Lipid nanoparticle formulation and preparation method therefor

Abstract

A lipid nanoparticle formulation having remarkably improved stability and a preparation method therefor. The lipid nanoparticle formulation comprises a pharmaceutically acceptable buffer solution, lipid nanoparticles and a nucleic acid encapsulated in the lipid nanoparticles.

Description

Lipid nanoparticle formulations and methods of making the same

[0001] This application claims priority to International Application No. PCT / CN2024 / 086570 filed on April 8, 2024. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine. Specifically, the present application relates to lipid nanoparticle formulations and methods of making the same. BACKGROUND

[0003] To combat the prevalence of diseases, mRNA-based vaccines have been rapidly developed and applied. Lipid nanoparticles (LNPs) can effectively deliver nucleic acids and protect nucleic acids from degradation by nucleases.

[0004] Although LNP technology is an effective way to deliver mRNA into the body, certain chemical actions such as oxidation, hydrolysis or transesterification during the storage of mRNA vaccines will lead to the degradation of the intact mRNA backbone into smaller fragments.

[0005] Many LNP compositions have common chemical properties, such as the use of ionizable amino cationic lipids. Packer et al. found that ionizable lipids in LNP components can generate aldehyde impurities through oxidation and hydrolysis pathways, which can covalently add to mRNA to form lipid addition products that form late eluting-peak (LP) after the main peak in HPLC detection, resulting in the loss of activity of this part of mRNA (Packer, M.; Gyawali, D.; Yerabolu, R.; Schariter, J.; White, P. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat. Commun. 12, 6777 (2021)).

[0006] The raw materials, production processes and packaging materials contacted by mRNA-LNP can introduce metal ions, which are not conducive to the long-term stability of the preparation. At the same time, it has been found that metal ions such as Mg(II), Zn(II), Ca(II) can hydrolyze the sugar-phosphate backbone of mRNA and have a tendency to accelerate the degradation of mRNA (Pogocki, D. & Schoneich, C. Chemical stability of nucleic acid-derived drugs. J. Pharm. Sci. 89, 443-456 (2000)).

[0007] Therefore, there is still a desire in the field of biomedicine to develop formulations that allow for a higher mRNA stability. SUMMARY

[0008] The present invention aims at developing new formulations comprising nucleic acids, wherein the nucleic acid stability is higher.

[0009] Therefore, according to a first aspect of the present invention, there is provided a lipid nanoparticle formulation, characterized in that it comprises:

[0010] a pharmaceutically acceptable buffer comprising an aldehyde scavenger and a chelating agent,

[0011] a lipid nanoparticle, and

[0012] a nucleic acid encapsulated in the lipid nanoparticle.

[0013] According to a second aspect of the present invention, there is provided a method for preparing the above-mentioned lipid nanoparticle formulation, characterized in that it comprises the following steps:

[0014] I) providing a lipid working solution and a nucleic acid working solution;

[0015] II) using the lipid working solution and the nucleic acid working solution to prepare a lipid nanoparticle encapsulating a nucleic acid; and

[0016] III) performing a dialysis exchange so that the buffer comprises an aldehyde scavenger and a chelating agent.

[0017] The inventors have found that by adding an aldehyde scavenger and a chelating agent to the buffer, the chemical stability of the resulting formulation is significantly improved.

[0018] According to a third aspect of the present invention, there is provided a pharmaceutical product, characterized in that it comprises: a lipid nanoparticle formulation of the present invention; and a container containing the lipid nanoparticle formulation. BRIEF DESCRIPTION OF DRAWINGS

[0019] The present invention is explained and illustrated in more detail below with reference to the enclosed drawings, in which:

[0020] Figure 1 shows the intact mRNA, adducts and degraded mRNA content of the formulation without aldehyde scavenger and the formulations with different types of aldehyde scavengers in Example 1 after 1 week at 25°C.

[0021] Figure 2 shows the mRNA purity results of the formulation without antioxidant and the formulations with different types and concentrations of antioxidants in Example 2 over a period of 2 weeks at 37°C.

[0022] Figure 3 shows the mRNA purity results of the formulation without chelating agent and the formulations with different types and concentrations of chelating agents in Example 3 over a period of 2 weeks at 37°C.

[0023] Figure 4 shows the mRNA purity results of the formulations with 0-0.1 mM DTPA added in Example 3 during 2 weeks at 37 °C.

[0024] Figure 5 shows the physicochemical properties (particle size, PDI and entrapment efficiency) of formulations 1, 3, 5, 7 and 9 in Example 4 during storage.

[0025] Figure 6 shows the mRNA purity results of formulations 1 to 10 in Example 4 during 2 weeks of storage.

[0026] Figure 7 shows the mRNA purity results of formulations containing different LNP-mRNA in Example 5 during 2 weeks at 37 °C.

[0027] Figure 8 shows the lipid adduct product results of formulations containing different LNP-mRNA in Example 5 during 2 weeks at 37 °C.

[0028] Figure 9 shows the physicochemical properties (particle size, PDI and entrapment efficiency) of formulations with or without propylene glycol added in Example 6 during 2 weeks at 37 °C.

[0029] Figure 10 shows the insoluble particulate results of formulations with or without propylene glycol added in Example 6 after 2 days of shaking.

[0030] Figure 11 shows the insoluble particulate results of formulations with additional sucrose in Example 7 after 2 days of shaking.

[0031] Figure 12 shows the physicochemical properties (particle size, PDI and entrapment efficiency) of formulations in Example 8 during long-term storage at 2-8 °C.

[0032] Figure 13 shows the mRNA purity results of formulations in Example 8 during long-term storage at 2-8 °C.

[0033] Figure 14 shows the adduct product results of formulations in Example 8 during long-term storage at 2-8 °C.

[0034] Figure 15 shows the physicochemical properties (particle size, PDI and entrapment efficiency) of formulations with different concentrations of Tris added in Example 9 during 1 week at 40 °C.

[0035] Figure 16 shows the mRNA purity results of formulations with different concentrations of Tris added in Example 9 during 2 weeks at 40 °C.

[0036] Figure 17 shows the lipid adduct product results of formulations with different concentrations of Tris added in Example 9 during 1 week at 40 °C. DETAILED DESCRIPTION

[0037] The aspects of the application will be more fully understood in connection with some specific embodiments, and further objects, features and advantages will become apparent when that description is read in conjunction with the accompanying drawings.

[0038] All patents, patent applications, scientific publications, manufacturer's specifications and descriptions, and the like, cited herein are hereby incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the patent disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. Also, all patents, patent applications, scientific publications, manufacturer's specifications and descriptions, and the like, cited herein are hereby incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the patent disclosure is not entitled to antedate such publication by virtue of prior disclosure. nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Also, for better understanding of the present application, definitions and explanations of relevant terms are provided below.

[0040] As used herein, the expressions "comprise", "comprising", "contain", "containing", and "have" are open-ended, meaning including but not limited to the listed elements, steps or components. The expression "consisting of" excludes any element, step or component not specified. The expression "consisting essentially of" means limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. It is understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising".

[0041] As used herein, the term "one or more" or "at least one" covers 1, 2, 3, 4, 5, 6, 7, 8, 9, or more, unless the context indicates otherwise.

[0042] As used herein, the conjunctive term "and / or" between elements of a plurality of recited elements is to be understood to include the options individually and in combination. In other words, "and / or" includes both "and" and "or". For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements defined with "and / or" are understood in a similar manner and include any of the same and any combination thereof.

[0043] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No implication exists, unless otherwise indicated, that the upper or lower limits of a range itinerated are also necessarily the limits of a range also itinerated. Unless specifically stated otherwise, nor does any representation imply that a given numerical property is a desired property.

[0044] In this document, "nucleotides" include deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, "ribonucleotides" are the constituent materials of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. While "deoxyribonucleotides" are the constituent materials of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydrogen instead of a hydroxyl group at the 2' position of the β-D-ribofuranosyl group, and is the primary chemical constituent of chromosomes. "Nucleotides" are generally referred to by a single letter representing the base therein: "A (a)" refers to a deoxyadenylate or adenylate containing adenine, "C (c)" refers to a deoxycytidylate or cytidylate containing cytosine, "G (g)" refers to a deoxyguanylate or guanylate containing guanine, "U (u)" refers to a uridylate containing uracil, "T (t)" refers to a deoxythymidylate containing thymine, and "Ψ" refers to a pseudouridylate monophosphate ΨMP.

[0045] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to indicate the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the exception of the deoxythymidylates in the DNA coding strand sequence corresponding to uridylates in the encoded RNA sequence.

[0046] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve production of a transcript and / or a polypeptide. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, in particular mRNA, in a system free of cells, for example in a suitable cell extract (see, e.g., Pardi N., Muramatsu H., Weissman D., Kariko K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as "transcription vector", which comprises regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription".

[0047] As used herein, "encoding" refers to the inherent property of specific sequences of nucleotides in a nucleic acid to serve as templates for synthesis of other polymers and macromolecules in biological processes. Such terms refer to the inherent property of specific sequences of nucleotides in a nucleic acid to serve as templates for synthesis of other polymers and macromolecules in biological processes. Thus, a gene encodes a protein if mRNA derived from that gene is translated into the protein in a cell or other biological system. The term "transgene" refers to a nucleic acid sequence that has been introduced into the genome by a process of genetic engineering.

[0048] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, which include both an amino functional group and an acid functional group and which are capable of being incorporated into a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs; amino acid analogs having varied side chains; and all stereoisomers of any of the foregoing. The term "amino acid" as used herein includes D- or L-optical isomers and peptidomimetics.

[0049] The terms "polypeptide chain", "polypeptide", "peptide", and "protein" (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be straight or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, for example, conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from a eukaryotic or prokaryotic host, or they can be the product of synthetic procedures.

[0050] As used herein, an "antibody" refers to a protein produced by the body in response to the stimulation of an antigen that has a protective effect. It is an immunoglobulin produced by B lymphocytes. The monomer of an antibody is a Y-shaped molecule consisting of 4 polypeptide chains. This includes two identical heavy chains, and two identical light chains, which are held together by disulfide bonds. Each heavy chain is approximately 50 kDa, and each light chain is approximately 25 kDa, with disulfide bonds linking the light and heavy chains. It is unique in its high affinity and specificity for binding partners.

[0051] The term "polymer-bound lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-bound lipid is a pegylated lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol.

[0052] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH value range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment for which a predetermined lipid use is intended, such as a physiological pH value. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(octyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP); sphingomyelin (SM); ceramides. The neutral lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0053] The term "steroid" refers to a lipid with a cyclopentanoperhydrophenanthrene as the parent nucleus, including sterols and sterol derivatives. Steroid compounds do not contain bound fatty acids and are non-saponifiable lipids. In the present context, a steroid can serve as a structural lipid in the lipid component of a nanoparticle composition. As non-limiting examples, steroids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, soysterol, and mixtures thereof.

[0054] The term“charged lipid” encompasses any lipid molecule that exists in a positively or negatively charged form at a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment for which a predetermined lipid is intended for use, such as a physiological pH value. By way of non-limiting example, charged lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, cholesteryl hemisuccinate, dialkyi trimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl cholesterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycero) sodium salt (DOPG-Na), and 1,2-dioleoyl-sn-glycero-3-phospho sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from a natural source or compound (isolated or modified therefrom).

[0055] As used herein and unless otherwise indicated, the term“alkyl” refers to a saturated straight chain or branched chain hydrocarbon chain radical consisting only of carbon and hydrogen atoms. In some embodiments, an alkyl group has, for example, 1 to 24 carbon atoms (Ci-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (Ci-C 15 alkyl), 1 to 12 carbon atoms (Ci-C 12 alkyl), 1 to 8 carbon atoms (Ci-C8 alkyl), or 1 to 6 carbon atoms (Ci-C6 alkyl), and is attached to the rest of the molecule by a single bond. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise indicated, alkyl groups are optionally substituted.

