid is

[0158] [ka]

[0159] More preferably, it is "DMG-PMOZ" in which n is 45 to 50, and most preferably "PMOZ2" in which n has an average value of 50. Further most preferred embodiments for the PMOZ[P] moiety (polymethyloxazoline): For PMOZ, the preferred average molecular mass of the [P] moiety is about 3.8 kDa to about 4.8 kDa, about 3.9 kDa to about 4.7 kDa, about 4 kDa to about 4.6 kDa, about 4.1 kDa to about 4.5 kDa, about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. Other preferred average molecular masses of the [P] moiety are (i) about 3.9 kDa to about 4.4 kDa, about 3.9 kDa to about 4.1 kDa, or about 4.2 kDa to about 4.4 kDa.

[0160] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than 4.3 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is between about 4.25 kDa and about 4.675 kDa, between about 4.675 kDa and about 5.1 kDa, between about 5.1 kDa and about 5.525 kDa, between about 5.525 kDa and about 5.95 kDa, between about 5.95 kDa and about 6.375 kDa, between about 6.375 kDa and about 6.8 kDa, or greater than 6.8 kDa.

[0161] [ka]

[0162] In other preferred embodiments for PMOZ according to the present invention, n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0163] In more preferred embodiments for PMOZ, n has an average value of 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.

[0164] Thus, the PMOZ moiety is preferably one having a molecular mass of about 4.3 kDa, although shorter and longer moieties can also be used. Further most preferred embodiments for the PEOZ[P] moiety (polyethyloxazoline): For PEOZ, the preferred average molecular mass of the [P] moiety is about 4.5 kDa to about 5.5 kDa, about 4.6 kDa to about 5.4 kDa, about 4.7 kDa to about 5.3 kDa, about 4.8 kDa to about 5.2 kDa, about 4.9 kDa to about 5.1 kDa, or most preferably about 5 kDa.

[0165] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than about 5 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is between about 4.95 kDa and about 5.445 kDa, between about 5.445 kDa and about 5.94 kDa, between about 5.94 kDa and about 6.435 kDa, between about 6.435 kDa and about 6.93 kDa, between about 6.93 kDa and about 7.425 kDa, between about 7.425 kDa and about 7.92 kDa, or greater than 7.92 kDa.

[0166] [ka]

[0167] In another preferred embodiment for PEOZ according to the present invention, n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0168] In more preferred embodiments for PEOZ, n has an average value of 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.

[0169] Thus, the PEOZ moiety is preferably a PEOZ moiety having a molecular mass of about 5 kDa, although shorter and longer moieties can also be used. Further most preferred embodiments for the PPOZ[P] moiety (polypropyloxazoline) or PIPOZ[P] moiety (poly-2-isopropyl-2-oxazoline): For PPOZ or, equivalently, PIPOZ, the preferred average molecular mass of the [P] moiety 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, or most preferably about 5.7 kDa.

[0170] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than 5.7 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is from about 5.65 kDa to about 6.215 kDa, from about 6.215 kDa to about 6.78 kDa, from about 6.78 kDa to about 7.345 kDa, from about 7.345 kDa to about 7.91 kDa, from about 7.91 kDa to about 8.475 kDa, from about 8.475 kDa to about 9.04 kDa, or greater than 9.04 kDa.

[0171] [ka]

[0172] In another preferred embodiment for PPOZ or equivalently PIPOZ (poly(2-isopropyl-2-oxazoline)), n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0173] In further preferred embodiments for PPOZ or equivalently PIPOZ, n has an average value of 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.

[0174] Thus, the PPOZ moiety or equivalently, the PIPOZ moiety is preferably a PPOZ moiety or equivalently, a PIPOZ moiety having a molecular mass of about 5.7 kDa, although shorter and longer moieties can also be used.

[0175] In other preferred embodiments, the "n" in the "P" portion of the novel polymer-conjugated lipids 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, 40, 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, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 1 9, 40, 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 more preferably 50. In a further preferred embodiment, "n" of the monomeric compound of [P] is selected so that the [P] moiety 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.

[0176] In certain further preferred embodiments, n is such that the [P] moiety is about 2 kDa; 2.1 kDa; 2.2 kDa; 2.3 kDa; 2.4 kDa; 2.5 kDa; 2.6 kDa; 2.7 kDa; 2.8 kDa; 2.9 kDa; 3 kDa; 3.1 kDa; 3.2 kDa; 3.3 kDa; 3.4 kDa; 3.5 kDa; 3.6 kDa; 3.7 kDa; 3.8 kDa; 3.9 kDa; 4 kDa; 4.1 kDa; 4.2 kDa; 4.3 kDa; 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;5.8k Da;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;8.7k Da;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; Novel polymer-conjugated lipids are selected to have an average molecular weight of 10.2 kDa; 10.3 kDa; 10.4 kDa; 10.5 kDa; 10.6 kDa; 10.7 kDa; 10.8 kDa; 10.9 kDa; 11 kDa; 11.1 kDa; 11.2 kDa; 11.3 kDa; 11.4 kDa; 11.5 kDa; 11.6 kDa; 11.7 kDa; 11.8 kDa; 11.9 kDa; 12 kDa or above 12 kDa.

[0177] In an even more preferred embodiment, the polymer-conjugated lipid comprises a poly(2-methyl-2-oxazoline) (PMOZ) moiety as [P], where n is the [P] moiety is about 2 kDa; 2.1 kDa; 2.2 kDa; 2.3 kDa; 2.4 kDa; 2.5 kDa; 2.6 kDa; 2.7 kDa; 2.8 kDa; 2.9 kDa; 3 kDa; 3.1 kDa; 3.2 kDa; 3.3 kDa; 3.4 kDa; 3.5 kDa; 3.6 kDa; 3.7 kDa; 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;5.3 kDa;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;8.7kDa;8.8kDa;8.9kDa;9kDa;9.1kDa;9.2kDa;9.3kDa;9.4kDa;9.5kDa;9.6kDa;9.7kDa;9 0.8kDa; 9.9kDa; 10kDa; 10kDa; 10.1kDa; 10.2kDa; 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 above.

[0178] In a further preferred embodiment, the polymer-conjugated lipid comprises a polyethyloxazoline (PEOZ) moiety as [P], and n is, in order of increasing preference: n has an average value in the range of about 40 to about 60; n has an average value in the range of about 45 to about 55; n has an average value ranging from about 46 to about 54; n has an average value ranging from about 47 to about 53; n has an average value ranging from about 48 to about 52; n has an average value ranging from about 49 to about 51; n is about 50; and n=50 is selected from the group consisting of:

[0179] In an even more most preferred embodiment, the polymer-conjugated lipid comprises a poly(2-methyl-2-oxazoline) (PMOZ) moiety as [P], where n is, in order of increasing preference: n has an average value in the range of about 40 to about 60; n has an average value in the range of about 45 to about 55; n has an average value ranging from about 46 to about 54; n has an average value ranging from about 47 to about 53; n has an average value ranging from about 48 to about 52; n has an average value ranging from about 49 to about 51; n is about 50; and n=50 is selected from the group consisting of:

[0180] Further highly preferred embodiments for the PMOZ[P] moiety (polymethyloxazoline): For PMOZ, the preferred average molecular mass of the [P] moiety is about 3.8 kDa to about 4.8 kDa, about 3.9 kDa to about 4.7 kDa, about 4 kDa to about 4.6 kDa, about 4.1 kDa to about 4.5 kDa, about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0181] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than 4.3 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is between about 4.25 kDa and about 4.675 kDa, between about 4.675 kDa and about 5.1 kDa, between about 5.1 kDa and about 5.525 kDa, between about 5.525 kDa and about 5.95 kDa, between about 5.95 kDa and about 6.375 kDa, between about 6.375 kDa and about 6.8 kDa, or greater than 6.8 kDa.

[0182] [ka]

[0183] In other preferred embodiments for PMOZ according to the present invention, n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0184] In more preferred embodiments for PMOZ, n has an average value of 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.

[0185] Thus, the PMOZ moiety is preferably one having a molecular mass of about 4.3 kDa, although shorter and longer moieties can also be used. Further highly preferred embodiments for the PEOZ[P] moiety (polyethyloxazoline): For PEOZ, the preferred average molecular mass of the [P] moiety is about 4.5 kDa to about 5.5 kDa, about 4.6 kDa to about 5.4 kDa, about 4.7 kDa to about 5.3 kDa, about 4.8 kDa to about 5.2 kDa, about 4.9 kDa to about 5.1 kDa, or most preferably about 5 kDa.

[0186] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than 5 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is from about 4.95 kDa to about 5.445 kDa, from about 5.445 kDa to about 5.94 kDa, from about 5.94 kDa to about 6.435 kDa, from about 6.435 kDa to about 6.93 kDa, from about 6.93 kDa to about 7.425 kDa, from about 7.425 kDa to about 7.92 kDa, or greater than 7.92 kDa.

[0187] [ka]

[0188] In another preferred embodiment for PEOZ according to the present invention, n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0189] In more preferred embodiments for PEOZ, n has an average value of 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.

[0190] Thus, the PEOZ moiety is preferably a PEOZ moiety having a molecular mass of about 5 kDa, although shorter and longer moieties can also be used. Further highly preferred embodiments for the PPOZ[P] moiety (polypropyloxazoline) or PIPOZ[P] moiety (poly-2-isopropyl-2-oxazoline): For PPOZ or, equivalently, PIPOZ, the preferred average molecular mass of the [P] moiety 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, or most preferably about 5.7 kDa.

[0191] In further preferred embodiments, the average molecular mass of the [P] moiety is greater than 5.7 kDa. In other preferred embodiments, the average molecular mass of the [P] moiety is from about 5.65 kDa to about 6.215 kDa, from about 6.215 kDa to about 6.78 kDa, from about 6.78 kDa to about 7.345 kDa, from about 7.345 kDa to about 7.91 kDa, from about 7.91 kDa to about 8.475 kDa, from about 8.475 kDa to about 9.04 kDa, or greater than 9.04 kDa.

[0192] [ka]

[0193] In another preferred embodiment for PPOZ or equivalently PIPOZ (poly(2-isopropyl-2-oxazoline)), n has an average value ranging from about 40 to about 80, preferably from about 45 to about 70, more preferably from about 50 to about 60, or most preferably, n has an average value of about 50.

[0194] In further preferred embodiments for PPOZ or equivalently PIPOZ, n has an average value of 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.

[0195] Thus, the PPOZ moiety or equivalently, the PIPOZ moiety is preferably a PPOZ moiety or equivalently, a PIPOZ moiety having a molecular mass of about 5.7 kDa, although shorter and longer moieties can also be used.

[0196] In one embodiment, the lipid moiety [L] shown in 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 optionally being interrupted by one or more biodegradable groups and / or optionally comprising one terminal biodegradable group, the biodegradable group being selected from the group consisting of, but not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 alkenyl), zwitterionic linkers, non-ester and ester-containing linker moieties (-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(R 5 )=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 sulfonate ester, and combinations thereof; R 3 , R 4 and R 5 are independently H or alkyl (e.g., C1-C4 alkyl).

[0197] In another embodiment, the lipid moiety [L] comprises at least one linear or branched, saturated or unsaturated alkyl chain comprising 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, most preferably 14 carbon atoms; All choices are independent of each other.

[0198] In one embodiment, the linker group [linker] shown in formula (I) ([P]-[linker]-[L]) can be selected from, but is not limited to, a pH-sensitive moiety, an alkyl or alkenyl moiety (C 1~9 Alkyl or C 2~9 alkenyl), zwitterionic linkers, non-ester and ester-containing linker moieties (-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(R 5 )=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—), and sulfonate ester, and combinations thereof; R 3 , R 4 and R 5 are independently H or alkyl (e.g., C1-C4 alkyl).

[0199] In another embodiment, the linker group [linker] comprises an amide linker moiety, preferably an ester linker moiety, or the linker group [linker] has the structure

[0200] [ka]

[0201] It has. In a further embodiment, the polymer-conjugated lipid is (i)

[0202] [ka]

[0203] or preferably

[0204] [ka]

[0205] [ka]

[0206] wherein the linker group [linker] is selected from any one of the linker groups disclosed herein, preferably the linker group [linker] comprises 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, or even more preferably 45 to 50, and most preferably 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. having the structure Most preferably, the polymer-conjugated lipid is DMG-PMOZ, where n has an average value of 45-50, most preferably 50.

[0207] In a highly preferred embodiment, the polymer-conjugated lipid is

[0208] [ka]

[0209] ["PMOZ1"], more preferably n=50, i.e., having the structure of "PMOZ1" having 50 monomer repeats. In an even more preferred embodiment, the polymer-conjugated lipid is

[0210] [ka]

[0211] ["PMOZ3"], more preferably n=50, i.e., having the structure of "PMOZ3" having 50 monomer repeats. In another preferred embodiment, the polymer-conjugated lipid is

[0212] [ka]

[0213] ["PMOZ5"], more preferably having the structure "PMOZ5" where n=50, i.e., having 50 monomer repeats. In another highly preferred embodiment, the polymer-conjugated lipid is

[0214] [ka]

[0215] ["PMOZ2"], more preferably n=50, i.e., having the structure of "PMOZ2" having 50 monomer repeats. In a most preferred embodiment, the polymer-conjugated lipid is:

[0216] [ka]

[0217] ["PMOZ4"], more preferably n=50, i.e., "PMOZ4" having 50 monomer repeats, i.e.,

[0218] [ka]

[0219] [n=50, i.e., "PMOZ4" having 50 monomer repeats] It has the following structure. For "PMOZ1" to "PMOZ5," preferably, 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, or even more preferably 45 to 50, and most preferably 50, or n is selected so that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0220] In another highly preferred embodiment, the linker group [linker] preferably comprises an amide linker moiety. In a further highly preferred embodiment, the linker group [linker] preferably comprises an ester linker moiety.

[0221] In a further highly preferred embodiment, the linker group [linker] preferably comprises a succinate linker moiety. In another highly preferred embodiment, the linker group [linker] comprises both an ester linker moiety and an amide linker moiety, hi another preferred embodiment, the linker group [linker] comprises all of an ester linker moiety, an amine linker moiety, and an amide linker moiety.

[0222] In another highly preferred embodiment, the linker group [linker] is preferably

[0223] [ka]

[0224] or the linker group [linker] is preferably an amine, preferably a secondary amine linker moiety. The inventors have surprisingly and advantageously found that the polymer-conjugated lipids, preferably "PMOZ1", "PMOZ2", "PMOZ3", "PMOZ4" or "PMOZ5", or each of the linker groups [linker]

[0225] [ka]

[0226] It has been found that polymer-conjugated lipids containing succinate, peptide bond (—CO—NH—), amine, or secondary amine (more preferably, the linker group [linker] contains succinamidyl (—NHC(O)CH2CH2C(O)—) or (—NHC(O)CH2CH2C(O)—)) have certain advantages in terms of productivity or general synthesis, preferably GMP productivity. In other words, the production of these polymer-conjugated lipids is easier to implement, more feasible, simpler, and / or can be carried out in a more cost-effective manner. In other words, the overall synthesis of these compounds containing the preferred linkers is easier and more feasible. Finally, polymer-conjugated lipids containing the [linker] group are more stable in terms of chemical stability. In other words, the polymer-conjugated lipids and [linkers] disclosed above have highly advantageous and unexpected behavior in terms of synthesis and manufacturing.

[0227] In further embodiments, the lipid nanoparticles comprise a polymer-conjugated lipid of the present disclosure. In a further preferred embodiment, the polymer-conjugated lipids of the present invention do not contain polyethylene glycol-(PEG)-moieties or residues; and / or do not contain sulfur groups (-S-); and / or do not contain terminal nucleophiles.

[0228] In a further preferred embodiment, the polymer-conjugated lipids of the present invention do not contain any polyethylene glycol-(PEG)-moieties or residues. In a further preferred embodiment, the polymer-conjugated lipids of the present invention do not contain a sulfur group (-S-).

[0229] In a further preferred embodiment, the polymer-conjugated lipids of the present invention do not contain a terminal nucleophile. In a further preferred embodiment, the polymer-conjugated lipids of the present invention are free of a sulfur group (-S-); and free of a terminal nucleophile.

[0230] In a further preferred embodiment, the polymer-conjugated lipid is not covalently coupled to a biologically active component that is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA.

[0231] In yet further embodiments, the lipid nanoparticles do not comprise polyethylene glycol-(PEG)-lipid conjugates or conjugates of PEG and lipid-like materials, preferably do not comprise PEG, and / or (ii) the polymer-conjugated lipids of the present invention do not comprise a sulfur group (-S-), a terminal nucleophile, and / or are covalently coupled to a biologically active component that is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof, preferably, the biologically active component is a chemically modified mRNA or a chemically unmodified mRNA, more preferably, the biologically active component is a chemically unmodified mRNA.

[0232] In another highly preferred embodiment, the polymer-conjugated lipids of the present invention do not contain sulfur (S) or sulfur groups (—S—). In a further embodiment, the lipid nanoparticles of the present invention further comprise a sterol or steroid, preferably selected from the group consisting of cholesterol, cholesteryl hemisuccinate (CHEMS) and derivatives thereof, preferably the lipid nanoparticles further comprise cholesterol.

