Nucleic acid compositions comprising multivalent anions such as inorganic polyphosphates and methods for making, storing and using same

Abstract

The present disclosure relates generally to the field of nucleic acid (e.g., DNA or RNA, in particular mRNA or inhibitory RNA, e.g., siRNA) compositions comprising a multivalent anion (e.g., inorganic polyphosphate), methods for preparing and storing such compositions, and use of such compositions in therapy.

Description

Technical Field

[0001] The present disclosure generally relates to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA or inhibitory RNA, such as siRNA) compositions comprising polyvalent anions (e.g., inorganic polyphosphate), methods for preparing and storing such compositions, and the use of such compositions in therapy. Background Art

[0002] The use of recombinant nucleic acids (e.g., DNA or RNA) for delivering foreign genetic information into target cells is well known. Recombinant nucleic acids can be administered in naked form to subjects in need thereof; however, recombinant nucleic acids are typically administered using compositions. For example, nucleic acids, such as RNA, can be delivered to subjects using different delivery vehicles, primarily based on cationic polymers or lipids that form nanoparticles with the nucleic acid. Nanoparticles are designed to protect nucleic acids (e.g., RNA) from degradation, enable delivery of nucleic acids (e.g., RNA) to target sites, and promote cellular uptake and processing by target cells. The efficiency of nucleic acid delivery depends in part on the molecular composition of the nanoparticles and can be affected by many parameters, including particle size, formulation, and charge or grafting with molecular moieties (e.g., polyethylene glycol (PEG) or other ligands). The fate of such nanoparticle formulations is controlled by a variety of key factors (e.g., the size and size distribution of the nanoparticles, etc.). For example, in the 2018 FDA "Liposome Drug Products Guidance," these factors are referred to as specific properties that should be analyzed and explained. Advantages of using RNA include transient expression and non-transformation characteristics. Furthermore, RNA does not need to enter the nucleus to be expressed, and furthermore RNA cannot integrate into the host genome, thus eliminating various risks such as tumorigenesis.

[0003] Consider grafting with PEG to reduce serum interactions, improve serum stability and extend circulation time, which may help certain targeting methods. Ligands that bind to receptors at the target site can help improve targeting efficacy. In addition, PEGylation can be used for particle modification. For example, if lipid nanoparticles (LNPs) are prepared by mixing an aqueous phase of nucleic acids (e.g., RNA) with an organic phase of lipids, a certain fraction of PEG-conjugated lipids is required in the lipid mixture, otherwise the particles will aggregate during or after the mixing step. It has been shown that by changing the molar fraction of PEG lipids containing different molar masses of PEG, the size of the particles can be adjusted. Similarly, the particle size can be adjusted by changing the molar mass of the PEG portion of the PEGylated lipid. The typical size that can be obtained is 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles thus formed have the additional advantage that, due to the PEG moiety, they interact less with serum components and have a longer circulation half-life, which is desirable in many drug delivery methods. Without PEG lipids, particles of discrete sizes cannot be formed; the particles form large aggregates and precipitate. Thus, one of the main functions of PEG lipids is to facilitate particle self-assembly by providing a steric barrier on the surface of nascent particles that form when nucleic acids are rapidly mixed in an ethanolic solution containing lipids to bind nucleic acids, such as RNA. PEG steric hindrance prevents interparticle fusion and promotes the formation of a uniform LNP population, wherein diameters <100 nm can be achieved.

[0004] PEG is the most widely used and gold standard "stealth" polymer for drug delivery. PEG lipids are often incorporated into systems to prepare uniform and colloidally stable populations of nanoparticles due to their hydrophilic steric properties (the PEG shell prevents electrostatic or van der Waals attraction that leads to aggregation). PEGylation enables the attraction of a water shell around the polymer, protecting the particles from opsonization by serum proteins and increasing serum half-life, which results in improved pharmacokinetic behavior. The lipid acyl chains (C 18 、C 16 or C 14 Variation in the length of the PEG lipids altered the stability of the PEG lipid incorporation in the particles, which led to modulation of the pharmacokinetics. 14The use of PEG lipids with acyl chains resulted in optimal gene silencing efficacy in hepatocytes (Chen et al., 2014, J. Control Release 196:106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163). In addition, strict control over particle size can be achieved by varying the PEG lipid parameters: a higher PEG MW or a higher PEG lipid mole fraction in the particle results in smaller particles.

[0005] In spite of these advantages, the PEGylation of nanoparticles may also lead to several effects that are unfavorable for the intended use of drug delivery. The PEGylation of known liposomes and LNPs has reduced cellular uptake and endosome escape, thereby ultimately reducing overall transfection efficiency. In fact, the PEG shell provides a spatial barrier for the effective combination of particles and cells, and also hinders endosome release by stopping the membrane fusion between liposomes and the endosomal membrane. This is why the type of PEG lipid and the amount of used PEG lipid must always be carefully adjusted. It should provide enough stealth effects in vivo and in terms of stability on the one hand, while not hindering transfection on the other hand. This phenomenon is referred to as the "PEG dilemma".

[0006] In addition to reduced transfection efficiency, PEGylation has been associated with the phenomenon of accelerated blood clearance (ABC) induced by anti-PEG antibodies and / or complement activation, as well as storage diseases (Bendele A et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; SM Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42: 463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes in rats significantly altered the pharmacokinetic behavior of a second dose when the second dose was administered several days later (Laverman P et al., 2001, J. Pharmacol. Exp. Ther. 298(2), 607-12; Ishida et al., 2006, J. Control Release 115(3), 251-8). The "accelerated blood clearance" (ABC) phenomenon was shown to be associated with the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma induced a higher clearance of particles by the mononuclear phagocyte system (MPS), which ultimately reduced the efficacy of the drug.

[0007] PEG is also thought to induce complement activation, which can lead to hypersensitivity reactions, also known as Complement-Activation Related Pseudo-Allergy (CARPA). It remains unclear from the literature whether the activation of complement is due to the presence of nanoparticles in general or PEG in particular.

[0008] The presence of PEG in other lipid nanoparticles can also induce specific immune responses. Semple et al. (2005, J.Pharmacol.Exp.Ther.312(3),1020-6) reported that liposomes containing PEG lipid derivatives and encapsulated antisense oligodeoxynucleotides or plasmid DNA triggered a strong immune response that resulted in rapid blood clearance of subsequent doses in mice. The magnitude of this response was sufficient to induce significant morbidity and, in some cases, mortality. The rapid elimination of liposome-encapsulated oligodeoxynucleotides from the blood depended on the presence of PEG lipids in the membrane, as the use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG lipids eliminated this response. The production of anti-PEG antibodies and presumed complement activation are possible explanations for the rapid elimination of vesicles from the blood.

[0009] Because PEG can induce an immune response, it needs to be avoided for certain applications that require multiple injections. Some examples are treatments using nucleic acids (e.g., RNA, particularly mRNA), such as for protein replacement therapy. Here, the risk may be particularly high due to the potential inherent immunogenicity of nucleic acids (particularly RNA). Other examples are protein knockdown treatments using inhibitory RNA (e.g., siRNA), antisense oligonucleotides, or DNA-based therapies.

[0010] Therefore, there remains a need in the art for effective compositions and methods for introducing nucleic acids (e.g., RNA) into cells that avoid the disadvantages associated with the use of PEG. Ideally, these compositions and methods should ensure that (i) the compositions are stable and can be stored within a temperature range consistent with conventional techniques in pharmaceutical practice, particularly at a temperature of about -20°C or even in liquid form at a temperature of +2 to +20°C; (ii) the compositions can be repeatedly frozen and thawed; (iii) the compositions are ready for use; (iv) the PEG-free compositions maintain high biological efficacy; and / or (v) the nucleic acids contained in the compositions are in a stable form and do not significantly degrade upon storage. The present disclosure addresses these and other needs.

[0011] The present inventors have unexpectedly discovered that the compositions and methods described herein meet the above requirements. In particular, it has been shown that by using multivalent anions, such as inorganic polyphosphate, inorganic phosphate or citrate, stable compositions can be prepared (particularly with respect to the colloidal size of the particles contained in the composition), which can be stored in liquid form, can be repeatedly frozen and thawed, contain nucleic acids in a stable form, and maintain high biological efficacy, even when the composition / particles do not contain PEG lipids or any other stealth lipids. Summary of the Invention

[0012] In a first aspect, the present disclosure provides a composition comprising (i) a nucleic acid; (ii) a lipid that can ionize into a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a multivalent anion, such as an inorganic polyphosphate.

[0013] As demonstrated herein, the aggregation of particles (e.g., lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), or mixtures thereof) comprised in a composition (particularly an aqueous composition) and formed from nucleic acids (particularly RNA, e.g., mRNA), lipids that ionize to release cations, steroids, and neutral lipids can be prevented by adding polyvalent anions, e.g., inorganic polyphosphates, to the composition, even when the composition / particles do not contain PEG lipids or any other stealth lipids. Furthermore, the present application demonstrates that, unexpectedly, in the presence of polyvalent anions (e.g., inorganic polyphosphates), and by adjusting the amounts of lipids relative to one another, nucleic acid (e.g., RNA) compositions can be obtained that exhibit different biological properties: when used to transfect cells, nucleic acid compositions comprising a higher relative amount of ionizable and neutral lipids and a lower relative amount of steroids result in higher expression of the nucleic acid in the transfected cells when the transfection is performed in the presence of serum, compared to the expression obtained when the transfection is performed in the absence of serum (this effect is referred to herein as "serum stimulation" and is similar to the biological properties of standard nucleic acid compositions comprising PEG lipids). In contrast, nucleic acid compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids used to transfect cells resulted in comparable or reduced expression of the nucleic acid in the transfected cells when transfection was performed in the presence of serum (i.e., these compositions exhibited some or no serum inhibition, but not serum stimulation). Thus, the claimed compositions are stable and can be stored within a temperature range consistent with conventional techniques in pharmaceutical practice, providing ready-to-use compositions and maintaining high biological efficacy, even when the compositions / particles do not contain PEG lipids or any other stealth lipids. Furthermore, depending on the relative amounts of the lipids relative to one another, the claimed compositions can exhibit different biological properties, i.e., serum-stimulable or non-serum-stimulable.

[0014] As used herein, the term "polyvalent anion" is understood to refer to an ion with multiple (i.e., more than one) negative charges. For example, a polyvalent anion can be a divalent anion, i.e., has a charge of 2- or has two negative charges. In another example, a polyvalent anion can be a trivalent anion, i.e., has a charge of 3- or has three negative charges. In another example, a polyvalent anion can be a tetravalent anion, i.e., has a charge of 4- or has four negative charges. In other examples, a polyvalent anion can have multiple negative charges. Generally speaking, a polyvalent anion is not or does not comprise nucleic acid, such as DNA or RNA. In some embodiments, a polyvalent anion has no more than 20 negative charges (i.e., a charge of 20-), preferably no more than 10 negative charges (i.e., a charge of 10-), or most preferably no more than 5 negative charges (i.e., a charge of 5-). The polyvalent anion can have 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 5, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5 negative charges, optionally 2 to 10 negative charges, preferably 2 to 5 negative charges.

[0015] The polyvalent anion may be selected from: inorganic polyphosphate (as further defined herein), inorganic phosphate (e.g. PO4 3- ), sulfate, sulfite, pyrosulfate, dithionite, dithionite, metabisulfite, thiosulfate, trithionite or tetrathionite, dicarboxylic acid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, sebacic acid, phthalic acid, isophthalic acid or terephthalic acid), substituted dicarboxylic acid (e.g., tartronic acid, mesooxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid or saccharinic acid), tricarboxylic acid (e.g., citric acid, isocitric acid, propane-1,2,3-tricarboxylic acid or trimesic acid), or mixtures thereof. Generally speaking, polyvalent anions in water are molecularly dissolved as solutes and do not form supramolecular assemblies such as micelles. Structurally, the polyvalent anion is not or does not comprise a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion (e.g., the polyvalent anion is not a negatively charged lipid).

[0016] In some embodiments, the multivalent anion is an inorganic polyphosphate. The inorganic polyphosphate can be any linear, cyclic or branched inorganic polyphosphate. In some embodiments of the first aspect, the inorganic polyphosphate is a linear inorganic polyphosphate (e.g., linear inorganic triphosphate).

[0017] In some embodiments of the first aspect, the inorganic polyphosphate comprises the formula [P x O (3x+1) ] y, wherein x is an integer and is at least 2, preferably at least 3; and y is an anionic charge. For example, if x is 3, the inorganic polyphosphate is a compound comprising the formula [P3O 10 ] 5- Similarly, if x is 4, then the inorganic polyphosphate is a linear inorganic triphosphate containing the formula [P4O 13 ] 6- Linear or branched inorganic tetraphosphate.

[0018] In some embodiments of the first aspect, the inorganic polyphosphate is selected from diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate and mixtures thereof, for example, selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate and mixtures thereof. In some preferred embodiments of the first aspect, the inorganic polyphosphate is selected from triphosphate, tetraphosphate, pentaphosphate and mixtures thereof. In some preferred embodiments of the first aspect, the inorganic polyphosphate is triphosphate.

[0019] In some embodiments, the multivalent anion is an inorganic phosphate (e.g., PO4 3- ), sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate, or tetrathionate. In some embodiments, the polyvalent anion is an inorganic phosphate (e.g., PO4 3- ).

[0020] In some embodiments, the polyvalent anion is a dicarboxylic acid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, sebacic acid, phthalic acid, isophthalic acid, or terephthalic acid) or a substituted dicarboxylic acid (e.g., tartronic acid, mesooxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid, or saccharinic acid).

[0021] In some embodiments, the multivalent anion is a tricarboxylic acid (eg, citric acid, isocitric acid, propane-1,2,3-tricarboxylic acid, or trimesic acid).

[0022] In some preferred embodiments, the polyvalent anion is selected from the group consisting of: inorganic polyphosphate (as defined herein), inorganic phosphate (e.g., PO4 3- ), sulfate, succinate, glutarate, tartrate, malate, citrate, or mixtures thereof. In some embodiments, the multivalent anion is an inorganic polyphosphate (as defined herein), an inorganic phosphate, or a citrate. In some most preferred embodiments, the multivalent anion is an inorganic polyphosphate (as defined herein).

[0023] In some embodiments of the first aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 1: 2. For example, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation can be at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0. In some preferred embodiments of the first aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 2: 3. In some preferred embodiments of the first aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 4:3.

[0024] In some embodiments of the first aspect, the composition is substantially free of lipids comprising polyethylene glycol (PEG). In some embodiments, the composition is substantially free of any compounds comprising PEG. In some embodiments, the composition is substantially free of PEG. Thus, in some embodiments of the first aspect, the composition comprises (i) a nucleic acid; (ii) a lipid that can ionize into a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion, wherein the composition is substantially free of lipids comprising PEG, substantially free of any compounds comprising PEG, or substantially free of PEG. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, and citrate. In some of these embodiments, the polyvalent anion is an inorganic polyphosphate.

[0025] In some embodiments of the first aspect, the composition is also substantially free of additional polymer-conjugated lipids. In some embodiments, the additional polymer-conjugated lipid is a polysarcosine-conjugated lipid. In some embodiments, the composition is substantially free of any polymer-conjugated lipids (including PEG lipids and polysarcosine-conjugated lipids). Thus, in some embodiments of the first aspect, the composition comprises (i) a nucleic acid; (ii) a lipid that can ionize out a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion, wherein the composition is substantially free of any polymer-conjugated lipids (including PEG lipids and polysarcosine-conjugated lipids). In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphates, inorganic phosphates (e.g., PO4 3- In some embodiments, the polyvalent anion is selected from an inorganic polyphosphate, an inorganic phosphate, a sulfate, a sulfite, a pyrosulfate, a dithionate, a dithionite, a metabisulfite, a thiosulfate, a trithionate or a tetrathionate, a dicarboxylic acid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, sebacic acid, phthalic acid, isophthalic acid or terephthalic acid), a substituted dicarboxylic acid (e.g., tartronic acid, mesooxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid or saccharinic acid) or a tricarboxylic acid (e.g., citric acid, isocitric acid, propane-1,2,3-tricarboxylic acid or trimesic acid). In some of these embodiments, the polyvalent anion is selected from an inorganic polyphosphate, an inorganic phosphate, a sulfate, a succinate, a glutarate, a tartrate, a malate, a citrate, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from an inorganic polyphosphate, an inorganic phosphate and a citrate. In some of these embodiments, the polyvalent anion is an inorganic polyphosphate.

[0026] In some embodiments of the first aspect, the pH of the composition is from about 4.0 to about 8.0. In some embodiments of the first aspect, the pH of the composition is from about 4.5 to about 8.0, e.g., from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, or from about 7.0 to about 7.8.