[0056] As used herein and unless otherwise indicated, the term“alkenyl” refers to a straight chain or branched chain hydrocarbon chain radical consisting only of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds. As understood by one of ordinary skill in the art, the term“alkenyl” also encompasses radicals having“cis” and“trans” configurations or, alternatively,“E” and“Z” configurations. In some embodiments, an alkenyl group has, for example, 2 to 24 carbon atoms (C2-C 24 alkenyl), 4 to 20 carbon atoms (C4-C 20 alkenyl), 6 to 16 carbon atoms (C6-C16 alkenyl), 6 to 9 carbon atoms (C6-C9alkenyl), 2 to 15 carbon atoms (C2-C 15 alkenyl), 2 to 12 carbon atoms (C2-C 12 alkenyl), 2 to 8 carbon atoms (C2-C8alkenyl), or 2 to 6 carbon atoms (C2-C6alkenyl), and is attached to the rest of the molecule by a single bond. Examples of alkenyl include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like. Unless otherwise specified, an alkenyl is optionally substituted.

[0057] As used herein, and unless otherwise specified, the term“alkynyl” refers to a straight or branched hydrocarbon chain radical group that contains one or more carbon-carbon triple bonds, and only carbon and hydrogen atoms. In some embodiments, an alkynyl group has, for example, 2 to 24 carbon atoms (C2-C 24 alkynyl), 4 to 20 carbon atoms (C4-C 20 alkynyl), 6 to 16 carbon atoms (C6-C 16 alkynyl), 6 to 9 carbon atoms (C6-C9alkynyl), 2 to 15 carbon atoms (C2-C 15 alkynyl), 2 to 12 carbon atoms (C2-C 12 alkynyl), 2 to 8 carbon atoms (C2-C8alkynyl), or 2 to 6 carbon atoms (C2-C6alkynyl), and is attached to the rest of the molecule by a single bond. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, an alkynyl is optionally substituted.

[0058] As used herein, and unless otherwise specified, the term“alkylene” or“alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, which consists only of carbon and hydrogen and is saturated. In some embodiments, an alkylene has, for example, 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12 alkylene), 1 to 8 carbon atoms (C1-C8alkylene), 1 to 6 carbon atoms (C1-C6alkylene), 2 to 4 carbon atoms (C2-C4alkylene), 1 to 2 carbon atoms (C1-C2alkylene). Examples of alkylene include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. An alkylene chain is attached to the rest of the molecule through a single bond, and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon, or any two carbons within the chain. Unless otherwise specified, an alkylene chain is optionally substituted.

[0059] As used herein and unless otherwise indicated, the term "alkylene" refers to a straight-chain or branched, bivalent hydrocarbon chain that only consists of carbon and hydrogen and contains one or more carbon-carbon double bonds. In some embodiments, the alkylene group has, for example, 2 to 24 carbon atoms (C2-C 24 alkylene), 2 to 15 carbon atoms (C2-C 15 alkylene), 2 to 12 carbon atoms (C2-C 12 alkylene), 2 to 8 carbon atoms (C2-C8alkylene), 2 to 6 carbon atoms (C2-C6alkylene), or 2 to 4 carbon atoms (C2-C4alkylene). Examples of alkylene groups include, but are not limited to, ethylene, propylene, n-butylene, and the like. The alkylene group is attached to the remainder of the molecule by a single or double bond and to the group by a single or double bond. The points of attachment of the alkylene group to the remainder of the molecule and to the group can be through one carbon or any two carbons within the chain. Unless otherwise indicated, the alkylene group is optionally substituted.

[0060] As used herein and unless otherwise indicated, the term "cycloalkyl" refers to a non-aromatic, saturated monocyclic or polycyclic hydrocarbon radical that only consists of carbon and hydrogen atoms. The cycloalkyl group can include fused or bridged ring systems. In some embodiments, the cycloalkyl group has, for example, 3 to 15 ring carbon atoms (C3-C 15 cycloalkyl), 3 to 10 ring carbon atoms (C3-C 10 cycloalkyl), or 3 to 8 ring carbon atoms (C3-C8cycloalkyl). The cycloalkyl group is attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. Unless otherwise indicated, the cycloalkyl group is optionally substituted.

[0061] As used herein and unless otherwise indicated, the term "cycloalkylene" refers to a divalent cycloalkyl group. Unless otherwise indicated, the cycloalkylene group is optionally substituted.

[0062] As used herein and unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical that only consists of carbon and hydrogen atoms and includes one or more carbon-carbon double bonds. The cycloalkenyl group can include fused or bridged ring systems. In some embodiments, the cycloalkenyl group has, for example, 3 to 15 ring carbon atoms (C3-C 15 cycloalkenyl), 3 to 10 ring carbon atoms (C3-C 10Cycloalkenyl) or 3 to 8 ring carbon atoms (C3-C8cycloalkenyl). Cycloalkenyl is attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise indicated, cycloalkenyl is optionally substituted.

[0063] As used herein and unless otherwise indicated, the term "cycloalkenyl" is a divalent cycloalkenyl. Unless otherwise indicated, cycloalkenyl is optionally substituted.

[0064] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a nonaromatic, monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur. Heterocyclyl can be attached to the main structure at any heteroatom or carbon atom. Heterocyclyl can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring system, which can be fused, bridged, or spiro. The heterocyclyl polycyclic ring system can contain one or more heteroatoms in one or more rings. Heterocyclyl can be saturated or partially unsaturated. A saturated heterocyclyl can be referred to as a "heterocycloalkyl." A partially unsaturated heterocyclyl can be referred to as a "heterocycloalkenyl" when the heterocyclyl contains at least one double bond, or a "heterocycloalkynyl" when the heterocyclyl contains at least one triple bond. In some embodiments, heterocyclyl has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). When appearing herein, a numerical range, such as "3 to 18," refers to each integer within the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, and so on, up to and including 18 ring atoms. Examples of heterocyclyl include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thiophenyl, pyridyl, piperidinyl, quinolinyl, and isoquinolinyl. Unless otherwise indicated, heterocyclyl is optionally substituted.

[0065] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a nonaromatic, monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur. Heterocyclyl can be attached to the main structure at any heteroatom or carbon atom. Heterocyclyl can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring system, which can be fused, bridged, or spiro. The heterocyclyl polycyclic ring system can contain one or more heteroatoms in one or more rings. Heterocyclyl can be saturated or partially unsaturated. A saturated heterocyclyl can be referred to as a "heterocycloalkyl." A partially unsaturated heterocyclyl can be referred to as a "heterocycloalkenyl" when the heterocyclyl contains at least one double bond, or a "heterocycloalkynyl" when the heterocyclyl contains at least one triple bond. In some embodiments, heterocyclyl has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). When appearing herein, a numerical range, such as "3 to 18," refers to each integer within the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, and so on, up to and including 18 ring atoms. Examples of heterocyclyl include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thiophenyl, pyridyl, piperidinyl, quinolinyl, and isoquinolinyl. Unless otherwise indicated, heterocyclyl is optionally substituted.

[0066] As used herein and unless otherwise indicated, the term "aryl" refers to a monocyclic aromatic radical and / or a polycyclic monovalent aromatic radical containing at least one aromatic hydrocarbon ring. In certain embodiments, aryl groups have from 6 to 18 ring carbon atoms (C6-C 18 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl), or 6 to 10 ring carbon atoms (C6-C 10 aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings wherein at least one ring is aromatic and the other rings can be saturated, partially unsaturated, or aromatic, such as indanyl, indenyl, dihydroindenyl, or tetrahydronaphthyl / tetralinyl. Unless otherwise indicated, aryl groups are optionally substituted.

[0067] As used herein and unless otherwise indicated, the term "arylene" is a divalent aryl group. Unless otherwise indicated, arylene groups are optionally substituted.

[0068] As used herein and unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic radical and / or a polycyclic aromatic radical containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. Heteroaryl groups can be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, heteroaryl groups have from 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic, or other polycyclic rings wherein at least one ring is aromatic and the other rings can be saturated, partially unsaturated, or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thiophenyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridyl, furopyridyl, thienopyridyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise indicated, heteroaryl groups are optionally substituted.

[0069] As used herein and unless otherwise indicated, the term "heteroaryl" means a monovalent aromatic group having 5 to 20 ring atoms, where one or more ring atoms is a heteroatom selected from N, O, and S. Unless otherwise indicated, a heteroaryl group is optionally substituted.

[0070] When a group described herein is termed "substituted," it can be substituted with one or more of any suitable substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: a halogen atom, such as F, Cl, Br, or I; a cyano group; an oxo group (=0); a hydroxyl group (-OH); an alkyl group; an alkenyl group; an alkynyl group; a cycloalkyl group; an aryl group; -(C=0)OR'; -0(C=0)R'; -C(=0)R'; -OR'; -S-SR'; -C(=0)SR'; -SC(=0)R'; -NR'R'; -NR'C(=0)R'; -C(=0)NR'R'; -NR'C(=0)NR'R'; -OC(=0)NR'R'; -NR'C(=0)OR'; -NR'S(O) x NR'R'; -NR'S(O) x NR'R'; -NR'S(O) x R'; and -S(O) x NR'R', where: R' is, at each occurrence, independently H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 alkyl. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR'R').

[0071] As used herein and unless otherwise indicated, the term "optionally present" or "optionally" (e.g., optionally substituted) means that the event or circumstance subsequently described can or can not occur, and the description includes both instances where the event or circumstance occurs and where it does not. For example, "optionally substituted alkyl" means that the alkyl group can or can not be substituted, and the description includes both substituted alkyl groups and alkyl groups without substitution.

[0072] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0073] Examples of pharmaceutically acceptable acid addition salts include, without limitation, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like.

[0074] Examples of pharmaceutically acceptable base addition salts include, without limitation, salts prepared by the addition of inorganic or organic bases to the free acid compounds. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts and the like. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines and basic ion-exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. In some embodiments, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0075] The compounds provided herein can contain one or more asymmetric centers and thus can exist in enantiomeric, diastereomeric, and other stereoisomeric forms. The absolute stereochemistry is not necessarily specified unless otherwise indicated. All such possible isomers, as well as their racemic and optically pure forms, are intended to be included within the scope of the present disclosure. The optically active ( + ) and ( - ) isomers, and racemic mixtures (+) and ( - ) can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation of / separation of isomers are applied as appropriate to the given substance. When the compounds described herein contain olefinic double bonds, other geometric isomers are possible. Unless otherwise specified, all geometric isomers are intended to be embraced within the scope of the present disclosure. Likewise, all tautomeric forms of the compounds described herein are also intended to be included.

[0076] As used herein and unless otherwise indicated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the way the atoms are arranged in space. "Atropisomers" are stereoisomers resulting from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of one another. A mixture of enantiomers in any ratio is referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric centers of which are not mirror images of each other.

[0077] " Stereoisomers" can also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as either the E or Z isomer. In other embodiments, the compounds described herein are a mixture of E and Z isomers.

[0078] " Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment the compound is in, and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0079] It is also noted that the compounds described herein can contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds can be radiolabeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I), sulfur-35 ( 35 S), or carbon-14 ( 14 C), or can be isotopically enriched, such as deuterium ( 2 H), carbon-13 (13 C) or nitrogen-15 15 N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" means that the isotopic composition of an atom differs from the natural isotopic composition of that atom. "Isotopically enriched" can also mean that a compound contains at least one atom that has an isotopic composition that differs from the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents; research reagents, e.g., binding assay reagents; and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, e.g., isotopologues that are deuterium, carbon-13, and / or nitrogen-15 enriched. As used herein, "deuterated" means that at least one hydrogen (H) in a compound has been replaced with deuterium (indicated as D or 2 H). In some embodiments, the compound is enriched with deuterium at at least one position.