[0233] In yet another embodiment, the lipid nanoparticles comprise, based on the molar percentage composition of all lipid components or excipients relative to 100%, (i) a polymer-conjugated lipid of the invention disclosed herein in an amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %; (ii) a polymer-conjugated lipid of the invention disclosed herein, preferably in an amount of 5 mol %; (iii) more preferably, the polymer-conjugated lipid of the invention disclosed herein in an amount of 2.5 mol %; or (iv) also preferably, in an amount of 1.7 mol % of a polymer-conjugated lipid of the invention disclosed herein Includes.

[0234] In a further embodiment, the lipid nanoparticles are (i) 59 mol % cationic or ionizable lipid, preferably one of the C1-C24 ionizable lipid structures, more preferably ionizable lipid structure C24 or formula III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) (formula III-3 is further described in the specification below in the section on cationic lipids), 29.3 mol % cholesterol, 10 mol % neutral lipid, and 1.7 mol % polymer-conjugated lipid of the present invention; (ii) 59 mol % cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably ionizable lipid structure C24 or formula III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 28.5 mol % cholesterol, 10 mol % neutral lipid, and 2.5 mol % polymer-conjugated lipid of the present invention; (iii) 59 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably ionizable lipid structure C24 or formula III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 28.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 1 mol% DHPC, and 2.5 mol% polymer-conjugated lipid of the present invention; (iv) 49 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably ionizable lipid structure C24 or formula III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 29.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 10 mol% DHPC, and 2.5 mol% polymer-conjugated lipid of the present invention; (v) 47.4 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C27, more preferably ionizable lipid structure C24 or formula III-3 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 40.9 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, and 1.7 mol% polymer-conjugated lipid of the present invention; (vi) 47.4 mol% of formula III-3 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 40.1 mol% of cholesterol, 10 mol% of DSPC and 2.5 mol% of a polymer-conjugated lipid of the present invention; (vii) 47.4 mol % of Formula III-3 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 40.9 mol % of cholesterol, 10 mol % of DSPC, and 1.7 mol % of a polymer-conjugated lipid of the present invention; (viii) 47.4 mol% of formula III-3 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 40.1 mol% of cholesterol, 10 mol% of DSPC, and 2.5 mol% of 2-[(PMOZ)] n -N,N-ditetradecylacetamide] (N,N-ditetradecylacetamide as further described herein below); and (ix) 47.4 mol% of formula III-3 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 40.9 mol% of cholesterol, 10 mol% of DSPC, and 1.7 mol% of 2-[(PMOZ)] n -N,N-ditetradecylacetamide] and the excipients are selected from the ratio selected from the group consisting of: n has an average value in the range of about 45 to about 55, preferably n is about 50, or n is selected so that the polymer portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0235] In a further preferred embodiment, the lipid nanoparticles comprise: (i) 59 mol % cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably the ionizable lipid structure C24, 29.3 mol % cholesterol, 10 mol % neutral lipid, and 1.7 mol % polymer-conjugated lipid of the present invention; (ii) 59 mol % cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably the ionizable lipid structure C24, 28.5 mol % cholesterol, 10 mol % neutral lipid, and 2.5 mol % polymer-conjugated lipid of the present invention; (iii) 59 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably the ionizable lipid structure C24, 28.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 1 mol% DHPC, and 2.5 mol% polymer-conjugated lipid of the present invention; (iv) 49 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24, more preferably the ionizable lipid structure C24, 29.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 10 mol% DHPC, and 2.5 mol% polymer-conjugated lipid of the present invention; (v) 47.4 mol % cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C27, more preferably ionizable lipid structure C24, 40.9 mol % cholesterol, 10 mol % DSPC or DPhyPE, preferably DPhyPE, and 1.7 mol % polymer-conjugated lipid of the present invention; (vi) 47.4 mol% C24, 40.1 mol% cholesterol, 10 mol% DSPC and 2.5 mol% polymer-conjugated lipid of the present invention; (vii) 47.4 mol% C24, 40.9 mol% cholesterol, 10 mol% DSPC, and 1.7 mol% polymer-conjugated lipid of the present invention; (viii) 47.4 mol% C24, 40.1 mol% cholesterol, 10 mol% DSPC, and 2.5 mol% 2-[(PMOZ)] n -N,N-ditetradecylacetamide] (N,N-ditetradecylacetamide as further described herein below); and (ix) 47.4 mol% C24, 40.9 mol% cholesterol, 10 mol% DSPC and 1.7 mol% 2-[(PMOZ)] n -N,N-ditetradecylacetamide] and the excipients are selected from the ratio selected from the group consisting of: n has an average value in the range of about 45 to about 55, preferably n is about 50, or n is selected so that the polymer portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0236] In a most preferred embodiment, the lipid nanoparticles comprise 59 mol% C24, 28.5 mol% cholesterol, 10 mol% DPhyPE and 2.5 mol% "PMOZ4".

[0237] In a further embodiment, the biologically active component contained in the lipid nanoparticle is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof, preferably, the biologically active component is chemically modified mRNA or chemically unmodified mRNA, more preferably, the biologically active component is chemically unmodified mRNA.

[0238] In a highly preferred embodiment, the nucleic acid compound is an artificial or isolated mRNA. In yet another embodiment, the lipid nanoparticle comprises at least one coding sequence encoding a pathogenic antigen, the pathogenic antigen being (i) SARS-CoV-2, nCov-2019 coronavirus, SARS-CoV (SARS-CoV), Bunyavirales, cytomegalovirus (CMV), dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), Epstein-Barr virus (EBV), flaviviruses, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (HMPV), human papillomavirus (HPV), human parainfluenza virus (HPV), and derived from HPIV, influenza virus, extraintestinal pathogenic Escherichia coli (ExPEC), Lassa-Mam arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia trachomatis that causes chlamydia), or malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale); and / or (ii) derived from a structural protein, accessory protein, or replicase protein from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), or an immunogenic fragment or immunogenic variant of any of these; and / or (iii) derived from the spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (N) from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), or an immunogenic fragment or immunogenic variant of any of these (preferably, the spike protein (S) comprises or consists of spike protein fragment S1 or spike protein fragment S2, more preferably spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof (e.g., receptor binding domain (RBD), primary neutralization domain (CND))); and / or (iv) Derived from a prefusion-stabilizing spike protein (S) (S_stab) from SARS-CoV-2, nCoV-2019 coronavirus, or SARS-CoV, containing at least one prefusion-stabilizing mutation.

[0239] In still further embodiments, the polymer-conjugated lipid is 2-[(PMOZ)] n -N,N-ditetradecylacetamide], 2-[(PEOZ)] n -N,N-ditetradecylacetamide], 2-[(PPOZ)] n -N,N-ditetradecylacetamide], 2-[(PBOZ)] n -N,N-ditetradecylacetamide], 2-[(PIPOZ)] n -N,N-ditetradecylacetamide], and preferably the polymer-conjugated lipid is selected from the group consisting of 2-[(PM(O)Z)] n -N,N-ditetradecylacetamide], where n has an average value in the range of about 45 to about 55, preferably n is about 50, or n is selected such that the polymer portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0240] In other preferred embodiments, the novel polymer-conjugated lipids may be derived from the polymer-conjugated lipids disclosed in WO2018078053 (i.e., N,N-ditetradecylacetamide-based compounds or lipids derived from claim 5 of WO2018078053), the entire disclosure of which is incorporated herein by reference.

[0241] In this specification, all chemical compounds referred to throughout the specification can be prepared by methods known to those skilled in the art; it is understood that the starting materials and / or reagents used in these methods can be obtained through the routine knowledge of those skilled in the art based on common general knowledge (e.g., from textbooks or patent applications WO 2022173667, WO 2009043027, WO 2013067199, WO 2010006282, WO 2009089542, WO 2016019340, WO 2008106186, WO 2020264505, and WO 2020023947, the complete disclosures of which are incorporated herein by reference).

[0242] lipid composition In some embodiments of the invention, the LNP comprises a lipid conjugate, preferably a polymer-conjugated lipid as described above, a cationic lipid, a steroid, and a neutral lipid.

[0243] Cationic, ionizable or cationizable lipids Cationic lipids are preferably ionizable or cationizable, i.e., the pH is greater than the pK of the ionizable group of the lipid. a When the pH drops below 100, the lipid becomes protonated, but at higher pH values ​​it becomes progressively more neutral.When positively charged, the lipid can associate with negatively charged nucleic acids.In certain embodiments, the cationic lipid comprises a zwitterionic lipid, which becomes positively charged as the pH drops.

[0244] In some embodiments, the lipid nanoparticles comprise a molar ratio of ionizable cationic lipid of 20-60%. For example, the lipid nanoparticles may comprise a molar ratio of ionizable cationic lipid 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 comprise a molar ratio of ionizable cationic lipid of 20%, 30%, 40%, 50%, or 60%.

[0245] In some embodiments, the lipid nanoparticles comprise a molar ratio of 5-25% non-cationic lipid. For example, the lipid nanoparticles may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

[0246] In some embodiments, the lipid nanoparticles comprise a molar ratio of sterol of 25-55%. For example, the lipid nanoparticles comprise a molar ratio of sterol 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 comprise a molar ratio of sterol of 25%, 30%, 35%, 40%, 45%, 50%, or 55%.

[0247] In some embodiments, the lipid nanoparticles comprise 0.5-15% molar ratio of a polymer-conjugated lipid of the present disclosure. For example, the lipid nanoparticles may comprise 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15% molar ratio. In some embodiments, the lipid nanoparticles comprise 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% molar ratio of a polymer-conjugated lipid of the present disclosure.

[0248] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid of the present disclosure.

[0249] Ionizable lipids The cationic lipids of LNPs are cationizable, i.e., they protonate when the pH is reduced below the pK of the ionizable group of the lipid, but become progressively more neutral at higher pH values.At pH values ​​below the pK, the lipids can associate with negatively charged nucleic acids.In certain embodiments, the cationic lipids comprise zwitterionic lipids, which become positively charged as the pH is reduced.

[0250] Preferred cationic lipids are compounds according to formula (Cat-I) R a -AR b Formula (Cat-I) (In the formula, R a teeth

[0251] [ka]

[0252] or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; Rb teeth

[0253] [ka]

[0254] -R 1 -N(H)-C(O)-R 3 -R 4 ,or -R 1 -N(CH3)2 Selected from; 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 is optionally substituted ethanediyl, propanediyl, butanediyl, or a linear or branched alkanediyl having 2 to 8 carbon atoms; R 2 is an alkanediyl having 2 to 8 carbon atoms; R 3 is optional and, if present, -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 -NH-C(O)O-; R 4 is a lipophilic substituent having 12 to 36 carbon atoms; R 5 is an alkanediyl having 1 to 6 carbon atoms; X is a carbon atom or a nitrogen atom; All choices are independent of each other However, in some cases, R 1 , R 2 and R 5 are all straight-chain unsubstituted ethanediyl, A is -SS-, and R a and R b If are identical, R 4 teeth

[0255] [ka]

[0256] isn't it) is defined as: In other embodiments, R 4 teeth

[0257] [ka]

[0258] is. In one preferred embodiment, A is -S- and R a and R b are identical, and R 4 teeth

[0259] [ka]

[0260] is. In another preferred embodiment, A is -S- and R 4 teeth

[0261] [ka]

[0262] is. R in formula (Cat-I) 4 R is defined as a lipophilic substituent having 12 to 36 carbon atoms. a and possibly also R b (R b Ga-R 1 This "tail" end of the -N(CH3)2 group (unless it is -N(CH3)2) is thought to provide the degree of lipophilicity typically required for the molecule to be able to cross biological membranes. 4In principle, R can be any structure that is substantially lipophilic. For example, hydrocarbon structures are lipophilic. In one embodiment, R 4 may consist exclusively of carbon and hydrogen atoms in at least one of its occurrences. 4 represents a straight or branched alkyl or alkenyl, preferably having 12 to 25 carbon atoms. The branched alkyl or alkenyl may optionally have multiple side chains, such as two, three, four or more methyl side chains. In another embodiment, R 4 can be, for example, an alkyl or alkenyl containing a single alkyl or alkenyl side chain having from 2 to 10 carbon atoms. For example, R 4 can be 1-n-hexyl-n-nonyl (or 7-n-pentadecyl), or 2-n-hexyl-n-decyl. In other embodiments, the lipophilic substituent can optionally contain one or more heteroatoms such as O, S, or N. In other embodiments, the lipophilic substituent can optionally contain one or more saturated, unsaturated, or aromatic ring structures, which can optionally contain one or more heteroatoms such as O, S, or N.

[0263] R 4 may also contain a small number of heteroatoms, such as oxygen atoms, so long as the predominantly lipophilic nature is maintained. 4 R contains one or more oxygen atoms and no other heteroatoms. 4 may also optionally contain cyclic structures, such as aromatic or aliphatic cyclic structures, containing one or more oxygen atoms. When present, the heteroatoms and / or cyclic structures are located within the optional R rather than towards the end of the "tail." 3 Preferably, R is located toward the structure. 4 is a lipophilic group derived from tocopherol or tocotrienol. In one embodiment, R 1 , R 2 and R 5 are not all straight-chain unsubstituted ethanediyl, A is -SS-, and R a and Rb If are identical, R 4 is a lipophilic group derived from alpha-tocopherol, especially

[0264] [ka]

[0265] is. The "lipophilic group derived from tocopherol or tocotrienol" referred to herein includes derivatives of tocopherol and tocotrienol, particularly those having the structure shown in Scheme 1 below, i.e., derivatives derived from alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol.

[0266] [ka]

[0267] [Table 1]

[0268] Scheme 1: Tocopherol derivatives have saturated phytyl chains, while tocotrienol derivatives have polyunsaturated phytyl chains. For both tocopherol and tocotrienol derivatives, isoforms are defined by R1 and R2, which are selected from CH3 and H. Thus, for example, when R1 is CH3 and R2 is CH3, as shown, the resulting derivatives are alpha isoforms of tocopherol and tocotrienol, respectively (referred to as alpha-tocopherol and alpha-tocotrienol derivatives, respectively). The OH group is naturally absent from the derivatives, since it is the point of attachment, as shown on the left in the two structures.

[0269] In a particularly preferred embodiment of aspect A above, R 4 is a straight or branched chain alkyl or alkenyl having 12 to 25 carbon atoms, or a lipophilic group selected from the group consisting of derivatives of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol shown herein in Scheme 1.

[0270] In yet another preferred embodiment, particularly in aspect A above, R 4 is a straight or branched alkyl or alkenyl having 12 to 25 carbon atoms, or

[0271] [ka]

[0272] is. In other preferred 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, thereby forming LNPs, wherein the cationic lipids of the LNPs are selected from lipids derived from structures C1-C23, or C1-C27, respectively, or formula (I) in Table 1 of PCT patent application PCT / EP2019 / 086825 or a subsequent patent application claiming priority to PCT / EP2019 / 086825, i.e., WO2021123332. In other 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, thereby forming an LNP, wherein the cationic lipid of the LNP is derived from structures C1-C23, or C1-C27, respectively, of Table 1 of PCT patent application PCT / EP2019 / 086825 or a subsequent patent application claiming priority to PCT / EP2019 / 086825, i.e., WO2021123332, and element "A" of formula (I) of PCT / EP2019 / 086825 is -S-. Accordingly, Formulas C1-C23, or C1-C27, respectively, of PCT / EP2019 / 086825 or subsequent patent applications claiming priority to PCT / EP2019 / 086825, i.e., WO 2021123332, and the specific disclosures relating thereto are incorporated herein by reference.

[0273] In still further embodiments, the cationic lipid is preferably selected from the cationic lipids listed herein in Table 1.

[0274] [Table 2-1]

[0275] [Table 2-2]

[0276] [Table 2-3]

[0277] [Table 2-4]

[0278] [Table 2-5]

[0279] [Table 2-6]

[0280] [Table 2-7]

[0281] [Table 2-8]

[0282] [Table 2-9]

[0283] [Table 2-10]

[0284] [Table 2-11]

[0285] Therefore, the present invention is directed to a composition comprising the above-mentioned cationic lipid. For example, the composition may comprise a cationic lipid selected from compounds C1 to C27 in Table 1. In other preferred 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, thereby forming an LNP, and the cationic lipid of the LNP has the structure "C24," which is the most preferred structure for cationic lipids contained in the lipid nanoparticle compositions of the present invention.

[0286] [ka]

[0287] 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-dioleoyltrimethylammonium propane 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-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (γ-DLenDMA), Noleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 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), 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-Dilinoleoyl-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(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-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-tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, En-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)(2-hydroxydodecyl)amine) (amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-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-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-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), GIBCO / BRL, Grand Island, NY, USA)

[0033] LIPOFECTAMINE® (a commercially available cationic liposome comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), available from GIBCO / BRL; and TRANSFECTAM® (a commercially available cationic lipid comprising dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, available from Promega Corp., Madison, Wisconsin, USA), or any combination of any of the foregoing. Further suitable cationic lipids for use in the compositions and methods of the present invention include the cationic lipids HGT4003, HGT5000, HGTS001, HGT5001, and HGT5002 (see U.S. Patent Application Publication No. 20150140070) described in International Patent Publications WO 2010053572 (and in particular CI2-200 described in paragraph

[0225] ) and WO 2012170930 (both of which are incorporated herein by reference).

[0288] In some embodiments, the cationic lipid can be an amino lipid. Representative amino lipids 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), 1, 2-Dilinoleyloxy-3-(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); MC3 (U.S. Patent Application Publication No. 20100324120).