[0027] In some embodiments of the first aspect, water is the major component in the composition, and / or the total amount of solvent other than water included in the composition is less than about 1.0% (v / v), for example, less than about 0.5% (v / v). For example, the amount of water included in the composition can be at least 50% (w / w), for example, at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). In particular, if the composition comprises a cryoprotectant, the amount of water included in the composition can be at least 50% (w / w), for example, at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the composition is substantially free of a cryoprotectant, the amount of water included in the composition can be at least 95% (w / w). Additionally or alternatively, the total amount of solvent other than water included in the composition can be less than about 0.5% (v / v), for example, less than about 0.4% (v / v), less than about 0.3% (v / v), less than about 0.2% (v / v), less than about 0.1% (v / v), less than about 0.05% (v / v), less than about 0.01% (v / v), or less than about 0.005% (v / v). In this regard, cryoprotectants that are liquid under normal conditions will not be considered solvents other than water, but rather will be considered cryoprotectants. In other words, the above optional limitation, i.e., the total amount of solvent other than water included in the composition may be less than about 0.5% (v / v), such as less than about 0.4% (v / v), does not apply to cryoprotectants that are liquid under normal conditions.

[0028] In some embodiments of the first aspect, the osmolarity of the composition is at most about 1000×10 -3 In some embodiments of the first aspect, the osmolarity of the composition is at most about 1000×10 -3 In some embodiments, the osmolality of the composition is at most about 500×10 -3 osmol / kg, for example, up to about 490×10 -3 osmol / kg, up to about 480×10 -3 osmol / kg, up to about 470×10 -3 osmol / kg, up to about 460×10 -3 osmol / kg, up to about 450×10 -3 osmol / kg, up to about 440×10-3 osmol / kg, up to about 430×10 -3 osmol / kg, up to about 420×10 -3 osmol / kg, up to about 410×10 -3 osmol / kg, up to about 400×10 -3 osmol / kg, up to about 390×10 - 3 osmol / kg, up to about 380×10 -3 osmol / kg, up to about 370×10 -3 osmol / kg, up to about 360×10 -3 osmol / kg, up to about 350×10 -3 osmol / kg, up to about 340×10 -3 osmol / kg, up to about 330×10 -3 osmol / kg, up to about 320×10 -3 osmol / kg, up to about 310×10 -3 osmol / kg, or up to about 300×10 -3 In some embodiments of the first aspect, the osmolarity of the composition is about 100×10 -3 osmol / kg to about 500×10 -3 osmol / kg, for example, about 300×10 -3 If the composition does not contain a cryoprotectant, the osmolality of the composition may be less than 300×10 -3 osmol / kg, for example, up to about 250×10 -3 osmol / kg, up to about 200×10 -3 osmol / kg, up to about 150×10 -3 osmol / kg, up to about 100×10 -3 osmol / kg, up to about 50×10 -3 osmol / kg, up to about 40×10 - 3 osmol / kg, or up to about 30×10 -3 osmol / kg. If the composition comprises a cryoprotectant, it is preferred that a major portion of the osmolality of the composition is provided by the cryoprotectant. For example, the cryoprotectant may provide at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the osmolality of the composition.

[0029] In some embodiments of the first aspect, the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 500 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 100 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 5 mg / l to about 500 mg / l, for example, from about 10 mg / l to about 400 mg / l, from about 10 mg / l to about 300 mg / l, from about 10 mg / l to about 200 mg / l, from about 10 mg / l to about 150 mg / l, or from about 10 mg / l to about 100 mg / l, preferably from about 10 mg / l to about 140 mg / l, more preferably from about 20 mg / l to about 130 mg / l, more preferably from about 30 mg / l to about 120 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 5 mg / l to about 150 mg / l, for example from about 10 mg / l to about 140 mg / l, from about 20 mg / l to about 130 mg / l, from about 25 mg / l to about 125 mg / l, from about 30 mg / l to about 120 mg / l, from about 35 mg / l to about 115 mg / l, from about 40 mg / l to about 110 mg / l, from about 45 mg / l to about 105 mg / l, or from about 50 mg / l to about 100 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from 1 mg / l to about 50 mg / l or from about 10 mg / l to about 100 mg / l. In some embodiments of the first aspect (particularly those wherein the composition is in frozen form), the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 50 mg / l. In some embodiments of the first aspect (particularly those wherein the composition is in liquid form), the concentration of nucleic acid (particularly RNA) in the composition is from about 10 mg / l to about 100 mg / l.

[0030] In some embodiments of the first aspect, the composition comprises a cryoprotectant. In some embodiments of the first aspect, the composition is substantially free of a cryoprotectant.

[0031] In some embodiments of the first aspect, the molar ratio of the polyvalent anion to the lipid that can ionize to release a cation is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 100 mg / l (e.g., 1 mg / l to about 50 mg / l, or about 10 mg / l to about 100 mg / l). In some embodiments of the first aspect (particularly those in which the composition is in frozen form), the molar ratio of the polyvalent anion to the lipid that can ionize to release a cation is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 50 mg / l. In some embodiments of the first aspect (particularly those wherein the composition is in liquid form), the molar ratio of the polyvalent anion to the lipid that can ionize out the cation is at least about 1:2 (preferably at least about 2:3, e.g., at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is from about 10 mg / l to about 100 mg / l. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, and citrate.

[0032] In some embodiments of the first aspect, the multivalent anion is an inorganic polyphosphate, the inorganic polyphosphate is a linear inorganic polyphosphate, in particular a triphosphate, and the molar ratio of the inorganic polyphosphate to the lipid that can ionize to release a cation is at least about 1:2, preferably at least about 2:3, for example at least about 1.00 or at least about 4:3. In some preferred embodiments of the first aspect, the inorganic polyphosphate is a linear inorganic polyphosphate (in particular a triphosphate), the molar ratio of the inorganic polyphosphate to the lipid that can ionize to release a cation is at least about 1:2 (preferably at least about 2:3, for example at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (in particular RNA) in the composition is about 1 mg / l to about 100 mg / l (e.g., 1 mg / l to about 50 mg / l, or about 10 mg / l to about 100 mg / l). In some embodiments of the first aspect (particularly those in which the composition is in frozen form), the inorganic polyphosphate is a linear inorganic polyphosphate (particularly triphosphate), the molar ratio of the inorganic polyphosphate to the lipid that can ionize to release cations is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 50 mg / l. In some embodiments of the first aspect (particularly those in which the composition is in liquid form), the inorganic polyphosphate is a linear inorganic polyphosphate (particularly triphosphate), the molar ratio of the inorganic polyphosphate to the lipid that can ionize to release cations is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is from about 10 mg / l to about 100 mg / l.

[0033] In some embodiments of the first aspect, the lipid ionizable to release a cation comprises a head group comprising at least one tertiary amine moiety.

[0034] In some embodiments of the first aspect, the lipid that can ionize to release a cation has the structure of formula (X):

[0035]

[0036] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 10 , L 20 , G 1 , G 2 , G 3 、R 35 、R 36 and R 37As defined herein. In some embodiments, the lipid that can ionize to release a cation is selected from the following: structures X-1 to X-36 (shown herein); structures A to G (shown herein); or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA) and 4-((di((9Z,12Z)-octadecane-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-1-amine (DPL-14). In some embodiments, the lipid that can ionize to release a cation is a lipid having structure X-3. In some embodiments, the lipid that can ionize to release a cation is DPL-14 (i.e., a lipid having structure G). In some embodiments, the lipid that ionizes to release a cation is a lipid having structure D.

[0037] In some embodiments of the first aspect, the lipid that can ionize into a cation has the structure of Formula (XI):

[0038]

[0039] Wherein R1, R2 R3, R4, L2, G2 and m are as defined herein. In some embodiments, the lipid that can ionize to give a cation is selected from structures (XIV-1), (XIV-2) and (XIV-3) (shown herein). In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-1. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-2. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-3.

[0040] In some embodiments of the first aspect, the lipid that can ionize out a cation is completely or partially replaced by a cationic lipid. In some embodiments, the cationic lipid is selected from Structures XV-1 to XV-6 (shown in this article). In those embodiments in which the lipid that can ionize out a cation is completely replaced by a cationic lipid, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the lipid that can ionize out a cation is replaced by the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the cationic lipid. In some embodiments, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the cationic lipid is at least about 1:2 (e.g., at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0, preferably the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the cationic lipid is at least about 2:3, e.g., at least about 4:3). In those embodiments in which the lipid ionizable to release cations is partially replaced by a cationic lipid, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the lipid ionizable to release cations is replaced by the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid ionizable to release cations. Thus, in some embodiments, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid that can ionize to release a cation is at least about 1:2 (e.g., at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0, preferably, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid that can ionize to release a cation is at least about 2:3, e.g., at least about 4:3).

[0041] In some embodiments of the first aspect, the lipids that can release cations comprise about 20 mol% to about 75 mol% of the total lipids present in the composition, for example, about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% (particularly for those embodiments with higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% (particularly for those embodiments with lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids). In those embodiments in which the lipids that can release cations are partially or completely replaced by cationic lipids, it is preferred that the same ranges as specified above for the lipids that can release cations (e.g., about 20 mol% to about 75 mol%, etc.) apply to the sum of the lipids that can release cations and the cationic lipids.

[0042] In some embodiments of the first aspect, the steroid comprises a sterol. In some preferred embodiments of the first aspect, the steroid comprises or is cholesterol.

[0043] In some embodiments of the first aspect, the steroid comprises about 15 mol% to about 60 mol% of the total lipids present in the composition, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% (particularly for those embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly for those embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids).

[0044] In some embodiments of the first aspect, the neutral lipid is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. In some embodiments, the phospholipid is selected from T g In some embodiments, the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE) and dipalmitoyl-phosphatidylethanolamine (DPPE). In some embodiments, the neutral lipid is DSPC.

[0045] In some embodiments of the first aspect, the neutral lipids comprise about 5 mol% to about 25 mol% of the total lipids present in the composition, e.g., about 15 mol% to about 25 mol%, or about 17 mol% to about 21 mol% (particularly for those embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 5 mol% to about 15 mol%, or about 7 mol% to about 14 mol% (particularly for those embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids).

[0046] In some embodiments of the first aspect, the lipids that ionize to release cations comprise about 20 mol% to about 70 mol% of the total lipids present in the composition; the steroids comprise about 15 mol% to about 60 mol% of the total lipids present in the composition; and the neutral lipids (e.g., phospholipids) comprise about 5 mol% to about 25 mol% of the total lipids present in the composition.

[0047] In some embodiments of the first aspect, the lipid that can ionize out cations accounts for about 40mol% to about 70mol% of the total lipids present in the composition, for example, about 45mol% to about 65mol% or about 50mol% to about 60mol%; Steroid (it is preferably cholesterol) accounts for about 15mol% to about 40mol% of the total lipids present in the composition, for example, about 20mol% to about 35mol% or about 20mol% to about 30mol%; and neutral lipid (it is preferably phospholipid) accounts for about 15mol% to about 25mol% of the total lipids present in the composition, for example, about 17mol% to about 21mol%. These embodiments of the composition of the first aspect, i.e., the compositions comprising higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids, are particularly suitable for transfection cells in the presence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0048] In some embodiments of the first aspect (which comprise a higher relative amount of ionizable and neutral lipids and a lower relative amount of steroids), the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. Thus, in some embodiments of the first aspect, the lipids that can ionize out cations account for about 40 mol% to about 70 mol% (e.g., about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%) of the total lipids present in the composition; the steroids account for about 15 mol% to about 40 mol% (e.g., about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%) of the total lipids present in the composition; the neutral lipids account for about 15 mol% to about 25 mol% (e.g., about 17 mol% to about 21 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0049] In some alternative embodiments of the first aspect, the lipid that can ionize out cations accounts for about 20mol% to about 40mol% of the total lipids present in the composition, for example, about 25mol% to about 40mol% or about 25mol% to about 35mol%; Steroid (it is preferably cholesterol) accounts for about 35mol% to about 60mol% of the total lipids present in the composition, for example, about 40mol% to about 60mol% or about 45mol% to about 60mol%; And neutral lipid (it is preferably phospholipid) accounts for about 5mol% to about 15mol% of the total lipids present in the composition, for example, about 7mol% to about 14mol%. These alternative embodiments of the composition of the first aspect, i.e., the composition comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids, are particularly suitable for transfection cells in the absence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0050] In some embodiments of the first aspect (which comprise lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids), the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is 3.0 to 10.0, for example 5.0 to 7.0. Thus, in some embodiments of the first aspect, lipids that can ionize to release cations account for about 20 mol% to about 40 mol% (e.g., about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%) of the total lipids present in the composition; steroids account for about 35 mol% to about 60 mol% (e.g., about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%) of the total lipids present in the composition; neutral lipids account for about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is 3.0 to 10.0, for example 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0051] In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids may comprise a cationic lipid. In these embodiments in which a cationic lipid is present, the amount of the lipid that can be ionized to produce a cation and the amount of the cationic lipid are used for calculation. For example, if the amount of the lipid that can be ionized to produce a cation in the composition should be from about 20 mol % to about 70 mol % and the composition should also comprise a cationic lipid, then the amount of the lipid that can be ionized to produce a cation and the amount of the cationic lipid will be from about 20 mol % to about 70 mol %.

[0052] In some embodiments of the first aspect, the only lipids contained in the composition are lipids that can ionize to release cations, steroids, and neutral lipids, particularly lipids that can ionize to release cations, steroids, and phospholipids.

[0053] In some embodiments of the first aspect, the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into a cation, at least a portion of the steroid, and at least a portion of the neutral lipid; and wherein at least a portion of the multivalent anion (e.g., inorganic polyphosphate) is associated with the particle. In some embodiments, the particle comprises or is selected from lipid nanoparticles (LNPs), liposomes, lipid complexes (LPXs), and mixtures thereof. In some embodiments, the particle comprises or is an LNP. In some embodiments, the particle comprises or is a liposome. In some embodiments, the particle comprises or is an LPX. In some embodiments, the particle comprises or is a mixture of LNPs and liposomes. In some embodiments, the particle comprises or is a mixture of LNPs and LPX. In some embodiments, the particle comprises or is a mixture of liposomes and LPX. In some embodiments, the particle comprises or is a mixture of LNPs, liposomes, and LPX.

[0054] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the particles comprise substantially all lipids present in the composition (particularly all lipids ionizable to release cations, steroids, and neutral lipids).

[0055] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of lipids that ionize into cations, steroids, and neutral lipids (eg, substantially free of lipids).

[0056] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the particles comprise at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acids (particularly RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85% of the nucleic acids (particularly RNA) present in the composition.

[0057] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of nucleic acids.

[0058] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the nucleic acid (eg, RNA) is encapsulated within or associated with the particles.

[0059] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%) of the polyvalent anions (e.g., polyphosphate) present in the composition are associated with the particles. In some embodiments, at least 20%, and more preferably at least 50%, of the polyvalent anions (e.g., polyphosphate) present in the composition are associated with the particles.

[0060] In some embodiments of the first aspect, wherein the composition comprises particles dispersed in an aqueous phase, the particles have a size of about 30 nm to about 500 nm. In some embodiments, the particles have a size of about 50 nm to about 150 nm.

[0061] In some embodiments of the first aspect, the nucleic acid is DNA.

[0062] In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA (eg, siRNA).

[0063] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and (i) comprises modified nucleosides instead of uridine; (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with increased G / C content compared to the wild-type coding sequence. In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0064] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and comprises at least one or more of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence. In some embodiments, the RNA (e.g., mRNA) comprises all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably interrupted by A nucleotides. In some embodiments, the 5' cap is a cap 1 or cap 2 structure.

[0065] In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and encodes one or more polypeptides. In some embodiments, the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response to an antigen in a subject.

[0066] In some embodiments of the first aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of a protein, or an immunogenic fragment of a protein or an immunogenic variant thereof. In some embodiments, the pathogen is a pathogen that causes an infectious disease.

[0067] In some embodiments of the first aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof. In some embodiments, the RNA (e.g., mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof.

[0068] In some embodiments of the first aspect, the nucleic acid is an inhibitory RNA (e.g., siRNA) and selectively hybridizes to and / or is specific for a target mRNA. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, particularly a pharmaceutically active peptide or polypeptide whose expression (particularly increased expression, e.g., increased expression compared to expression in healthy subjects) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (particularly increased expression, e.g., increased expression compared to expression in healthy subjects) is associated with a cancer.

[0069] In some embodiments of the first aspect, the composition is in liquid form, preferably at a temperature of about 2°C to about 10°C.