[0080] It should be noted that if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure is intended to govern.

[0081] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes any and all solvents, dispersion media, diluents, or other

[0082] Lipid nanoparticle formulation

[0083] According to a first aspect of the present application, there is provided a lipid nanoparticle formulation, characterized by comprising:

[0084] a pharmaceutically acceptable buffer comprising an aldehyde scavenger and a chelator,

[0085] a lipid nanoparticle, and

[0086] a nucleic acid encapsulated in the lipid nanoparticle.

[0087] buffer

[0088] The lipid nanoparticle formulation of the present application is a liquid formulation comprising a pharmaceutically acceptable buffer.

[0089] As used herein, "pharmaceutically acceptable" means physiologically acceptable and useable in pharmacy. As used herein, "buffer" refers to a solution comprising a buffering agent (any conjugate acid-base pair suitable for maintaining a desired pH range), and optionally comprising a divalent or monovalent cation. The pH and osmolality of the buffer can be adjusted to suit the lipid nanoparticle formulation of the application for administration to a subject. In some embodiments, the lipid nanoparticle formulation of the application is injectable.

[0090] In some embodiments, the pharmaceutically acceptable buffer is selected from the group consisting of phosphate buffer, citrate buffer, Tris buffer, acetate buffer, histidine buffer, HEPES buffer, and MES buffer. As used herein, "the pharmaceutically acceptable buffer is selected from the group consisting of buffer A, buffer B, and buffer C" means that the pharmaceutically acceptable buffer comprises the buffering system represented by buffer A, buffer B, and / or buffer C.

[0091] In some embodiments, the pharmaceutically acceptable buffer is selected from the group consisting of Tris buffer and phosphate buffer. Suitable Tris buffers can include, but are not limited to, Tris-HCl buffer and Tris-phosphate buffer. In some particular embodiments, the Tris buffer is Tris-HCl buffer. In some preferred embodiments, the pharmaceutically acceptable buffer is or comprises a Tris buffer. In some embodiments, the buffer further comprises sodium chloride. In some embodiments, the final concentration of sodium chloride is 4.0 mg / ml - 12.0 mg / ml, preferably 5.0 mg / ml - 11.0 mg / ml or 6.0 mg / ml - 10.0 mg / ml, more preferably 7.0 mg / ml - 9.0 mg / ml. In some particular embodiments, the final concentration of sodium chloride is 7.0 mg / ml, 8.0 mg / ml, or 9.0 mg / ml.

[0092] In some other embodiments, the pharmaceutically acceptable buffer is or comprises a Tris buffer and a phosphate buffer.

[0093] In some embodiments, the buffer comprises 1-40 mM, such as 3-40 mM, 3-35 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-15 mM, 10-40 mM, 10-35 mM, 10-30 mM, 10-25 mM, 10-20 mM, 15-40 mM, 15-35 mM, 15-30 mM, 15-25 mM, 20-40 mM, 20-35 mM, or 20-30 mM of tromethamine. In some preferred embodiments, the buffer comprises 3-40 mM, 5-40 mM, or 10-40 mM, preferably 3-35 mM, 5-35 mM, or 10-35 mM, more preferably 5-30 mM or 10-30 mM, most preferably 10-25 mM of tromethamine.

[0094] In yet other embodiments, the pharmaceutically acceptable buffer is or comprises a phosphate buffer.

[0095] In some embodiments, the phosphate buffer comprises sodium chloride, potassium chloride, sodium phosphate dibasic, and potassium phosphate monobasic.

[0096] In some embodiments, the sodium chloride has a final concentration of 4.0 mg / ml - 12.0 mg / ml, preferably 5.0 mg / ml - 11.0 mg / ml or 6.0 mg / ml - 10.0 mg / ml, more preferably 7.0 mg / ml - 9.0 mg / ml. In some specific embodiments, the sodium chloride has a final concentration of 7.0 mg / ml, 8.0 mg / ml, or 9.0 mg / ml.

[0097] In some embodiments, the potassium chloride has a final concentration of 0.05 mg / ml - 0.5 mg / ml, preferably 0.08 mg / ml - 0.4 mg / ml, more preferably 0.1 mg / ml - 0.3 mg / ml. In some embodiments, the potassium chloride has a final concentration of 0.1 mg / ml, 0.2 mg / ml, or 0.3 mg / ml.

[0098] In some embodiments, the sodium phosphate dibasic has a final concentration of 0.5 mg / ml - 2.0 mg / ml, preferably 0.8 mg / ml - 1.5 mg / ml, more preferably 1.0 mg / ml - 1.2 mg / ml. In some embodiments, the sodium phosphate dibasic has a final concentration of 1.11 mg / ml, 1.12 mg / ml, 1.13 mg / ml, 1.14 mg / ml, 1.15 mg / ml, 1.16 mg / ml, 1.17 mg / ml, 1.18 mg / ml, 1.19 mg / ml, or 1.20 mg / ml.

[0099] In some embodiments, the final concentration of the potassium dihydrogen phosphate is 0.05 mg / ml - 0.5 mg / ml, preferably 0.08 mg / ml - 0.4 mg / ml, more preferably 0.1 mg / ml - 0.3 mg / ml. In some embodiments, the final concentration of the potassium dihydrogen phosphate is 0.12 mg / ml, 0.18 mg / ml, 0.24 mg / ml or 0.3 mg / ml.

[0100] The buffer comprises an aldehyde absorber and a chelating agent.

[0101] Preferably, the buffer further comprises a pH adjusting agent, e.g. in the form of HC1.

[0102] Preferably, the pH of the buffer is in the range of 6.0 - 8.0. More preferably, the pH of the buffer is in the range of 6.5 - 7.8. Particularly preferably, the pH of the buffer is in the range of 7.0 - 7.5, e.g. 7.4.

[0103] Preferably, the buffer does not comprise an antioxidant.

[0104] Preferably, the buffer does not comprise propylene glycol in an amount of 6% by weight or more. Preferably, the buffer does not comprise propylene glycol.

[0105] Preferably, the buffer does not comprise sucrose in an amount of 7% by weight or more. Preferably, the buffer does not comprise sucrose.

[0106] Aldehyde absorber

[0107] Preferably, the aldehyde absorber is selected from the group consisting of straight-chain or branched aliphatic polyhydroxy amine compounds and amidine group-containing amino acids.

[0108] Preferably, the aldehyde absorber is selected from the group consisting of straight-chain or branched aliphatic polyhydroxy amine compounds.

[0109] Preferably, the straight-chain or branched aliphatic polyhydroxy amine compound comprises 1 - 6 carbon atoms.

[0110] Preferably, the straight-chain or branched aliphatic polyhydroxy amine compound comprises at least 3 hydroxyl groups.

[0111] As an example of a straight-chain or branched aliphatic polyhydroxy amine compound, tromethamine (Tris) can be cited.

[0112] As an example of an amidine group-containing amino acid, arginine can be cited.

[0113] Particularly preferably, tromethamine is comprised in the buffer.

[0114] The inventors found that the presence of an aldehyde absorber can reduce the generation of nucleic acid addition products.

[0115] Advantageously, the aldehyde absorbent is present in the buffer at a concentration of 1-40 mM, such as 3-40 mM, 3-35 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-15 mM, 10-40 mM, 10-35 mM, 10-30 mM, 10-25 mM, 10-20 mM, 15-40 mM, 15-35 mM, 15-30 mM, 15-25 mM, 20-40 mM, 20-35 mM, or 20-30 mM, based on the volume of the buffer. In some preferred embodiments, the aldehyde absorbent is present in the buffer at a concentration of 3-40 mM, 5-40 mM, or 10-40 mM, preferably 3-35 mM, 5-35 mM, or 10-35 mM, more preferably 5-30 mM or 10-30 mM, most preferably 10-25 mM, based on the volume of the buffer.

[0116] Chelating agent

[0117] Preferably, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA or pentetic acid), citric acid (citrates), edetate disodium, edetate dipotassium, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.

[0118] More preferably, the buffer comprises a chelating agent selected from diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, and citric acid.

[0119] Advantageously, the chelating agent is present in the buffer at a concentration of 0.001-10 mM, preferably 0.005-5 mM, more preferably 0.01-1 mM, based on the volume of the buffer.

[0120] In some preferred embodiments, the buffer comprises 0.005-1 mM of ethylenediaminetetraacetic acid.

[0121] In some preferred embodiments, the buffer comprises 1-10 mM of citric acid.

[0122] In some preferred embodiments, the buffer comprises 0.005-1 mM of diethylenetriaminepentaacetic acid.

[0123] The skilled person will appreciate that tromethamine can be used as a biological buffer. Thus, in some embodiments, tromethamine can be used as both a buffer and an aldehyde absorbent.

[0124] In some embodiments, the pharmaceutically acceptable buffer is or comprises a Tris buffer, which further comprises a chelating agent, and optionally sodium chloride. In other embodiments, the pharmaceutically acceptable buffer is or comprises a Tris buffer and a phosphate buffer, which further comprises a chelating agent. In some embodiments, the Tris buffer is a Tris-HCl buffer. In some embodiments, the buffer comprises 1-40 mM, e.g., 3-40 mM, 3-35 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-15 mM, 10-40 mM, 10-35 mM, 10-30 mM, 10-25 mM, 10-20 mM, 15-40 mM, 15-35 mM, 15-30 mM, 15-25 mM, 20-40 mM, 20-35 mM, or 20-30 mM of tromethamine. In some preferred embodiments, the buffer comprises 3-40 mM, 5-40 mM, or 10-40 mM, preferably 3-35 mM, 5-35 mM, or 10-35 mM, more preferably 5-30 mM or 10-30 mM, most preferably 10-25 mM of tromethamine. In some embodiments, the buffer comprises a chelating agent selected from the group consisting of diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, and citric acid. In some preferred embodiments, the buffer comprises 0.005-1 mM of ethylenediaminetetraacetic acid. In some preferred embodiments, the buffer comprises 1-10 mM of citric acid. In some preferred embodiments, the buffer comprises 0.005-1 mM of diethylenetriaminepentaacetic acid.

[0125] Lipid nanoparticle

[0126] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the nanometer (nm) scale (e.g., 1 to 1,000 nm) that contains one or more types of lipid molecules.

[0127] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that includes, but is not limited to, fatty acid esters, and is generally characterized by poor solubility in water but solubility in many nonpolar organic solvents. Although lipids generally have weak water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be classified into at least three categories: (1) "simple lipids" including fats and oils, and waxes; (2) "compound lipids" including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derived lipids" such as sterol-like compounds. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound" also referred to simply as "lipidoid," refers to a lipid-like compound (e.g., an amphiphilic compound having lipid-like physical properties).

[0128] Cationic Lipid

[0129] Preferably, the lipid nanoparticle comprises a cationic lipid.

[0130] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or is capable of becoming positively charged in response to the pH value or hydrogen ion activity of its environment, e.g., the environment of its intended use. Thus, the term "cationic" encompasses "permanent cationic" and "cationizable." In certain embodiments, the positive charge in a cationic lipid is caused by the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use, e.g., at physiological pH.

[0131] For example, the cationic lipid is a cationic lipid described in International Patent Application Publication No. WO2021204175, which is incorporated herein by reference in its entirety.