[0289] In embodiments, the cationic lipid may be an amino alcohol lipidoid. Aminoalcohol lipidoids that can be used in the present invention can be prepared by the methods described in U.S. Patent No. 8,450,298, which is incorporated herein by reference in its entirety. Suitable (ionizable) lipids can also be the compounds disclosed in Tables 1, 2 and 3 of WO2017075531, which is incorporated herein by reference, and defined in claims 1 to 24 thereof.

[0290] In another embodiment, suitable lipids may also be compounds disclosed in WO2015074085 (i.e., ATX-001 to ATX-032 or compounds specified in claims 1-26), U.S. Patent Application Publication Nos. 61 / 905,724 and 15 / 614,499, or U.S. Patent Nos. 9,593,077 and 9,567,296, which are incorporated herein by reference in their entirety.

[0291] In other embodiments, suitable cationic lipids may also be compounds disclosed in WO2017117530 (i.e., lipids 13, 14, 15, 16, 17, 18, 19, 20, or compounds specified in the claims), which are incorporated herein by reference in their entirety.

[0292] In a preferred embodiment, the ionizable or cationic lipid may also be selected from the lipids disclosed in WO 2018078053 (i.e., lipids derived from Formulas I, II, and III of WO 2018078053, or lipids specified in Claims 1 to 12 of WO 2018078053) (the entire disclosure of WO 2018078053 is incorporated herein by reference). In this context, the lipids disclosed in Table 7 of WO 2018078053 (e.g., lipids derived from Formulas I-1 to I-41) and the lipids disclosed in Table 8 of WO 2018078053 (e.g., lipids derived from Formulas II-1 to II-36) may be suitably used in the context of the present invention. Therefore, Formulas I-1 to I-41 and II-1 to II-36 of WO 2018078053, and specific disclosures related thereto, are incorporated herein by reference.

[0293] In a preferred embodiment, the cationic lipid can be derived from Formula III of published PCT patent application WO 2018078053. Formula III of WO 2018078053, and the specific disclosures relating thereto, are hereby incorporated by reference.

[0294] In particularly preferred 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, thereby forming an LNP, wherein the cationic lipid of the LNP is selected from structures III-1 through III-36 in Table 9 of published PCT patent application WO 2018078053. Accordingly, formulas III-1 through III-36 of WO 2018078053, and the specific disclosures relating thereto, are incorporated herein by reference.

[0295] In a particularly preferred embodiment of the second aspect, at least one nucleic acid (e.g., DNA or RNA), preferably at least one RNA, is complexed with one or more lipids, thereby forming an LNP, the LNP comprising:

[0296] [ka]

[0297] Or most preferably Formula III-3 of WO 2018078053, i.e., (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate):

[0298] [ka]

[0299] The cationic lipids include those derived from The lipid of formula III-3 suitable for use herein has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315, i.e., CAS number 2036272-55-4.

[0300] In certain embodiments, a cationic lipid as defined herein, more preferably cationic lipid compound III-3 ((4-hydroxybutyl)azanediyl)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%, more preferably about 47.4 mol%, relative to the total lipid content of the LNP. When two or more cationic lipids are incorporated within the LNP, these percentages apply to the combined cationic lipids.

[0301] In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 mol% to about 70 mol%. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 mol% to about 60 mol%, e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%, respectively. In another embodiment, the cationic lipid is present in the LNP in an amount of about 47 mol% to about 48 mol%, e.g., about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 50.0 mol%, respectively, with 47.4 mol% being particularly preferred.

[0302] In some embodiments, the cationic lipid is present in a ratio of about 20 mol% to about 70 mol%, or 75 mol%, or about 45 mol% to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the LNP. In further embodiments, the LNP comprises about 25% to about 75% cationic lipid on a molar basis (based on 100% total moles of lipid in the lipid nanoparticle), e.g., 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% cationic lipid on a molar basis. In some embodiments, the ratio of cationic lipid to nucleic acid (e.g., coding RNA or DNA) is about 3 to about 15, e.g., about 5 to about 13, or about 7 to about 11.

[0303] Other suitable (cationic or ionizable) lipids are described in WO 2009086558, WO 2009127060, WO 2010048536, WO 2010054406, WO 2010088537, WO 2010129709, WO 2011153493, WO 2013063468, U.S. Patent Application Publication No. 20110256175, U.S. Patent Application Publication No. 20120 128760, U.S. Patent Application Publication No. 20120027803, U.S. Patent No. 8158601, International Publication No. 2016118724, International Publication No. 2016118725, International Publication No. 2017070613, International Publication No. 2017070620, International Publication No. 2017099823, International Publication No. 2012040184, International Publication No. 2011153120, International Publication No. 2011149733, International Publication No. 2011090965, International Publication No. 2011043913, International Publication No. 2011022460, International Publication No. 2012061259, International Publication No. 2012054365, International Publication No. 2012044638, International Publication No. 2010080724, International Publication No. 201021865, International Publication No. 2008103276, International Publication No. 2013086373, International Publication No. 2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, and 7,404,969. Nos. 8,283,333, 8,466,122, and 8,569,256, as well as U.S. Patent Application Publication Nos. 20100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, 20140039032, and WO 2017112865.In that context, WO 2009086558, WO 2009127060, WO 2010048536, WO 2010054406, WO 2010088537, WO 2010129709, WO 2011153493, WO 2013063468, U.S. Patent Application Publication No. 20110256175, U.S. Patent Application Publication No. 20110256176, U.S. Patent Application Publication No. 20110256177, U.S. Patent Application Publication No. 20110256178, U.S. Patent Application Publication No. 20110256179 ... 0120128760, U.S. Patent Application Publication No. 20120027803, U.S. Patent No. 8158601, International Publication No. 2016118724, International Publication No. 2016118725, International Publication No. 2017070613, International Publication No. 2017070620, International Publication No. 2017099823, International Publication No. 2012040184, International Publication No. 2011153120, International Publication No. 2011149733, International Publication No. 2011090965, International Publication No. 2011043913, International Publication No. 2011022460, International Publication No. 2012061259, International Publication No. 2012054365, International Publication No. 2012044638, International Publication No. 2010080724, International Publication No. 201021865, International Publication No. 2008103276, International Publication No. 2013086373, International Publication No. 2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,222,232, and 8,232. The disclosures of U.S. Patent Application Publication Nos. 20100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, and 20140039032, and International Publication No. WO 2017112865 are incorporated herein by reference.

[0304] In other embodiments, the cationic or ionizable lipid is

[0305] [ka]

[0306] [ka]

[0307] is. In embodiments, amino or cationic lipids as defined herein have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species and does not require that all of the lipids be present in a charged or neutral form. Lipids with two or more protonatable or deprotonatable groups or that are zwitterionic are not excluded and may also be suitable in the context of the present invention. In some embodiments, the protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., about 5 to about 7.

[0308] LNPs can contain two or more (different) cationic lipids as defined herein. Cationic lipids can be selected to contribute various advantageous properties. For example, cationic lipids with different properties, such as amine pKa, chemical stability, circulation half-life, tissue half-life, net accumulation in tissue, or toxicity, can be used in LNPs. In particular, cationic lipids can be selected so that the properties of the mixed LNP are more desirable than those of a single LNP of each individual lipid.

[0309] The amount of persistent cationic lipid, lipidoid, or preferably ionizable cationic lipid, can be selected taking into account the amount of nucleic acid cargo. In one embodiment, these amounts are selected to provide an N / P ratio of the nanoparticle or composition in the range of about 0.1 to about 20, or (i) an amount to achieve an N / P ratio in the range of from about 1 to about 20, preferably from about 2 to about 15, more preferably from about 3 to about 10, even more preferably from about 4 to about 9, and most preferably about 6; (ii) an amount to achieve an N / P ratio in the range of about 5 to about 20, more preferably about 10 to about 18, even more preferably about 12 to about 16, and most preferably about 14; (iii) an amount to achieve a lipid:mRNA weight ratio in the range of 20 to 60, preferably about 3 to about 15, 5 to about 13, about 4 to about 8, or about 7 to about 11; or (iv) an amount sufficient to achieve an N / P ratio in the range of about 6 for lipid nanoparticles according to the present invention, particularly lipid nanoparticles comprising cationic lipid III-3. is selected.

[0310] In other preferred embodiments, the N / P ratio can range from about 1 to about 50. In other embodiments, the range is from about 1 to about 20, preferably from about 1 to about 15. For the lipid nanoparticles of the present invention, a preferred N / P (molar ratio of lipid to RNA) is about 14 or about 17. An even more preferred N / P, i.e., a molar ratio of lipid to RNA, is about 6. Another preferred N / P ratio is about 4.85 or 5 (molar ratio of lipid to RNA).

[0311] In a highly preferred embodiment, the amount of ionizable cationic lipid is selected taking into account the amount of nucleic acid cargo, in an amount to achieve an N / P ratio in the range of about 12 to about 16, most preferably about 14.

[0312] In this context, the N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid or lipidoid to the phosphate group ("P") of the nucleic acid used as cargo. The N / P ratio can be calculated, for example, based on the fact that 1 μg of RNA typically contains about 3 nmol of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The "N" value of a cationic lipid or lipidoid can be calculated based on its molecular weight and the relative content of persistent cationic groups and, if present, cationizable groups. If two or more cationic lipids are present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticle.

[0313] In other embodiments, the ionizable lipid of the present disclosure is a compound of formula (Cat-II):

[0314] [ka]

[0315] or their N-oxides, or their salts or isomers (In the formula, 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 independently H, C 1~14 Alkyl, C 2~14 Alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is hydrogen, C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, and Q is selected from the group consisting of carbocycle, heterocycle, -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-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, wherein each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; Each R6 is independently 1~3 Alkyl, C 2~3 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-, an aryl group, and a heteroaryl group; M'' is a bond, C 1~13 Alkyl or C 2~13 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 carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1~18 Alkyl, C2~18 Alkenyl, -R * selected from the group consisting of YR″, -YR″, and H; Each R'' is independently C 3~15 Alkyl and C 3~15 alkenyl; Each R * is independently C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic 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) n When n is CHQR, -CHQR, or -CQ(R), (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R), or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl. It can be one or more of:

[0316] As used herein, the term "ionizable lipid" has its ordinary 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 or negatively charged. The ionizable lipid may be positively charged, in which case it may be referred to as a "cationic lipid." In certain embodiments, the ionizable lipid molecule may contain an amine group and may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety having a formal charge, for example, 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 (primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, etc. The charge of a charged moiety can sometimes change with environmental conditions; for example, a change in pH can change the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of a molecule can be selected as desired. It should be understood that the term "charged" or "charged moiety" does not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art.When a functional group contains a bond that is polarized so that electron density is pulled toward one atom of the bond, creating a partial negative charge on that atom, a "partial negative charge" can occur.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 amino lipid, which is sometimes referred to in the art as an "ionizable cationic lipid."In one embodiment, the ionizable amino lipid can have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0317] Interestingly, the present inventors have found that one of the advantageous features of the composition of the present invention and lipid nanoparticles, for example, GN01 formulations comprising polymer-conjugated lipids according to formula (I), for example, PMOZ, is that they can induce a strong CD8+ T cell response.This is due to the fact that, for example, for malaria, CD8+ T cells are the main protective immune mechanism against intracellular infection caused by malaria parasites, so an effective malaria vaccine should induce a strong CD8+ T cell response.

[0318] A particularly preferred embodiment of the lipid nanoparticles of the present invention is provided when the following combination of excipients is used to formulate the lipid nanoparticles designated "GN01": 59 mol % of the cationic lipid C23 disclosed in Table 1, i.e., COATSOME® SS-EC (formerly named SS-33 / 4PE-15; NOF Corporation, Tokyo, Japan), 29.3 mol % of cholesterol as the steroid, 10 mol % of DPhyPE (4ME 16:0PE) as the neutral lipid / phospholipid, and 1.7 mol % of PMOZ-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE; 14:0PE) or preferably DMG-PMOZ (i.e., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, i.e., the PMOZ equivalent of DMG-PEG2000) as the polymer-conjugated lipid. Such LNPs containing the cationic lipid C23 are designated herein as "GN01." With respect to the molar ratios mentioned in this paragraph, any GN01-LNP composition containing a polymer-conjugated lipid according to Formula (I) is referred to herein and in the Examples as "GN01-PMOZ." SS-EC has a positive charge at pH 4 and a neutral charge at pH 7, which is advantageous for the LNPs and formulations / compositions of the present invention. For "GN01-PMOZ," the N / P (molar ratio of lipid to mRNA) is preferably 14, and the total lipid / mRNA mass ratio is preferably 40 (m / m).

[0319] A further particularly preferred embodiment of the lipid nanoparticles of the present invention is provided when the following combination of excipients is used to formulate the lipid nanoparticles: 59 mol% of the C2 or C24 lipid disclosed in Table 1 as the cationic lipid (i.e., HEXA-C5DE-PipSS, which is the cationic lipid compound C2 in Table 1, or VitE-C4DE-piperidine-thioether, which is the cationic lipid compound C24 in Table 1, respectively), 29.3 mol% of cholesterol as the steroid, 10 mol% of DPhyPE as the neutral lipid / phospholipid, and 1.7 mol% of PMOZ-DMPE or preferably DMG-PMOZ as the polymer-conjugated lipid. Such LNPs containing the cationic lipid C2 are designated herein as "GN02." With respect to the molar ratios mentioned in this paragraph, any GN02-LNP composition containing a polymer-conjugated lipid according to Formula (I) is referred to herein and in the Examples as "GN02-PMOZ." For "GN02-PMOZ", N / P (molar ratio of lipid to mRNA) is 17.5, more preferably 14, and the total lipid / mRNA mass ratio is preferably 40 (m / m).

[0320] Further, preferred compositions include: (i) the cationic lipid may be selected from the compounds in Table 1; and / or (ii) The neutral lipid or neutral phospholipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also known as 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine ( DPPC, also known as 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), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 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-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 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-Palmitoyl 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-hetero ... Xadecanyl-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-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesteryl hemisuccinoyl a zwitterionic compound selected from the group consisting of xiñoyl-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 (edelfosine); and / or (iii) the polymer-conjugated lipid is a polymer-conjugated lipid according to formula (I): [P]-[linker]-[L] Formula (I) (In the formula, [P] is at least one polyoxazoline (POZ) monomer unit

[0321] [ka]

[0322] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value ranging from about 45 to about 55, preferably n is about 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. is a homopolymer moiety comprising [linker] is an optional linker group; [L] is the lipid moiety) is.

[0323] In other preferred embodiments, the lipid-based carrier comprises a cationic or ionizable lipid. The cationic or ionizable lipid of lipid-based carrier can be cationizable or ionizable, that is, when pH is reduced below the pK of the ionizable group of lipid, it becomes protonated, but at higher pH values, it becomes progressively more neutral.At pH values ​​below pK, lipid can associate with negatively charged nucleic acid.In certain embodiments, cationic lipid comprises zwitterionic lipid, which becomes positively charged when pH is reduced.

[0324] In a preferred embodiment, the lipid-based carrier comprises a cationic or ionizable lipid, preferably having a net positive charge at physiological pH, and more preferably the cationic or ionizable lipid comprises a tertiary or quaternary nitrogen group. Thus, in a preferred embodiment, the lipid-based carrier comprises a cationic or ionizable lipid selected from amino lipids.

[0325] In a further embodiment, the lipid formulation comprises a cationic or ionizable lipid as defined by formula I in paragraph

[0251] of WO2021222801, or a lipid selected from the disclosures in paragraphs

[0260] or

[0261] of WO2021222801. In another embodiment, 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 WO2021183563. The disclosures of WO2021222801 and WO2021183563, in particular the lipids described above, are incorporated herein by reference.

[0326] Further suitable cationic lipids may be selected from or derived from cationic lipids according to PCT Claims 1 to 14 of published patent application WO 2021123332 or Table 1 of WO 2021123332, the disclosures relating to Claims 1 to 14 or Table 1 of WO 2021123332 are incorporated herein by reference. Accordingly, suitable cationic lipids may be selected from or derived from cationic lipids according to Compounds 1 to 27 (C1 to C27) of Table 1 of WO 2021123332.

[0327] In other preferred embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from (COATSOME® SS-EC) SS-33 / 4PE-15 (see C23 in Table 1 of WO2021123332).

[0328] In another preferred embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from HEXA-C5DE-PipSS (see C2 in Table 1 of WO2021123332). In a most preferred embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises compound C26 disclosed in Table 1 of WO2021123332:

[0329] [ka]

[0330] The cationic lipids include those selected from or derived from: In another embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, also known as SM-102. Other preferred lipid-based carriers (e.g., LNP) of the pharmaceutical composition are squaramide ionizable amino lipids, more preferably those of formula (M1) and (M2):

[0331] [ka]

[0332] (wherein substituents (e.g., R1, R2, R3, R5, R6, R7, R 10 , M, M1, m, n, o, l) are defined in claims 1-13 of U.S. Pat. No. 10,392,341; U.S. Pat. No. 10,392,341 is incorporated herein by reference in its entirety. The cationic lipid comprises a cationic lipid selected from the group consisting of:

[0333] Thus, in a preferred embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from ALC-0315, SM-102, SS-33 / 4PE-15, HEXA-C5DE-PipSS, or compound C26 (see C26 in Table 1 of WO2021123332).

[0334] In a particularly preferred embodiment, the lipid-based carrier of the pharmaceutical composition, preferably the LNP, comprises a cationic lipid selected from or derived from ALC-0315. In some embodiments, the lipid-based carriers of the present invention comprise two or more (different) cationic lipids as defined herein.