[0070] In some embodiments of the first aspect, after storage for at least one week, preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the nucleic acid integrity (e.g., RNA integrity) of the composition is such that the desired effect, such as induction of an immune response, can be achieved. In some embodiments, after storage for at least one week (e.g., at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, or at least 6 months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the nucleic acid integrity (e.g., RNA integrity) of the composition is at least 90% compared to the nucleic acid integrity before storage. In some embodiments, after storage for at least four weeks, preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the nucleic acid integrity (e.g., RNA integrity) of the composition is at least 90% compared to the nucleic acid integrity before storage. In some embodiments, after storage for at least three months, preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the nucleic acid integrity (e.g., RNA integrity) of the composition is at least 90% compared to the nucleic acid integrity before storage.

[0071] In some embodiments of the first aspect, the initial nucleic acid integrity (e.g., initial RNA integrity) of the composition (i.e., after its preparation but before storage) is at least 50%, and the nucleic acid integrity (e.g., RNA integrity) of the composition is at least 90% of the initial RNA integrity after storage for at least one week (e.g., at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, or at least 3 months), preferably at a temperature of 0°C or higher, e.g., from about 2°C to about 8°C.

[0072] Additionally or alternatively, in some embodiments of the first aspect, after storage (e.g., for at least one week), preferably at 0°C or higher, such as about 2°C to about 8°C, the size (Z) of the nucleic acid particles (e.g., RNA particles) of the liquid composition is 平均值 ) (and / or size distribution and / or polydispersity index (PDI)) such that a desired effect, such as induction of an immune response, can be achieved. In some embodiments, after storage (e.g., for at least one week), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the size (Z) of the nucleic acid particles (e.g., RNA particles) of the liquid composition is 平均值 ) ) and / or size distribution and / or polydispersity index (PDI)) are substantially equal to the size (Z) of the nucleic acid particles (e.g., RNA particles) of the initial composition (i.e., before storage). 平均值) ) ) and / or size distribution and / or PDI. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles) is changed after storage for at least one week (e.g., at least four weeks or at least three months), preferably at a temperature of 0°C or higher, e.g., about 2°C to about 8°C, for said period of time. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In some embodiments, the PDI of the nucleic acid particles (e.g., RNA particles) is less than 0.3, preferably less than 0.2, more preferably less than 0.1 after storage for at least one week (e.g., at least four weeks or at least three months), preferably at a temperature of 0°C or higher, such as from about 2°C to about 8°C. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In some embodiments, the PDI of the nucleic acid particles (e.g., RNA particles) is less than 0.3, preferably less than 0.2, more preferably less than 0.1 after storage for at least one week (e.g., at least four weeks or at least three months), preferably at a temperature of 0°C or higher, such as from about 2°C to about 8°C. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the size (Z) of the nucleic acid particles (e.g., RNA particles) after storage of the liquid composition for at least one week (e.g., at least four weeks or at least three months), preferably at a temperature of 0° C. or higher, e.g., from about 2° C. to about 8° C., for said period of time. 平均值 ) (and / or size distribution and / or PDI) is substantially equal to the size (Z) of the nucleic acid particles (e.g., RNA particles) prior to storage. 平均值 ) ) ) and / or size distribution and / or PDI. In one embodiment, the size (Z) of the nucleic acid particles (e.g., RNA particles) of the liquid composition after storage for at least one week (e.g., at least four weeks or at least three months), preferably at a temperature of 0°C or higher, e.g., about 2°C to about 8°C, for said period of time. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the PDI of the nucleic acid particles (e.g., RNA particles) is less than 0.3 (preferably less than 0.2, more preferably less than 0.1) after storage of the liquid composition for at least one week (e.g., at least four weeks or at least three months), preferably at 0°C or higher, e.g., from about 2°C to about 8°C, e.g., at 0°C or higher for at least one week.

[0073] In some embodiments of the first aspect, the composition is in frozen form (e.g., at -20°C). In some embodiments, the nucleic acid integrity (e.g., RNA integrity) after thawing the frozen composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100% compared to the nucleic acid integrity (e.g., RNA integrity) before the composition was frozen. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles), particularly the LNPs, after thawing the frozen composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100%. 平均值 ) and / or a size distribution and / or polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles (e.g., RNA particles) before the composition is frozen. 平均值 ) and / or size distribution and / or PDI.

[0074] In some embodiments of the first aspect, the initial nucleic acid integrity (e.g., initial RNA integrity) of the composition (i.e., after its preparation but before freezing) is at least 50%, and the nucleic acid integrity (e.g., RNA integrity) of the composition after thawing the frozen composition is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably essentially 100% of the initial nucleic acid integrity (e.g., initial RNA integrity).

[0075] Additionally or alternatively, in some embodiments of the first aspect, after thawing the frozen composition, the size (Z 平均值 ) ) and / or size distribution and / or polydispersity index (PDI)) are substantially equal to the size (Z) of the nucleic acid particles (e.g., RNA particles) before the composition is frozen. 平均值 ) ) and / or size distribution and / or PDI. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In some embodiments, the PDI of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the size (Z) of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition is 平均值 ) (and / or size distribution and / or PDI) is substantially equal to the size (Z) of the nucleic acid particles (e.g., RNA particles) before freezing. 平均值) ) and / or size distribution and / or PDI. In some embodiments, the size (Z) of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the PDI of the nucleic acid particles (e.g., RNA particles) after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0076] In some embodiments of the first aspect, the size of the nucleic acid particles (e.g., RNA particles) and the nucleic acid integrity (e.g., RNA integrity) of the composition are substantially equivalent to the size and nucleic acid integrity (e.g., RNA integrity) of the nucleic acid particles (e.g., RNA particles) of the initial composition (i.e., before the composition is first frozen) after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, and more preferably after five freeze / thaw cycles or more.

[0077] In a second aspect, the present disclosure provides a method of preparing a composition comprising particles dispersed in a final aqueous phase, wherein the composition comprises (i) a nucleic acid; (ii) a lipid that can ionize to release a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion (e.g., an inorganic polyphosphate); wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize to release a cation, and at least a portion of the steroid; wherein at least a portion of the polyvalent anion (e.g., an inorganic polyphosphate) is associated with the particles; and wherein the final aqueous phase comprises a final buffer system;

[0078] The methods include:

[0079] (I) preparing a formulation comprising particles dispersed in a final aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into a cation, at least a portion of the steroid, and at least a portion of the neutral lipid, and wherein at least a portion of the multivalent anion (e.g., inorganic polyphosphate) is associated with the particles; and

[0080] (II) optionally freezing the formulation to about -10°C or below,

[0081] Thus, a composition is obtained,

[0082] Wherein step (1) comprises:

[0083] (a) providing (e.g., preparing) a nucleic acid solution comprising water and a first buffer system;

[0084] (b) providing (e.g., preparing) an organic solution comprising a lipid that can ionize to release a cation, a steroid, and a neutral lipid;

[0085] (c) mixing the nucleic acid solution provided under (a) with the organic solution provided under (b), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system;

[0086] (d) mixing the first intermediate formulation prepared under (c) with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby preparing a second intermediate formulation comprising particles dispersed in a second aqueous phase comprising a second buffer system, wherein at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is associated with the particles; and

[0087] (e) filtering (e.g., dialysis, tangential flow filtration, or diafiltration) and / or diluting the second intermediate formulation prepared under (d) using a final aqueous buffer solution comprising the final buffer system,

[0088] A formulation comprising particles dispersed in a final aqueous phase is thus prepared.

[0089] As demonstrated herein, by using the methods of the present application, compositions comprising particles dispersed in a final aqueous phase can be prepared, wherein the composition comprises (i) a nucleic acid; (ii) a lipid that can ionize to release a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion (e.g., an inorganic polyphosphate); and wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize to release a cation, and at least a portion of the steroid, wherein aggregation of the particles is prevented due to the presence of the polyvalent anion (e.g., an inorganic polyphosphate), even when the composition / particles do not comprise PEG lipids or any other stealth lipids (polymer-conjugated lipids). Furthermore, the present application demonstrates that, unexpectedly, by using the methods of the present application, in particular by adjusting the amounts of lipids relative to one another, nucleic acid (e.g., RNA) compositions can be obtained that exhibit different biological properties: when used to transfect cells, nucleic acid compositions comprising higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids result in higher expression of the nucleic acid in the transfected cells when transfection is performed in the presence of serum (these compositions are similar to standard nucleic acid compositions comprising PEG lipids), compared to the expression obtained when transfection is performed in the absence of serum. In contrast, nucleic acid compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids and used to transfect cells result in comparable or reduced expression of the nucleic acid in the transfected cells when transfection is performed in the presence of serum (i.e., these compositions exhibit some or no serum inhibition, but not serum stimulation).

[0090] Figure 1AAn exemplary flow chart of steps (c) to (e) of the method according to the second aspect is depicted in FIG. First, an aqueous nucleic acid solution (comprising a first buffer system, e.g., a buffer system having a pH lower than 6.0, e.g., a pH of about 3.5 to about 5.9) is mixed with an organic (e.g., ethanol) lipid solution ( Figure 1A In another embodiment, the first intermediate preparation is prepared by mixing a first intermediate preparation with a plurality of particles dispersed in a first aqueous phase comprising a first buffer system. Afterwards, the first intermediate preparation is mixed with a multivalent anion (e.g., an inorganic polyphosphate) or a salt thereof to prepare a second intermediate preparation comprising particles dispersed in a second aqueous phase comprising a second buffer system, wherein at least a portion of the multivalent anion (e.g., an inorganic polyphosphate) is associated with the particles. The second intermediate preparation is then filtered (e.g., using dialysis, tangential flow filtration, and / or diafiltration) and / or diluted using the final aqueous buffer solution comprising the final buffer system to prepare a preparation comprising particles dispersed in the final aqueous phase. For example, the second intermediate preparation is filtered to remove undesirable compounds (e.g., organic solvents (e.g., ethanol) and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally corresponding counter cations for maintaining a pH lower than 6.0) and / or to increase nucleic acid (e.g., RNA) concentration and / or change the pH and / or change the buffer system to a final buffer system.

[0091] In some embodiments of the second aspect, step (d) is performed at most about 20 minutes after step (c). In some embodiments, step (d) is performed at most about 19 minutes (e.g., at most about 18 minutes, at most about 17 minutes, at most about 16 minutes, at most about 15 minutes, at most about 14 minutes, at most about 13 minutes, at most about 12 minutes, at most about 11 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, or at most about 5 minutes) after step (c).

[0092] In some embodiments of the second aspect, after step (c) and / or step (d) and / or step (e) (preferably only after step (d) and / or step (e)), step (i) also includes one or more optional processing steps, which are selected from dilution and filtration, such as dialysis, tangential flow filtration and diafiltration. For example, the dilution step may include adding a dilution solution (such as water) to the intermediate preparation. Such a dilution solution may include one or more other compounds (such as cryoprotectants) and optional buffer systems (such as final buffer systems). A dilution step may be performed to dilute undesirable compounds (such as organic solvents (such as ethanol) and / or one or more dibasic and / or polybasic organic acids) in the intermediate preparation and / or change pH and / or change buffer systems and / or add one or more other compounds (such as cryoprotectants). One or more filtration steps (including steps (e), (h'), (i'), and (j') as described herein) can be used to remove undesirable compounds (e.g., organic solvents (e.g., ethanol) and / or one or more dibasic and / or polybasic organic acids) from the intermediate formulation and / or to increase the nucleic acid (e.g., RNA) concentration of the intermediate formulation and / or to change the pH and / or to change the buffer system of the intermediate formulation. To this end, an aqueous buffer solution can be used that does not contain undesirable compounds (so that undesirable compounds are filtered or washed out of the intermediate formulation and into the aqueous buffer solution) and / or is hypertonic compared to the aqueous buffer solution (so that water flows from the intermediate formulation to the aqueous buffer solution) and / or has a pH and / or buffer system that is different from the pH and / or buffer system of the intermediate formulation.

[0093] In some embodiments of the second aspect, step (a) includes (a') providing an aqueous nucleic acid solution; (b') providing a first aqueous buffer solution comprising a first buffer system; and (c') mixing the aqueous nucleic acid solution provided under (a') with the first aqueous buffer solution provided under (b'), thereby providing (e.g., preparing) a nucleic acid solution comprising water and the first buffer system.

[0094] In some embodiments of the second aspect, the organic solution comprises an organic solvent selected from lower alcohols, for example, alcohols (particularly fatty alcohols) having up to 6 carbon atoms, and mixtures of two or more of these alcohols. In some preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from ethanol, propanol, isopropanol, 1,2-propylene glycol, and mixtures of two or more of these alcohols.

[0095] In some embodiments of the second aspect, step (I) comprises:

[0096] (a') providing an aqueous nucleic acid solution;

[0097] (b') providing a first aqueous buffer solution comprising a first buffer system;

[0098] (c') mixing the aqueous nucleic acid solution provided under (a') with the first aqueous buffer solution provided under (b'), thereby preparing a nucleic acid solution comprising water and the first buffer system;

[0099] (d') providing (e.g., preparing) an organic solution comprising a lipid that can ionize to release a cation, a steroid, and a neutral lipid;

[0100] (e') mixing the nucleic acid solution prepared under (c') with the organic solution provided (e.g., prepared) under (d'), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system;

[0101] (f') optionally diluting the first intermediate formulation prepared under (e') with water or an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising particles dispersed in an additional aqueous phase, the additional aqueous phase comprising the first buffer system or the additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution;

[0102] (g') mixing the first intermediate formulation obtained in step (e') with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof (as described herein) if step (f') is not present, or mixing the further intermediate formulation obtained in step (f') with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof (as described herein) if step (f') is present, thereby preparing a second intermediate formulation comprising particles dispersed in a second aqueous phase, wherein at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is associated with the particles;

[0103] (h') optionally filtering the second first intermediate formulation prepared under (g') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising particles dispersed in an additional aqueous phase, the additional aqueous phase comprising an additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first and / or second aqueous buffer solution;

[0104] (i') optionally repeating step (h') one or two or more times, wherein the further intermediate preparation comprising particles dispersed in a further aqueous phase comprising a further buffer system obtained after step (h') of one cycle is used as the second intermediate preparation of the next cycle, wherein in each cycle the further aqueous buffer solution may be the same as or different from the first and / or second aqueous buffer solution;

[0105] (j') filtering using the final aqueous buffer solution comprising the final buffer system: the second intermediate preparation obtained in step (g') if step (h') is absent, or the further intermediate preparation obtained in step (h') if step (h') is present and step (i') is absent, or the further intermediate preparation obtained after step (i') if steps (h') and (i') are present; and

[0106] (k') optionally diluting the preparation obtained in step (j') with a dilution solution;

[0107] A formulation comprising particles dispersed in a final aqueous phase is thus prepared.

[0108] Figure 1BAn exemplary flow chart of steps (e'), (g') and (j') according to these embodiments of the second aspect is depicted in (optional processing steps (f'), (h'), (i') and (k') are also shown). First, in step (e'), an aqueous nucleic acid solution (comprising a first buffer system, e.g., a buffer system having a pH below 6.0, e.g., a buffer system having a pH of about 3.5 to about 5.9) is mixed with an organic lipid solution to form a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system. Thereafter, optionally, the first intermediate formulation is diluted (f') with water or another aqueous buffer solution comprising another buffer system to prepare another intermediate formulation comprising particles dispersed in another aqueous phase comprising the first buffer system or another buffer system, wherein the other aqueous buffer solution can be the same as or different from the first aqueous buffer solution. Optional step (f') can be performed to dilute undesirable compounds in the first intermediate formulation (e.g., an organic solvent (e.g., ethanol) and / or one or more dibasic and / or polybasic organic acids) and / or to change the pH and / or to change the buffer system. In some embodiments, step (f') is used to change the pH of the first intermediate formulation to a pH of 7 to 9, preferably 7.5 to 8.5, and even more preferably 7.5 to 8.0. Then, in step (g'), the first intermediate formulation (if step (f') is absent) or the corresponding further intermediate formulation (if step (f') is present) is mixed with a polyvalent anion (e.g., an inorganic polyphosphate) or a salt thereof, thereby preparing a second intermediate formulation comprising particles dispersed in a second aqueous phase comprising a second buffer system, wherein at least a portion of the polyvalent anion (e.g., an inorganic polyphosphate) is associated with the particles. Then, optionally, the second intermediate preparation is subjected to (h') filtration (e.g., dialysis, tangential flow filtration, or diafiltration) using another aqueous buffer solution comprising another buffer system to prepare another intermediate preparation comprising particles dispersed in another aqueous phase comprising another buffer system, wherein the additional aqueous buffer solution may be the same or different from the first and / or second aqueous buffer solution; and optionally, under (i'), step (h') is repeated one or two or more times. Optional steps (h') and (i') may be performed to remove undesirable compounds (e.g., organic solvents (e.g., ethanol) and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally their corresponding countercations) from the second intermediate preparation and / or to increase the nucleic acid (e.g., RNA) concentration and / or change the pH and / or change the buffer system. Then, in step (j'), the second intermediate preparation (if step (h') is absent) or the corresponding further intermediate preparation (if step (h') is present or both steps (h') and (i') are present) is filtered (e.g. dialyzed, tangential flow filtration or diafiltered) using the final aqueous buffer solution comprising the final buffer system.Step (j') is performed to remove undesirable compounds (e.g., organic solvents (e.g., ethanol) and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally their corresponding countercations) from the intermediate formulation and / or to increase the concentration of nucleic acid (e.g., RNA) and / or to change the pH and / or to change the buffer system to the final buffer system. Optionally, in step (k'), the formulation obtained in step (j') is diluted with a diluent solution (e.g., for the addition of a cryoprotectant).