[0132] In some embodiments, the cationic lipid is a compound of Formula (01-I):

[0133] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0134] G 1 and G 2 each independently is a bond, C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in said alkylene or alkenylene is optionally replaced with -O-;

[0135] L 1 为-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C6-C 10 亚芳基)-R 1 、-(6元至10元亚杂芳基)-R 1 或R 1 ;

[0136] L 2 为-OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0137] R 1 and R 2 Each independently is C6-C 32 Alkyl or C6-C 32 alkenyl;

[0138] R a 、R b 、R d and R e Each independently represents H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0139] R c and R f Each independently is C1-C 32 Alkyl or C2-C 32 alkenyl;

[0140] G 3 C2-C 24 Alkylene, C2-C 24 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0141] R 3 -N(R 4 )R 5 ;

[0142] R 4 is a C3-C8 cycloalkyl, a C3-C8 cycloalkenyl, a 4-membered to 8-membered heterocyclic group or a C6-C 10 Aryl; or R 4 , G 3 or G 3 A portion of together with the nitrogen to which it is attached forms a cyclic portion;

[0143] R 5 C1-C12 alkyl or C3-C8cycloalkyl; or R 4 , R 5 together with the nitrogen to which they are attached form a cyclic moiety;

[0144] x is 0, 1, or 2; and

[0145] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0146] In some embodiments, the cationic lipid is a compound of Formula (01-II):

[0147] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0148] is a single or double bond;

[0149] G 1 and G 2 each independently is a bond, C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in said alkylene or alkenylene is optionally replaced with -O-;

[0150] L 1 is -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C6-C 10 arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0151] L 2 -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0152] R 1 and R 2 Each independently is C6-C 32 Alkyl or C6-C 32 alkenyl;

[0153] R a 、R b 、R d and R e Each independently represents H, C1-C24 alkyl or C2-C 24 alkenyl;

[0154] R c and R f each independently is C1-C 32 alkyl or C2-C 32 alkenyl;

[0155] G 4 is a bond, C1-C 23 alkylene, C2-C 23 alkenylene, C3-C8cycloalkylene, or C3-C8cycloalkenylene;

[0156] R 3 is -N(R 4 )R 5 ;

[0157] R 4 is C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, 4- to 8-membered heterocyclyl, or C6-C 10 aryl; or R 4 , G 3 , or G 3 together with the nitrogen to which it is attached forms a cyclic moiety;

[0158] R 5 is C1-C 12 alkyl or C3-C8cycloalkyl; or R 4 , R 5 together with the nitrogen to which it is attached forms a cyclic moiety;

[0159] x is 0, 1, or 2; and

[0160] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0161] In some embodiments, the compound is a compound of Formula (01-I-B), (01-I-B’), (01-I-B”), (01-I-C), (01-I-D), or (01-I-E):

[0162] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0163] In some embodiments, G 1 and G 2 each independently is C3-C7alkylene. In some embodiments, G 1 and G2 Each is independently C5 alkylene. In some embodiments, G 3 In some embodiments, G 3 In some embodiments, G 3 It is a C4 alkylene group.

[0164] In some embodiments, R 3 Has one of the following structures:

[0165] In some embodiments, R 1 、R 2 、R c and R f Each independently a branched C6-C 32 Alkyl or branched C6-C 32 In some embodiments, R 1 、R 2 、R c and R f Each independently a branched C6-C 24 Alkyl or branched C6-C 24 In some embodiments, R 1 、R 2 、R c and R f Each independently is -R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C5 alkylene, and R 8 and R 9 Independently C2-C 10 In some embodiments, R 1 、R 2 、R c and R f Each independently is -R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C1 alkylene, and R 8 and R 9 are independently C4-C8 alkyl.

[0166] In some embodiments, the compound is a compound listed in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0167] Table I

[0168] For example, the cationic lipid is a cationic lipid described in International Patent Application Publication No. WO 2023 / 138611, which is incorporated herein by reference in its entirety. In some embodiments, the cationic lipid is a compound of Formula (02-I):

[0169] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0170] G 1 and G 2 each independently is C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in G 1 and G 2 is optionally replaced by -O-, -C(=O)O-, or -OC(=O)-;

[0171] each L 1 independently is -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ) or -NR a P(=O)(OR b )(OR c );

[0172] each L 2independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ) or -NR d P(=O)(OR e )(OR f );

[0173] R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl;

[0174] R a , R b , R d and R e are each independently H, C1-C 24 alkyl or C2-C 24 alkenyl;

[0175] R c and R f are each independently C1-C 24 alkyl or C2-C 24 alkenyl;

[0176] G 3 is C2-C 12 alkylene or C2-C 12alkylene or part or all of the alkenylene is optionally replaced with C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0177] R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 ;

[0178] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0179] R 5 is H, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0180] R 6 is hydrogen, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, or C6-C 10 aryl;

[0181] x is 0, 1, or 2; and

[0182] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0183] In some embodiments, the cationic lipid is a compound of Formula (02-II):

[0184] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0185] G 1 and G 2 are each independently C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in G 1 and G 2 is optionally replaced with -O-, -C(=O)O-, or -OC(=O)-;

[0186] each L 1 is independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ) or -NR a P(=O)(OR b )(OR c );

[0187] each L 2 is independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ) or -NR d P(=O)(OR e )(OR f );

[0188] R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl;

[0189] R a , R b , R d and R e are each independently H, C1-C 24 alkyl or C2-C 24 alkenyl;

[0190] R c and R f are each independently C1-C 24 alkyl or C2-C 24 alkenyl;

[0191] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene, wherein a part or all of the alkylene or alkenylene is optionally replaced by C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0192] R 3 is -N(R 4 )R 5 , -OR 6 or -SR 6 ;

[0193] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0194] R 5 is H, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0195] R 6is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl;

[0196] x is 0, 1, or 2; and

[0197] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0198] In some embodiments, the compound is of formula (O2-VA), (O2-VB), (O2-VC), (O2-VD), (O2-VE), (O2-VF):

[0199] wherein z is an integer from 2 to 12,

[0200] x0 is an integer from 1 to 11;

[0201] y0 is an integer from 1 to 11;

[0202] x1 is an integer from 0 to 9;

[0203] y1 is an integer from 0 to 9;

[0204] x2 is an integer from 2 to 5;

[0205] x3 is an integer from 1 to 5;

[0206] x4 is an integer from 0 to 3;

[0207] y2 is an integer from 2 to 5;

[0208] y3 is an integer from 1 to 5; and

[0209] y4 is an integer from 0 to 3;

[0210] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0211] In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 and y0 are independently 2 to 6. In some embodiments, x0 and y0 are independently 4 or 5. In some embodiments, x1 and y1 are independently 2 to 6. In some embodiments, x1 and y1 are independently 4 or 5. In some embodiments, x2 and y2 are independently an integer from 2 to 5. In some embodiments, x2 and y2 are independently 3 or 5. In some embodiments, x3 and y3 are both 1. In some embodiments, x4 and y4 are independently 0 or 1.

[0212] In some embodiments, each L 1 is independently -OR 1 , -OC(=O)R 1 , or -C(=O)OR 1 , and each L 2 is independently -OR 2 , -OC(=O)R 2 , or -C(=O)OR 2 . In some embodiments, R 1 and R 2 are independently linear C6-C 10 alkyl or -R 7 -CH(R 8 )(R 9 ), wherein R 7 is C0-C5 alkylene, and R 8 and R 9 are independently C2-C 10 alkyl or C2-C 10 alkenyl.

[0213] In some embodiments, the compound is a compound of Formula (02-VI-A), (02-VI-B), (02-VI-C), (02-VI-D), (02-VI-E), or (02-VI-F):

[0214] wherein z is an integer from 2 to 12;

[0215] y is an integer from 2 to 12;

[0216] x0 is an integer from 1 to 11;

[0217] x1 is an integer from 0 to 9;

[0218] x2 is an integer from 2 to 5;

[0219] x3 is an integer from 1 to 5; and

[0220] x4 is an integer from 0 to 3;

[0221] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0222] In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 is 4 or 5. In some embodiments, x1 is 4 or 5. In some embodiments, x2 is an integer from 2 to 5. In some embodiments, x2 is 3 or 5. In some embodiments, x3 is 0 or 1. In some embodiments, y is an integer from 2 to 6. In some embodiments, y is 5.

[0223] In some embodiments, each L 1 is independently -OR 1 , -OC(=O)R 1 , or -C(=O)OR 1 , and L 2 is -OC(=O)R 2 or -C(=O)OR 2 , -NR d C(=O)R 2 , or -C(=O)NR e R f . In some embodiments, R 1 is a linear C6-C 10 alkyl or -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 are independently a C2-C 10 alkyl or C2-C 10 alkenyl. In some embodiments, R 2 and R f are each independently a linear C6-C 18 alkyl, C6-C 18 alkenyl, or -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 are independently a C2-C 10 alkyl or C2-C 10 alkenyl. In some embodiments, R d and R e are each independently H.

[0224] In some embodiments, the compound is a compound in Table II, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0225] Table II

[0226] For example, the cationic lipid is a cationic lipid described in International Patent Application Publication No. WO2022152109, which is incorporated herein by reference in its entirety.

[0227] In some embodiments, the cationic lipid is a compound of Formula (03-I):

[0228] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0229] G 1 and G 2 each independently is a bond, C2-C 12 alkylene, or C2-C 12 alkenylene, wherein one or more -CH2- in G 1 and G 2 is optionally replaced with -O-;

[0230] each L 1 independently is -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ), -(C6-C 10 arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 , -(4- to 8-membered heterocyclylene)-R 1 or R 1 ;

[0231] each L 2 independently is -OC(=O)R2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 、-(4- to 8-membered heterocyclylene)-R 2 or R 2 ;

[0232] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0233] R a 、R b 、R d and R e Each independently represents H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0234] R c and R f Each independently is C1-C 24alkyl or C2-C 24 alkenyl;

[0235] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced with C3-C8cycloalkylene, C3-C8cycloalkenylene, C3-C8cycloalkynylene, 4- to 8-membered heterocyclylene, C6-C 10 arylene or 5- to 10-membered heteroarylene;

[0236] R 3 is hydrogen, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C3-C8cycloalkynyl, 4- to 8-membered heterocyclyl, C6-C 10 aryl or 5- to 10-membered heteroaryl; or part of R 3 , G 1 or G 1 together with the nitrogen to which it is attached forms a cyclic moiety; or part of R 3 , G 3 or G 3 together with the nitrogen to which it is attached forms a cyclic moiety;

[0237] R 4 is C1-C 12 alkyl or C3-C8cycloalkyl;

[0238] x is 0, 1, or 2;

[0239] n is 1 or 2;

[0240] m is 1 or 2; and

[0241] wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0242] In some embodiments, the compound is a compound of Formula (03-II-A):

[0243] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0244] In some embodiments, the compound is a compound of Formula (03-II-B):

[0245] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0246] In some embodiments, the compound is a compound of Formula (03-II-C):

[0247] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0248] In some embodiments, the compound is a compound of Formula (03-II-D):

[0249] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0250] In some embodiments, G 1 and G 2 each independently is C2-C 12 alkylene. In some embodiments, G 1 and G 2 each independently is C5 alkylene. In some embodiments, G 3 is C2-C6 alkylene.

[0251] In some embodiments, R 3 is C1-C 12 alkyl, C2-C 12 alkenyl, or C3-C8 cycloalkyl. In some embodiments, R 3 is C3-C8 cycloalkyl. In some embodiments, R 3 is unsubstituted. In some embodiments, R 4 is substituted C1-C 12 alkyl. In some embodiments, R 4 is -CH2CH2OH.

[0252] In some embodiments, L 1 is -OC(=O)R 1 , -C(=O)OR 1 , -NR a C(=O)R 1 , or -C(=O)NR b R c ; and L 2 is -OC(=O)R 2 , -C(=O)OR 2 , -NR d C(=O)R 2 , or -C(=O)NR e R f . In some embodiments, R 1 , R 2, R c , and R f each independently is a linear C6-C 18 alkyl, a linear C6-C 18 alkenyl, or -R 7 -CH(R 8 )(R 9 ), wherein R 7 is a C0-C5 alkylene, and R 8 and R 9 independently are C2-C 10 alkyl or C2-C 10 alkenyl. In some embodiments, R 1 , R 2 , R c , and R f each independently is a linear C7-C 15 alkyl, a linear C7-C 15 alkenyl, or -R 7 -CH(R 8 )(R 9 ), wherein R 7 is a C0-C1 alkylene, and R 8 and R 9 independently are C4-C8 alkyl or C6-C 10 alkenyl. In some embodiments, R a , R b , R d , and R e each independently is H.