[0335] In certain embodiments, the cationic lipid as defined herein, more preferably the cationic lipid ALC-0315, is present in the lipid-based carrier in an amount of about 30 mol% to about 95 mol%, based on the total lipid content of the lipid-based carrier. When two or more cationic lipids are incorporated into the lipid-based carrier, such percentages apply to the combined cationic lipids.

[0336] In one embodiment, the cationic lipid is present in the lipid-based carrier in an amount of about 30 mol% to about 70 mol%. In one embodiment, the cationic lipid is present in the lipid-based carrier in an amount of about 40 mol% to about 60 mol%, for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%, respectively. In another embodiment, the cationic lipid is present in the lipid-based carrier in an amount 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, or 50.0 mol%, respectively, with 47.4 mol% being particularly preferred. In other preferred embodiments, the cationic lipid is present in the lipid-based carrier in an amount of about 55 mol % to about 65 mol %, for example about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mol %, respectively, with 59 mol % being particularly preferred.

[0337] In some embodiments, the cationic lipid is present in a ratio of about 20 mol% to about 70 mol%, or 75 mol%, or about 45 mol% to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the lipid-based carrier. In further embodiments, the LNP comprises about 25% to about 75% on a molar basis, e.g., 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% cationic lipid on a molar basis (based on 100% total moles of lipid in the lipid nanoparticle).

[0338] In some embodiments, the ratio of cationic lipid to RNA is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11. steroid A "steroid" is an organic compound that has four rings arranged in a specific molecular configuration. Steroids contain the following carbon skeleton:

[0339] [ka]

[0340] Steroids and neutral steroids include both naturally occurring steroids and their analogs (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" provided herein, a neutral steroid can be a steroid that does not have any ionizable atoms or groups under physiological conditions, or can be a zwitterionic steroid. In a preferred embodiment, a neutral steroid does not contain any ionizable atoms or groups under physiological conditions. In some preferred embodiments, the steroid or steroid analog is cholesterol. The terms "steroid" and "neutral steroid" are used interchangeably herein.In other embodiments, the sterol is a phytosterol, e.g., β-sitosterol, campesterol, stigmasterol, fucosterol, stigmastanol, dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, 3β-[N-(N′N′-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, ... Cholesterol, 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-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol, cholesteryl- 3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol The cholesterol ester may be selected from the group consisting of cholesterol, fecosterol or fecosterol, or salts or esters thereof, 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.

[0341] In a further embodiment, the steroid is an imidazole cholesterol ester or "ICE," as disclosed in paragraphs

[0320] and

[0339] -

[0340] of International Publication No. WO2019226925, the entire contents of which are incorporated herein by reference.

[0342] In other embodiments, the polymer-conjugated lipids of the present invention are POZ-steroid or POZ-sterol conjugates. In further embodiments, the lipid portion [L] of Formula (I) (i.e., [P]-[Linker]-[L]) is a phytosterol, e.g., β-sitosterol, campesterol, stigmasterol, fucosterol, stigmastanol, dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol ... Sterols (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-cholest-7-en-3β-ol, 3,6,9-Trioxaoctan-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, or fecosterol or a salt or ester thereof, 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, imidazole cholesterol ester or "ICE," or a derivative thereof.

[0343] In other preferred embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, a steroid analog, or a sterol. Suitably, the steroid, steroid analog, or sterol may be derived from or selected from cholesterol, cholesteryl hemisuccinate (CHEMS), and derivatives thereof. In another embodiment, the lipid-based carrier of the pharmaceutical composition comprises a steroid, steroid analog, or sterol derived from a phytosterol (e.g., a sitosterol, such as β-sitosterol), preferably a compound having the structure of Formula I disclosed in Claim 1 of WO2020061332; the disclosure of WO2020061332, particularly the disclosure of Formula I and phytosterols, is incorporated herein by reference. In a further embodiment, the steroid is an imidazole cholesterol ester or "ICE," as disclosed in paragraphs

[0320] and

[0339] -

[0340] of WO2019226925; the entire disclosure of WO2019226925 is incorporated herein by reference.

[0344] In a particularly preferred embodiment, the lipid-based carrier of the pharmaceutical composition comprises cholesterol. The molar ratio of cationic lipid to cholesterol in the lipid-based carrier can range from about 2:1 to about 1:1.

[0345] In some embodiments, the lipid-based carrier comprises about 10 mol% to about 60 mol%, or about 25 mol% to about 40 mol% of sterols (based on 100% total moles of lipids in the lipid-based carrier). In one embodiment, the sterols are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipids present in the lipid-based carrier. In another embodiment, the lipid-based carrier comprises about 5% to about 50% on a molar basis, e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5%, or about 30% on a molar basis (based on 100% total moles of lipids in the lipid-based carrier). In preferred embodiments, the lipid-based carrier comprises about 28%, about 29%, or about 30% sterol (based on 100% total moles of lipids in the lipid-based carrier). In the most preferred embodiment, the lipid-based carrier comprises about 40.9% sterol (based on 100% total moles of lipids in the lipid-based carrier).

[0346] References to other suitable cationic or ionizable, neutral, steroid / sterol or aggregation-reducing lipids: Other suitable cationic or ionizable, neutral, steroid / sterol or aggregation-reducing lipids are described in WO2010053572, WO2011068810, WO2012170889, WO2012170930, WO2013052523, WO2013090648, WO2013149140, WO2013149141, WO2013151663, WO2013151664, WO2013151665, WO2013151666 , International Publication No. 2013151667, International Publication No. 2013151668, International Publication No. 2013151669, International Publication No. 2013151670, International Publication No. 2013151671, International Publication No. 2013151672, International Publication No. 2013151736, International Publication No. 2013185069, International Publication No. 2014081507, International Publication No. 2014089486, International Publication No. 2014093924, International Publication No. 2014144196, International Publication No. 2014152211, International Publication No. 2014152774, International Publication No. 201415 2940, International Publication No. 2014159813, International Publication No. 2014164253, International Publication No. 2015061461, International Publication No. 2015061467, International Publication No. 2015061500, International Publication No. 2015074085, International Publication No. 2015105926, International Publication No. 2015148247, International Publication No. 2015164674, International Publication No. 2015184256, International Publication No. 2015199952, International Publication No. 2015200465, International Publication No. 2016004318, International Publication No. 2016022914, International Publication No. 2016036902, International Publication No. 2016081029, International Publication No. 2016118724, International Publication No. 2016118725, International Publication No. 2016176330, International Publication No. 2017004143, International Publication No. 2017019935, International Publication No. 2017023817, International Publication No. 2017031232, International Publication No. 2017049074, International Publication No. 2017049245, International Publication No. 2017070601, International Publication No. 2017070613, International Publication No. 2017070616, International Publication No. 2017070618,International Publication No. 2017070620, International Publication No. 2017070622, International Publication No. 2017070623, International Publication No. 2017070624, International Publication No. 2017070626, International Publication No. 2017075038, International Publication No. 2017075531, International Publication No. 2017099823, International Publication No. 2017106799, International Publication No. 2017112865, International Publication No. 2017117528, International Publication No. 2017117530, International Publication No. 2017180917, International Publication No. 2017201325, International Publication No. 201 7201340, International Publication No. 2017201350, International Publication No. 2017201352, International Publication No. 2017218704, International Publication No. 2017223135, International Publication No. 2018013525, International Publication No. 2018081480, International Publication No. 2018081638, International Publication No. 2018089540, International Publication No. 2018089790, International Publication No. 2018089801, International Publication No. 2018089851, International Publication No. 2018107026, International Publication No. 2018118102, International Publication No. 2018119163, International Publication No. 2018157009, International Publication No. 2018165257, International Publication No. 2018170245, International Publication No. 2018170306, International Publication No. 2018170322, International Publication No. 2018170336, International Publication No. 2018183901, International Publication No. 2018187590, International Publication No. 2018191657, International Publication No. 2018191719, International Publication No. 2018200943, International Publication No. 2018231709, International Publication No. 2018231990, International Publication No. 2018232120, International Publication No. 201823213 8232357, International Publication No. 2019036000, International Publication No. 2019036008, International Publication No. 2019036028, International Publication No. 2019036030, International Publication No. 2019040590, International Publication No. 2019089818, International Publication No. 2019089828, International Publication No. 2019140102, International Publication No. 2019152557, International Publication No. 2019152802, International Publication No. 2019191780, International Publication No. 2019222277, International Publication No. 2019222424, International Publication No. 2019226650,International Publication No. 2019226925, International Publication No. 2019232095, International Publication No. 2019232097, International Publication No. 2019232103, International Publication No. 2019232208, International Publication No. 2020061284, International Publication No. 2020061295, International Publication No. 2020061332, International Publication No. 2020061367, International Publication No. 2020081938, International Publication No. 2020097376, International Publication No. 2020097379, International Publication No. 2020097384, International Publication No. 2020102172, International Publication No. 20202 0106903, International Publication No. 2020146805, International Publication No. 2020214946, International Publication No. 2020219427, International Publication No. 2020227085, International Publication No. 2020232276, International Publication No. 2020243540, International Publication No. 2020257611, International Publication No. 2020257716, International Publication No. 2021007278, International Publication No. 2021016430, International Publication No. 2021022173, International Publication No. 2021026358, International Publication No. 2021030701, International Publication No. 2021046260 , International Publication No. 2021050986, International Publication No. 2021055833, International Publication No. 2021055835, International Publication No. 2021055849, International Publication No. 2021127394, International Publication No. 2021127641, International Publication No. 2021202694, International Publication No. 2021231697, International Publication No. 2021231901, International Publication No. 2008103276, International Publication No. 2009086558, International Publication No. 2009127060, International Publication No. 2010048536, International Publication No. 2010054406, International Publication No. 20 10080724, International Publication No. 2010088537, International Publication No. 2010129709, International Publication No. 201021865, International Publication No. 2011022460, International Publication No. 2011043913, International Publication No. 2011090965, International Publication No. 2011149733, International Publication No. 2011153120, International Publication No. 2011153493, International Publication No. 2012040184, International Publication No. 2012044638, International Publication No. 2012054365, International Publication No. 2012061259, International Publication No. 2013063468,International Publication No. 2013086354, International Publication No. 2013086373, U.S. Patent No. 7893302B2, U.S. Patent No. 7404969B2, U.S. Patent No. 8158601B2, U.S. Patent No. 8283333B2, U.S. Patent No. 8466122B2, U.S. Patent No. 8569256B2, U.S. Patent Application Publication No. 20100036115, U.S. Patent Application Publication No. 20110256175, U.S. Patent Application Publication No. 20120202871, U.S. Patent Application Publication No. 20120027803, U.S. Patent Application Publication No. 20120027803, U.S. Patent Application Publication No. 20120027804, U.S. Patent Application Publication No. 20120027805, U.S. Patent Application Publication No. 20120027806, U.S. Patent Application Publication No. 20120027807, U.S. Patent Application Publication No. 20120027808, U.S. Patent Application Publication No. 20120027809 ... and US Patent Application Publication Nos. 20120128760, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, and 20140039032; the disclosures of said publications specifically relating to cationic or ionizable, neutral, sterol, or aggregation-reducing lipids suitable for lipid-based carriers are incorporated herein by reference.

[0347] For example, suitable cationic lipids or cationizable or ionizable 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-dioleoyltrimethylammonium propane 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-E 12 (WO 2015200465), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 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-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3, U.S. Patent Application Publication No. 20100324120), 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)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N,N 16-Diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z (1,9Z,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), GIBCO / BRL, Grand Island, NY, USA

[0023] LIPOFECTAMINE® (a commercially available cationic liposome comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), available from GIBCO / BRL; and TRANSFECTAM® (a commercially available cationic lipid comprising dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, available from Promega Corp., Madison, Wisconsin, USA), or any combination of any of the foregoing. Further suitable cationic or ionizable lipids include those described in International Patent Publication WO 2010053572 (and in particular, paragraph

[0225] of WO 2010053572).1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol C12-200) and cationic or ionizable lipids described in WO 2012170930 (both of which are incorporated herein by reference), HGT4003, HGT5000, HGTS001, HGT5001, HGT5 002 (see U.S. Patent Application Publication No. 20150140070), 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-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-diiodopropyl)-1,2-propanediol (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-diiodopropyl)-1,2-propanediol (DLin-AP), 3-(N,N-diiodopropyl)-1,2-propanediol (DLin-S-DMA ... Examples of such compounds include 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA, International Publication No. 2010042877); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA); 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA, International Publication No. 2010042877); and dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA).

[0348] Neutral lipids, neutral phospholipids The "neutral lipids," also referred to as "helper lipids" according to the present invention, are preferably phospholipids or neutral phospholipids. As used herein, "neutral phospholipids" are amphipathic compounds consisting of molecules typically 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 constitute a significant proportion of the additives in biological membranes. As used herein, the terms "phospholipid" or "neutral phospholipid" encompass both natural and synthetic phospholipids.

[0349] The terms "neutral lipid," "neutral phospholipid," or "zwitterionic compound," as used interchangeably herein, refer to any one of several lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Exemplary neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, which are further described herein below.

[0350] According to one preferred embodiment, the composition comprises zwitterionic neutral lipid, such as phosphatidylcholine or phosphatidylethanolamine.Examples of suitable phosphatidylcholine include natural or purified mixtures, which are usually derived from egg yolk or soybean, and are sometimes called "lecithin" or "phosphatidylcholine"; or highly purified or semi-synthetic compounds, such as phosphatidylcholine having two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc.

[0351] In another preferred embodiment, the neutral lipid or neutral phospholipid is, but is not limited to, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also known as 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPhyPE ... Dioleoyl-sn-glycero-3-phosphocholine (DPPC, also known as 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 (DSPE), Phosphatidylethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 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-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-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-O-hexadecanyl-2-O-(9Z-o (Cutadecenyl)-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-dicholesterylhemisuccinoyl The zwitterionic compound is selected from the group consisting of 1-O-octadecyl-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 (edelfosine).

[0352] In other preferred embodiments, the lipid nanoparticles of the present invention further comprise "DPhyPS" or "WT-PS" (i.e., 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine or 18:0-18:1PS, which are derived from two different fatty acid / alkyl chains of WT-PS, widely distributed among animals, plants, and microorganisms), 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS, and 18:1-PS DOPS, in which serine is bound to the first carbon atom of glycerol via a phosphodiester, and the second and third carbon atoms of glycerol are each bound to a fatty acid via an ester. The structure of the above phosphatidylserine is as follows (note that all of these lipids are commercially available, for example, from Avanti Polar Lipids):

[0353] [ka]

[0354] [ka]

[0355] Within this group, the two fatty acids may be the same (see, e.g., DPhyPS, 16:0PS, 14:0-PS, 10:0-PS, 6:0-PS, and 18:1-PS DOPS) or different (see, e.g., WT-PS or 18:0-18:1PS). In other examples, such as 18:1-Lyso PS and 18:0-Lyso PS, serine is similarly linked to the first carbon atom of glycerin via a phosphodiester, and only one additional carbon atom of glycerin is linked to a fatty acid via an ester, leaving a single OH group on the remaining carbon atom of glycerin. Such groups are typically referred to as "lysophosphatidylserines," which, in light of the above definition, are included in the term "phosphatidylserine" as used herein. Preferred embodiments related to DPhyPS are described herein in the "Fourth Set of Embodiments" section.

[0356] In another highly preferred embodiment, the lipid nanoparticles of the present invention comprise a phosphatidylserine 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. Most preferably, the phosphatidylserine is either DPhyPS or WT-PS (18:0-18:1PS).

[0357] 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-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC). In an even more preferred, particularly preferred embodiment, the neutral lipid according to the present invention is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE). An advantage of the present invention related to the use of DPhyPE is its high membrane fusion ability due to its bulky tail, which allows for high levels of fusion with endosomal lipids. Thus, in another embodiment, the present invention provides a lipid-based carrier or nucleic acid-lipid particle, preferably DPhyPE, as shown herein:

[0358] [ka]

[0359] The present invention relates to the use of lipids having high membrane fusion properties in the Specifically, the advantageous use of 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) disclosed herein, preferably in combination with the lipids of the present invention disclosed herein, to deliver mRNA vaccines in vivo, resulting in significantly enhanced immune responses, is a surprising discovery made by the inventors and resembles specific aspects and embodiments of the present invention. In other words, the inventors surprisingly discovered that the use of DPhyPE offers distinct advantages over mRNA and also siRNA, particularly, but not limited to, DSPC, which has been used to date in the art as the standard neutral lipid in nearly all prior art LNP compositions for vaccination settings. In other words, the compositions of the present invention have highly advantageous and unpredictable behavior in vivo, resulting in highly enhanced immune responses.

[0360] Furthermore, the data presented in the Examples demonstrate that the compositions of the present invention, i.e., all of the RNA vaccines of the present invention, are useful in significantly enhancing immune responses. Surprisingly, in contrast to prior art knowledge indicating that DSPC is the most common and undisputed neutral lipid for lipid nanoparticles, the present inventors have found that it is preferable to use DPhyPE in mRNA formulations of compositions for producing vaccines.