[0109] In some embodiments of the second aspect, step (f') is used to change the pH of the first intermediate formulation to a pH of 7 to 9, preferably 7.5 to 8.5, and even more preferably 7.5 to 8.0, and is performed no later than 12 hours, preferably no later than 4 hours, and more preferably no later than 30 minutes after step (e'). In some specific embodiments of the second aspect, step (f') is used to change the pH of the first intermediate formulation to a pH of 7 to 9, followed by addition of the polyvalent anion in step (g'), and the combined duration of steps (f') and (g') is no longer than 12 hours, preferably no longer than 4 hours, and more preferably no longer than 30 minutes after step (e').

[0110] In some embodiments of the second aspect, step (g') is performed at most about 20 minutes after step (e'). In some embodiments, step (g') is performed at most about 19 minutes (e.g., at most about 18 minutes, at most about 17 minutes, at most about 16 minutes, at most about 15 minutes, at most about 14 minutes, at most about 13 minutes, at most about 12 minutes, at most about 11 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, or at most about 5 minutes) after step (e').

[0111] In some embodiments of the second aspect, step (f') is absent, and step (g') is performed at most about 20 minutes after step (e'). In some embodiments, step (g') is performed at most about 19 minutes (e.g., at most about 18 minutes, at most about 17 minutes, at most about 16 minutes, at most about 15 minutes, at most about 14 minutes, at most about 13 minutes, at most about 12 minutes, at most about 11 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, or at most about 5 minutes) after step (e').

[0112] In some embodiments of the second aspect, step (f') is absent. Figure 1CAn exemplary flow diagram of steps (e'), (g'), and (j') according to these embodiments of the second aspect (i.e., wherein step (f') is absent) is depicted in (i.e., in the absence of step (f')) (optional processing steps (h'), (i'), and (k') are also shown). These embodiments are particularly preferred when step (g') is performed up to about 20 minutes after step (e').

[0113] In some embodiments of the second aspect, step (f') is present.

[0114] In some embodiments of the second aspect, the organic solution provided in step (e') comprises an organic solvent selected from lower alcohols, such as alcohols having up to 6 carbon atoms (particularly fatty alcohols), and mixtures thereof (e.g., mixtures of two or more of these alcohols). In some preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from ethanol, propanol, isopropanol, 1,2-propylene glycol, and mixtures of two or more of these alcohols.

[0115] In some embodiments of the second aspect, the polyvalent anion or its salt is an inorganic phosphate, sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate, tetrathionate, or a salt thereof. In some embodiments, the polyvalent anion is an inorganic phosphate (e.g., PO4 3- ).

[0116] In some embodiments of the second aspect, the polyvalent anion or salt thereof is a dicarboxylic acid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, sebacic acid, phthalic acid, isophthalic acid, or terephthalic acid), a substituted dicarboxylic acid (e.g., tartronic acid, mesooxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid, or saccharinic acid), or a salt thereof.

[0117] In some embodiments, the multivalent anion or salt thereof is a tricarboxylic acid (eg, citric acid, isocitric acid, propane-1,2,3-tricarboxylic acid, or trimesic acid) or a salt thereof.

[0118] In some preferred embodiments of the second aspect, the multivalent anion or its salt is selected from the group consisting of: inorganic polyphosphate, inorganic phosphate (e.g., PO4 3- ), sulfate, succinate, glutarate, tartrate, malate, citrate, salts thereof, or mixtures thereof. In some embodiments, the multivalent anion or its salt is an inorganic polyphosphate, an inorganic phosphate, citrate, or a salt thereof.

[0119] In some most preferred embodiments of the second aspect, the multivalent anion or salt thereof is an inorganic polyphosphate or salt thereof.

[0120] In some embodiments of the second aspect, wherein the polyvalent anion or its salt is an inorganic polyphosphate or its salt, the inorganic polyphosphate or its salt can be any linear, cyclic or branched inorganic polyphosphate or its salt. In some embodiments of the second aspect, the inorganic polyphosphate or its salt is a linear inorganic polyphosphate (e.g., linear inorganic triphosphate) or its salt.

[0121] In some embodiments of the second aspect, wherein the multivalent anion or salt thereof is an inorganic polyphosphate or salt thereof, the inorganic polyphosphate or salt thereof comprises or has the formula [P x O (3x+1) ]M y’ , wherein x is an integer and is at least 2, preferably at least 3; each M is independently H + or cations; and y' is the number of cations required for charge balance. M can be of only one type (e.g., only Na + ), or may be of two or more types (e.g. Na + and K + mixture, or Na + and H + mixture, or Na + K + and H + In some embodiments, each M is independently selected from H + , basic cations (such as Li + 、Na + , K + ), ammonium (NH4 + ) and a monovalent organic cation (e.g., a monovalent organic amine, such as trimethylamine, triethylamine, etc.). In some embodiments, each M is independently selected from H + 、Na + , K + 、Li + and NH4 + For example, if x is 3, the inorganic polyphosphate or its salt is a compound comprising or having the formula [P3O 10 ]M y’ (For example [P3O 10 ]Na5) linear inorganic triphosphate or its salt. Similarly, if x is 4, then the inorganic polyphosphate or its salt is a linear inorganic triphosphate or its salt comprising or having the formula [P4O 13 ]M y (For example [P4O 13 ]Na6 or [P4O 13 ]Na4K2) linear or branched inorganic tetraphosphate or its salt.

[0122] In some embodiments of the second aspect, wherein the polyvalent anion or its salt is an inorganic polyphosphate or its salt, the inorganic polyphosphate or its salt is selected from diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, its salt and mixture, for example, selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate and mixture. In some preferred embodiments of the second aspect, the inorganic polyphosphate or its salt is selected from triphosphate, tetraphosphate, pentaphosphate, its salt and mixture. In some preferred embodiments of the second aspect, the inorganic polyphosphate or its salt is triphosphate or its salt.

[0123] In some embodiments of the second aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 1: 2. For example, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation can be at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0. In some preferred embodiments of the second aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 2: 3. In some preferred embodiments of the second aspect, the molar ratio of (v) the polyvalent anion (e.g., inorganic polyphosphate) to (ii) the lipid that can ionize to release a cation is at least about 4:3.

[0124] In some embodiments of the second aspect, particularly if it is desired to prepare the composition in frozen form, the method of the second aspect comprises (II) freezing the formulation to about -10° C. or below. Thus, in these embodiments, performing the method of the second aspect results in a frozen form of the composition.

[0125] In some alternative embodiments, particularly if it is desired to prepare a composition in liquid form, the method of the second aspect does not include step (II). Thus, in these embodiments, the method of the second aspect is performed to obtain a composition in liquid form.

[0126] In some embodiments of the second aspect, the organic solution provided (e.g., prepared) under (b) or (d'), respectively, further comprises an acid. In some embodiments, the acid is an inorganic acid (e.g., a monobasic inorganic acid such as hydrochloric acid, hydrobromic acid, or nitric acid) or an organic acid (e.g., a monobasic, dibasic, or polybasic organic acid such as a monocarboxylic acid (e.g., acetic acid, propionic acid, or lactic acid), a dicarboxylic acid (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, tartaric acid, or malic acid), or a polycarboxylic acid (e.g., citric acid, isocitric acid, or trimesic acid). In some preferred embodiments, the acid is a monobasic acid, such as a monobasic inorganic acid (e.g., hydrochloric acid) or a monobasic organic acid (e.g., acetic acid).

[0127] In some embodiments of the second aspect, the composition is substantially free of lipids comprising polyethylene glycol (PEG). In some embodiments, the composition is substantially free of any compound comprising PEG. In some embodiments, the composition is substantially free of PEG. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, its salt and mixture. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, citrate and its salt. In some of these embodiments, the polyvalent anion or its salt is inorganic polyphosphate or its salt.

[0128] In some embodiments of the second aspect, the composition is also substantially free of other polymer-conjugated lipids. In some embodiments, the other polymer-conjugated lipids are polysarcosine-conjugated lipids. Therefore, in some embodiments, the composition is substantially free of PEG lipids and substantially free of polysarcosine-conjugated lipids. In some embodiments, the composition is substantially free of any polymer-conjugated lipids. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, its salt and mixture. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, citrate and its salt. In some of these embodiments, the polyvalent anion or its salt is inorganic polyphosphate or its salt.

[0129] In some embodiments of the second aspect, the pH of the final buffer system (and the pH of the composition) is from about 4.0 to about 8.0. For example, the pH of the final buffer system (and the pH of the composition) can be from about 4.5 to about 8.0, such as from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, from about 7.0 to about 7.8, or about 7.5.

[0130] In some embodiments of the second aspect, the pH of the first buffer system (and the pH of the nucleic acid (e.g., RNA) solution provided / obtained in step (a) or (c')) is less than 6.0, preferably at most about 5.5, for example, at most about 5.0, at most about 4.9, at most about 4.8, at most about 4.7, at most about 4.6, or at most about 4.5. For example, the pH of the first buffer system (and the pH of the nucleic acid (e.g., RNA) solution provided / obtained in step (a) or (c')) can be from about 3.5 to about 5.9, for example, from about 4.0 to about 5.5, or from about 4.5 to about 5.0. To this end, the nucleic acid (e.g., RNA) solution provided / obtained in step (a) or (c') may further comprise one or more acids (e.g., selected from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, or nitric acid) and organic acids (e.g., monobasic, dibasic, or polybasic organic acids, such as monocarboxylic acids (e.g., acetic acid, propionic acid, or lactic acid), dicarboxylic acids (e.g., oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, tartaric acid, or malic acid), or polycarboxylic acids (e.g., citric acid, isocitric acid, or trimesic acid)). In some preferred embodiments, the acid is a monobasic acid, For example, a monobasic inorganic acid (such as hydrochloric acid) or a monobasic organic acid (such as acetic acid). In some embodiments, it is preferred that step (e) is carried out under conditions that remove one or more undesirable substances (e.g., organic solvents (such as ethanol) and / or one or more acids) to obtain a formulation comprising particles dispersed in a final aqueous phase, wherein the final aqueous phase is substantially free of such one or more undesirable substances. For example, such conditions may include using a final buffer system (i.e., a final buffer substance) The intermediate preparation obtained in step (d) comprising particles dispersed in the second intermediate aqueous phase is subjected to at least one filtration step, such as dialysis, tangential flow filtration or diafiltration, wherein the final buffer solution does not contain the one or more undesirable substances. Alternatively, such conditions may include (1) diluting the intermediate preparation (i.e., the second intermediate preparation) obtained in step (d) comprising particles dispersed in the second intermediate aqueous phase with water or another aqueous buffer solution comprising another buffer system, thereby preparing another intermediate preparation comprising particles dispersed in another aqueous phase comprising the first buffer system or another buffer system, wherein the other buffer system of the other aqueous buffer solution may be the same as or different from the buffer system used in step (a); and (2) filtering the other intermediate preparation obtained in step (1) with the final aqueous buffer solution, such as dialysis, tangential flow filtration or diafiltration, wherein at least the final aqueous buffer solution (preferably the intermediate aqueous buffer solution and the final aqueous buffer solution) does not contain the one or more undesirable substances.

[0131] Similarly, in some embodiments of the second aspect, wherein step (I) comprises steps (a' to (e'), (g') and (j') (and optionally one or more of steps (f'), (h'), (i') and (k')), the pH of the first aqueous buffer solution (and the pH of the nucleic acid (e.g., RNA) solution obtained in step (c')) is less than 6.0, preferably at most about 5.5, e.g., at most about 5.0, at most about 4.9, at most about 4.8, at most about 4.7, at most about 4.6, or at most about 4.5. For example, the pH of the first aqueous buffer solution (and the pH of the nucleic acid (e.g., RNA) solution obtained in step (c')) can be from about 3.5 to about 5.9, e.g., from about 4.0 to about 5.5, or from about 4.5 to about 5.0. To this end, the first aqueous buffer solution (and the first aqueous phase) provided under (b') can further comprise one or more acids (e.g., one or more monobasic, dibasic or polybasic acids). Acid). In these embodiments, it is preferred that at least one of steps (f' to (j') is carried out under conditions wherein one or more undesirable substances (e.g., organic solvent (e.g., ethanol) and / or one or more monobasic, dibasic, or polybasic acids) are removed from the first intermediate formulation and / or from the second intermediate formulation and / or from the further intermediate formulation, thereby obtaining a further intermediate formulation comprising particles dispersed in a further aqueous phase or a final aqueous phase, wherein the further aqueous phase and / or the final aqueous phase are substantially free of the one or more undesirable substances. For example, such conditions may comprise the use of a further aqueous buffer solution and / or a final buffer solution, wherein at least one of the further aqueous buffer solution and the final buffer solution (preferably both of the further aqueous buffer solution and the final buffer solution) is free of the one or more undesirable substances. In some embodiments, the filtration step may be independently selected from dialysis, tangential flow filtration, and diafiltration, preferably selected from dialysis and tangential flow filtration.

[0132] In some embodiments of the second aspect, the first buffer system used in step (a) comprises the final buffer substance used in step (e), and preferably, the buffer system of the first buffer system used in step (a) and the buffer system and pH of the final aqueous buffer solution used in step (e) are identical. For example, in this embodiment of the second aspect, only a kind of aqueous buffer solution is used. In this case, the pH of the second intermediate preparation can be adjusted by adding a polyvalent anion (such as an inorganic polyphosphate) and combining its corresponding counter cation. For example, the pH of the second intermediate preparation can be realized by adding a solution of pentasodium triphosphate, tetrasodium diphosphate, disodium hydrogen phosphate or trisodium citrate.

[0133] Similarly, in some embodiments of the second aspect, wherein step (I) comprises steps (a') to (e'), (g') and (j') (and optionally one or more of steps (f'), (h'), (i') and (k')), each first buffer system and each additional buffer system used in steps (b'), (f'), (h') and (i') comprises the final buffer substance used in step (j'), preferably, the buffer system and pH of each first aqueous buffer solution and each additional aqueous buffer solution used in steps (b'), (f'), (h') and (i') are the same as the buffer system and pH of the final aqueous buffer solution. In some embodiments, for example, the homogeneous buffer system (i.e., each first buffer system and each additional buffer system used in the method) can comprise acetic acid and tris, wherein acetic acid predominates in steps (a') to (d'), and tris is added in (e') in an amount to achieve a pH of 7 to 9, preferably 7.5 to 8.5, before the polyvalent anion is added in step (f'). In another example, the polyvalent anion can be a component of the buffer system, as in the case of a buffer composed of citric acid and tris, wherein in steps (a') to (d'), citric acid is combined with 0.1 to 2, preferably 0.2 to 1 equivalents of tris, and in (e') tris is added in an amount to achieve a pH of 7 to 9, preferably 7.5 to 8.5, and step (f') is used to add water or step (f') is absent. In this example, the materials used in steps (e') and (f') are interchangeable, such that after diluting the first intermediate formulation with water, a solution of tris(hydroxymethyl)aminomethane is used to adjust the pH in step (f'). In some embodiments of the second aspect, the formulation and / or composition obtained in step (i) comprises a cryoprotectant. In some embodiments of the second aspect, the formulation and / or composition obtained in step (i) is substantially free of a cryoprotectant.