[0253] In some embodiments, the compound is a compound in Table III, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0254] Table III

[0255] For example, the cationic lipid is a cationic lipid described in International Patent Application Publication No. WO 2022 / 247755, which is incorporated herein by reference in its entirety.

[0256] In some embodiments, the cationic lipid is a compound of Formula (04-I):

[0257] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0258] G 1 and G 2 each independently is a bond, C2-C 12 alkylene, or C2-C 12alkenylene;

[0259] L 1 -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR b )(OR c ) -(C6-C 10 arylene)-R 1 -(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0260] L 2 -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NRe R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0261] R 1 and R 2 Each independently is C5-C 32 Alkyl or C5-C 32 alkenyl;

[0262] R a 、R b 、R d and R e Each independently represents H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0263] R c and R f Each independently is C1-C 32 Alkyl or C2-C 32 alkenyl;

[0264] R 0 C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0265] G 3 C2-C 12 Alkylene or C2-C 12 alkenylene;

[0266] R 4 C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0267] R 5 is C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0268] x is 0, 1, or 2;

[0269] s is 0 or 1; and

[0270] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.

[0271] In some embodiments, the cationic lipid is a compound of Formula (04-III):

[0272] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0273] R 1 and R 2 each independently is C5-C 32 alkyl or C5-C 32 alkenyl;

[0274] R 0 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0275] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene;

[0276] G 4 is C2-C 12 alkylene or C2-C 12 alkenylene;

[0277] R 3 is -N(R 4 )R 5 or -OR 6 ;

[0278] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0279] R5 C1-C6alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C10aryl, or 4- to 8-membered 12 heterocycloalkyl; or R 10 and the nitrogen to which it is attached form a cyclic moiety; 4 , R 5 and the nitrogen to which it is attached form a cyclic moiety;

[0280] R 6 is hydrogen, C1-C6alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C10aryl, or 4- to 8-membered 12 heterocycloalkyl; or R 10 and the nitrogen to which it is attached form a cyclic moiety;

[0281] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.

[0282] In some embodiments, the compound is a compound of Formula (04-IV):

[0283] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0284] In some embodiments, G 3 is C2-C4alkylene. In some embodiments, G 4 is C2-C4alkylene.

[0285] In some embodiments, R 0 is C1-C6alkyl. In some embodiments, R 3 is -OH. In some embodiments, R 3 is -N(R 4 )R 5 . In some embodiments, R 4 is C3-C8cycloalkyl. In some embodiments, R 4 is unsubstituted. In some embodiments, R 5 is -CH2CH2OH.

[0286] In some embodiments, L 1 is -OC(=O)R 1 , -C(=O)OR 1 , -C(=O)R 1 , -C(=O)NR b R c , or R 1 ; and L 2 is -OC(=O)R 2 , -C(=O)OR 2 , -C(=O)R 2 , -C(=O)NRe R f or R 2 In some embodiments, R 1 and R 2 each independently is a branched C6-C 24 alkyl or branched C6-C 24 alkenyl. In some embodiments, R 1 and R 2 each independently is -R 7 -CH(R 8 )(R 9 ), wherein R 7 is a C1-C5 alkylene, and R 8 and R 9 independently are C2-C 10 alkyl or C2-C 10 alkenyl. In some embodiments, R 1 is a linear C6-C 24 alkyl and R 2 is a branched C6-C 24 alkyl. In some embodiments, R 1 is a linear C6-C 24 alkyl and R 2 is -R 7 -CH(R 8 )(R 9 ), wherein R 7 is a C1-C5 alkylene, and R 8 and R 9 independently are C2-C 10 alkyl.

[0287] In some embodiments, the compound is a compound in Table IV, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0288] Table IV

[0289] In some embodiments, the lipid nanoparticle comprises 20 mol% to 65 mol% of the cationic lipid. Preferably, the lipid nanoparticle comprises 25 mol% to 60 mol% of the cationic lipid. More preferably, the lipid nanoparticle comprises 30 mol% to 55 mol% of the cationic lipid. Still more preferably, the lipid nanoparticle comprises 35 mol% to 55 mol% of the cationic lipid. Still more preferably, the lipid nanoparticle comprises 40 mol% to 52 mol% of the cationic lipid.

[0290] In some embodiments, the lipid nanoparticle comprises about 22 mol%, 24 mol%, 26 mol%, 28 mol%, 32 mol%, 34 mol%, 36 mol%, 38 mol%, 42 mol%, 44 mol%, 46 mol%, 48.0 mol%, or 50.0 mol% of a cationic lipid.

[0291] Polymer-bound lipids

[0292] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more polymer-bound lipids, such as a PEGylated lipid (PEG lipid).

[0293] Exemplary polymer-bound lipids include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0294] In some embodiments, the polymer-bound lipid is a PEGylated lipid. For example, some embodiments include a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); a PEGylated phosphatidylethanolamine (PEG-PE); a PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-ditetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG); a PEGylated ceramide (PEG-cer); or a PEG dialkoxylpropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-ditetradecyloxypropyl)carbamate or 2,3-ditetradecyloxypropyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.

[0295] In some embodiments, the PEGylated lipid has the following formula:

[0296] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0297] R 12 and R 13each independently is a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

[0298] w has an average value ranging from 30 to 60.

[0299] In some embodiments, R 12 and R 13 each independently is a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, w has an average value ranging from 42 to 55, e.g., w has an average value of 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some particular embodiments, w has an average value of about 49.

[0300] In some embodiments, the pegylated lipid has the following formula:

[0301] wherein w has an average value of about 49.

[0302] In some embodiments, the polymer-bound lipid (preferably, the pegylated lipid) is present at a concentration ranging from 0.8 mol% to 3 mol%. Preferably, the polymer-bound lipid is present at a concentration ranging from about 1.0 mol% to 2.5 mol%. More preferably, the polymer-bound lipid is present at a concentration ranging from about 1.2 mol% to 2 mol%. More preferably, the polymer-bound lipid is present at a concentration ranging from about 1.4 mol% to 1.8 mol%.

[0303] In some embodiments, the polymer-bound lipid is present at a concentration of 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, or 3.0 mol%.

[0304] structural lipid

[0305] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more structural lipids.

[0306] Exemplary structural lipids include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, a-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone, or a combination thereof.

[0307] In some embodiments, the lipid nanoparticles provided herein comprise a steroid or steroid analog. In some embodiments, the steroid or steroid analog is cholesterol.

[0308] In some embodiments, the structural lipid is present at a concentration ranging from 30 mol% to 50 mol%. Preferably, the structural lipid is present at a concentration ranging from 32 mol% to 46 mol%. More preferably, the structural lipid is present at a concentration ranging from 34 mol% to 44 mol%. Still more preferably, the structural lipid is present at a concentration ranging from 36 mol% to 42 mol%.

[0309] In some embodiments, the structural lipid is present at a concentration of about 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 38.5 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol%.

[0310] Phospholipids

[0311] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more phospholipids, such as one or more (poly)unsaturated lipids.

[0312] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and the like. Exemplary phospholipids can be found, for example, in PCT Publication No. WO 2023 / 098842, the contents of which are incorporated herein in their entirety. In certain embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0313] Still other exemplary phospholipids include, for example, dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidyl ethanolamine (POPE), and dioleoyl phosphatidyl ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidyl ethanolamine (SOPE), and 1,2-diretro-oleoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0314] In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0315] Further, phospholipids that can form part of the lipid nanoparticles of the present application also include those described in WO 2017 / 112865, the entirety of which is incorporated by reference herein in its entirety.

[0316] In some embodiments, the phospholipid is present at a concentration ranging from 5 mol% to 15 mol%. Preferably, the phospholipid is present at a concentration ranging from 7 mol% to 13 mol%. More preferably, the phospholipid is present at a concentration ranging from 9 mol% to 11 mol%.

[0317] In some embodiments, the phospholipid is present at a concentration of about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol%.

[0318] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more of the lipids of Series 01, 02, 03, and 04 described herein, for example one or more of the lipids according to formulae (01-I), (01-II), (02-I), (03-I), and (04-I) (and subformulae thereof), a phospholipid (such as an unsaturated lipid, preferably DOPE or DSPC), a polymer-bound lipid (preferably a PEG lipid), and a structural lipid (preferably cholesterol).

[0319] In the above embodiments, preferably, the lipid nanoparticle comprises 20 mol% to 65 mol% of the cationic lipid. More preferably, the lipid nanoparticle comprises 25 mol% to 60 mol% of the cationic lipid. Still more preferably, the lipid nanoparticle comprises 30 mol% to 55 mol% of the cationic lipid. Still more preferably, the lipid nanoparticle comprises 35 mol% to 55 mol% of the cationic lipid. Still more preferably, the lipid nanoparticle comprises 40 mol% to 52 mol% of the cationic lipid.

[0320] In the above embodiments, preferably, the phospholipid is present at a concentration ranging from 5 mol% to 15 mol%. More preferably, the phospholipid is present at a concentration ranging from 7 mol% to 13 mol%. Still more preferably, the phospholipid is present at a concentration ranging from 9 mol% to 11 mol%.

[0321] In the above embodiments, preferably, the PEG lipid is present at a concentration ranging from 0.8 mol% to 3 mol%. More preferably, the PEG lipid is present at a concentration ranging from about 1.0 mol% to 2.5 mol%. Still more preferably, the PEG lipid is present at a concentration ranging from about 1.2 mol% to 2 mol%. Still more preferably, the polymer-bound lipid is present at a concentration ranging from about 1.4 mol% to 1.8 mol%.

[0322] In the above embodiments, preferably, the structural lipid is present at a concentration ranging from 30 mol% to 50 mol%. Preferably, the structural lipid is present at a concentration ranging from 32 mol% to 46 mol%. More preferably, the structural lipid is present at a concentration ranging from 34 mol% to 44 mol%. Still more preferably, the structural lipid is present at a concentration ranging from 36 mol% to 42 mol%.

[0323] In the above embodiments, preferably, the molar ratio of cationic lipid to structural lipid is in the range of 5: 1 to 1 : 1. More preferably, the molar ratio of cationic lipid to structural lipid is in the range of 3: 1 to 1 : 1. Still more preferably, the molar ratio of cationic lipid to structural lipid is in the range of 2: 1 to 1 : 1.

[0324] In the above embodiments, preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 1 : 1 to about 10: 1. More preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 2: 1 to about 8: 1. Still more preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 3: 1 to 6: 1.

[0325] In the above embodiments, preferably, the molar ratio of cationic lipid to polymer- conjugated lipid is in the range of about 100: 1 to about 10: 1. More preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 80: 1 to about 15: 1. More preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 60: 1 to about 20: 1. Still more preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 40: 1 to about 25: 1. Still more preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 35: 1 to about 30: 1.

[0326] In some embodiments, the molar ratio of cationic lipid, phospholipid (preferably, DSPC), structural lipid (preferably, cholesterol), and polymer-conjugated lipid (preferably, PEG lipid) in the lipid nanoparticle is 50: 10: 38.5: 1.5.

[0327] In some preferred embodiments, the cationic lipid comprises compound CI or C2.

[0328] In some preferred embodiments, the PEG-lipid comprises DMG-PEG, such as DMG-PEG2000.

[0329] In some most preferred embodiments, the lipid nanoparticle comprises 50 mol% of compound CI or C2, 1.5 mol% of DMG-PEG (such as DMG-PEG2000), 10 mol% of DSPC, and 38.5 mol% of cholesterol.

[0330] As used herein, "mol%" described in reference to lipids refers to the mole percentage of a component relative to the total moles of all lipid components in the LNP. Unless specifically indicated, the sum of the amounts (mol%) of all lipids in a lipid nanoparticle is 100 mol%.