[0361] DSPC, DOPC or DOPE, which are routinely used in the art as phospholipids in LNPs, each have the structure shown herein below:

[0362] [ka]

[0363] As is clear from the two C 18 It has chain side arms. Surprisingly, in a further aspect of the present invention, the present inventors have found that the addition of a phospholipid having a shorter alkyl chain than, for example, the prior art DSPC or DOPE is highly beneficial to the efficacy of the lipid nanoparticles of the present invention comprising a polymer-conjugated lipid according to formula (I), compared to lipid nanoparticles that do not comprise such a phospholipid. Specifically, the advantageous use of, for example, (07:0)PC (DHPC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine), which has a shorter alkyl chain than the prior art DSPC disclosed herein, preferably in combination with the polymer-conjugated lipid of the present invention disclosed herein, for delivering mRNA vaccines in vivo to produce significantly enhanced immune responses, is another very surprising discovery made by the present inventors and resembles a specific aspect and embodiment of the present invention.

[0364] The structure of (07:0)PC (DHPC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine) is shown herein below:

[0365] [ka]

[0366] The present inventors have further surprisingly found that adding at least one additional neutral lipid, particularly a second neutral lipid, to the neutral lipid can also enhance immune responses (see corresponding Examples). As mentioned above, it is preferred for the (first) neutral lipid of the present invention to have two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc. (This particularly means that the fatty acyl moiety is a fairly long moiety starting from a moiety with 14 carbon atoms). The present inventors have found that adding a neutral lipid with a shorter fatty acyl moiety provides beneficial effects, particularly when the additional neutral lipid has two fatty acid moieties selected from pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, and decanoyl, i.e., a moiety with a maximum of 10 carbon atoms. A particularly preferred additional neutral lipid is 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC). Neutral lipids related to DHPC, such as 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 06:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine), and 10:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), are alternatively included in the present disclosure.

[0367] Therefore, in one aspect of the present invention, the lipid nanoparticles of the present invention are selected from the group consisting of C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 or C14 Preferably, the length is C6, C7, C8, C9, or C 10 In another embodiment of the present invention, the lipid nanoparticles of the present invention comprise a neutral lipid or phospholipid having at least one alkyl chain having a length of C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60 10 , C 11 , C 12 , C 13 or C 14 Preferably, the length is C6, C7, C8, C9, or C 10 The lipid nanoparticles of the present invention further comprise a neutral lipid or phospholipid having at least two alkyl chains, each having a length of C6, C7, or C8, and most preferably having a length of C7. In a preferred embodiment, the lipid nanoparticles of the present invention further comprise DHPC. In a further embodiment, one or more alkyl chains may contain a carbon-carbon double bond.

[0368] In other embodiments, the lipid nanoparticles comprise an additional phospholipid selected from the group consisting of 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 04:0PC (1,2-dibutyryl-sn-glycero-3-phosphocholine), 06:0PC (DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), and 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine).

[0369] In other preferred embodiments, the lipid-based carrier (eg, LNP) comprises a neutral lipid or a phospholipid. The term "neutral lipid" refers to any one of several lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Suitable neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of a neutral lipid for use in the particles described herein is generally guided by considerations, for example, of lipid particle size and stability of the lipid particle in the bloodstream. Preferably, the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipid contains saturated fatty acids with carbon chain lengths ranging from C10 to C20. In another embodiment, neutral lipids with mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C10 to C20 are used. Furthermore, neutral lipids with a mixture of saturated and unsaturated fatty acid chains can be used.

[0370] 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), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), or a mixture thereof.

[0371] In a preferred embodiment, the neutral lipid of the lipid-based carrier (eg, LNP) of the pharmaceutical composition is selected from or derived from 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).

[0372] In other preferred embodiments, the neutral lipid of the lipid-based carrier (eg, LNP) of the pharmaceutical composition is selected from or derived from 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE).

[0373] Thus, in a preferred embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a neutral lipid selected from or derived from DSPC, DHPC, or DPhyPE.

[0374] In a particularly preferred embodiment, the lipid-based carrier of the pharmaceutical composition, preferably LNP, comprises a neutral lipid selected from or derived from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0375] In various embodiments, the molar ratio of cationic lipid to neutral lipid in the lipid-based carrier ranges from about 2:1 to about 8:1. The neutral lipids preferably comprise about 5 mol% to about 90 mol%, about 5 mol% to about 10 mol%, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 mol% of the total lipids present in the lipid-based carrier. In one embodiment, the lipid-based carrier comprises about 0% to about 15% or 45% neutral lipids on a molar basis, e.g., about 3% to about 12%, or about 5% to about 10%. For example, the lipid-based carrier may comprise about 15%, about 10%, about 7.5%, or about 7.1% neutral lipids on a molar basis (based on 100% total moles of lipids in the lipid-based carrier).

[0376] Lipid Nanoparticle Composition The terms " lipid nanoparticle composition " and " composition " are used interchangeably herein.In the context of the present invention, lipid nanoparticle is not limited to any specific form, and should be interpreted as including any form that is produced when cationic lipid and optionally one or more additional lipids are combined, for example, in aqueous environment and / or in the presence of nucleic acid compound.For example, liposome, lipid complex, lipoplex etc. are within the scope of lipid nanoparticle.

[0377] In the context of the present invention, "composition" refers to any type of composition in which the specified components can be incorporated, optionally with any additional excipients, usually with at least one pharmaceutically acceptable carrier or excipient. Thus, the composition can be a dry composition such as a powder or granules, or a solid unit such as a lyophilized form or tablet. Alternatively, the composition can be in liquid form, and each excipient can be incorporated independently in dissolved or dispersed (e.g., suspended 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 the versions of the composition containing nucleic acid cargos, which are described in more detail below.

[0378] In some embodiments, the lipid nanoparticles disclosed herein encapsulating nucleic acids are lyophilized lipid nanoparticles. Lyophilized lipid nanoparticles are obtained by freeze-drying, in which 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 comprise a polymer-conjugated lipid of the present invention, preferably a lipid comprising a polyoxazoline, more preferably a PMOZ-lipid. In some embodiments, the lyophilized lipid nanoparticles disclosed herein comprise a nucleic acid. In some embodiments, the lyophilized lipid nanoparticles disclosed herein comprise a nucleic acid encapsulated in the lipid nanoparticle. In some embodiments, the lyophilized lipid nanoparticles disclosed herein comprise a compound of Formula I. In some embodiments, the lyophilized lipid nanoparticles disclosed herein comprise a PMOZ. In some embodiments, the lyophilized lipid nanoparticles disclosed herein comprise a lipid, a nucleic acid, a compound of Formula I, or any mixture thereof.

[0379] In the compositions of the present invention, the cationic lipid may be present within or as part of a lipid nanoparticle (LNP). In other words, such compositions comprise lipid nanoparticles and the cationic lipid is present in the lipid nanoparticle.

[0380] As used herein, a "nanoparticle" is a submicron particle having any structure or morphology. Submicron particles may also be referred to as colloids or colloidal. With respect to the material on which the nanoparticles are based and the structure or morphology, nanoparticles can be classified as, for example, nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polyplexes, mixed or hybrid complexes, to mention just a few of the possible designations for specific types of nanoparticles.

[0381] As defined above, lipid nanoparticles include any kind 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.

[0382] In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I)) and one or more excipients selected from a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid (e.g., a polymer-conjugated lipid such as the polymer-conjugated lipid described above having Formula (I)). It is presently believed by the inventors that compositions comprising cationic lipids, steroids, neutral lipids, and polymer-conjugated lipids according to Formula (II), as defined herein, exist, at least in an aqueous environment, as compositions comprising lipid nanoparticles typically formed by these excipients.

[0383] LNP can comprise any lipid that can form particles that one or more nucleic acid molecules are bound to or that one or more nucleic acid molecules are encapsulated in.In some embodiments, mRNA, or a part thereof, is encapsulated in the aqueous space that is enclosed by the lipid portion of lipid nanoparticles, or by part or all of the lipid portion of lipid nanoparticles, thereby protecting it from enzymatic degradation or other undesirable effects, such as adverse immune response, that are induced by the mechanism of host organisms or cells.In some embodiments, mRNA, or a part thereof, is associated with lipid nanoparticles.

[0384] As noted, compositions containing the lipid excipients described herein typically form lipid nanoparticles, at least in an aqueous environment. As defined herein, 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, mean diameter may be expressed as the z-average determined by dynamic light scattering. In one embodiment, the composition is a sterile liquid composition containing lipid nanoparticles having a mean hydrodynamic diameter (or mean particle size) of about 30 nm to about 800 nm, as determined by dynamic laser scattering. In various embodiments, the lipid nanoparticles are from about 30 nm to about 150 nm, from about 50 nm to about 200 nm, from about 60 nm to about 200 nm, from about 70 nm to about 200 nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm, from about 90 nm to about 190 nm, from about 90 nm to about 180 nm, from about 90 nm to about 170 nm, from about 90 nm to about 160 nm, from about 90 nm to about 150 nm, from about 90 nm to about 140 nm, from about 90 nm to about 130 nm, from about 90 nm to about 120 nm, from about 90 nm to about 100 nm, from about 70 nm to about 9 ... 0 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 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, or 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, or 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 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 the range of about 60 nm to about 80 nm is also preferred.

[0385] The lipid nanoparticles of the present invention can be produced by the lipid excipient-containing composition described herein and be relatively homogeneous.Polydispersity index (PDI) can be used to indicate the homogeneity of nanoparticle composition, for example, the particle size distribution of nanoparticle composition.A small polydispersity index (for example, less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle compositions of the invention can have a polydispersity index of 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 compositions can be about 0.1 to about 0.2.

[0386] Various optional features, choices, and preferences for the compositions of the present invention are generally described herein; as will be clearly understood by those skilled in the art, all of these also apply to lipid nanoparticles. Similarly, the choices and preferences also apply to compositions comprising such lipid nanoparticles.

[0387] For example, a lipid nanoparticle according to one preferred embodiment comprises a cationic lipid as defined above, a neutral lipid, which may be DPhyPE, a steroid, which may be cholesterol, and a polymer-conjugated lipid according to formula (I): [P]-[linker]-[L] Formula (I) (In the formula, [P] is at least one polyoxazoline (POZ) monomer unit

[0388] [ka]

[0389] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value ranging from about 45 to about 55, preferably n is about 50, or n is selected such that the [P] moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. is a homopolymer moiety comprising [linker] is an optional linker group; [L] is the lipid moiety) a polymer-conjugated lipid, which may be The cationic lipid may optionally be selected from compounds C1-C27 listed in Table 1, or preferably, the cationic lipid is preferably the ionizable lipid structure C24 or a cationic lipid according to "Formula III-3" (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)).

[0390] Thus, in the context of the present invention, the mRNA is preferably comprised in a liquid or semi-liquid composition, wherein the mRNA is complexed or associated with lipid nanoparticles according to one of the preferred embodiments, i.e., in a preferred embodiment, said liquid or semi-liquid composition comprises a complex comprising the mRNA, wherein the complex is preferably present as a lipid nanoparticle as defined herein.

[0391] With regard to the amount of each excipient, it is preferred that cationic lipid is incorporated into lipid nanoparticles or compositions according to the present invention in a relatively high molar amount compared to the molar amount of polymer-conjugated lipid according to formula (I) present.Furthermore, the molar amount of cationic lipid is also preferably higher than the molar amount of neutral lipid in each composition or nanoparticle.Furthermore, the molar amount of steroid is optionally higher than the molar amount of polymer-conjugated lipid according to formula (I).

[0392] In certain embodiments, the polymer-conjugated lipid according to Formula (I) is present in the LNP in an amount of about 1 mol% to about 10 mol%, based on the total lipid content of the nanoparticle. In one embodiment, the polymer-conjugated lipid according to Formula (I) is present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, the polymer-conjugated lipid according to Formula (I) is present in the LNP at about 1 mol% or about 1.5 mol%. In a preferred embodiment, the polymer-conjugated lipid according to Formula (I) is present in the LNP in an amount of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%, preferably 5 mol%, more preferably 2.5 mol%, or also preferably 1.7 mol%, based on the molar percentage composition of all lipid components or excipients relative to 100%.

[0393] In various embodiments, the molar ratio of cationic lipid (e.g., a lipid of Formula (I)) to polymer-conjugated lipid according to Formula (I) ranges from about 100:1 to about 25:1, from about 50:1 to about 25:1, or from about 40:1 to about 25:1.

[0394] In certain embodiments, the LNPs contain one or more additional lipids that stabilize the particle during its formation. Suitable stabilizing lipids include neutral lipids and anionic lipids. In various embodiments, the molar ratio of cationic lipid (e.g., lipid of Formula (I)) to neutral lipid ranges from about 2:1 to about 8:1, from about 3:1 to about 7:1, or from about 4:1 to about 6:1.

[0395] As used herein, lipid nanoparticles are typically formed with each excipient and reflect the same quantitative ratio of excipient as in the overall composition containing the nanoparticles, and therefore references to the molar amount of lipid excipient in a composition of the invention should also be understood to describe the molar amount of each excipient in the lipid nanoparticles contained in the composition.

[0396] Generally, the amount of cationic lipid in the composition (and thus in the lipid nanoparticles) is typically at least about 20 mol%, relative to the total molar amount of all lipid excipients in the composition (or nanoparticles). In another embodiment, the amount of cationic lipid is at least about 25 mol%, or at least 30 mol%, respectively. In other preferred embodiments, the amount of cationic lipid 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, 35, 40, 45, 50, 55, 60, 65, or 70 mol%, or about 40 mol% to about 60 mol%, respectively; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%, respectively.

[0397] The amount of steroid in the composition may optionally be at least about 10 mol%, or may range from about 10 mol% to about 60 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 45 mol%, respectively; e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol%, respectively. For the avoidance of doubt, the molar percentages are relative to the total molar amount of all lipid excipients in the composition.

[0398] Neutral lipid can optionally be present in an amount of at least about 5mol%.In some embodiments, the amount of neutral lipid in the composition is, using the same standard of mole percentage, respectively, about 5mol% to about 25mol%, or about 5mol% to about 15mol%, or about 8mol% to about 12mol%; for example, respectively about 5mol%, 6mol%, 7mol%, 8mol%, 9mol%, 10mol%, 11mol%, 12mol%, 13mol%, 14mol%, 15mol%, 16mol%, 17mol%, 18mol%, 19mol%, 20mol%, 21mol%, 22mol%, 23mol%, 24mol% or 25mol%.

[0399] The amount of polymer-conjugated lipid according to Formula (I) in the composition or lipid nanoparticle can be selected to be, for example, about 0.1 mol% or more. In certain embodiments, the amount of polymer-conjugated lipid according to Formula (I) is about 1 mol% to about 15 mol%, or about 2 mol% to about 12 mol%, also using the total molar amount of all lipid excipients as a molar percentage basis. In certain other embodiments, the composition or lipid nanoparticle contains 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8;2.9;3.0;3.1;3.2;3.3;3.4;3.5;3.6;3.7;3.8;3.9;4.0;4.1;4.2;4.3;4.4;4.5;4.6;4.7;4.8;4.9;5.0;5.1;5.2;5.3;5.4;5.5;5.6;5.7;5.8;5.9;6;6.1;6.2;6 .3;6.4;6.5;6.6;6.7;6.8;6.9;7;7.1;7.2;7.3;7.4;7.5;7.6;7.7;7.8;7.9;8;8.1;8.2;8.3;8.4;8.5;8.6;8.7;8.8;8.9;9;9.1;9.2;9.3;9.4;9.5;9.6;9.7;9.8;9 10.9; 10; 10.1; 10.2; 10.3; 10.4; 10.5; 10.6; 10.7; 10.8; 10.9; 11; 11.1; 11.2; 11.3; 11.4; 11.5; 11.6; 11.7; 11.8; 11.9; or 12 mol% or more of polymer-conjugated lipid. In a preferred embodiment, the content of the polymer-conjugated lipid according to Formula (I) of the present invention is about 1 to 5 mol%, preferably 1.7 mol% or 2.5 mol%, of the total lipid content of the formulation. As a non-limiting preferred example, the lipid nanoparticle contains 5% polymer-conjugated lipid. As another non-limiting preferred example, the lipid nanoparticle contains 10% polymer-conjugated lipid. As another non-limiting example, the lipid nanoparticle contains 7.5% polymer-conjugated lipid.

[0400] In one embodiment, the composition comprises: (a) cationic lipids in an amount of 30–70 mol %; (b) steroids in amounts of 20–50 mol %; (c) neutral lipids in an amount of 5 to 25 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 10 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0401] In another embodiment, the composition comprises: (a) cationic lipids in an amount of 40–70 mol %; (b) steroids in amounts of 20–50 mol %; (c) neutral lipids in an amount of 5 to 15 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 10 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0402] In one embodiment, the composition comprises: (a) cationic lipids in an amount of 20–60 mol %; (b) steroids in amounts of 25–55 mol %; (c) neutral lipids in an amount of 5 to 25 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 15 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0403] In a further embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) steroids in amounts of 25–45 mol %; (c) neutral lipids in an amount of 8 to 12 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 10 mol %, preferably 1.7 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0404] In a further preferred embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) steroids in amounts of 25–45 mol %; (c) neutral lipids in an amount of 8 to 12 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 10 mol %, preferably 1.7 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0405] In a further preferred embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) cholesterol in an amount of 25–45 mol%; (c) a neutral lipid in an amount of 8 to 12 mol% and optionally DHPC in an amount of 1 to 10 mol%; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 3 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0406] In a further preferred embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) cholesterol in an amount of 25–45 mol%; (c) DPhyPE in an amount of 8 to 12 mol % and optionally DHPC in an amount of 1 to 10 mol %; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1 to 3 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0407] In a further preferred embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) cholesterol in an amount of 25–45 mol%; (c) DPhyPE in an amount of 8 to 12 mol % and optionally DHPC in an amount of 1 to 10 mol %; and (d) DMG-PMOZ or DMPE-PMOZ-v1 in an amount of 1–10 mol% (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0408] In a further preferred embodiment, the composition comprises: (a) cationic lipids in an amount of 45–65 mol %; (b) cholesterol in an amount of 25–45 mol%; (c) DPhyPE in an amount of 8 to 12 mol % and optionally DHPC in an amount of 1 to 10 mol %; and (d) DMG-PMOZ or DMPE-PMOZ-v1 in an amount of 1–3 mol% (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0409] In these embodiments, the cationic lipid is preferably a compound selected according to any one of the preferences disclosed herein. For example, the cationic lipid can be selected from the compounds listed in Table 1. Furthermore, these embodiments can also include a steroid, a neutral lipid, and / or a polymer-conjugated lipid selected according to any one of the preferences disclosed herein. In all embodiments in which the compositions or lipid nanoparticles described herein are listed and mol% values ​​are given for each excipient, each amount should be considered relative to the total molar amount of all lipid excipients in the lipid nanoparticle.