[0134] In some embodiments of the second aspect, the formulation and / or composition comprises water as a major component, and / or the total amount of solvent other than water included in the composition is less than about 1.0% (v / v), for example, less than about 0.5% (v / v). For example, the amount of water included in the formulation and / or composition can be at least 50% (w / w), for example, at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). If the formulation and / or composition comprises a cryoprotectant, the amount of water included in the formulation and / or composition may be at least 50% (w / w), e.g., at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the formulation and / or composition is substantially free of a cryoprotectant, the amount of water included in the formulation and / or composition may be at least 95% (w / w). Additionally or alternatively, the total amount of solvent other than water included in the composition can be less than about 1.0% (v / v), such as less than about 0.9% (v / v), less than about 0.8% (v / v), less than about 0.7% (v / v), less than about 0.6% (v / v), less than about 0.5% (v / v), less than about 0.4% (v / v), less than about 0.3% (v / v), less than about 0.2% (v / v), less than about 0.1% (v / v), less than about 0.05% (v / v), less than about 0.01% (v / v), or less than about 0.005% (v / v). In this regard, a cryoprotectant that is liquid under normal conditions will not be considered a solvent other than water, but rather a cryoprotectant. In other words, the above optional limitation, namely that the total amount of solvent other than water included in the composition may be less than about 1.0% (v / v), such as less than about 0.5% (v / v), does not apply to cryoprotectants that are liquid under normal conditions.

[0135] In some embodiments of the second aspect, the osmolarity of the composition is at most about 1000×10 -3 In some embodiments, the osmolality of the composition is at most about 500×10 -3 osmol / kg, for example, up to about 490×10 - 3 osmol / kg, up to about 480×10 -3 osmol / kg, up to about 470×10 -3 osmol / kg, up to about 460×10 -3osmol / kg, up to about 450×10 -3 osmol / kg, up to about 440×10 -3 osmol / kg, up to about 430×10 -3 osmol / kg, up to about 420×10 -3 osmol / kg, up to about 410×10 -3 osmol / kg, up to about 400×10 -3 osmol / kg, up to about 390×10 -3 osmol / kg, up to about 380×10 -3 osmol / kg, up to about 370×10 -3 osmol / kg, up to about 360×10 - 3 osmol / kg, up to about 350×10 -3 osmol / kg, up to about 340×10 -3 osmol / kg, up to about 330×10 -3 osmol / kg, up to about 320×10 -3 osmol / kg, up to about 310×10 -3 osmol / kg, or up to about 300×10 -3 If the composition does not contain a cryoprotectant, the osmolality of the composition may be less than 300×10 -3 osmol / kg, for example, up to about 250×10 -3 osmol / kg, up to about 200×10 -3 osmol / kg, up to about 150×10 -3 osmol / kg, up to about 100×10 -3 osmol / kg, up to about 50×10 -3 osmol / kg, up to about 40×10 -3 osmol / kg, or up to about 30×10 - 3 osmol / kg. If the composition comprises a cryoprotectant, it is preferred that a major portion of the osmolality of the composition is provided by the cryoprotectant. For example, the cryoprotectant may provide at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the osmolality of the composition.

[0136] In some embodiments of the second aspect, the concentration of the nucleic acid (e.g., RNA) in the composition is from about 1 mg / l to about 500 mg / l, for example, from about 1 mg / l to about 100 mg / l. In some embodiments, the concentration of the nucleic acid (particularly RNA) in the composition is from about 5 mg / l to about 500 mg / l, for example, from about 10 mg / l to about 400 mg / l, from about 10 mg / l to about 300 mg / l, from about 10 mg / l to about 200 mg / l, from about 10 mg / l to about 150 mg / l, or from about 10 mg / l to about 100 mg / l, preferably from about 10 mg / l to about 140 mg / l, more preferably from about 20 mg / l to about 130 mg / l, and more preferably from about 30 mg / l to about 120 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 5 mg / l to about 150 mg / l, for example, from about 10 mg / l to about 140 mg / l, from about 20 mg / l to about 130 mg / l, from about 25 mg / l to about 125 mg / l, from about 30 mg / l to about 120 mg / l, from about 35 mg / l to about 115 mg / l, from about 40 mg / l to about 110 mg / l, from about 45 mg / l to about 105 mg / l, or from about 50 mg / l to about 100 mg / l. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from 1 mg / l to about 50 mg / l, or from about 10 mg / l to about 100 mg / l.

[0137] In some embodiments of the second aspect (particularly those wherein the method comprises (II) freezing the formulation to about -10°C or below and whereby the composition prepared by the method is in frozen form), the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 50 mg / l.

[0138] In some embodiments of the second aspect (particularly those wherein the method does not include step (II) and thus the composition produced by the method is in liquid form), the concentration of nucleic acid (particularly RNA) in the composition is from about 10 mg / l to about 100 mg / l.

[0139] In some embodiments of the second aspect, the molar ratio of the polyvalent anion or salt thereof to the lipid that can ionize to release a cation is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 100 mg / l (e.g., 1 mg / l to about 50 mg / l, or about 10 mg / l to about 100 mg / l). In some embodiments of the second aspect (particularly those wherein the method comprises (II) freezing the formulation to about -10°C or below and the composition prepared by the method is thus in frozen form), the molar ratio of the polyvalent anion or salt thereof to the lipid that can ionize to release a cation is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 50 mg / l. In some embodiments of the second aspect (particularly those in which the method does not include step (II) and the composition prepared by the method is in liquid form), the molar ratio of the polyvalent anion or its salt to the lipid that can ionize into a cation is at least about 1:2 (preferably at least about 2:3, e.g., at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is from about 10 mg / l to about 100 mg / l. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, citrate, and salts thereof.

[0140] In some embodiments of the second aspect, the multivalent anion is an inorganic polyphosphate or a salt thereof. In some embodiments, the inorganic polyphosphate or a salt thereof is a linear inorganic polyphosphate or a salt thereof as defined herein (e.g., a linear triphosphate or a salt thereof), and the molar ratio of the inorganic polyphosphate or a salt thereof to the lipid that can ionize out a cation is at least about 1:2, preferably at least about 2:3, such as at least about 1.00 or at least about 4:3. In some preferred embodiments of the second aspect, the inorganic polyphosphate or a salt thereof is a linear inorganic polyphosphate (particularly triphosphate) or a salt thereof, and the molar ratio of the inorganic polyphosphate or a salt thereof to the lipid that can ionize out a cation is at least about 1:2 (preferably at least about 2:3, such as at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is from about 1 mg / l to about 100 mg / l (e.g., 1 mg / l to about 50 mg / l or from about 10 mg / l to about 100 mg / l). In some embodiments of the second aspect (particularly those wherein the method comprises (II) freezing the formulation to about -10°C or below and the composition prepared by the method is thereby in frozen form), the inorganic polyphosphate is a linear inorganic polyphosphate (particularly triphosphate) or a salt thereof, the molar ratio of the inorganic polyphosphate or its salt to the lipid that can ionize into a cation is at least about 1:2 (preferably at least about 2:3, e.g., at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 1 mg / l to about 50 mg / l. In some embodiments of the second aspect (particularly those wherein the method does not include step (II) and the composition prepared by the method is therefore in liquid form), the inorganic polyphosphate or salt thereof is a linear inorganic polyphosphate (particularly triphosphate) or a salt thereof, the molar ratio of the inorganic polyphosphate or salt thereof to the lipid that can ionize into a cation is at least about 1:2 (preferably at least about 2:3, for example at least about 1.00 or at least about 4:3), and the concentration of the nucleic acid (particularly RNA) in the composition is about 10 mg / l to about 100 mg / l.

[0141] In some embodiments of the second aspect, the lipid ionizable to release a cation comprises a head group comprising at least one tertiary amine moiety.

[0142] In some embodiments of the second aspect, the lipid that can ionize into a cation has the structure of Formula (X):

[0143]

[0144] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 10 , L 20 , G 1 , G 2 , G 3 、R 35 、R36 and R 37 As defined herein. In some embodiments, the lipid that can ionize out a cation is selected from the following: structures X-1 to X-36 (shown herein); and / or structures A to G (shown herein); and / or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA) and 4-((di((9Z,12Z)-octadecane-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-1-amine (DPL-14). In some embodiments, the lipid that can ionize out a cation is a lipid having structure X-3. In some embodiments, the lipid that can ionize out a cation is DPL-14 (i.e., a lipid having structure G). In some embodiments, the lipid that ionizes to release a cation is a lipid having structure D.

[0145] In some embodiments of the second aspect, the lipid that can ionize into a cation has the structure of Formula (XI):

[0146]

[0147] Wherein R1, R2 R3, R4, L2, G2 and m are as defined herein. In some embodiments, the lipid that can ionize to give a cation is selected from structures (XIV-1), (XIV-2) and (XIV-3) (shown herein). In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-1. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-2. In some embodiments, the lipid that can ionize to give a cation is a lipid having structure XIV-3.

[0148] In some embodiments of the second aspect, the lipid that can ionize out a cation is replaced in whole or in part by a cationic lipid. In some embodiments, the lipid that can ionize out a cation is selected from Structures XV-1 to XV-6 (shown in this article). In those embodiments in which the lipid that can ionize out a cation is completely replaced by a cationic lipid, the molar ratio of a polyvalent anion (e.g., an inorganic polyphosphate) or a salt thereof to the lipid that can ionize out a cation is replaced by the molar ratio of a polyvalent anion (e.g., an inorganic polyphosphate) or a salt thereof to the cationic lipid. In some embodiments, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) or salt thereof to the cationic lipid is at least about 1:2 (e.g., at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0, preferably, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) or salt thereof to the cationic lipid is at least about 2:3, e.g., at least about 4:3). In those embodiments in which the lipid ionizable to release cations is partially replaced by a cationic lipid, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the lipid ionizable to release cations is replaced by the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid ionizable to release cations. In some embodiments, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid that can ionize to release a cation is at least about 1:2 (e.g., at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0, preferably, the molar ratio of the polyvalent anion (e.g., inorganic polyphosphate) to the sum of the cationic lipid and the lipid that can ionize to release a cation is at least about 2:3, e.g., at least about 4:3).

[0149] In some embodiments of the second aspect, the lipids that can ionize to release cations comprise about 20 mol% to about 75 mol% of the total lipids present in the organic solution, e.g., about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% (particularly for those embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% (particularly for those embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids). In some embodiments of the second aspect, the lipids that can release cations comprise about 20 mol% to about 75 mol% of the total lipids present in the composition, for example, about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% (particularly for embodiments with higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% (particularly for embodiments with lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids). In those embodiments in which the lipids that can release cations are completely or partially replaced by cationic lipids, it is preferred that the same ranges as specified above for the lipids that can release cations (e.g., about 20 mol% to about 75 mol%, etc.) apply to the sum of the lipids that can release cations and the cationic lipids.

[0150] In some embodiments of the second aspect, the steroid comprises a sterol. In some preferred embodiments of the second aspect, the steroid comprises or is cholesterol.

[0151] In some embodiments of the second aspect, the steroid comprises about 15 mol% to about 60 mol% of the total lipids present in the organic solution, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% (particularly for those embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly for those embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids). In some embodiments of the second aspect, the steroid comprises about 15 mol% to about 60 mol% of the total lipids present in the composition, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% (particularly for those embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly for those embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids).

[0152] In some embodiments of the second aspect, the neutral lipid is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. In some embodiments, the phospholipid is selected from T g In some embodiments, the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE) and dipalmitoyl-phosphatidylethanolamine (DPPE). In some embodiments, the neutral lipid is DSPC.

[0153] In some embodiments of the second aspect, the neutral lipids comprise from about 5 mol% to about 25 mol% of the total lipids present in the organic solution, e.g., from about 15 mol% to about 25 mol%, or from about 17 mol% to about 21 mol% (particularly for embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or from about 5 mol% to about 15 mol%, or from about 7 mol% to about 14 mol% (particularly for embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids). In some embodiments of the second aspect, the neutral lipids comprise from about 5 mol% to about 25 mol% of the total lipids present in the composition, e.g., from about 15 mol% to about 25 mol%, or from about 17 mol% to about 21 mol% (particularly for embodiments having higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroids); or from about 5 mol% to about 15 mol%, or from about 7 mol% to about 14 mol% (particularly for embodiments having lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids).

[0154] In some embodiments of the second aspect, the lipids that ionize to release cations comprise about 20 mol% to about 70 mol% of the total lipids present in the organic solution; the steroids comprise about 15 mol% to about 60 mol% of the total lipids present in the organic solution; and the neutral lipids (e.g., phospholipids) comprise about 5 mol% to about 25 mol% of the total lipids present in the organic solution.

[0155] In some embodiments of the second aspect, the lipids that can ionize to release cations account for about 40 mol% to about 70 mol% of the total lipids present in the organic solution, for example, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%; the steroid (which is preferably cholesterol) accounts for about 15 mol% to about 40 mol% of the total lipids present in the organic solution, for example, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%; and the neutral lipids (which are preferably phospholipids) account for about 15 mol% to about 25 mol% of the total lipids present in the organic solution, for example, about 17 mol% to about 21 mol%. In some embodiments of the second aspect, the lipid that can ionize out cation accounts for about 40mol% to about 70mol% of the total lipid present in the composition, for example, about 45mol% to about 65mol% or about 50mol% to about 60mol%; Steroid (it is preferably cholesterol) accounts for about 15mol% to about 40mol% of the total lipid present in the composition, for example, about 20mol% to about 35mol% or about 20mol% to about 30mol%; And neutral lipid (it is preferably phospholipid) accounts for about 15mol% to about 25mol% of the total lipid present in the composition, for example, about 17mol% to about 21mol%. These embodiments of the second aspect (that is, for the preparation of the nucleic acid composition comprising higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroid) are particularly suitable for transfection cells in the presence of serum. In some of these embodiments, steroid is cholesterol and neutral lipid is phospholipid.

[0156] In some embodiments of the second aspect (which comprise a higher relative amount of ionizable and neutral lipids and a lower relative amount of steroids), the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is from 1 to 2.5. Thus, in some embodiments of the second aspect, lipids that can ionize into cations account for about 40 mol% to about 70 mol% (e.g., about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%) of the total lipids present in the organic solution (or in the composition); steroids account for about 15 mol% to about 40 mol% (e.g., about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%) of the total lipids present in the organic solution (or in the composition); neutral lipids account for about 15 mol% to about 25 mol% (e.g., about 17 mol% to about 21 mol%) of the total lipids present in the organic solution (or in the composition); and the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is from 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0157] In some alternative embodiments of the second aspect, the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% of the total lipids present in the organic solution, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%; the steroid (which is preferably cholesterol) accounts for about 35 mol% to about 60 mol% of the total lipids present in the organic solution, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%; and the neutral lipids (which are preferably phospholipids) account for about 5 mol% to about 15 mol% of the total lipids present in the organic solution, for example, about 7 mol% to about 14 mol%. In some embodiments of the second aspect, the lipid that can ionize out cation accounts for about 20mol% to about 40mol% of the total lipid present in the composition, for example, about 25mol% to about 40mol% or about 25mol% to about 35mol%; Steroid (it is preferably cholesterol) accounts for about 35mol% to about 60mol% of the total lipid present in the composition, for example, about 40mol% to about 60mol% or about 45mol% to about 60mol%; And neutral lipid (it is preferably phospholipid) accounts for about 5mol% to about 15mol% of the total lipid present in the composition, for example, about 7mol% to about 14mol%. These embodiments of the second aspect (that is, for the preparation of the nucleic acid composition comprising lower relative amount of ionizable and neutral lipid and higher relative amount of steroid) are particularly suitable for transfection cells in the absence of serum. In some of these embodiments, steroid is cholesterol and neutral lipid is phospholipid.

[0158] In some embodiments of the second aspect comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids, the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0. Thus, in some embodiments of the second aspect, the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% (e.g., about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%) of the total lipids present in the organic solution (or in the composition); the steroids account for about 35 mol% to about 60 mol% (e.g., about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%) of the total lipids present in the organic solution (or in the composition); the neutral lipids account for about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipids present in the organic solution (or in the composition); and the molar ratio of steroid to neutral lipids is at least 3.0, preferably the ratio is 3.0 to 10.0, for example, 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0159] In some embodiments of the second aspect, the organic solution / composition further comprises one or more additional lipids. In these embodiments in which a cationic lipid is present, the sum of (1) the amount of lipid that can ionize to give a cation and (2) the amount of the cationic lipid is used for calculation. For example, if the amount of lipid that can ionize to give a cation in the organic solution / composition should be from about 20 mol % to about 70 mol % and the organic solution / composition should also comprise a cationic lipid, then the sum of (1) the amount of lipid that can ionize to give a cation and (2) the amount of the cationic lipid would be from about 20 mol % to about 70 mol %.