[0331] In some embodiments, the lipid nanoparticle has an average diameter in the range of 50 nm to 180 nm. Preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 150 nm. More preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 120 nm. Still more preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 100 nm. Still more preferably, the lipid nanoparticle has an average diameter in the range of 60 nm to 85 nm.

[0332] nucleic acid

[0333] The lipid nanoparticle encapsulates a nucleic acid as a therapeutic payload.

[0334] Exemplary forms of the nucleic acid include, but are not limited to, combinations of one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0335] In some embodiments, the nucleic acid comprises RNA. Useful RNAs include, but are not limited to, shortmers, agomirs, antagomirs, antisenses, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), and messenger RNAs (mRNAs).

[0336] In some embodiments, the nucleic acid comprises mRNA.

[0337] In some embodiments, the nucleic acid is mRNA.

[0338] In particular, the mRNA encodes a peptide or polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified peptide or polypeptide. The peptide or polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA payload can have a therapeutic effect when expressed in a cell.

[0339] In some embodiments, the mRNA comprises at least one coding region (e.g., open reading frame (ORF)) encoding a peptide or polypeptide of interest. In some embodiments, the mRNA further comprises at least one untranslated region (UTR). In some embodiments, an untranslated region (UTR) is located upstream (5’ end) of the coding region, and is referred to herein as a 5’-UTR. In some embodiments, an untranslated region (UTR) is located downstream (3’ end) of the coding region, and is referred to herein as a 3’-UTR. In some embodiments, the mRNA comprises both a 5’-UTR and a 3’-UTR. In some embodiments, the 5’-UTR comprises a 5’-cap structure. In some embodiments, the mRNA comprises a Kozak sequence (e.g., in the 5’-UTR). In some embodiments, the mRNA comprises a poly-A region (e.g., in the 3’-UTR). In some embodiments, the mRNA comprises a polyadenylation signal (e.g., in the 3’-UTR). In some embodiments, the mRNA comprises a stabilization region (e.g., in the 3’-UTR). In some embodiments, the mRNA comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the mRNA comprises a stem-loop sequence (e.g., in the 5’-UTR and / or 3’-UTR). In some embodiments, the mRNA comprises one or more intronic regions capable of being excised during splicing. In some embodiments, the mRNA comprises one or more regions selected from the 5’-UTR and the coding region. In some embodiments, the mRNA comprises one or more regions selected from the coding region and the 3’-UTR. In some embodiments, the mRNA comprises one or more regions selected from the 5’-UTR, the coding region, and the 3’-UTR.

[0340] In some embodiments, the nucleic acid has a molecular length of at least about 30 nucleotides (nt). In some embodiments, the nucleic acid has a molecular length of at least about 35 nt. In some embodiments, the nucleic acid has a molecular length of at least about 40 nt. In some embodiments, the nucleic acid has a molecular length of at least about 45 nt. In some embodiments, the nucleic acid has a molecular length of at least about 50 nt. In some embodiments, the nucleic acid has a molecular length of at least about 55 nt. In some embodiments, the nucleic acid has a molecular length of at least about 60 nt. In some embodiments, the nucleic acid has a molecular length of at least about 65 nt. In some embodiments, the nucleic acid has a molecular length of at least about 70 nt. In some embodiments, the nucleic acid has a molecular length of at least about 75 nt. In some embodiments, the nucleic acid has a molecular length of at least about 80 nt. In some embodiments, the nucleic acid has a molecular length of at least about 85 nt. In some embodiments, the nucleic acid has a molecular length of at least about 90 nt. In some embodiments, the nucleic acid has a molecular length of at least about 95 nt. In some embodiments, the nucleic acid has a molecular length of at least about 100 nt. In some embodiments, the nucleic acid has a molecular length of at least about 120 nt. In some embodiments, the nucleic acid has a molecular length of at least about 140 nt. In some embodiments, the nucleic acid has a molecular length of at least about 160 nt. In some embodiments, the nucleic acid has a molecular length of at least about 180 nt. In some embodiments, the nucleic acid has a molecular length of at least about 200 nt. In some embodiments, the nucleic acid has a molecular length of at least about 250 nt. In some embodiments, the nucleic acid has a molecular length of at least about 300 nt. In some embodiments, the nucleic acid has a molecular length of at least about 400 nt. In some embodiments, the nucleic acid has a molecular length of at least about 500 nt. In some embodiments, the nucleic acid has a molecular length of at least about 600 nt. In some embodiments, the nucleic acid has a molecular length of at least about 700 nt. In some embodiments, the nucleic acid has a molecular length of at least about 800 nt. In some embodiments, the nucleic acid has a molecular length of at least about 900 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1000 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1100 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1200 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1300 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1400 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1500 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1600 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1700 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1800 nt. In some embodiments, the nucleic acid has a molecular length of at least about 1900 nt.In some embodiments, the nucleic acid has a molecular length of at least about 2000 nt.

[0341] Advantageously, the concentration of the nucleic acid is 0.001 to 2 mg / ml, preferably 0.003 to 1 mg / ml, more preferably 0.01 to 0.5 mg / ml, based on the volume of the lipid nanoparticle formulation.

[0342] Advantageously, the molar ratio of the lipid to the nucleic acid is 1 : 1 to 10: 1, preferably 2: 1 to 10: 1, more preferably 4: 1 to 8: 1. The lipid nanoparticle formulation of the present application has good chemical stability. In particular, the lipid nanoparticle formulation of the present application has less nucleic acid addition product and higher nucleic acid integrity after being left for a certain period of time (e.g. 1 week, 2 weeks under accelerated (i.e. higher than regular storage temperature (2-8°C)) conditions).

[0343] The method for preparing the lipid nanoparticle formulation of the present application can use methods known in the art.

[0344] According to a second aspect of the present application, there is provided a method for preparing the above-mentioned lipid nanoparticle formulation, characterized in that it comprises the following steps:

[0345] I) providing a lipid working solution and a nucleic acid working solution;

[0346] II) using the lipid working solution and the nucleic acid working solution to prepare lipid nanoparticles encapsulating nucleic acid; and

[0347] III) performing dialysis to make the buffer solution contain an aldehyde absorbent and a chelating agent.

[0348] The above steps can all be performed by means known in the art. The skilled person will appreciate that tromethamine can be used as a buffering agent in the lipid working solution and / or the nucleic acid working solution. Thus, in some embodiments, the pharmaceutically acceptable buffer used in the present application is or comprises a Tris buffer, and the purpose of the above step of dialysis can be to only make the buffer solution contain a chelating agent.

[0349] In some embodiments, the method for preparing further comprises a step of performing filtration after dialysis.

[0350] In some embodiments, the method for preparing further comprises a step of packaging the resulting lipid nanoparticle formulation into a packaging material after filtration.

[0351] The packaging material can be a vial, a reservoir bag, or a syringe. Exemplary packaging materials can include, but are not limited to, glass bottles (e.g., a vial and an ampoule) and a pre-filled syringe (also known as a "prefilled needle"). In some preferred embodiments, the packaging material is a vial. In some preferred embodiments, the packaging material is an ampoule. In some preferred embodiments, the packaging material is a pre-filled needle.

[0352] Preferably, the step of packaging the resulting lipid nanoparticle formulation into a packaging material is performed under nitrogen protection.

[0353] The inventors have found that nitrogen protection can reduce the rate of formation of nucleic acid addition products.

[0354] Pharmaceutical product

[0355] According to a third aspect of the present application, there is provided a pharmaceutical product, characterized by comprising: the lipid nanoparticle formulation of the present application; and a container that contains the lipid nanoparticle formulation.

[0356] The container can be a vial, a reservoir bag, or a syringe. Exemplary containers can include, but are not limited to, glass bottles (e.g., a vial and an ampoule) and a pre-filled syringe (also known as a "prefilled needle"). In some preferred embodiments, the container is a vial. In some preferred embodiments, the container is an ampoule. In some preferred embodiments, the container is a pre-filled needle. In some preferred embodiments, the container is a vial, an ampoule, or a pre-filled needle that is filled with nitrogen.

[0357] The description of each feature in the present application can be combined with each other feature in the present application, as long as they are not mutually contradictory, and the technical solutions thus obtained all fall within the scope of the present application. For example, any description of an aldehyde absorbent can be combined with any description of a chelating agent.

[0358] The "comprising" and "including" described in the present application encompass the case where other elements not explicitly mentioned are further included or composed, as well as the case where they are composed of the mentioned elements.

[0359] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions of terms in the present specification and the meanings commonly understood by those skilled in the art, the definitions described in the present specification shall prevail.

[0360] Unless otherwise indicated, all numbers expressing quantities of ingredients, temperatures and so forth as used in the specification and claims are to be understood as being approximations based on the desired properties sought to be obtained by the employ of said values. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that can vary from the numerical values stated in the specification and claims in different instances.

[0361] Examples

[0362] The concept and the resulting technical effects of the present application will be further described in conjunction with the embodiments and the accompanying drawings, so that those skilled in the art can fully understand the purposes, features and effects of the present application. Those skilled in the art will understand that the embodiments herein are only for illustrative purposes, and the scope of the present application is not limited thereto.

[0363] Preparation of formulations

[0364] In the following examples, mRNA-LNP formulations in PBS buffer were prepared by microfluidic method. Specifically, the mixed-lipid working solution in ethanol (the mixed-lipid consists of 50 mol.% cationic lipid (exemplary cationic lipids: compounds C1 and C2; unless otherwise specified, the cationic lipid is compound C1), 1.5 mol.% DMG-PEG2000, 10 mol.% DSPC and 38.5 mol.% cholesterol) and mRNA working solution in aqueous citric acid solution (exemplary mRNA: Delta mRNA (SEQ ID NO: 2) and IL-12 mRNA (SEQ ID NO: 1); unless otherwise specified, the mRNA is Delta mRNA) were mixed at a certain flow rate ratio for encapsulation; then dialyzed against buffer solution (composition: NaCl: 0.137 mM; KCl: 2.683 mM; Na2HPO4: 8.1 mM; KH2PO4: 1.76 mM); and finally filtered to obtain the formulation.

[0365] Other formulations were prepared by dialyzing this formulation into buffer solutions containing different excipients as follows:

[0366] The formulation was loaded into a 20K MWCO dialysis cassette (15 mL, Thermo Fisher Scientific Inc) and placed in 5 L of pre-prepared buffer solution, respectively, and dialyzed at 4°C for 24 h or more.

[0367] The mRNA was extracted by the following method:

[0368] mRNA was extracted from mRNA-LNP formulations using the RNeasy kit (Qiagen). A 10 pg mRNA amount of formulation was added to a 1.5 ml centrifuge (eP) tube, then the LNP was lysed by adding 350 pL RLT buffer to the eP tube, followed by adding an equal volume of 70% ethanol solution to the eP tube and mixing well by pipetting. 700 pL of the mixture (including precipitate) was transferred to a 2 ml ultrafiltration in-tube. Centrifugation at 8000 g for 15 s, and the lower liquid was discarded. 700 pL of RW1 buffer was added to the ultrafiltration in-tube. Centrifugation at 8000 g for 15 s, and the lower liquid was discarded. 500 pL of RPE buffer was added to the ultrafiltration in-tube. Centrifugation at 8000 g for 15 s, and the lower liquid was discarded. 500 pL of RPE buffer was added to the ultrafiltration in-tube. Centrifugation at 8000 g for 2 min, and the lower liquid was discarded. The ultrafiltration in-tube was placed in a new 2 ml outer tube. Centrifugation at 8000 g for 1 min, and the filter membrane was dried. The ultrafiltration in-tube was placed in a new 1.5 ml eP tube, and 30 pL of nuclease-free water was added to the in-tube filter membrane. Centrifugation at 8000 g for 1 min, and the mRNA was eluted, and the lower layer was the extracted mRNA solution.