[0410] In a further preferred embodiment, the compositions or lipid nanoparticles described herein comprise 59 mol % cationic lipid, 10 mol % neutral lipid, 29.3 mol % steroid and 1.7 mol % polymer-conjugated lipid according to formula (I).

[0411] In one embodiment, a composition or lipid nanoparticle described herein comprises 59 mol% cationic lipid, 10 mol% DPhyPE, 29.3 mol% cholesterol, and 1.7 mol% polymer-conjugated lipid according to formula (I). In one embodiment, a composition or lipid nanoparticle described herein comprises 59 mol% cationic lipid, 10 mol% DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% polymer-conjugated lipid according to formula (I). In one embodiment, a composition or lipid nanoparticle described herein comprises 59 mol% cationic lipid, 10 mol% DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% "DMG-PMOZ"

[0412] [ka]

[0413] Includes. In another embodiment, the composition or lipid nanoparticle described herein comprises 47.4 mol% cationic lipid, 10 mol% neutral lipid, 40.9 mol% steroid, and 1.7 mol% polymer-conjugated lipid according to Formula (I).

[0414] In a further embodiment, the compositions or lipid nanoparticles described herein comprise 47.4 mol% cationic lipid, 10 mol% DPhyPE, 40.9 mol% cholesterol, and 1.7 mol% polymer-conjugated lipid according to Formula (I). In one embodiment, the compositions or lipid nanoparticles described herein comprise 47.4 mol% cationic lipid, 10 mol% DPhyPE, 40.1 mol% cholesterol, and 2.5 mol% polymer-conjugated lipid according to Formula (I). In one embodiment, the compositions or lipid nanoparticles described herein comprise 47.4 mol% cationic lipid, 10 mol% DPhyPE, 40.1 mol% cholesterol, and 2.5 mol% "DMG-PMOZ".

[0415] In a most preferred embodiment, the compositions or lipid nanoparticles described herein comprise 59 mol% cationic lipid (preferably "THIOETHER"), 10 mol% neutral lipid, preferably DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% of a polymer-conjugated lipid described herein above or below, preferably according to formula (I), also preferably selected from the group consisting of PMOZ1, PMOZ2, PMOZ3, PMOZ4, and PMOZ5. In another most preferred embodiment, the compositions or lipid nanoparticles described herein comprise 49 mol% cationic lipid (preferably "THIOETHER"), 10 mol% neutral lipid, preferably DPhyPE, 40.9 mol% cholesterol, and 1.7 mol% of a polymer-conjugated lipid described herein above or below, preferably according to formula (I), also preferably selected from the group consisting of PMOZ1, PMOZ2, PMOZ3, PMOZ4, and PMOZ5. In another most preferred embodiment, the composition or lipid nanoparticle described herein comprises 59 mol% cationic lipid (preferably "THIOETHER"), 10 mol% neutral lipid, preferably DPhyPE, 28.5 mol% cholesterol and 2.5 mol% of a polymer-conjugated lipid described herein above or below, preferably according to formula (I), also preferably selected from the group consisting of PMOZ1, PMOZ2, PMOZ3, PMOZ4 and PMOZ5.

[0416] In yet a most preferred embodiment, the compositions or lipid nanoparticles described herein comprise 59 mol% cationic lipid (preferably "THIOETHER"), 10 mol% neutral lipid, preferably DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% of a polymer-conjugated lipid according to formula (I) described herein above or below, also preferably selected from the group consisting of PMOZ2 and PMOZ4. In another most preferred embodiment, the compositions or lipid nanoparticles described herein comprise 59 mol% C24, 10 mol% DPhyPE, 28.5 mol% cholesterol, and 2.5 mol% PMOZ4.

[0417] In any of the above embodiments in this section disclosing specific compositions or lipid nanoparticles having distinct % values ​​for excipients, where 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as the neutral lipid, in further embodiments, DPhyPE can be replaced with another neutral lipid, preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC). Furthermore, in any of the above embodiments in this section disclosing specific compositions or lipid nanoparticles having distinct % values ​​for excipients, where 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as the neutral lipid, in still further embodiments, DPhyPE can be replaced with another neutral lipid, preferably 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine) or alternatively 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0418] Further preferred lipid compositions according to further specific embodiments of the present invention comprise at least four lipid excipients as disclosed herein in Table E. For example, a preferred lipid composition comprises the excipients disclosed in the "E1" row, which are "C1" (disclosed herein in Table 1) as the cationic lipid, DPhyPE as the neutral lipid, cholesterol as the sterol, and DMG-PMOZ as the polymer-conjugated lipid excipient. As another example, a preferred lipid composition comprises the excipients disclosed in the "E35" row, which are "C12" (disclosed herein in Table 1) as the cationic lipid, DPhyPE as the neutral lipid, cholesterol as the sterol, and DMPE-PMOZ-v1 as the polymer-conjugated lipid excipient.

[0419] [Table 3-1]

[0420] [Table 3-2]

[0421] [Table 4-1]

[0422] [Table 4-2]

[0423] Further, preferred lipid formulations of the present invention are shown in Table F, which shows the distinct mol percentages of at least four lipid excipients of the compositions of the present invention. For example, a preferred lipid composition comprises the mol percentages of lipids disclosed in row "F1," i.e., 59 mol% cationic lipid, 29.3 mol% sterol, 10 mol% neutral lipid, and 1.7 mol% polymer-conjugated lipid. As another example, a preferred lipid composition comprises the mol percentages of lipids disclosed in row "F31," i.e., 45 mol% cationic lipid, 43.5 mol% sterol, 10 mol% neutral lipid, and 1.5 mol% polymer-conjugated lipid.

[0424] [Table 5]

[0425] Therefore, in a further preferred embodiment of the present invention, the composition of the present invention is 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, F30, 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, F67, F68 and F69 The excipient combination designations, in discrete mol percentages, disclosed in Table F, are selected from the group consisting of: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, E19, E20, E21, E22, E23, E24, E25, E26, E27, E28, E29, E 30, E31, E32, E33, E34, E35, E36, E37, E38, E39, E40, E41, E42, E43, E44, E45, E46, E47, E48, E49, E50, E51, E52, E53, E54, E55, E56, E 57, E58, E59, E60, E61, E62, E63, E64, E65, E66, E67, E68, E69, E70, E71, E72, E73, E74, E75, E76, E77, E78, E79, E80, E81, E82, E83, E84, E85, E86, E87, E88, E89, E90, E91, E92, E93, E94, E95, E96, E97, E98, E99, E100, E101, E102, E103, E104, E105, E106, E107 and E108 The excipients disclosed in Table E are selected from the group consisting of:

[0426] The most preferred embodiment is lipid excipient combination E1 with "PMOZ4" as the conjugate lipid and the molar percentages shown in formulation F65. Alternatively, composition can be provided in solid form.Particularly, composition can be provided as sterile solid composition for reconstitution with sterile liquid carrier;In this case, solid composition can further comprise one or more inactive ingredients selected from pH corrector, bulking agent, stabilizer, nonionic surfactant and antioxidant.In this embodiment, sterile liquid carrier is preferably aqueous carrier.

[0427] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic 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 can 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.; Malvern, UK). Depending on the amount and nature of the charged compounds in the lipid nanoparticle, the nanoparticle can be characterized by its zeta potential. In a preferred embodiment, the zeta potential is in the range of about -50 mV to about +50 mV. In another preferred embodiment, the zeta potential is in the range of about -25 mV to about +25 mV. In some embodiments, the zeta potential of the lipid nanoparticles of the present invention can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV. Preferably, the lipid nanoparticles of the present invention exhibit a zeta potential 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.

[0428] In certain embodiments, the LNPs comprise one or more targeting moieties that can target the LNPs to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand that directs the LNPs to a receptor found on the cell surface.

[0429] In certain embodiments, LNPs comprise one or more internalization domains. For example, in one embodiment, LNPs comprise one or more domains that bind to cells and induce internalization of the LNPs. 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 LNPs. In certain embodiments, LNPs can bind to biomolecules in vivo, whereby the LNP-bound biomolecules can be recognized by cell surface receptors and induce internalization. For example, in one embodiment, LNPs bind to systemic ApoE, which leads to uptake of the LNPs and associated cargo. In certain embodiments of the present invention, ApoE can be supplemented into the vehicle or pharmaceutical composition used.

[0430] Preferably, in one embodiment, the composition of the present invention further comprises a bioactive ingredient. In a preferred embodiment of the present invention, the lipid nanoparticles comprise at least one polyoxazoline (POZ) monomer unit.

[0431] [ka]

[0432] wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 or C2 alkyl, and n has an average value in the range of 2 to 200, preferably 20 to 100, more preferably 24 to 26 or 45 to 50, or n is selected such that the [P] moiety has an average molecular weight of 1.5 to 22 kDa, more preferably 2 to 19 kDa, even more preferably about 7.5 kDa or about 15 kDa, preferably 1 to 15 kDa, more preferably 2 to 12.5 kDa, more preferably about 5 kDa or about 10 kDa, and even more preferably about 2 kDa to 2.5 kDa or about 4 kDa to 5 kDa. a polymer-conjugated lipid comprising Preferably, the homopolymer moiety comprising a plurality of monomer units is selected from the group consisting of 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), and poly(2-dimethylamino-2-oxazoline) (PDMAOx), More preferably, it comprises any of the polymer-conjugated lipids described herein above or below, most preferably "PMOZ4."

[0433] Encapsulation / complexation into LNPs In preferred embodiments 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, encapsulated in, partially encapsulated in, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome.

[0434] The nucleic acid (e.g., DNA or RNA) incorporated into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be located completely or partially within the interior space, lipid layer / membrane of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, or may be associated with the outer surface of the lipid layer / membrane. The incorporation of nucleic acids into liposomes / LNPs is referred to herein as "encapsulation," and the nucleic acid, e.g., RNA, is completely contained within the interior space of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. The purpose of incorporating nucleic acids into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes is to protect the nucleic acid, preferably RNA, from an environment that may contain enzymes, chemicals, or conditions that degrade the nucleic acid and / or systems or receptors that cause the nucleic acid to be rapidly excreted. Furthermore, incorporating nucleic acids, preferably RNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes can facilitate uptake of the nucleic acid and thus enhance the therapeutic effect of nucleic acids, e.g., RNA encoding antigenic SARS-CoV-2 (nCoV-2019) proteins. Thus, incorporating nucleic acids, e.g., RNA or DNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes can be particularly suitable for coronavirus vaccines (e.g., SARS-CoV-2 vaccines), e.g., for intramuscular and / or intradermal administration.

[0435] In this context, the terms "complexed" or "associated" refer to the essentially stable combination of a nucleic acid and one or more lipids into a larger complex or aggregate without the use of covalent bonds.

[0436] The term "lipid nanoparticle," also referred to as "LNP," is not limited to any particular form and includes any form produced when cationic lipids and optionally one or more additional lipids are combined, for example, in an aqueous environment and / or in the presence of nucleic acid, e.g., RNA. For example, liposomes, lipid complexes, SNALPs, lipoplexes, etc., all fall within the scope of lipid nanoparticles (LNPs). Thus, a "lipid nanoparticle" (LNP) is a nanoparticle formed by lipids that typically contain at least one amphiphilic membrane-forming lipid, and optionally other lipids, and optionally further contains a cargo material, such as a nucleic acid compound. As used herein, the term "lipid nanoparticle" or "LNP" includes any subtype and form of nanoparticle formed or co-formed by lipids, such as the liposomes and lipoplexes described above.

[0437] Liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes can be of various diameters, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be between 50 nm and 500 nm in diameter.

[0438] The LNPs of the present invention are preferably characterized as microvesicles having an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of LNPs is typically formed by amphiphilic molecules, such as synthetic or natural lipids, containing spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, LNPs typically serve to transport at least one nucleic acid, preferably at least one RNA, to target tissues.

[0439] Thus, 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, thereby forming a lipid nanoparticle (LNP), preferably said LNP being particularly suitable for intramuscular and / or intradermal administration.

[0440] bioactive ingredients As used herein, bioactive ingredient means any compound or material that has biological activity, such that the compound or material is potentially useful in the prevention, management, amelioration, treatment or cure of a disease or condition in a subject, such as an animal, particularly a human subject.

[0441] In one preferred embodiment, active compound is nucleic acid compound.The example of nucleic acid compound that can be potentially used for carrying out the present invention includes: the nucleic acid compound selected from the group consisting of chemically modified or unmodified messenger RNA (mRNA), chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA, viral RNA, replicon RNA and self-replicating RNA or any combination thereof; preferably, bioactive component is mRNA.

[0442] In a preferred embodiment, the nucleic acid compound is complexed or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids) to thereby form a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome. In this context, the term "complexed" or "associated" refers to the essentially stable combination of the nucleic acid compound of the first aspect and one or more lipids into a larger complex or aggregate without the use of a covalent bond.

[0443] In a specific embodiment, the active ingredient may comprise a CRISPR RNA (crRNA) plus tracer RNA (tracrRNA), guide RNA (gRNA) or single guide RNA (sgRNA), and / or donor DNA, combined with a CRISPR endonuclease. Suitably, the CRISPR endonuclease may be provided as a protein or polypeptide, or as an mRNA encoding the CRISPR endonuclease. A composition or formulation comprising this combination is suitable for delivering CRISPR gene editing activity to target cells. In one embodiment, the composition according to the present invention may provide a gRNA and an mRNA encoding a CRISPR endonuclease for separate, sequential, or simultaneous administration. That is, the gRNA and the mRNA may be provided in the same formulation or lipid nanoparticle according to the present invention, or in separate lipid nanoparticles for separate, simultaneous, or sequential administration. Suitably, the ratio of gRNA to mRNA for administration is, for example, 1:1, 1:3, 1:9, or 1:19 (i.e., 50%, 25%, 10%, and 5% guide RNA). In one embodiment, gRNA and mRNA encoding a CRISPR endonuclease, such as cas9, are co-loaded into a formulation according to the present invention. Advantageously, co-loading allows for a higher encapsulation efficiency (EE). Suitably, a formulation or pharmaceutical composition according to the present invention co-loaded with gRNA and mRNA comprises LNPs having an average diameter of between 80 and 160 nm. In one embodiment, the gRNA can be a modified gRNA sequence. Suitable modifications are described, for example, in International Publication Nos. WO 2016089433, WO 2017068377, and PCT / GB2016 / 053312. Other suitable modifications are well known to those skilled in the art.

[0444] "CRISPR endonuclease" refers to an endonuclease that can be used in a CRISPR gene editing composition. Suitable "CRISPR endonucleases" include cas9 and its mutant and modified forms. Thus, the mRNA for use in combination with gRNA encodes a CRISPR endonuclease, preferably cas9. Other "CRISPR endonucleases" include, for example, cpfl. Those skilled in the art will recognize that gRNAs pair with specific "CRISPR endonucleases." Thus, the present invention contemplates compositions using suitable gRNA / endonuclease pairings. Suitably, the gRNA is specific to a target gene, and preferably, the target gene is a gene associated with liver disease.

[0445] In another embodiment, the peptide or protein expressed by the nucleic acid compound is a therapeutic protein, or a fragment or variant thereof, which is useful in the treatment or prevention of a genetic or acquired disease or improves the condition of an individual. In particular, therapeutic proteins play an important role in the design of new therapeutic agents that can, among other functions, modify and repair genetic defects, destroy cancer cells or pathogen-infected cells, treat or prevent immune system disorders, or treat or prevent metabolic or endocrine disorders.

[0446] In another embodiment, the peptide or protein expressed by the nucleic acid compound is an antigen. As defined in more detail herein above, an antigen is a compound or material that can be recognized by the immune system, preferably the adaptive immune system, and elicit, for example, an antigen-specific immune response.

[0447] In some embodiments, the active ingredient is siRNA. siRNA is a small interfering RNA, as described, for example, in International Patent Application No. 2004015107 and PCT / EP03 / 08666. These molecules typically consist of a double-stranded RNA structure containing 15-25, preferably 18-23, nucleotide pairs that can base pair with each other, i.e., are essentially complementary to each other, mediated by Watson-Crick base pairing. One strand of this double-stranded RNA molecule is essentially complementary to a target nucleic acid, preferably mRNA, and the second strand of the double-stranded RNA molecule is essentially identical to the target nucleic acid extension. The siRNA molecule may be flanked by several additional nucleotides on each side and in each extension, which do not necessarily base pair with each other.