[0160] In some embodiments of the second aspect, the organic solution, composition or its two are substantially free of lipids comprising polyethylene glycol (PEG). In some embodiments of the second aspect, the organic solution, composition or its two are substantially free of any compound comprising PEG. In some embodiments of the second aspect, the organic solution, composition or its two are substantially free of PEG. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, its salt and mixture. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, citrate and its salt. In some embodiments of these embodiments, the polyvalent anion is inorganic polyphosphate or its salt.

[0161] In some embodiments of the second aspect, the organic solution is also substantially free of other polymer-conjugated lipids. In some embodiments, the other polymer-conjugated lipids are polysarcosine-conjugated lipids. Therefore, in some embodiments, the organic solution is substantially free of PEG lipids and substantially free of polysarcosine-conjugated lipids. In some embodiments, the organic solution is substantially free of any polymer-conjugated lipids (including PEG lipids and polysarcosine-conjugated lipids). In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, its salt and mixture. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, citrate and its salt. In some embodiments of these embodiments, the polyvalent anion is inorganic polyphosphate or its salt.

[0162] In some embodiments of the second aspect, the only lipids contained in the organic solution are lipids, steroids and neutral lipids that can ionize out cations, particularly lipids, steroids and phospholipids that can ionize out cations. In some embodiments of the second aspect, the composition is substantially free of any polymer-conjugated lipids. In some embodiments of the second aspect, the only lipids contained in the composition are lipids, steroids and neutral lipids that can ionize out cations, particularly lipids, steroids and phospholipids that can ionize out cations. In some of these embodiments, the polyvalent anion or its salt is selected from inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, its salt and mixture. In some of these embodiments, the polyvalent anion is selected from inorganic polyphosphate, inorganic phosphate, citrate and its salt. In some embodiments of these embodiments, the polyvalent anion is an inorganic polyphosphate or its salt.

[0163] In some embodiments of the second aspect, the particle comprises or is selected from lipid nanoparticles (LNPs), liposomes, lipid complexes (LPXs), and mixtures thereof. In some embodiments, the particle comprises or is LNP. In some embodiments, the particle comprises or is liposomes. In some embodiments, the particle comprises or is LPX. In some embodiments, the particle comprises or is a mixture of LNP and liposomes. In some embodiments, the particle comprises or is a mixture of LNP and LPX. In some embodiments, the particle comprises or is a mixture of liposomes and LPX. In some embodiments, the particle comprises or is a mixture of LNP, liposomes, and LPX.

[0164] In some embodiments of the second aspect, the particle comprises substantially all lipids present in the composition (particularly all lipids ionizable to release cations, steroids, and neutral lipids).

[0165] In some embodiments of the second aspect, the aqueous phase is substantially free of lipids, steroids, and neutral lipids that ionize into cations (eg, substantially free of lipids).

[0166] In some embodiments of the second aspect, the particles comprise at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acids (particularly RNA) present in the composition. In some embodiments, the particles comprise at least 75%, preferably at least 85% of the nucleic acids (particularly RNA) present in the composition.

[0167] In some embodiments of the second aspect, the aqueous phase is substantially free of nucleic acids.

[0168] In some embodiments of the second aspect, the nucleic acid (eg, RNA) is encapsulated within or associated with the particle.

[0169] In some embodiments of the second aspect, at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%) of the multivalent anions (e.g., inorganic polyphosphate) present in the composition are associated with the particles. In some embodiments, at least 20%, and more preferably at least 50% of the multivalent anions (e.g., inorganic polyphosphate) present in the composition are associated with the particles.

[0170] In some embodiments of the second aspect, the size of the particles is from about 30 nm to about 500 nm. In some embodiments, the size of the particles is from about 50 nm to about 150 nm.

[0171] In some embodiments of the second aspect, the nucleic acid is DNA.

[0172] In some embodiments of the second aspect, the nucleic acid is RNA (eg, mRNA or inhibitory RNA, eg, siRNA).

[0173] In some embodiments of the second aspect, the RNA (e.g., mRNA) (i) comprises modified nucleosides instead of uridine; (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with increased G / C content compared to the wild-type coding sequence. In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0174] In some embodiments of the second aspect, the RNA (e.g., mRNA) comprises at least one or more of the following: (a) a 5' cap, such as a cap 1 or cap 2 structure; (b) a 5' UTR; (c) a 3' UTR; and (d) a poly-A sequence. In some embodiments, the RNA (e.g., mRNA) comprises all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably interrupted by a sequence of A nucleotides.

[0175] In some embodiments of the second aspect, the RNA (e.g., mRNA) encodes one or more polypeptides. In some embodiments, the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response to an antigen in a subject.

[0176] In some embodiments of the second aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of a protein, or an immunogenic fragment of a protein or an immunogenic variant thereof. In some embodiments, the pathogen is a pathogen that causes an infectious disease.

[0177] In some embodiments of the second aspect, the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof. In some embodiments, the RNA (e.g., mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof.

[0178] In some embodiments of the second aspect, the nucleic acid is an inhibitory RNA (e.g., siRNA) and selectively hybridizes to and / or is specific for a target mRNA. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, particularly a pharmaceutically active peptide or polypeptide whose expression (particularly increased expression, e.g., increased expression compared to expression in healthy subjects) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (particularly increased expression, e.g., increased expression compared to expression in healthy subjects) is associated with a cancer.

[0179] It will be understood that any embodiment described herein in the context of the first aspect may also apply to any embodiment of the second aspect.

[0180] In a third aspect, the present disclosure provides a method of storing a composition, comprising preparing a composition according to the method of the second aspect and storing the composition at a temperature of about -90°C to about -10°C, for example, about -90°C to about -40°C, or about -40°C to about -25°C, or about -25°C to about -10°C, or at a temperature of about -20°C. In some embodiments of the third aspect, the frozen composition is stored for at least 1 week, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. In some embodiments of the third aspect, the frozen composition is stored at -20°C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months. In some embodiments of the third aspect, the composition can be stored at -70°C.

[0181] In some embodiments of the third aspect, the composition comprises a cryoprotectant. In some embodiments of the third aspect, the composition is substantially free of a cryoprotectant.

[0182] In some embodiments of the third aspect, the method of storing the composition comprises preparing the composition according to the method of the second aspect comprising step (II) (i.e., freezing the formulation to about -10°C or below); storing the frozen composition at a temperature of about -90°C to about -10°C for a certain period of time (e.g., at least one week); and storing the frozen composition at a temperature of about 0°C to about 20°C for a certain period of time (e.g., at least four weeks).

[0183] It will be understood that any embodiment described herein in the context of the first or second aspect may also apply to any embodiment of the third aspect.

[0184] In a fourth aspect, the present disclosure provides a method of storing a composition, comprising preparing a liquid composition according to the method of the second aspect and storing the liquid composition at a temperature of about 0°C to about 20°C, e.g., about 1°C to about 15°C, about 2°C to about 10°C, or about 2°C to about 8°C, or at a temperature of about 5°C. In some embodiments of the fourth aspect, the liquid composition is stored for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months, preferably at least 4 weeks. In some embodiments of the fourth aspect, the liquid composition is stored at 5°C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months.

[0185] In some embodiments of the fourth aspect, the composition comprises a cryoprotectant. In some preferred embodiments of the fourth aspect, the composition is substantially free of a cryoprotectant.

[0186] It will be appreciated that any embodiment described herein in the context of the first or second aspects may also apply to any embodiment of the fourth aspect.

[0187] In a fifth aspect, the present disclosure provides compositions that can be prepared by the method for the second, third or fourth aspects. In some embodiments of the fifth aspect, the composition can be in a frozen form, which can preferably be stored at a temperature of about -90 ° C or higher, such as about -90 ° C to about -10 ° C. For example, the frozen composition of the fifth aspect can be stored at a temperature of about -90 ° C to about -40 ° C, or about -40 ° C to about -25 ° C, or about -25 ° C to about -10 ° C, or at a temperature of about -20 ° C. In some embodiments of the fifth aspect, the composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen composition can be stored at -20 ° C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months.

[0188] In some embodiments of the fifth aspect, the composition comprises a cryoprotectant. In some embodiments of the fifth aspect, the composition is substantially free of a cryoprotectant.

[0189] In some embodiments of the fifth aspect, wherein the composition is in frozen form, the nucleic acid integrity (particularly RNA integrity) after thawing the frozen composition is at least 90%, at least 95%, at least 97%, at least 98% or essentially 100% compared to the nucleic acid integrity (particularly RNA integrity) of the composition before the composition was frozen, for example, after thawing a frozen composition stored at -20°C.

[0190] In some embodiments, the initial nucleic acid integrity (particularly initial RNA integrity) of the composition (i.e., after its preparation but before freezing) is at least 50%, and the nucleic acid integrity (particularly RNA integrity) of the composition after thawing the frozen composition is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably essentially 100% of the initial nucleic acid integrity (particularly initial RNA integrity).

[0191] Additionally or alternatively, in some embodiments of the fifth aspect wherein the composition is in frozen form, the size (Z) of the nucleic acid (e.g., RNA) particles after thawing the frozen composition 平均值 ) ) and / or size distribution and / or polydispersity index (PDI)) are substantially equal to the size (Z) of the nucleic acid (e.g., RNA) particles before the composition is frozen. 平均值 ) ) and / or size distribution and / or PDI. In some embodiments, the size (Z) of the nucleic acid (eg, RNA) particles after thawing the frozen composition is 平均值) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In some embodiments, the PDI of the nucleic acid (e.g., RNA) particles after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In some embodiments, the size (Z) of the nucleic acid (e.g., RNA) particles after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the size (Z) of the nucleic acid (e.g., RNA) particles after thawing the frozen composition is 平均值 ) ) and / or size distribution and / or PDI) are substantially equal to the size (Z) of the nucleic acid (e.g., RNA) particles before freezing. 平均值 ) ) and / or size distribution and / or PDI. In some embodiments, the size (Z) of the nucleic acid (eg, RNA) particles after thawing the frozen composition is 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the PDI of the nucleic acid (e.g., RNA) particles after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0192] In some embodiments, after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, and more preferably after five freeze / thaw cycles or more, the nucleic acid (e.g., RNA) particle size and nucleic acid integrity (particularly RNA integrity) of the composition are substantially equivalent to the nucleic acid (e.g., RNA) particle size and nucleic acid integrity (particularly RNA integrity) of the initial composition (i.e., before the composition was first frozen).

[0193] In an alternative embodiment of the fifth aspect, the composition is in liquid form.

[0194] In some embodiments of the fifth aspect, wherein the composition is in liquid form, the nucleic acid integrity (particularly RNA integrity) of the liquid composition is such that the desired effect, such as induction of an immune response, is achieved when stored, for example, at 0°C or higher for at least one week. For example, when stored, for example, at 0°C or higher for at least one week (e.g., at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, or at least six months), the nucleic acid integrity (particularly RNA integrity) of the liquid composition can be at least 90%, at least 95%, at least 97%, or at least 98% compared to the nucleic acid integrity (particularly RNA integrity) before storage. In some embodiments, after storage for at least four weeks, preferably at 0°C or higher, for example, at a temperature of about 2°C to about 8°C, the nucleic acid integrity (particularly RNA integrity) of the composition is at least 90%, at least 95%, at least 97%, or at least 98% compared to the nucleic acid integrity (particularly RNA integrity) before storage.

[0195] In some embodiments, the initial nucleic acid integrity (particularly initial RNA integrity) of the liquid composition (i.e., after its preparation but before storage) is at least 50%, and after storage for at least one week (e.g., at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, or at least 3 months), preferably at a temperature of 0°C or higher, e.g., from about 2°C to about 8°C, the nucleic acid integrity (particularly RNA integrity) of the liquid composition is at least 90% of the initial nucleic acid integrity (particularly initial RNA integrity).

[0196] Additionally or alternatively, in some embodiments of the fifth aspect wherein the composition is in liquid form, the size (Z) of the nucleic acid (e.g., RNA) particles of the liquid composition when stored at, for example, 0° C. or higher for at least one week is 平均值 ) ) and / or size distribution and / or polydispersity index (PDI)) such that a desired effect, such as induction of an immune response, can be achieved. For example, when stored at, for example, 0° C. or higher for at least one week, the size (Z) of the nucleic acid (e.g., RNA) particles of the liquid composition may be 平均值 ) ) and / or size distribution and / or polydispersity index (PDI)) substantially equal to the size (Z) of the nucleic acid (e.g., RNA) particles of the initial composition (i.e., before storage). 平均值 ) ) and / or size distribution and / or PDI. In some embodiments, after storage of the liquid composition, for example, at 0°C or higher for at least one week, the size (Z) of the nucleic acid (e.g., RNA) particles is 平均值) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In some embodiments, after storage of the liquid composition, for example, at 0° C. or higher for at least one week, the PDI of the nucleic acid (e.g., RNA) particles is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In some embodiments, after storage of the liquid composition, for example, at 0° C. or higher for at least one week, the size (Z) of the nucleic acid (e.g., RNA) particles is less than 0.3, preferably less than 0.2, more preferably less than 0.1. 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the size (Z) of the nucleic acid (e.g., RNA) particles of the liquid composition after storage, for example, at 0° C. or higher for at least one week. 平均值 ) ) and / or size distribution and / or PDI) are substantially equal to the size (Z) of the nucleic acid (e.g., RNA) particles prior to storage. 平均值 ) ) and / or size distribution and / or PDI. In some embodiments, after storage of the liquid composition, for example, at 0°C or higher for at least one week, the size (Z) of the nucleic acid (e.g., RNA) particles is 平均值 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the PDI of the nucleic acid (e.g., RNA) particles after storage of the liquid composition, for example, at 0° C. or higher for at least one week, is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0197] It will be understood that any embodiment described herein in the context of the first, second, third or fourth aspects may also apply to any embodiment of the fifth aspect.

[0198] In a sixth aspect, the present disclosure provides a method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a frozen composition prepared by the method of the second, third or fourth aspect and thawing the frozen pharmaceutical composition to obtain the ready-to-use pharmaceutical composition.

[0199] It will be understood that any embodiment described herein in the context of the first, second, third, fourth or fifth aspects may also apply to any embodiment of the sixth aspect.

[0200] In a seventh aspect, the present disclosure provides a method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a liquid composition prepared by the method of the second, third or fourth aspect, thereby obtaining the ready-to-use pharmaceutical composition.

[0201] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspects may also apply to any embodiment of the seventh aspect.

[0202] In an eighth aspect, the present disclosure provides a ready-to-use pharmaceutical composition that can be prepared by the method of the sixth or seventh aspect.

[0203] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth or seventh aspects may also be applicable to any embodiment of the eighth aspect.

[0204] In a ninth aspect, the present disclosure provides the composition of any one of the first, fifth and eighth aspects for use in therapy.

[0205] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspects may also be applicable to any embodiment of the ninth aspect.

[0206] In a tenth aspect, the present disclosure provides the composition of any one of the first, fifth, eighth and ninth aspects for use in inducing an immune response.

[0207] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect may also be applicable to any embodiment of the tenth aspect.

[0208] In the 11th aspect, present disclosure provides the method for transfection cell, it includes adding the composition of any one in the first, fifth or eighth aspect to cell, and the mixture of composition and cell is hatched and continues the time of sufficient amount.In some embodiments (particularly those, wherein nucleic acid is DNA or RNA (such as mRNA) and encodes pharmaceutically active protein), the mixture of composition and cell is hatched and continues the time that is enough to make pharmaceutically active protein expression.In some embodiments (particularly those, wherein nucleic acid is inhibitory RNA (such as siRNA) for target mRNA), the mixture of composition and cell is hatched and continues the time that is enough to make the transcription and / or translation of suppressing target mRNA.In some embodiments, the time of sufficient amount is at least one hour (such as at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or up to about 48 hours (such as up to about 36 or up to about 24 hours).In some embodiments of the 11th aspect, hatching composition and cell mixture is carried out when there is serum (such as human serum).

[0209] In some embodiments of the eleventh aspect, the lipid that can ionize out cation accounts for about 40mol% to about 70mol% of the total lipid present in the composition, for example, about 45mol% to about 65mol% or about 50mol% to about 60mol%; Steroid (it is preferably cholesterol) accounts for about 15mol% to about 40mol% of the total lipid present in the composition, for example, about 20mol% to about 35mol% or about 20mol% to about 30mol%; And neutral lipid (it is preferably phospholipid) accounts for about 15mol% to about 25mol% of the total lipid present in the composition, for example, about 17mol% to about 21mol%. For these embodiments (that is, the composition of the ionizable and neutral lipid comprising a higher relative amount and a lower relative amount of steroid), it is preferred that the mixture of incubation compositions and cells is carried out in the presence of serum (for example, human serum). In some of these embodiments, steroid is cholesterol and neutral lipid is phospholipid.