[0369] Capillary electrophoresis (CE): Extracted mRNA samples were tested for purity on a Fragment Analyzer (from Agilent), and RNA was quantified and qualitatively analyzed using RNA Analysis Reagents (from Agilent). RNA separation gels were mixed with dye (AATI) at a 10,000: 1 v / v ratio as separation matrix. Extracted mRNA was denatured at 70 °C for 2 min, and after cooling on ice, the denatured sample was diluted 12-fold with RNA dilution solution before loading. Data were analyzed using ProSize data analysis software.

[0370] Reverse phase ion pair chromatography (RP-HPLC): Samples were separated on a DNAPac RP analytical column with mobile phase A being dibutylammonium acetate (DBAA, 50 mM) and triethylammonium acetate (TEAA, 100 mM) and mobile phase B consisting of 50% acetonitrile, DBAA (50 mM) and TEAA (100 mM). Separation was achieved using a gradient elution. Adduct peaks were quantified as a relative percentage of total chromatographic peak area.

[0371] Particle size and polydispersity index (PDI) of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode.

[0372] Encapsulation efficiency of lipid nanoparticles was determined using Quant-it Ribogreen RNA quantitation assay kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions.

[0373] Osmotic pressure was determined according to the method of osmotic pressure molar concentration determination in Chinese Pharmacopoeia 2020 edition.

[0374] Insoluble microparticles of the formulations were detected using Micro-flow imaging (Protein Simple) according to the instrument instruction.

[0375] Example 1

[0376] This example aims to study the effect of primary amine-containing small molecule compounds on the adduct. Seventeen batches of formulations were prepared, as shown in Table 1, wherein the concentration of the formulation is the concentration of mRNA based on the volume of the obtained formulation.

[0377] In formulations 1-17, the mRNA used was Delta mRNA, and the mRNA-LNP in PBS buffer was prepared by microfluidic method, and the mRNA-LNP in other buffers was prepared by dialysis, and the molar ratio of lipid to mRNA was 5.67.

[0378] Table 1. Formulation prescription composition

[0379] The prepared formulations were placed at 25°C for 1 week, and then the LP was determined using RP-HPLC.

[0380] The contents of intact mRNA, adduct and degraded mRNA of the formulations without aldehyde absorbent and the formulations with different types of aldehyde absorbent after being placed at 25°C for 1 week are shown in Figure 1.

[0381] The adduct content of formulations 1-17 after being placed at 25°C for 1 week is summarized in Table 2.

[0382] Table 2. Adduct content of formulations 1-17 after being placed at 25°C for 1 week

[0383] As can be seen from Figure 1 and Table 1, under accelerated conditions, Tris and arginine are more effective in reducing the generation of adducts. The adduct in formulation 1 without Tris is 6.3%, while the adduct in formulation 4 containing arginine is 4.9%, and the adduct in formulation 2 containing Tris is 2.7%.

[0384] Example 2

[0385] This example aims to study the effect of traditional antioxidants on mRNA purity. Five batches of formulations were prepared, and the composition is shown in Table 3. The concentration of the antioxidant used is based on the maximum amount of the auxiliary allowed in the unit formulation according to the FDA.

[0386] In Formulations 1-5, the mRNA used was Delta mRNA, mRNA-LNP in PBS buffer was prepared by microfluidic method, mRNA-LNP in other buffers was prepared by dialysis, and the molar ratio of lipid to mRNA was 5.67.

[0387] Table 3. Types and concentrations of antioxidants

[0388] The mRNA was extracted during the incubation of the prepared formulations at 37°C for 2 weeks, and the purity of mRNA was detected by capillary electrophoresis.

[0389] The results of mRNA purity during the incubation of formulations without antioxidants and formulations with different types and concentrations of antioxidants at 37°C for 2 weeks are shown in Figure 2.

[0390] The test results show that neither sodium bisulfite nor ascorbic acid can maintain the integrity of mRNA, and the addition of antioxidants even causes a decrease in purity.

[0391] Example 3

[0392] This example aims to study the effects of metal ions and the types and concentrations of chelating agents on the integrity of mRNA. Fifteen batches of formulations were prepared, and the compositions are shown in Tables 4 and 5.

[0393] In Formulations 1-15, the mRNA used was Delta mRNA, mRNA-LNP in PBS buffer was prepared by microfluidic method, mRNA-LNP in other buffers was prepared by dialysis, and the molar ratio of lipid to mRNA was 5.67.

[0394] Table 4. Types and concentrations of chelating agents

[0395] Table 5. Concentrations of DTPA in the formulations

[0396] The mRNA was extracted during the incubation of the prepared formulations at 37°C for 2 weeks, and the purity of mRNA was detected by capillary electrophoresis.

[0397] The results of mRNA purity during the incubation of formulations without chelating agents and formulations with different types and concentrations of chelating agents at 37°C for 2 weeks are shown in Figure 3.

[0398] The mRNA purity of Formulations 1-12 after incubation at 37°C for 2 weeks is summarized in Table 6.

[0399] Table 6. Results of mRNA purity % of Formulations 1-12

[0400] As can be seen from Figure 3 and Table 6, the addition of zinc ions (Formulation 2) exacerbates mRNA degradation compared to Formulation 1.

[0401] At the same molar concentration of chelating agents, DTPA has a better chelating effect on metal ions (Formulation 10) than EDTA and citric acid (CA) (Formulation 4 and Formulation 5).

[0402] The mRNA purity results of Formulations with 0-0.1 mM DTPA during 2 weeks storage at 37°C are shown in Figure 4.

[0403] As can be seen from Figure 4, reducing the concentration of DTPA to 0.01 mM (Formulation 14) has a similar chelating effect as 0.1 mM (Formulation 15), and the addition of DTPA has a certain protective effect on mRNA purity compared to the buffer of 1X PBS, 20 mM Tris (Formulation 13). Overall, the mRNA purity in all formulations with DTPA (Formulations 10, 11, 12, 14, 15) is better than that in the group of 1X PBS, 20 mM Tris (Formulation 1, Formulation 13).

[0404] Example 4

[0405] This example aims to study the effect of the combination of aldehyde absorbent and chelating agent on the stability of the formulation, and whether the added components affect the physicochemical properties of the formulation. At the same time, the effect of N2 protection on the addition product and mRNA purity is studied.

[0406] According to the information shown in Table 7, samples under different formulations, different headspace gases, and different storage conditions were obtained.

[0407] Table 7. Formulation information

[0408] The physicochemical properties (particle size, PDI, and encapsulation efficiency) of Formulations 1, 3, 5, 7, and 9 during storage are shown in Figure 5.

[0409] As can be seen from Figure 5, the addition of aldehyde absorbent and chelating agent does not affect the particle size and encapsulation efficiency of the formulation.

[0410] The mRNA purity of Formulations 1 to 10 during 2 weeks storage is shown in Figure 6.

[0411] As can be seen from Figure 6, the addition of 20 mM Tris in PBS buffer causes a slight decrease in purity, and the addition of 10 mM CA and 0.01 mM DTPA can improve the purity decrease problem caused by Tris.

[0412] As can also be seen from Figure 6, the headspace N2 protection measure has no effect on maintaining mRNA purity.

[0413] The addition product content after 2 weeks of storage is summarized in Table 8.

[0414] Table 8. Growth rate of lipid addition product

[0415] As can be seen from Table 8, the addition of 20 mM Tris can significantly slow down the generation rate of LP impurities compared to 1XPBS. On the basis of Tris, the combination of chelating agent CA affects the inhibitory effect of Tris on addition product, which may be related to the too high concentration of CA. On the basis of Tris, the combination of chelating agent DTPA does not affect the inhibitory effect of Tris on addition product.

[0416] As can be seen from Table 8, nitrogen protection can further reduce the generation rate of addition product.

[0417] Example 5

[0418] This example aims to study the physicochemical performance on different mRNA-LNP formulations through 37°C accelerated experiment. The formulation information is shown in Table 9, wherein the cationic lipid compound C1 has the following structure:

[0419] The cationic lipid compound C2 has the following structure:

[0420] Table 9. Formulation information

[0421] The mRNA purity results of formulations containing different LNP-mRNA during 2 weeks of storage at 37°C are shown in Figure 7.

[0422] As can be seen from Figure 7, for the IL-12 / C1 formulation group, compared with only 1XPBS (formulation 1), the addition of 20 mM Tris (formulation 2) does not reduce the purity of mRNA, and the purity of mRNA is equivalent (on day 14, 32% of formulation 2 vs. 26% of formulation 1, with an instrument detection error of about 5%), and the addition of 0.01 mM DTPA (formulation 3) has a certain protective effect on the degradation of mRNA. For the Delta / C2 formulation group, compared with only 1XPBS (formulation 4), the addition of 20 mM Tris (formulation 5) will cause the purity of mRNA to decrease, and 0.01 mM DTPA (formulation 6) can improve the purity decrease problem caused by Tris.

[0423] The addition product results of formulations containing different LNP-mRNA during 2 weeks of storage at 37°C are shown in Figure 8.

[0424] As can be seen from Figure 8, the addition of 20 mM Tris can significantly reduce the formation of lipid adducts, and the addition of 0.01 mM DTPA to the 20 mM Tris does not affect the Tris inhibition of adduct formation.

[0425] Example 6

[0426] Alcohols stabilizers are common protective agents in frozen, lyophilized biological products. This example aims to investigate the effect of alcohols stabilizers on the physicochemical properties (particle size, encapsulation efficiency and osmolality) of the water for injection formulation by adding propylene glycol (PG) to the 0.01 mM DTPA, 20 mM Tris, 1X PBS formulation. The formulation information is summarized in Table 10.

[0427] Table 10. Formulation information

[0428] The physicochemical properties (particle size, PDI and encapsulation efficiency) of the formulations with or without propylene glycol added are shown in Figure 9 during the 2-week period at 37°C.

[0429] As can be seen from Figure 9, the addition of 7% PG causes a slight increase in particle size, and 7% PG does not affect the encapsulation efficiency.

[0430] The osmolality of the formulations is summarized in Table 11.

[0431] Table 11. Osmolality of the formulations

[0432] As can be seen from Table 11, the addition of 7% PG causes a significant increase in the osmolality of the formulation. However, the osmolality of normal human blood ranges from 285 to 310 mOsmol / kg, and a solution with too high an osmolality can cause discomfort or adverse reactions at the injection site.

[0433] The stability of the product during transportation was simulated by accelerated shaking experiments (accelerated shaking conditions are shown in Table 12).

[0434] Table 12. Accelerated shaking conditions for the formulations

[0435] The results of the insoluble particulate test for the formulations with or without propylene glycol added after 2 days of shaking are shown in Figure 10.

[0436] As can be seen from Figure 10, the insoluble particulates increased significantly during the shaking process, and the addition of 7% PG to the buffer compared to 0.01 mM DTPA, 20 mM Tris, 1X PBS caused a further increase in insoluble particulates, especially particles in the Feret≥25um range.

[0437] Example 7

[0438] The effect of the alcohol stabilizer propylene glycol on the physicochemical properties of the liquid formulation was investigated in Example 6, and the results showed that the addition of propylene glycol would cause an increase in insoluble particles, and was not compatible with the water needle formulation. This example was extended to another type of stabilizer: sugar, and sucrose, which is widely used in biological products at present, was selected to investigate its effect on the insoluble particles of the formulation.

[0439] The amount of sucrose added was selected to be the concentration commonly used in the marketed mRNA-LNP product: 8% (m / v).

[0440] The results of the insoluble particle test of the formulation after adding sucrose and shaking for 2 days are shown in Figure 11. It can be seen that after adding 8% sucrose in addition to the combination of DTPA and Tris, the initial (shaking for 0 days) insoluble particles of the formulation were higher, and the particle count after shaking for 2 days also increased significantly.