[0448] In some embodiments, active ingredient is RNAi.RNAi has essentially the same design as siRNA, but the molecule is significantly longer than siRNA.RNAi molecule typically comprises more than 50 nucleotides and base pairs each.

[0449] In some embodiments, the active ingredient is an antisense nucleic acid. Antisense nucleic acids as used herein are preferably oligonucleotides that hybridize to target RNA, preferably mRNA, based on base complementarity, thereby activating RNase H. RNase H is activated by both phosphodiester-linked and phosphothioate-linked DNA. However, phosphodiester-linked DNA is rapidly degraded by cellular nucleases, whereas phosphothioate-linked DNA is not. Therefore, antisense polynucleotides are only effective as DNA-RNA hybrid complexes. The preferred length of antisense nucleic acids is in the range of 16 to 23 nucleotides. Examples of this type of antisense oligonucleotide are described, inter alia, in U.S. Patent Nos. 5,849,902 and 5,989,912.

[0450] In some embodiments, the active component is a ribozyme. Ribozymes are preferably catalytically active nucleic acids that are essentially composed of RNA and include two parts. The first part exhibits catalytic activity, and the second part is responsible for specific interaction with target nucleic acid. Typically, the interaction between target nucleic acid and the part of the ribozyme is caused by hybridization and Watson-Crick base pairing of essentially complementary stretches of bases on the two hybridizing strands, and the catalytically active part can become active, which means that if the catalytic activity of the ribozyme is phosphodiesterase activity, it will cleave the target nucleic acid intramolecularly or intermolecularly. Ribozymes, their use and design principles are known to those skilled in the art and are described, for example, in Doherty and Doudna (Annu.Ref.Biophys.Biomolstruc.2000:30:457-75).

[0451] In some embodiments, the active ingredient is an aptamer. Aptamers are single- or double-stranded D-nucleic acids that specifically interact with target molecules. The production or selection of aptamers is described, for example, in European Patent No. 0533838. In contrast to RNAi, siRNA, antisense nucleotides, and ribozymes, aptamers do not degrade any target mRNA but specifically interact with the secondary and tertiary structures of target compounds, such as proteins. Upon interacting with a target, the target typically exhibits a change in its biological activity. The length of an aptamer typically ranges from a minimum of 15 to a maximum of about 80 nucleotides, preferably from about 20 to about 50 nucleotides.

[0452] In some embodiments, the active ingredient is a spiegelmer. Spiegelmers are described, for example, in International Publication No. 1998008856. Spiegelmers are molecules similar to aptamers. However, in contrast to aptamers, spiegelmers are entirely or almost entirely composed of L-nucleotides, not D-nucleotides. In other respects, the same as outlined for aptamers applies to spiegelmers, especially with regard to the possible length of spiegelmers.

[0453] Formulation into lipid nanoparticles or lipid-based carriers: In the context of the present invention, a typical "lipid-based carrier" is selected from liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. In the context of the present invention, formulation into lipid nanoparticles refers to the term "lipid-based carrier," which encompasses lipid-based delivery systems for RNA that include a lipid component. The lipid nanoparticles or lipid-based carriers may further include other components suitable for encapsulating / incorporating / complexing RNA, including cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof.

[0454] The RNA of the pharmaceutical composition can be fully or partially incorporated or encapsulated in a lipid-based carrier, and the RNA can be located in 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 can be referred to as "encapsulation." The term "lipid-based carrier" is not limited to any particular form and includes, for example, any form produced when an aggregation-reducing lipid and at least one additional lipid are combined in an aqueous environment, for example, in the presence of RNA. For example, LNPs, liposomes, lipid complexes, lipoplexes, etc., are within the scope of the term "lipid-based carrier." Lipid-based carriers can be of various sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be between 50 and 500 nm in diameter. Liposomes, a specific type of lipid-based carrier, are characterized as microvesicles with an internal aqueous space separated from the external medium by one or more bilayer membranes. In liposomes, at least one RNA is typically located within the internal aqueous space, which is typically surrounded by a portion or the entire lipid portion of the liposome. The liposome bilayer membrane is typically formed by amphiphilic molecules, such as synthetic or natural lipids, which contain spatially separated hydrophilic and hydrophobic domains. Lipid nanoparticles (LNPs), a specific type of lipid-based carrier, are characterized as microscopic lipid particles with a solid or partially solid core. Typically, LNPs do not contain an internal aqueous space separated from the external medium by a bilayer. In LNPs, at least one RNA can be encapsulated or incorporated into a lipid portion of the LNP, or the lipid portion of the LNP can be entirely covered by the lipid portion of the LNP. LNPs can include any lipid capable of forming particles to which RNA can be attached or in which RNA can be encapsulated.Preferably, the lipid-based carrier is particularly suitable for intramuscular and / or intradermal administration.

[0455] 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 a lipid nanoparticle (LNP). In a particularly preferred embodiment, the lipid nanoparticle of the pharmaceutical composition encapsulates at least one RNA of the present invention.

[0456] The terms "encapsulated," such as incorporated, complexed, encapsulated, partially encapsulated, associated, and partially associated, refer to an essentially stable combination of RNA and one or more lipids in a lipid-based carrier (e.g., a larger complex or aggregate), preferably without covalent binding of RNA. RNA encapsulated in a lipid-based carrier may be completely or partially located inside the lipid-based carrier (e.g., in the lipid portion and / or the 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 "incorporation," since the RNA is preferably contained inside the lipid-based carrier. Without 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 an environment that may contain enzymes, chemicals, or conditions that degrade the RNA. Furthermore, incorporating RNA into a lipid-based carrier may facilitate RNA uptake and thus enhance the therapeutic effect of the RNA when administered to a cell or subject.

[0457] The terms "fusogenic" or "fusogenicity" are intended to refer to lipids that aid in the fusion of a lipid-based carrier or nucleic acid-lipid particle with a cell membrane, thereby aiding in the entry of the nucleic acid contained in the lipid-based carrier or nucleic acid-lipid particle into the cell.

[0458] In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition comprises at least one or more lipids selected from at least one aggregation-reducing lipid, at least one cationic lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog.

[0459] In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid, a cationic or ionizable lipid, a neutral lipid or phospholipid, and a steroid or steroid analog.

[0460] The term "PMOZ-LNP" refers to a lipid nanoparticle comprising a polyoxazophospholipid of the present invention, preferably a PMOZ-lipid, as a polymer-conjugated lipid. In a preferred embodiment, the PMOZ-LNP does not comprise a PEG-lipid. In another preferred embodiment, the PMOZ-LNP does not comprise a polymer-conjugated lipid comprising a sulfur (-S-) group. In another preferred embodiment, the PMOZ-LNP does not comprise a lipid covalently coupled to a bioactive component, and the bioactive component is mRNA.

[0461] Aggregation-reducing lipids or polymer-conjugated lipids of the present invention: Under storage conditions or during formulation, lipid-based carriers may undergo charge-induced aggregation, which may be undesirable for the stability of lipid-based carriers.Therefore, it may be desirable to include lipid compounds that can reduce aggregation, for example, by sterically stabilizing lipid-based carriers.Such steric stabilization may occur when the compound has sterically bulky but uncharged moieties, which shield or block the charged moieties of lipid-based carriers from close proximity to other lipid-based carriers in composition.In the context of the present invention, the stabilization of lipid-based carriers is achieved by including lipids, which may include lipids with sterically bulky groups that are preferably located on the outside of lipid-based carriers after the formation of lipid-based carriers.

[0462] The term "aggregation-reducing lipid" or "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a moiety suitable for reducing or preventing aggregation of a lipid-based carrier containing a cargo, preferably mRNA. Thus, an "aggregation-reducing lipid," also referred to herein as a "polymer-conjugated lipid," is a lipid comprising a polymer as an aggregation-reducing group. A polymer, as is clear from the context of the present invention, should be understood as a substance or material consisting of a very large molecule or macromolecule composed of many repeating subunits. A suitable polymer in the context of the present invention may be a hydrophilic polymer. In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid selected from polymer-conjugated lipids.

[0463] In some embodiments, the lipid-based carrier comprises less than about 3 mol%, 2 mol%, or 1 mol% of the aggregation-reducing lipid, based on the total moles of lipid in the lipid-based carrier. In further embodiments, the lipid-based carrier comprises about 0.1% to about 10% on a molar basis, e.g., 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% of the aggregation-reducing lipid or polymer-conjugated lipid, based on 100% total moles of lipid in the lipid-based carrier. In another preferred embodiment, the lipid-based carrier comprises about 1.0% to about 2.0% on a molar basis, e.g., 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%, and most preferably 1.7% of the aggregation-reducing lipid or polymer-conjugated lipid (based on 100% total moles of lipid in the lipid-based carrier). In another preferred embodiment, the lipid-based carrier comprises about 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, preferably 2.5%, of the aggregation-reducing lipid or polymer-conjugated lipid (based on 100% total moles of lipid in the lipid-based carrier). In a highly preferred embodiment, the lipid-based carrier comprises about 2.5% polymer-conjugated lipid on a molar basis (based on 100% total moles of lipid in the lipid-based carrier).

[0464] In various embodiments, the molar ratio of cationic lipid to aggregation-reducing lipid or polymer-conjugated lipid ranges from about 100:1 to about 25:1. Lipid-based carrier composition: In a preferred embodiment, the lipid-based carrier, preferably an LNP, of the pharmaceutical composition comprises at least one RNA as defined in the first aspect, a cationic lipid as defined herein, an aggregation-reducing lipid as defined herein, optionally a neutral lipid as defined herein, and optionally a steroid or steroid analogue as defined herein.

[0465] In a preferred embodiment, the lipid-based carrier comprising at least one RNA of the first aspect is (i) at least one cationic or ionizable lipid, preferably as defined herein; (ii) at least one neutral lipid or phospholipid, preferably as defined herein; (iii) at least one steroid or steroid analogue, preferably as defined herein; and (iv) at least one aggregation-reducing lipid, preferably as defined herein Includes.

[0466] In a preferred embodiment, the lipid-based carrier comprising at least one RNA of the first aspect is (i) at least one cationic lipid selected from or derived from ALC-0315, SM-102, SS-33 / 4PE-15, HEXA-C5DE-PipSS, or compound C26 (see C26 in Table 1 of WO2021123332); (ii) at least one neutral lipid selected from or derived from DSPC, DHPC, or DPhyPE; (iii) at least one steroid or steroid analog selected from or derived from cholesterol; and (iv) at least one aggregation-reducing lipid Includes; The lipid-based carrier encapsulates the RNA.

[0467] In preferred embodiments, cationic lipids (as defined herein), neutral lipids (as defined herein), steroids or steroid analogs (as defined herein), and / or aggregation-reducing lipids (as defined herein) may be combined in various relative ratios.

[0468] In a preferred embodiment, the lipid-based carrier comprises about 20-60% cationic or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analog, and about 0.5-15% aggregation-reducing lipid, e.g., polymer-conjugated lipid, in molar ratios (i)-(iv), preferably, the lipid-based carrier encapsulates RNA.

[0469] For example, the ratio of cationic or ionizable lipid to neutral lipid to steroid or steroid analog to aggregation-reducing lipid can be between about 30-60:20-35:20-30:1-15, or in ratios of about 40:30:25:5, 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3, or 40:33:25:2, respectively.

[0470] In a preferred embodiment, the lipid-based carrier, preferably LNP, comprising at least one RNA according to the first aspect comprises: (i) at least one cationic lipid selected from SM-102: (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid Includes; the lipid-based carrier encapsulates the RNA, preferably (i)-(iv) in a weight ratio of about 50% cationic lipid, about 10% neutral lipid, about 38.5% steroid or steroid analog, and about 1.5% aggregation-reducing lipid; Preferably, the lipid-based carrier encapsulates the RNA.

[0471] In a preferred embodiment, the lipid-based carrier, preferably LNP, comprising at least one RNA according to the first aspect comprises: (i) at least one cationic lipid selected from SM-102; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid Includes; the lipid-based carrier encapsulates the RNA, preferably (i)-(iv) in a weight ratio of about 48.5% cationic lipid, about 11.1% neutral lipid, about 38.9% steroid or steroid analog, and about 1.5% aggregation-reducing lipid; Preferably, the lipid-based carrier encapsulates the RNA. The preferred N / P ratio for this formulation is about 4.85 (molar ratio of lipid to RNA).

[0472] In a preferred embodiment, the lipid-based carrier, preferably LNP, comprising at least one RNA according to the first aspect comprises: (i) at least one cationic lipid selected from SS-33 / 4PE-15, HEXA-C5DE-PipSS, or compound C26 (see C26 in Table 1 of WO2021123332); (ii) at least one neutral lipid selected from DPhyPE; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid Includes; The lipid-based carrier encapsulates the RNA. Such LNPs are referred to herein as GN-LNPs.

[0473] In a preferred embodiment in that context, the RNA-containing lipid-based carrier, preferably GN-LNP, comprises 59 mol% HEXA-C5DE-PipSS lipid (see compound C2 in Table 1 of WO2021123332) or preferably 59 mol% compound C26 (see C26 in Table 1 of WO2021123332) as cationic lipid, 10 mol% DPhyPE as neutral lipid, 29.3% cholesterol as steroid and 1.7 mol% aggregation-reducing lipid.

[0474] In another preferred embodiment in that context, the RNA-containing lipid-based carrier, preferably GN-LNP, comprises 59 mol% of compound C26 (see C26 in Table 1 of WO2021123332) as a cationic lipid, 10 mol% of DPhyPE as a neutral lipid, 28.5 mol% of cholesterol as a steroid and 2.5 mol% of an aggregation-reducing lipid.

[0475] In a preferred embodiment, the lipid to RNA wt / wt ratio in the lipid-based carrier is about 10:1 to about 60:1, e.g., about 40:1. In a particularly preferred embodiment, the lipid to RNA wt / wt ratio is about 20:1 to about 30:1, e.g., about 25:1. In other preferred embodiments, the lipid to RNA wt / wt ratio is in the range of 20 to 60, preferably about 3 to about 15, about 5 to about 13, about 4 to about 8, or about 7 to about 11.

[0476] The amount of lipid included in the lipid-based carrier can be selected taking into account the amount of RNA cargo. In one embodiment, these amounts are selected to result in an N / P ratio of the lipid-based carrier encapsulating RNA ranging from about 0.1 to about 20. The N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid to the phosphate group ("P") of the RNA used as cargo. The N / P ratio can be calculated, for example, based on the fact that 1 μg of RNA typically contains about 3 nmol of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The "N" value of a lipid or lipidoid can be calculated based on its molecular weight and the relative content of persistent cationic groups and, if present, cationizable groups.

[0477] In embodiments, the N / P ratio can range from about 1 to about 50. In other embodiments, the range is from about 1 to about 20, preferably from about 1 to about 15. For "GN-LNP," a preferred N / P (molar ratio of lipid to RNA) is about 14 or about 17. A more preferred N / P, i.e., a molar ratio of lipid to RNA, is about 6. Another preferred N / P ratio is about 4.85 or 5 (molar ratio of lipid to RNA).

[0478] In various embodiments, the pharmaceutical composition comprises a lipid-based carrier (encapsulating the RNA) having a defined size (particle size, uniform size distribution). The diameter of the lipid-based carrier of a pharmaceutical composition is typically described herein as the Z-average diameter. The terms "average diameter," "mean diameter," "diameter," or "size" of particles (e.g., lipid-based carriers) are used synonymously with the Z-average value. The term "Z-average size" refers to the average diameter of particles measured by dynamic light scattering (DLS) using data analysis using the so-called cumulant algorithm, resulting in the so-called Z-average, which has a length dimension, and a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321).

[0479] The term "dynamic light scattering" or "DLS" refers to a method of analyzing particles in a liquid by illuminating the liquid with a typically monochromatic light source and detecting the light scattered by the particles in the liquid. DLS can thus be used to measure particle size in a liquid. Suitable DLS protocols are known in the art. DLS instruments are commercially available (e.g., Zetasizer Nano series, Malvern Instruments, Worcestershire, UK). DLS instruments employ either a 90° detector (e.g., Wyatt Technology's DynaPro® NanoStar® or Malvern Instruments' Zetasizer Nano S90®) or a backscattering detection system with 173° (e.g., Malvern Instruments' Zetasizer Nano S®) and 158° (Malvern Instruments' DynaPro Plate Reader®) near 180° incident light. DLS measurements are typically performed at a temperature of approximately 25°C. DLS is also used in the context of the present invention to determine the polydispersity index (PDI) and / or main peak diameter of lipid-based carriers incorporating RNA.

[0480] In various embodiments, the lipid-based carrier of the pharmaceutical composition encapsulating RNA has a diameter of about 50 nm to about 200 nm, about 50 nm to about 190 nm, about 50 nm to about 180 nm, about 50 nm to about 170 nm, about 50 nm to about 160 nm, 50 nm to about 150 nm, 50 nm to about 140 nm, 50 nm to about 130 nm, 50 nm to about 120 nm, 50 nm to about 110 nm, 50 nm to about 100 nm, 50 nm to about 90 nm, 50 nm to about 80 nm, 50 nm to about 70 nm, 50 nm to about 60 nm, 60 nm to about 200 nm, about 60 nm to about 190 nm, about 60 nm to about 180 nm, about 60 nm to about 170 nm, about 60 nm to about The Z-average diameter may be in the range of 160 nm, 60 nm to about 150 nm, 60 nm to about 140 nm, 60 nm to about 130 nm, 60 nm to about 120 nm, 60 nm to about 110 nm, 60 nm to about 100 nm, 60 nm to about 90 nm, 60 nm to about 80 nm, or 60 nm to about 70 nm, for example, about 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.