[0210] In some embodiments of the eleventh aspect (which relate to compositions comprising a higher relative amount of ionizable and neutral lipids and a lower relative amount of steroids), the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. Thus, in some embodiments of the eleventh aspect, the lipids that can ionize to give cations account for about 40 mol% to about 70 mol% (e.g., about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%) of the total lipids present in the composition; the steroids account for about 15 mol% to about 40 mol% (e.g., about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%) of the total lipids present in the composition; the neutral lipids account for about 15 mol% to about 25 mol% (e.g., about 17 mol% to about 21 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0211] In some alternative embodiments of the eleventh aspect, the lipid that can ionize out cations accounts for about 20mol% to about 40mol% of the total lipids present in the composition, for example, about 25mol% to about 40mol%, or about 25mol% to about 35mol%; the steroid (which is preferably cholesterol) accounts for about 35mol% to about 60mol% of the total lipids present in the composition, for example, about 40mol% to about 60mol%, or about 45mol% to about 60mol%; and the neutral lipid (which is preferably phospholipid) accounts for about 5mol% to about 15mol% of the total lipids present in the composition, for example, about 7mol% to about 14mol%. For these embodiments (i.e., compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids), incubating the mixture of the composition and the cells can be carried out in the presence or absence of serum, for example, in the absence of serum.

[0212] In some embodiments of the eleventh aspect (which relate to compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids), the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is 3.0 to 10.0, for example 5.0 to 7.0. Thus, in some embodiments of the eleventh aspect, lipids that can ionize into cations account for about 20 mol% to about 40 mol% (e.g., about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%) of the total lipids present in the composition; steroids account for about 35 mol% to about 60 mol% (e.g., about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%) of the total lipids present in the composition; neutral lipids account for about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is 3.0 to 10.0, for example 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0213] In some embodiments of the eleventh aspect, the method is performed in vivo (i.e., the cells form a part of an organ, tissue, and / or organism of a subject). In some embodiments of the eleventh aspect, the method is performed in vitro (i.e., the cells do not form a part of an organ, tissue, and / or organism of a subject, e.g., the cells are an ex vivo cell culture).

[0214] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspect may also be applicable to any embodiment of the eleventh aspect.

[0215] In a twelfth aspect, the present disclosure provides use of the composition of any one of the first, fifth, or eighth aspects for transfecting cells. In some embodiments of the twelfth aspect, transfecting the cells is performed in the presence of serum (eg, human serum).

[0216] In some embodiments of the 12th aspect, the lipid that can ionize out cations accounts for about 40mol% to about 70mol% of the total lipids present in the composition, for example, about 45mol% to about 65mol% or about 50mol% to about 60mol%; Steroid (which is preferably cholesterol) accounts for about 20mol% to about 40mol% of the total lipids present in the composition; and neutral lipid (which is preferably phospholipid) accounts for about 15mol% to about 25mol% of the total lipids present in the composition. For these embodiments (that is, compositions comprising higher relative amounts of ionizable and neutral lipids and lower relative amounts of steroid), it is preferred that the mixture of incubation compositions and cells is carried out in the presence of serum (e.g., human serum). In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0217] In some embodiments of the twelfth aspect (which relate to compositions comprising a higher relative amount of ionizable and neutral lipids and a lower relative amount of steroids), the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. Thus, in some embodiments of the twelfth aspect, lipids that can ionize into cations account for about 40 mol% to about 70 mol% (e.g., about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%) of the total lipids present in the composition; steroids account for about 15 mol% to about 40 mol% (e.g., about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%) of the total lipids present in the composition; neutral lipids account for about 15 mol% to about 25 mol% (e.g., about 17 mol% to about 21 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at most 2.5, preferably the ratio is 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0218] In some alternative embodiments of the twelfth aspect, the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% of the total lipids present in the composition, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%; the steroid (which is preferably cholesterol) accounts for about 35 mol% to about 60 mol% of the total lipids present in the composition, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%; and the neutral lipids (which are preferably phospholipids) account for about 5 mol% to about 15 mol% of the total lipids present in the composition, for example, about 7 mol% to about 14 mol%. For these embodiments (i.e., compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids), incubating the mixture of the composition and the cells can be carried out in the presence or absence of serum, for example, in the absence of serum.

[0219] In some embodiments of the twelfth aspect (which relate to compositions comprising lower relative amounts of ionizable and neutral lipids and higher relative amounts of steroids), the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is between 3.0 and 10.0, for example, between 5.0 and 7.0. Thus, in some embodiments of the twelfth aspect, lipids that can ionize to release cations account for about 20 mol% to about 40 mol% (e.g., about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%) of the total lipids present in the composition; steroids account for about 35 mol% to about 60 mol% (e.g., about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%) of the total lipids present in the composition; neutral lipids account for about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipids present in the composition; and the molar ratio of steroids to neutral lipids is at least 3.0, preferably the ratio is between 3.0 and 10.0, for example, between 5.0 and 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0220] In some embodiments of the twelfth aspect, the use is an in vivo use (i.e., the cells form a part of an organ, tissue, and / or organism of a subject). In some embodiments of the twelfth aspect, the use is an in vitro use (i.e., the cells do not form a part of an organ, tissue, and / or organism of a subject, e.g., the cells are an ex vivo cell culture).

[0221] It should be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh aspect may also be applicable to any embodiment of the twelfth aspect.

[0222] In another aspect, the present disclosure provides a kit comprising the composition of any one of the first, fifth, eighth, ninth or tenth aspects or the pharmaceutical composition described herein. In some embodiments, the kit is used for treatment, for example, for inducing an immune response. In some embodiments, the kit is used for inducing an immune response against a pathogen, for example, for treating or preventing an infectious disease.

[0223] Other itemized implementation options are as follows:

[0224] 1. A composition comprising (i) a nucleic acid; (ii) a lipid that can ionize into a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a multivalent anion.

[0225] 2. The composition of claim 1 , wherein the polyvalent anion comprises 2 to 10 negative charges, optionally 2 to 5 negative charges.

[0226] 3. The composition of item 1 or 2, wherein the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, or a mixture thereof.

[0227] 4. The composition of any one of items 1 to 3, wherein the multivalent anion is an inorganic polyphosphate.

[0228] 4a. The composition of item 4, wherein the inorganic polyphosphate comprises the formula [P x O (3x+1) ] y , where x is an integer and is at least 3; and y is the anionic charge.

[0229] 4b. The composition of item 4 or 4a, wherein the inorganic polyphosphate is selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof, preferably selected from triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof, more preferably, the inorganic polyphosphate is triphosphate.

[0230] 4c. The composition of any one of items 4 to 4b, wherein the inorganic polyphosphate is a linear inorganic polyphosphate, such as a linear inorganic triphosphate.

[0231] 5. The composition of any one of items 1 to 4c, wherein the molar ratio of (v) polyvalent anion (e.g., inorganic polyphosphate) to (ii) lipid that can ionize into a cation is at least about 1:2, preferably at least about 2:3, for example at least about 4:3.

[0232] 6. The composition of any one of items 1 to 5, which is substantially free of lipids comprising polyethylene glycol (PEG), preferably substantially free of any PEG-containing compound, more preferably substantially free of PEG.

[0233] 6a. The composition of any one of items 1 to 6, which is substantially free of any polymer-conjugated lipids.

[0234] 7. The composition of any one of items 1 to 6a, wherein the pH of the composition is from about 4.0 to about 8.0, preferably from about 4.5 to about 8.0, for example, from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, or from about 7.0 to about 7.8.

[0235] 8. The composition of any one of items 1 to 7, wherein water is the major component of the composition and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

[0236] 9. The composition of any one of items 1 to 8, wherein the osmolarity of the composition is at most about 1000×10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg to about 500×10 -3 osmol / kg, more preferably about 300×10 - 3 osmol / kg.

[0237] 10. The composition of any one of items 1 to 9, wherein the concentration of the nucleic acid in the composition is from about 1 mg / l to about 500 mg / l, for example, from about 1 mg / l to about 100 mg / l, from about 5 mg / l to about 100 mg / l, or from about 10 mg / l to about 100 mg / l.

[0238] 11. The composition of any one of items 1 to 10, wherein the lipid that can ionize to release cations comprises a head group comprising at least one tertiary amine moiety.

[0239] 12. The composition of any one of items 1 to 11, wherein the lipid capable of ionizing cations has a structure of formula (X)

[0240]

[0241] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0242] L 10 and L 20 One of them is

[0243] -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-,SC(=O)-, -NR a C(=O)-, -C(=O)NR a -,NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O-,

[0244] And L 10 and L 20 The other one is

[0245] -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -,-SS--C(=O)S-,SC(=O)-,-NR a C(=O)-, -C(=O)NR a -,NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O- or direct bond;

[0246] G 1 and G 2 Each independently is an unsubstituted C1-C 12 Alkylene or C 2-12 alkenylene;

[0247] G 3 It is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 cycloalkenylene;

[0248] R a Is H or C 1-12 alkyl;

[0249] R 35 and R 36 Each independently is C 6-24 Alkyl or C 6-24 alkenyl;

[0250] R 37 It's H, OR 50 ,CN,-C(=O)OR 40 -OC(=O)R 40 or -NR 50 C(=O)R40 ;

[0251] R 40 It is C 1-12 alkyl;

[0252] R 50 Is H or C 1-6 alkyl; and

[0253] x is 0, 1, or 2.

[0254] 13. The composition of any one of items 1 to 11, wherein the cationic lipid or the lipid that can ionize cations has the structure of formula (XI):

[0255]

[0256] in

[0257] Each of R1 and R2 is independently R5 or -G1-L1-R6, wherein at least one of R1 and R2 is -G1-L1-R6;

[0258] Each of R3 and R4 is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 Cycloalkyl;

[0259] Each of R5 and R6 is independently an acyclic hydrocarbon group having at least 10 carbon atoms;

[0260] Each of G1 and G2 is independently an unsubstituted C 1-12 Alkylene or C 2-12 alkenylene;

[0261] Each of L1 and L2 is independently selected from

[0262] -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)-, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;

[0263] Ra is H or C 1-12 alkyl;

[0264] m is 0, 1, 2, 3 or 4; and

[0265] x is 0, 1, or 2.

[0266] 14. The composition of any one of items 1 to 13, wherein the lipid that can ionize to release cations accounts for about 20 mol% to about 75 mol% of the total lipids present in the composition, for example, about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, about 50 mol% to about 60 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%.

[0267] 15. The composition of any one of items 1 to 14, wherein the steroid comprises a sterol, such as cholesterol.

[0268] 16. The composition of any one of items 1 to 15, wherein the steroid comprises about 15 mol% to about 60 mol% of the total lipids present in the composition, for example, about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, about 20 mol% to about 30 mol%, about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%.

[0269] 17. The composition of any one of items 1 to 16, wherein the neutral lipid is a phospholipid, preferably selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE) and dipalmitoyl-phosphatidylethanolamine (DPPE).

[0270] 18. The composition of any one of items 1 to 17, wherein the neutral lipids account for about 5 mol% to about 25 mol% of the total lipids present in the composition, for example, about 10 mol% to about 25 mol%, about 15 mol% to about 25 mol%, about 17 mol% to about 21 mol%, about 5 mol% to about 15 mol%, or about 7 mol% to about 14 mol%.

[0271] 19. The composition of any one of items 1 to 18, wherein the lipids that can ionize to release cations account for about 20 mol% to about 70 mol% of the total lipids present in the composition; the steroids account for about 15 mol% to about 60 mol% of the total lipids present in the composition; and the neutral lipids (e.g., phospholipids) account for about 5 mol% to about 25 mol% of the total lipids present in the composition.

[0272] 20. The composition of any one of items 1 to 19, wherein the lipids that can ionize to release cations account for about 40 mol% to about 70 mol% of the total lipids present in the composition, for example, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%; the steroids account for about 15 mol% to about 40 mol% of the total lipids present in the composition, for example, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%; and the neutral lipids account for about 15 mol% to about 25 mol% of the total lipids present in the composition, for example, about 17 mol% to about 21 mol%.

[0273] 20a. The composition of any one of items 1 to 20, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0274] 21. The composition of any one of items 1 to 20a, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.5.

[0275] 22. A composition according to any one of items 1 to 19, wherein the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% of the total lipids present in the composition, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%; the steroids account for about 35 mol% to about 60 mol% of the total lipids present in the composition, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%; and the neutral lipids account for about 5 mol% to about 15 mol% of the total lipids present in the composition, for example, about 7 mol% to about 14 mol%.

[0276] 22a. The composition of claim 22, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0277] 23. The composition of any one of items 1 to 19, 22 and 22a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0.

[0278] 24. The composition of any one of items 1 to 23, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into cations, at least a portion of the steroid, and at least a portion of the neutral lipid; and wherein at least a portion of the multivalent anions (e.g., the inorganic polyphosphate) are associated with the particles.

[0279] 25. The composition of claim 24, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipid complexes (LPXs), and mixtures thereof.

[0280] 25a. The composition of item 24 or 25, wherein the particle comprises substantially all of the ionizable lipid, the steroid, and the neutral lipid present in the composition.

[0281] 25b. The composition of any one of items 24 to 25a, wherein the aqueous phase is substantially free of the lipid that can ionize into a cation, the steroid, and the neutral lipid.

[0282] 26. The composition of any one of items 24 to 25b, wherein the particles account for at least 50%, preferably at least 75%, more preferably at least 85% of the nucleic acid present in the composition.

[0283] 26a. The composition of any one of items 24 to 26, wherein the aqueous phase is substantially free of the nucleic acid.

[0284] 27. The composition of any one of items 24 to 26a, wherein at least 10%, preferably at least 20%, and more preferably at least 50% of the multivalent anions (e.g., the inorganic polyphosphate) present in the composition are associated with the particles.

[0285] 28. The composition of any one of items 24 to 27, wherein the particles have a size of about 30 nm to about 500 nm, such as about 50 nm to about 150 nm.

[0286] 29. The composition of any one of items 1 to 28, wherein the nucleic acid is RNA, preferably mRNA.

[0287] 29a. The composition of any one of items 1 to 28, wherein the nucleic acid is DNA.

[0288] 29b. The composition of any one of items 1 to 28, wherein the nucleic acid is an inhibitory RNA, such as an siRNA.

[0289] 30. The composition of claim 29, wherein the RNA (1) comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U); (2) has a codon-optimized coding sequence; and / or (3) has a coding sequence with an increased G / C content compared to the wild-type coding sequence.

[0290] 31. The composition of item 29 or 30, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

[0291] 32. The composition of item 31, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted A nucleotide sequence.

[0292] 33. The composition of item 31 or 32, wherein the 5'-cap is a cap 1 or cap 2 structure.

[0293] 34. The composition of any one of items 29 and 30 to 33, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response against an antigen in a subject.

[0294] 35. The composition of claim 34, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or its immunogenic variant.

[0295] 36. The composition of claim 34 or 35, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0296] 37. The composition of any one of items 1 to 36, wherein the composition is in liquid form, preferably at a temperature of about 2°C to about 10°C.

[0297] 37a. The composition of claim 37, wherein the concentration of the nucleic acid in the composition is from about 10 mg / l to about 100 mg / l.

[0298] 38. The composition of any one of items 1 to 37a, wherein after storage for at least one week, preferably at a temperature of about 2°C to about 8°C for at least one week, the nucleic acid integrity of the composition is at least 90% compared to the nucleic acid integrity before storage.

[0299] 38a. The composition of any one of items 1 to 38, wherein after storage for at least four weeks, preferably at a temperature of about 2°C to about 8°C, the nucleic acid integrity of the composition is at least 90% compared to the nucleic acid integrity before storage.

[0300] 38b. The composition of any one of items 1 to 38a, wherein after storage for at least three months, preferably at a temperature of about 2°C to about 8°C for at least three months, the nucleic acid integrity of the composition is at least 90% compared to the nucleic acid integrity before storage.

[0301] 39. The composition of any one of items 24 to 38b, wherein the size of the nucleic acid particles (Z 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles before storage 平均值 ) and / or size distribution and / or PDI.

[0302] 40. The composition of any one of items 1 to 36, wherein the composition is in frozen form.

[0303] 40a. The composition of item 40, wherein the concentration of the nucleic acid in the composition is about 1 mg / l to about 50 mg / l.

[0304] 40b. The composition of item 40 or 40a, wherein the nucleic acid integrity after thawing the frozen composition is at least 50% compared to the nucleic acid integrity before the composition was frozen.

[0305] 41. The composition of item 40, 40a or 40b, wherein the nucleic acid integrity after thawing of the frozen composition is at least 90% or substantially 100% compared to the nucleic acid integrity before the composition was frozen.