[0441] Example 8

[0442] This example aims to study the effect of adding an aldehyde absorber and a chelating agent in the formulation on the long-term stability of mRNA-LNP. In addition, the results in Example 3 showed that for the formulation with a concentration of 30 pg / mL (see Figure 3 and Table 6 Formulations 10, 11 and 12), the addition of DTPA from 0.01 mM to 1 mM under accelerated conditions did not significantly affect the mRNA purity in the formulation. In this study, the concentration of the formulation was increased, and the effect of 0.1 mM and 1 mM DTPA on the mRNA purity in the higher concentration formulation was investigated, respectively.

[0443] In addition, considering that the aldehyde absorber Tris itself can be used as a buffer component to simplify the composition of the buffer, the effect on the long-term stability of the formulation was investigated. The formulation prescription information is shown in Table 13. In Formulations 1-4, the mRNA used was IL-12 mRNA.

[0444] Table 13. Formulation prescription information

[0445] The mRNA purity of Formulations 1-4 during long-term storage at 2-8°C is summarized in Table 14.

[0446] Table 14. mRNA purity % results of Formulations 1-4

[0447] The content of the addition product of Formulations 1-4 during long-term storage at 2-8°C is summarized in Table 15.

[0448] Table 15. Lipid addition product % results of Formulations 1-4

[0449] Figure 12 shows the physicochemical properties (particle size, PDI and encapsulation efficiency) of the formulation during long-term storage at 2-8°C. As can be seen from Figure 12, the particle size, encapsulation efficiency and stability of the formulation are not affected after simplifying the buffer components from Tris, IX PBS (sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride) (Formulation 2) to Tris, sodium chloride (Formulation 3). In addition, the addition of DTPA does not affect the particle size, encapsulation efficiency of the formulation during long-term storage.

[0450] As can be seen from Tables 14 and 15 and Figures 13 and 14, the mRNA of the mRNA-LNP formulation (Formulation 1) is extremely degradable during long-term storage, and the lipid adduct product also gradually increases with the storage time. After adding 20 mM Tris and 1 mM DTPA in IX PBS buffer (Formulation 2), the degradation of mRNA is significantly delayed (57.5% for Formulation 1 vs. 81.1% for Formulation 2 at 9 months) (Table 14 and Figure 13). Compared with the gradual increase of the lipid adduct product of Formulation 1 group with storage time, the group with the addition of aldehyde absorbers (Formulations 2-4) has reached a plateau from the 6th month to the 9th month (Tables 15 and 14).

[0451] At the 9th month of long-term storage, there is no significant difference in particle size, encapsulation efficiency, and lipid adduct product between the addition of 0.1 mM and 1 mM DTPA (Formulations 4 and 3) (Figure 12). The purity of the formulation with the addition of 1 mM DTPA (Formulation 3) is slightly higher than that of the formulation with the addition of 0.1 mM DTPA (Formulation 4) (Table 14 and Figure 13), but the detection error of the instrument is about 5%, and no significant difference is observed, and further storage is needed to investigate the effect of DTPA addition on mRNA purity in different concentrations of formulations.

[0452] As can also be seen from Tables 14 and 15 and Figures 13 and 14, at the same DTPA concentration, the formulation with Tris as the buffer and aldehyde absorber (Formulation 3) is comparable to the formulation with IX PBS as the buffer and additional Tris as the aldehyde absorber (Formulation 2) in terms of mRNA purity and lipid adduct product. The results show that simplifying the components of the buffer can reduce the amount of excipients while not affecting the long-term stability of the formulation.

[0453] Example 9

[0454] The concentration of DTPA was screened in Example 3, and it was found that the chelation effect was comparable when the concentration of DTPA was in the range of 0.01 mM-1 mM. In this example, the concentration of Tris was screened in the formulation with simplified components. By detecting the mRNA purity and the physicochemical parameters of the addition product of the formulations with different concentrations of Tris under the condition of accelerated storage, the appropriate concentration of Tris was screened. Table 16 summarizes the formulation information. In Formulations 1-5, the mRNA used was IL-12 mRNA.

[0455] Table 16. Concentration information of aldehyde absorbent-Tris

[0456] The physicochemical properties (particle size, PDI and encapsulation efficiency) of the formulations with different concentrations of Tris during the process of storage at 40°C for 1 week are shown in Figure 15. It can be seen that there is no significant difference between the particle size and encapsulation efficiency of the formulations with different concentrations of Tris.

[0457] The mRNA purity results of the formulations with different concentrations of Tris during the process of storage at 40°C for 2 weeks are shown in Figure 16. It can be seen that the mRNA purity gradually decreases as the concentration of Tris increases from 10 mM to 80 mM under the condition of accelerated storage for 14 days.

[0458] The lipid addition product results of the formulations with different concentrations of Tris during the process of storage at 40°C for 1 week are shown in Figure 17. It can be seen that the increase of lipid addition product is inhibited as the concentration of Tris increases.

[0459] The above results show that the effect of the concentration of Tris on mRNA purity and addition product is contradictory, so the concentration of Tris cannot be too high or too low.

[0460] Sequence information:

[0461] Exemplary IL-12 mRNA sequence: 5’- 7Me GpppG 2’OMe7Me GpppG 2’Ome is a cap structure.

[0462] Exemplary DELTA mRNA sequence: 5'- 7Me G ppp G 2'OMe7Me GpppG 2’Ome is a cap structure.

[0463] The above merely describes exemplary embodiments or examples of the present application, and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application is included in the scope of the claims of the present application.

Claims

1. A lipid nanoparticle preparation, characterized in that Include: a pharmaceutically acceptable buffer comprising an aldehyde absorber and a chelating agent, lipid nanoparticles, and Nucleic acids are encapsulated in the lipid nanoparticles.

2. The lipid nanoparticle preparation according to claim 1, characterized in that The aldehyde absorbent is selected from a linear or branched aliphatic polyhydroxyamine compound and an amidine-containing amino acid; preferably, the aldehyde absorbent is selected from a linear or branched aliphatic polyhydroxyamine compound containing 1 to 6 carbon atoms and arginine; more preferably, the buffer contains tromethamine.

3. The lipid nanoparticle preparation according to claim 1 or 2, characterized in that The aldehyde absorber is present in the buffer at a concentration of 1-40 mM, preferably 3-40 mM, 5-40 mM or 10-40 mM, particularly preferably 3-35 mM, 5-35 mM or 10-35 mM, more preferably 5-30 mM or 10-30 mM, most preferably 10-25 mM, based on the volume of the buffer.

4. The lipid nanoparticle preparation according to any one of claims 1 to 3, characterized in that The chelating agent is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, citric acid, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and trisodium edetate; preferably, the buffer contains a chelating agent selected from diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid and citric acid.

5. The lipid nanoparticle preparation according to any one of claims 1 to 4, characterized in that The chelating agent is present in the buffer at a concentration of 0.001-10 mM, preferably 0.005-5 mM, more preferably 0.01-1 mM, based on the volume of the buffer.

6. The lipid nanoparticle preparation according to any one of claims 1 to 3, characterized in that The buffer contains 0.005-1 mM ethylenediaminetetraacetic acid; The buffer contains 1-10 mM citric acid; or The buffer contains 0.005-1 mM diethylenetriaminepentaacetic acid.

7. The lipid nanoparticle preparation according to any one of claims 1 to 6, characterized in that The buffer is selected from phosphate buffer, citrate buffer, Tris buffer, acetate buffer, histidine buffer, HEPES buffer and MES buffer; preferably, the buffer is selected from Tris buffer and phosphate buffer.

8. The lipid nanoparticle preparation according to any one of claims 1 to 6, characterized in that The buffer is or comprises Tris buffer, and optionally comprises sodium chloride; or The buffer is or comprises Tris buffer and phosphate buffer.

9. The lipid nanoparticle preparation according to claim 8, characterized in that The buffer comprises 1-40 mM, preferably 3-40 mM, 5-40 mM or 10-40 mM, particularly preferably 3-35 mM, 5-35 mM or 10-35 mM, more preferably 5-30 mM or 10-30 mM, most preferably 10-25 mM tromethamine.

10. The lipid nanoparticle preparation according to any one of claims 1 to 9, characterized in that The pH of the buffer solution is in the range of 6.0-8.0; preferably, the pH of the buffer solution is in the range of 6.5-7.8; more preferably, the pH of the buffer solution is in the range of 7.0-7.

5.

11. The lipid nanoparticle preparation according to any one of claims 1 to 10, characterized in that The buffer satisfies at least one of the following: The buffer does not contain an antioxidant; The buffer does not contain 6% by weight or more of propylene glycol, preferably, the buffer does not contain propylene glycol; The buffer does not contain 7 wt% or more of sucrose, preferably, the buffer does not contain sucrose.

12. The lipid nanoparticle preparation according to any one of claims 1 to 11, characterized in that The lipid nanoparticles comprise cationic lipids.

13. The lipid nanoparticle preparation according to any one of claims 1 to 12, characterized in that The nucleic acid comprises RNA, preferably, the nucleic acid is selected from the group consisting of short polymer, agomir, antagomir, antisense, ribozyme, siRNA, aiRNA, miRNA, dsRNA, shRNA, tRNA and mRNA.

14. The lipid nanoparticle preparation according to any one of claims 1 to 13, characterized in that The nucleic acid has a molecular length of at least about 30 nucleotides (nt), at least about 35 nt, at least about 40 nt, at least about 45 nt, at least about 50 nt, at least about 55 nt, at least about 60 nt, at least about 65 nt, at least about 70 nt, at least about 75 nt, at least about 80 nt, at least about 85 nt, at least about 90 nt, at least about 95 nt, at least about 100 nt, at least about 120 nt, at least about 140 nt, at least about 160 nt, at least about 180 nt, at least about 200 nt, At least about 250nt, at least about 300nt, at least about 400nt, at least about 500nt, at least about 600nt, at least about 700nt, at least about 800nt, at least about 900nt, at least about 1000nt, at least about 1100nt, at least about 1200nt, at least about 1300nt, at least about 1400nt, at least about 1500nt, at least about 1600nt, at least about 1700nt, at least about 1800nt, at least about 1900nt or at least about 2000nt.

15. The lipid nanoparticle preparation according to any one of claims 1 to 14, characterized in that The concentration of the nucleic acid is 0.001 to 2 mg / ml, preferably 0.003 to 1 mg / ml, more preferably 0.01 to 0.5 mg / ml, based on the volume of the lipid nanoparticle preparation.

16. The lipid nanoparticle preparation according to any one of claims 1 to 15, characterized in that The molar ratio of the lipid to the nucleic acid is 1:1 to 10:1, preferably 2:1 to 10:1, more preferably 4:1 to 8:

1.

17. The method for preparing the lipid nanoparticle preparation according to any one of claims 1 to 16, characterized in that: The following steps are involved: 1) providing lipid working solution and nucleic acid working solution; II) preparing lipid nanoparticles encapsulating nucleic acids using a lipid working solution and a nucleic acid working solution; and III) Performing dialysis exchange so that the buffer contains the aldehyde absorbent and the chelating agent.

18. The preparation method according to claim 17, characterized in that: The method also includes a filtration step after the dialysis fluid exchange.

19. The preparation method according to claim 18, characterized in that The preparation method further comprises the step of packaging the obtained lipid nanoparticle preparation into packaging materials after filtration. Preferably, this step is performed under nitrogen protection.

20. The preparation method according to claim 19, characterized in that The packaging material is a vial, an ampoule or a prefilled needle.

21. A pharmaceutical product, characterized in that Include: The lipid nanoparticle formulation according to any one of claims 1 to 16; and A container containing the lipid nanoparticle preparation.

22. The pharmaceutical product according to claim 21, characterized in that The container is a vial, an ampoule or a prefilled syringe. Optionally, the container is a vial, an ampoule or a prefilled syringe filled with nitrogen.

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