[0481] In a preferred embodiment, the lipid-based carrier of the pharmaceutical composition encapsulating the RNA has a Z-average diameter in the range of about 50 nm to about 200 nm, preferably about 50 nm to about 150 nm, more preferably about 50 nm to about 120 nm, and also more preferably about 65 nm to about 90 nm.

[0482] Preferably, the pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of lipid-based carriers having a particle size greater than about 500 nm. Preferably, the pharmaceutical composition contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of LNPs having a particle size smaller than about 20 nm.

[0483] Preferably, at least about 80%, 85%, 90%, 95% of the lipid-based carrier of the composition has a spherical morphology. In preferred embodiments, the polydispersity index (PDI) of the lipid-based carrier is typically in the range of 0.1 to 0.5. In certain embodiments, the PDI is less than 0.2. Typically, the PDI is determined by dynamic light scattering.

[0484] In a preferred embodiment, 80% of the RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, preferably 85% of the RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, more preferably 90% of the RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, and most preferably 95% of the RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier.The encapsulation percentage can be determined by RiboGreen assay known in the art.

[0485] According to a preferred embodiment, the lipid-based carrier, preferably encapsulating or containing RNA, is purified by at least one purification step, preferably by at least one TFF step and / or at least one clarification step and / or at least one filtration step.

[0486] mRNA In one preferred embodiment, the nucleic acid compound is mRNA or mRNA compound.As the present inventors have found, the lipid and composition of the present invention are particularly suitable for the in vivo delivery of mRNA compound expressing antigen, thus enabling the highly effective, potent, versatile and safe vaccine that can be developed quickly and at reasonable cost....

Claims

1. Polymer-conjugated lipids according to formula (I): [P]-[linker]-[L] Formula (I) or a pharmaceutically acceptable salt, prodrug, tautomer or stereoisomer thereof (In the formula, [P] is a homopolymer moiety containing poly(2-methyl-2-oxazoline) (PMeOz50) having 50 repeats; [Linker] is 【Chemistry 1】 having the structure [L] is a lipid moiety containing ditetradecylamine, and the linker moiety [linker] (-NHC(O)CH ２ CH ２ C(O)?) forms an amide bond by bonding to the N atom of ditetradecylamine).

2. The polymer-conjugated lipid may be "PMOZ4" having an average value of n between 45 and 50, most preferably 50. 【Chemistry 2】 That is, The polymer-conjugated lipid of claim 1.

3. A lipid nanoparticle comprising the polymer-conjugated lipid of claim 1.

4. 4. The lipid nanoparticle of claim 3, comprising a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% neutral lipid, about 25-55% sterol or steroid analog, and about 0.5-15% aggregation-reducing lipid that is a polymer conjugate of claim 1, preferably encapsulating RNA.

5. (i) 59 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 29.3 mol% cholesterol, 10 mol% neutral lipid, and 1.7 mol% polymer-conjugated lipid of claim 1; (ii) 59 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 28.5 mol% cholesterol, 10 mol% neutral lipid, and 2.5 mol% polymer-conjugated lipid of claim 1; (iii) 59 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 28.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 1 mol% DHPC, and 2.5 mol% polymer-conjugated lipid according to claim 1; (iv) 49 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C24 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 29.3 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, 10 mol% DHPC, and 2.5 mol% polymer-conjugated lipid according to claim 1; (v) 47.4 mol% cationic lipid or ionizable lipid, preferably one of the ionizable lipid structures C1 to C27 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 40.9 mol% cholesterol, 10 mol% DSPC or DPhyPE, preferably DPhyPE, and 1.7 mol% polymer-conjugated lipid according to claim 1; (vi) 47.4 mol% cationic or ionizable lipid, preferably one of the ionizable lipid structures C1 to C27 listed in Table 1, more preferably ionizable lipid structure C24 listed in Table 1, 40.1 mol% cholesterol, 10 mol% DSPC, and 2.5 mol% polymer-conjugated lipid according to claim 1; and most preferably, (vii) 59 mol% of a C24 listed in Table 1, 28.5 mol% of cholesterol, 10 mol% of DPhyPE, and 2.5 mol% of the polymer-conjugated lipid of claim 1. and the excipients are selected from the ratio selected from the group consisting of:

4. The lipid nanoparticle of claim 3, wherein the polymer-conjugated lipid has an average n in the range of about 45 to about 55, preferably n is about 50, or n is selected such that the polymer moiety has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa. Table 1

6. The lipid nanoparticle of claim 3, further comprising a biologically active component, preferably the biologically active component is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof, more preferably the biologically active component is chemically modified mRNA or chemically unmodified mRNA.

7. the mRNA is associated with lipid nanoparticles, preferably the mRNA is encapsulated in lipid nanoparticles, and / or and / or comprising an amount of said mRNA to achieve an N / P ratio in the range of about 5 to about 20, more preferably about 10 to about 18, even more preferably about 12 to about 16, and most preferably about 14; and / or a sterile solid composition for reconstitution with a sterile liquid carrier, further comprising one or more inactive ingredients selected from pH modifiers, bulking agents, stabilizers, non-ionic surfactants, and antioxidants, wherein the sterile liquid carrier is an aqueous carrier; and / or a sterile liquid composition having a mean hydrodynamic diameter as determined by dynamic light scattering of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, or about 60 nm to about 200 nm, or about 70-200 nm, or about 75 nm to about 160, or about 85 nm to about 140 nm, or about 90 nm to about 130 nm, or about 50 nm to about 120 nm; and / or exhibiting 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, most preferably in the range of -5 mV to +5 mV; and / or the mRNA compound is a mono-, bi-, or multicistronic mRNA, and / or said mRNA compound comprises at least one chemical modification, said chemical modification being selected from the group consisting of a base modification, a sugar modification, a backbone modification and a lipid modification, preferably said chemical modification is a base modification, more preferably said base modification being preferably selected from the group consisting of pseudouridine (psi or ψ), N1-methylpseudouridine (N1MPU, N1Mpsi or N1Mψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof; and / or the mRNA compound comprises a coding region encoding a peptide or protein, the coding region exhibiting a sequence modification, the sequence modification being selected from a G / C content modification of the sequence, a codon modification, codon optimization or C optimization; preferably, compared to the coding region of the corresponding wild-type mRNA, - the coding region has an increased G / C content; - the coding region has an increased C content; - the codon usage of said coding region is adapted to human codon usage; and / or The lipid nanoparticle of claim 3, wherein the codon adaptation index (CAI) is increased or maximized in the coding region.

8. the mRNA compound a) a 5' cap structure, preferably m7GpppN, more preferably cap1 or m7G(5')ppp(5')(2'OMeA)pG; b) optionally, but preferably, at least one miRNA sequence, wherein the microRNA binding site is for a microRNA 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) a coding sequence; e) at least one 3'-UTR element; f) at least one poly(A) sequence; g) at least one poly(C) sequence; or any combination of these The lipid nanoparticle of claim 3, further comprising:

9. the at least one coding RNA comprises a 5' cap structure, preferably an m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, and / or the at least one coding RNA comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR, preferably wherein the at least one heterologous 5'-UTR comprises a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homologue, fragment or variant of any one of these genes; and / or Preferably, said at least one heterologous 3'-UTR comprises a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homologue, fragment or variant of any one of these genes; and / or the at least one coding RNA comprises (i) HSD17B4 5'-UTR and PSMB3 3'-UTR, or (ii) RPL32 5'-UTR and ALB7 3'-UTR, preferably a mutant alpha-globin 3'-UTR (SEQ ID NO: 11 / 12), more preferably HSD17B4 5'-UTR (SEQ ID NO: 21 / 22) and PSMB3 3'-UTR (SEQ ID NO: 19 / 20); and / or In the 5' to 3' direction, the following elements: a) a 5' cap structure, preferably selected from the group consisting of m7G(5'), m7G(5')ppp(5')(2'OMeA)pG and m7G(5')ppp(5')(2'OMeG)pG; b) a 5'-UTR element comprising a nucleic acid sequence derived from the 5'-UTR of a TOP gene, said nucleic acid sequence preferably comprising an RNA sequence corresponding to a nucleic acid sequence according to SEQ ID NO: 22, 24, 26, or a homologue, fragment or variant thereof, most preferably a nucleic acid sequence according to SEQ ID NO: 22 (HSD17B4); c) at least one coding sequence; d) a 3'-UTR element comprising a nucleic acid sequence derived from an α-globin gene, said nucleic acid sequence preferably comprising an RNA sequence corresponding to a nucleic acid sequence according to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, or a homolog, fragment or variant thereof; and / or a 3'-UTR element comprising a nucleic acid sequence derived from an albumin gene, said nucleic acid sequence preferably comprising an RNA sequence corresponding to a nucleic acid sequence according to SEQ ID NO: 18, or a homolog, fragment or variant thereof, most preferably a nucleic acid sequence according to SEQ ID NO: 20 (PSMB3); e) optionally at least one poly(A) sequence, preferably consisting of 10 to 200, 10 to 100, 40 to 80, or 50 to 70 adenosine nucleotides; f) optionally at least one poly(C) sequence, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides; and g) optionally at least one histone stem loop, preferably comprising an RNA sequence according to SEQ ID NO: 4 The lipid nanoparticle of claim 3, comprising:

10. The bioactive ingredient (a) an mRNA comprising at least one coding sequence encoding a peptide or protein, or a fragment or variant thereof, wherein said peptide or protein is an antigen, and said antigen is preferably derived from a pathogen antigen, a tumor antigen, an allergen antigen, or an autoimmune autoantigen, or a fragment or variant thereof; or (b) an mRNA comprising at least one coding sequence encoding a therapeutic protein, or a fragment or variant thereof, wherein said therapeutic protein is (i) therapeutic proteins for use in enzyme replacement therapy to treat metabolic, endocrine or amino acid disorders, or for use in replacing absent, defective or mutated proteins; (ii) a therapeutic protein for use in treating a blood disorder, a disease of the circulatory system, a disease of the respiratory system, an infectious disease or an immune deficiency; (iii) a therapeutic protein for use in the treatment of cancer or tumor diseases; (iv) therapeutic proteins for use in hormone replacement therapy; (v) therapeutic proteins for use in reprogramming somatic cells into pluripotent or totipotent stem cells; (vi) therapeutic proteins for use as adjuvants or immunostimulants; (vii) a therapeutic protein that is a therapeutic antibody; (viii) a therapeutic protein that is a gene editing agent; and (ix) A therapeutic protein for use in the treatment or prevention of a liver disease selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer. mRNA selected from the group consisting of and Preferably, said at least one coding sequence encoding a pathogenic antigen is selected from the group consisting of bacterial, viral, fungal and protozoan antigens, more preferably said at least one coding sequence encoding a pathogenic antigen is selected from the group consisting of: (i) SARS coronavirus 2 (SARS-CoV-2), nCov-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue viruses (DENV-1, DENV-2, DENV-3 and DENV-4), Ebola virus, Epstein-Barr virus (EBV), flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (HMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extraintestinal pathogenic E. coli (ExPEC), Lassa-Mam Arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes chlamydia), or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale); and / or (ii) derived from a structural protein, accessory protein, or replicase protein from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), or an immunogenic fragment or immunogenic variant of any of these; and / or (iii) derived from the spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (N) from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), or an immunogenic fragment or immunogenic variant of any of these (preferably, said spike protein (S) comprises or consists of spike protein fragment S1 or spike protein fragment S2, more preferably spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof (e.g., receptor binding domain (RBD), primary neutralization domain (CND))); and / or (iv) The lipid nanoparticle of claim 6, which is derived from a prefusion-stabilizing spike protein (S) (S_stab) from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, or SARS coronavirus (SARS-CoV), comprising at least one prefusion-stabilizing mutation.

11. (i) in the treatment or prevention of an infectious disease; a cancer or neoplastic disease, disorder or condition; a liver disease selected from the group consisting of liver fibrosis, cirrhosis and liver cancer; an allergy; or an autoimmune disease, disorder or condition; and / or (ii) for use in enzyme replacement therapy to treat metabolic or endocrine disorders or for use in replacing absent, defective or mutated proteins; and at least one coding RNA, which comprises at least one coding sequence encoding at least one peptide or protein for use, preferably for use in the treatment or prevention of a disease, disorder or condition, and which is administered to a subject in need thereof by local or locoregional injection, infusion or implantation, in particular by intradermal, subcutaneous, intramuscular, intracameral, subconjunctival, suprachoroidal injection, subretinal, subtenon, retrobulbar, topical, retropleural administration, or by pulmonary inhalation, interstitial, locoregional, intravitreal, intratumoral, intralymphatic, intranodal, intraarticular, intrasynovial, periarticular, intraperitoneal, intraabdominal, intracardiac, intralesional, intrapericardial, intraventricular, intrapleural, perineural, 11. The lipid nanoparticle of any one of claims 3 to 10, which is administered via intrathoracic, epidural, intradural, peridural, intrathecal, intramedullary, intracerebral, intracavity, intracavernosal, intraprostatic, intratesticular, intrachondral, intraosseous, intradiscal, intraspinal, intrasacral, intracapsular, intragingival, intraovarian, intrauterine, intraocular, periocular, periodontal, retrobulbar, subarachnoid, subconjunctival, suprachoroidal injection, infusion, implantation, nasal, buccal, sublingual, auricular, ocular, conjunctival, vaginal, rectal, intracervical, intranasal, laryngeal, oropharyngeal, ureteral, urethral administration, more preferably administered intramuscularly, intravenously, intradermal, subcutaneous, intratumoral, intranasal, or by inhalation, most preferably intramuscularly, to a subject in need thereof.

12. A kit or kit-of-parts comprising the lipid nanoparticles described in claim 3, optionally including a liquid vehicle for solubilization and optionally technical instructions providing information on the use and dosage of the components.

13. Lipid nanoparticles according to any one of claims 3 to 10 or kit or kit-of-parts according to claim 12 for use in in vivo drug delivery, preferably for use in delivering nucleic acids, preferably mRNA.

14. The lipid nanoparticles according to any one of claims 3 to 10 or the kit or kit-of-parts according to claim 12, for use as a medicament, preferably for preventing, prophylactically, treating and / or ameliorating a disease selected from infectious diseases including viral, bacterial or protozoological infectious diseases, cancer or tumor diseases, liver diseases, autoimmune diseases, allergies, monogenic diseases including genetic diseases, genetic diseases in general, diseases that have a genetic background and are typically caused by defined gene defects and are inherited according to Mendelian laws; cardiovascular diseases, neurological diseases, diseases of the respiratory system, diseases of the digestive system, diseases of the skin, musculoskeletal disorders, connective tissue disorders, neoplasms, immunodeficiencies, endocrine, nutritional and metabolic diseases, eye diseases, ear diseases, and diseases associated with peptide or protein deficiencies.

15. The lipid nanoparticle for use as a medicament according to claim 14, wherein the medicament is a vaccine composition.

16. A vaccine composition comprising the lipid nanoparticles of any one of claims 3 to 10 or the kit or kit-of-parts of claim 12 for use as a medicament and / or for preventing, prophylactic, treating and / or ameliorating a disease selected from infectious diseases, including viral, bacterial or protozoological infectious diseases, cancer or tumor diseases.

17. C ５ , C ６ , C ７ , C ８ , C ９ , C １０ , C １１ , C １２ , C １３ Or C １４ Preferably, C ６ , C ７ , C ８ , C ９ , or C １０ More preferably, C ６ , C ７ , C ８ most preferably C ７ or even most preferably, a combination of two neutral lipids, said combination being a neutral lipid or phospholipid having at least two alkyl chains, each alkyl chain independently, preferably C ６ , C ７ , C ８ , C ９ , or C １０ More preferably, C ６ , C ７ , C ８ most preferably C ７ and / or most preferably a phospholipid selected from the group consisting of 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 04:0PC (1,2-dibutyryl-sn-glycero-3-phosphocholine), 06:0PC (DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine), 07:0PC (DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), and 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine), preferably 07:0PC (DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine); and / or The lipid nanoparticles comprise a neutral lipid or a phospholipid having at least two alkyl chains, each alkyl chain independently selected from the group consisting of C ５ , C ６ , C ７ , C ８ , C ９ , C １０ , C １１ , C １２ , C １３ Or C １４ Preferably, C ６ , C ７ , C ８ , C ９ , or C １０ More preferably, C ６ , C ７ , C ８ most preferably C ７ or even most preferably, the lipid nanoparticles comprise a combination of two neutral lipids, said combination being a neutral lipid or phospholipid having at least two alkyl chains, each alkyl chain independently having a length of preferably C ６ , C ７ , C ８ , C ９ , or C １０ More preferably, C ６ , C ７ , C ８ most preferably C ７ and most preferably a phospholipid selected from the group consisting of 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 04:0PC (1,2-dibutyryl-sn-glycero-3-phosphocholine), 06:0PC (DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine), 07:0PC (DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), and 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine), preferably 07:0PC (DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine).

18. A vaccine composition comprising the lipid nanoparticles of claim 3 or 4 or the polymer-conjugated lipids of claim 1 or 2.

Images