[0306] 42. The composition of any one of items 40 to 41, wherein the size of the nucleic acid particles (Z 平均值 ) and / or a size distribution and / or polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles before the composition is frozen 平均值 ) and / or size distribution and / or PDI.

[0307] 42a. The composition of any one of items 40 to 42, wherein after one freeze / thaw cycle, preferably after two freeze / thaw cycles, the particle size and nucleic acid integrity of the composition are substantially equivalent to the particle size and nucleic acid integrity before the composition was first frozen.

[0308] 43. A method of preparing a composition comprising particles dispersed in a final aqueous phase, wherein the composition comprises (i) a nucleic acid; (ii) a lipid that ionizes to release a cation; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion (e.g., an inorganic polyphosphate); wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that ionizes to release a cation, and at least a portion of the steroid; wherein at least a portion of the polyvalent anion (e.g., the inorganic polyphosphate) is associated with the particles; and wherein the final aqueous phase comprises a final buffer system;

[0309] The method comprises:

[0310] (I) preparing a formulation comprising particles dispersed in the final aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into a cation, at least a portion of the steroid, and at least a portion of the neutral lipid, and wherein at least a portion of the multivalent anion (e.g., the inorganic polyphosphate) is associated with the particles; and

[0311] (II) optionally freezing the formulation to about -10°C or below,

[0312] Thus, the composition is obtained,

[0313] Wherein step (1) comprises:

[0314] (a) providing a nucleic acid solution comprising water and a first buffer system;

[0315] (b) providing an organic solution comprising the lipid capable of ionizing cations, the steroid, and the neutral lipid;

[0316] (c) mixing the nucleic acid solution provided under (a) with the organic solution provided under (b), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system;

[0317] (d) mixing the first intermediate formulation prepared under (c) with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby preparing a second intermediate formulation comprising particles dispersed in a second aqueous phase comprising a second buffer system, wherein at least a portion of the polyvalent anion (e.g., the inorganic polyphosphate) is associated with the particles; and

[0318] (e) filtering and / or diluting the second intermediate formulation prepared under (d) using a final aqueous buffer solution comprising the final buffer system,

[0319] The formulation comprising the particles dispersed in the final aqueous phase is thus prepared.

[0320] 43a. The method of item 43, wherein step (a) comprises (a') providing an aqueous nucleic acid solution; (b') providing a first aqueous buffer solution comprising a first buffer system; and (c') mixing the aqueous nucleic acid solution provided under (a') with the first aqueous buffer solution provided under (b') to provide a nucleic acid solution comprising water and the first buffer system.

[0321] 43b. The method of item 43 or 43a, wherein the organic solution comprises an organic solvent selected from alcohols having up to 6 carbon atoms and mixtures of two or more of these alcohols, preferably selected from ethanol, propanol, isopropanol, 1,2-propylene glycol, and mixtures of two or more of these alcohols.

[0322] 44. The method of any one of items 43 to 43b, wherein step (I) further comprises one or more steps selected from dilution and filtration.

[0323] 45. The method of any one of items 43 to 44, wherein step (I) comprises:

[0324] (a') providing an aqueous nucleic acid solution;

[0325] (b') providing a first aqueous buffer solution comprising a first buffer system;

[0326] (c') mixing the aqueous nucleic acid solution provided under (a') with the first aqueous buffer solution provided under (b'), thereby preparing a nucleic acid solution comprising water and the first buffer system;

[0327] (d') providing (e.g., preparing) an organic solution comprising the lipid that can ionize to release a cation, the steroid, and the neutral lipid;

[0328] (e') mixing the nucleic acid solution prepared under (c') with the organic solution provided under (d'), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system;

[0329] (f') optionally diluting the first intermediate formulation prepared under (e') with water or another aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising particles dispersed in an additional aqueous phase, the additional aqueous phase comprising the first buffer system or an additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution;

[0330] (g') mixing the first intermediate preparation obtained in step (e') with a polyvalent anion (e.g., an inorganic polyphosphate) or a salt thereof if step (f') is absent, or mixing the further intermediate preparation obtained in step (f') with a polyvalent anion (e.g., an inorganic polyphosphate) or a salt thereof if step (f') is present, thereby preparing a second intermediate preparation comprising particles dispersed in a second aqueous phase, wherein at least a portion of the polyvalent anion (e.g., the inorganic polyphosphate) is associated with the particles;

[0331] (h') optionally filtering the second first intermediate formulation prepared under (g') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution and / or the second aqueous buffer solution;

[0332] (i') optionally repeating step (h') once or twice or more times, wherein the further intermediate preparation comprising particles dispersed in the further aqueous phase comprising the further buffer system obtained after step (h') of one cycle is used as the second intermediate preparation of the next cycle, wherein in each cycle, the further aqueous buffer solution may be the same as or different from the first aqueous buffer solution and / or the second aqueous buffer solution;

[0333] (j') filtering using the final aqueous buffer solution comprising the final buffer system: the second intermediate preparation obtained in step (g') if step (h') is absent, or the further intermediate preparation obtained in step (h') if step (h') is present and step (i') is absent, or the further intermediate preparation obtained after step (i') if steps (h') and (i') are present; and

[0334] (k') optionally diluting the preparation obtained in step (j') with a dilution solution;

[0335] The formulation comprising the particles dispersed in the final aqueous phase is thereby prepared.

[0336] 45a. The method of item 45, wherein the organic solution comprises an organic solvent selected from alcohols having up to 6 carbon atoms and mixtures of two or more of these alcohols, preferably selected from ethanol, propanol, isopropanol, 1,2-propylene glycol, and mixtures of two or more of these alcohols.

[0337] 45b. The method of item 45 or 45a, wherein step (f') is absent.

[0338] 45c. The method of item 45 or 45a, wherein step (f') is present.

[0339] 45d. The method of any one of items 45 to 45c, wherein step (g') is performed at most about 20 minutes after step (e').

[0340] 45e. The method of any one of items 43 to 45d, wherein the organic solution provided under (b) or (d'), respectively, further comprises an acid.

[0341] 45f. The method of any one of items 43 to 45e, wherein step (f') comprises changing the pH of the first intermediate formulation to a pH of about 7.0 to about 9.0, optionally to a pH of about 7.5 to about 8.5, preferably to a pH of about 7.5 to about 8.0.

[0342] 45g. The method of item 45f, wherein step (f') is performed not later than 12 hours, preferably not later than 4 hours, and more preferably not later than 30 minutes after step (e').

[0343] 45h. The method of claim 45f or 45g, wherein step (f') is used to change the pH of the first intermediate formulation to a pH of 7 to 9, and then the polyvalent anion is added in step (g'), and the combined duration of steps (f') and (g') is no longer than 12 hours after step (e'), preferably no longer than 4 hours, and more preferably no longer than 30 minutes.

[0344] 46. ​​The method of any one of items 43 to 45h, wherein the filtration is dialysis, tangential flow filtration or diafiltration, preferably dialysis or tangential flow filtration.

[0345] 47. The method of any one of items 43 to 46, wherein the polyvalent anion comprises 2 to 10 negative charges, optionally 2 to 5 negative charges.

[0346] 47a. The method of any one of items 43 to 47, wherein the polyvalent anion is selected from: inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartrate, malate, citrate, or a mixture thereof.

[0347] 47b. The method of any one of items 43 to 46, wherein the polyvalent anion is an inorganic polyphosphate or a salt thereof.

[0348] 47c. The method of item 47b, wherein the inorganic polyphosphate or its salt comprises or has the formula P x O (3x+1) M y’, wherein x is an integer and is at least 3; each M is independently H + or cations; and y' is the number of cations required for charge balance.

[0349] 48. The method of item 47c, wherein each M is independently selected from H + , basic cations, ammonium and monovalent organic cations, preferably each M is independently selected from H + 、Na + , K + 、Li + and NH4 + .

[0350] 49. The method of any one of items 47b to 48, wherein the inorganic polyphosphate or its salt is selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, their salts and mixtures, preferably selected from triphosphate, tetraphosphate, pentaphosphate, their salts and mixtures, more preferably, the inorganic polyphosphate or its salt is triphosphate or its salt.

[0351] 50. The method of any one of items 47b to 49, wherein the inorganic polyphosphate or salt thereof is a linear inorganic polyphosphate or salt thereof, such as a linear triphosphate or salt thereof.

[0352] 51. The method of any one of items 43 to 50, wherein the molar ratio of (v) polyvalent anion (e.g., inorganic polyphosphate) to (ii) lipid that can ionize into a cation is at least about 1:2, preferably at least about 2:3, for example at least about 4:3.

[0353] 52. The method of any one of items 43 to 51, wherein the composition is substantially free of PEG-containing lipids, preferably substantially free of any PEG-containing compound, more preferably substantially free of PEG.

[0354] 52a. The method of any one of items 43 to 52, wherein the composition is substantially free of any polymer-conjugated lipids.

[0355] 53. The method of any one of items 43 to 52a, wherein (1) the pH of the nucleic acid solution obtained in step (a) is less than 6.0, preferably at most about 5.0, more preferably at most about 4.5; or (2) the pH of the first aqueous buffer solution is less than 6.0, preferably at most about 5.0, more preferably at most about 4.5.

[0356] 54. The method of any one of items 43 to 53, wherein the pH of the composition is from about 4.0 to about 8.0, preferably from about 4.5 to about 8.0, for example, from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, or from about 7.0 to about 7.8.

[0357] 55. The method of any one of items 43 to 54, wherein water is the major component of the formulation and / or composition, and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

[0358] 56. The method of any one of items 43 to 55, wherein the osmolarity of the composition is at most about 1000×10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg to about 500×10 -3 osmol / kg, more preferably about 300×10 - 3 osmol / kg.

[0359] 57. The method of any one of items 43 to 56, wherein the concentration of the nucleic acid in the composition is about 1 mg / l to about 500 mg / l, for example, about 1 mg / l to about 100 mg / l, about 5 mg / l to about 100 mg / l, or about 10 mg / l to about 100 mg / l.

[0360] 58. The method of any one of items 43 to 57, wherein the lipid that can ionize to release cations comprises a head group containing at least one tertiary amine moiety.

[0361] 59. The method of any one of items 43 to 58, wherein the lipid that can ionize to release cations, the steroid and the neutral lipid are present in the organic solution in the following molar ratios: about 20 mol% to about 70 mol% of the lipid that can ionize to release cations; about 15 mol% to about 60 mol% of the steroid; and about 5 mol% to about 25 mol% of the neutral lipid (e.g., phospholipid).

[0362] 60. The method of any one of items 43 to 59, wherein the lipid that can be ionized to release cations, the steroid and the neutral lipid are present in the organic solution in the following molar ratios: about 40 mol% to about 70 mol%, for example, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% of the lipid that can be ionized to release cations; about 15 mol% to about 40 mol%, for example, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% of the steroid; and about 15 mol% to about 25 mol%, for example, about 17 mol% to about 21 mol% of the neutral lipid.

[0363] 60a. The method of any one of items 43 to 60, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0364] 61. The method of any one of items 43 to 60a, wherein the molar ratio of steroid to neutral lipid in the organic solution is at most 2.5, preferably the ratio is from 1 to 2.5.

[0365] 62. The method of any one of items 43 to 59, wherein the lipid that can be ionized to release cations, the steroid and the neutral lipid are present in the organic solution in the following molar ratios: about 20 mol% to about 40 mol%, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% of the lipid that can be ionized to release cations; about 35 mol% to about 60 mol%, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% of the steroid; and about 5 mol% to about 15 mol%, for example, about 7 mol% to about 14 mol% of the neutral lipid.

[0366] 62a. The method of claim 62, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0367] 63. The method of any one of items 43 to 59, 62 and 62a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0.

[0368] 64. The method of any one of items 43 to 63, wherein the particles have a size of about 30 nm to about 500 nm, for example about 50 nm to about 150 nm.

[0369] 65. The method of any one of items 43 to 64, wherein the particle is selected from lipid nanoparticles (LNPs), liposomes, lipid complexes (LPXs), and mixtures of two or more thereof.

[0370] 65a. The method of any one of items 43 to 65, wherein the particle comprises substantially all of the ionizable lipid, the steroid, and the neutral lipid present in the composition.

[0371] 65b. The method of any one of items 43 to 65a, wherein the final aqueous phase is substantially free of the lipid that can ionize into a cation, the steroid, and the neutral lipid.

[0372] 65c. The method of any one of items 43 to 65b, wherein the particles account for at least 50%, preferably at least 75%, more preferably at least 85% of the nucleic acid present in the composition.

[0373] 65d. The method of any one of items 43 to 65c, wherein the final aqueous phase is substantially free of the nucleic acid.

[0374] 65e. The method of any one of items 43 to 65d, wherein at least 10%, preferably at least 20%, and more preferably at least 50% of the multivalent anions (e.g., the inorganic polyphosphate) present in the composition are associated with the particles.

[0375] 66. The method of any one of items 43 to 65e, wherein the nucleic acid is RNA, preferably mRNA.

[0376] 66a. The method of any one of items 43 to 65e, wherein the nucleic acid is DNA.

[0377] 66b. The method of any one of items 43 to 65e, wherein the nucleic acid is an inhibitory RNA, such as an siRNA.

[0378] 67. The method described in item 66, wherein the RNA (i) comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U); (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with an increased G / C content compared to the wild-type coding sequence.

[0379] 68. The method of item 66 or 67, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

[0380] 69. The method according to item 68, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted A nucleotide sequence.

[0381] 70. The method of item 68 or 69, wherein the 5' cap is a cap 1 or cap 2 structure.

[0382] 71. The method of any one of items 66 and 67 to 70, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response against an antigen in a subject.

[0383] 72. The method of claim 71, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or its immunogenic variant.

[0384] 73. The method of claim 71 or 72, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is the SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0385] 74. The method of any one of items 43 to 73, comprising (II) freezing the formulation to about -10°C or below.

[0386] 74a. The method of claim 74, wherein the concentration of the nucleic acid in the composition is from about 1 mg / l to about 50 mg / l.

[0387] 74b. The method of item 74 or 74a, wherein the final aqueous buffer solution comprises the multivalent anion (eg, the inorganic polyphosphate) or a salt thereof.

[0388] 75. The method of any one of items 43 to 73, which does not include step (II).

[0389] 75a. The method of claim 75, wherein the concentration of the nucleic acid in the composition is from about 10 mg / l to about 100 mg / l.

[0390] 76. A method of storing a composition, comprising preparing a composition according to the method of any one of items 43 to 74b, and storing the composition at a temperature of about -90°C to about -10°C, for example about -90°C to about -40°C, or about -25°C to about -10°C.

[0391] 77. The method of claim 76, wherein the composition is stored for at least 1 month, such as at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0392] 78. A method of storing a composition, comprising preparing a composition according to the method of any one of items 43 to 75a, and storing the composition at a temperature of about 0°C to about 20°C, for example, about 1°C to about 15°C, about 2°C to about 10°C, or about 2°C to about 8°C, or at a temperature of about 5°C.

[0393] 79. The method of claim 78, wherein the composition is stored for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months.

[0394] 80. A composition preparable by the method of any one of items 43 to 79.

[0395] 81. The composition of claim 80, which is in frozen form.

[0396] 81a. The composition of claim 81, wherein the concentration of the nucleic acid in the composition is from about 1 mg / l to about 50 mg / l.

[0397] 81b. The composition of item 81 or 81a, wherein the nucleic acid integrity of the frozen composition after thawing is at least 50% compared to the nucleic acid integrity of the composition before the composition was frozen.

[0398] 81c. The composition of any one of items 81, 81a and 81b, wherein the nucleic acid integrity after thawing the frozen composition is at least 50% compared to the nucleic acid integrity before the composition was frozen.

[0399] 82. The composition of items 81, 81a, 81b and 81c, wherein the nucleic acid integrity after thawing of the frozen composition is at least 90% or substantially 100% compared to the nucleic acid integrity before the composition was frozen.

[0400] 83. The composition of any one of items 81 to 82, wherein the size (Z 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles before the composition is frozen 平均值 ) and / or size distribution and / or PDI.

[0401] 83a. The composition of any one of items 81 to 83, wherein after one freeze / thaw cycle, preferably after two freeze / thaw cycles, the particle size and nucleic acid integrity of the composition are substantially equivalent to the particle size and nucleic acid integrity before the composition was first frozen.

[0402] 84. The composition of claim 80, which is in liquid form.

[0403] 84a. The composition of claim 84, wherein the concentration of the nucleic acid in the composition is from about 10 mg / l to about 100 mg / l.

[0404] 85. The composition of item 84 or 84a, wherein the nucleic acid integrity of the composition after storage for at least one week, preferably at a temperature of about 2°C to about 8°C for at least one week, is at least 90% compared to the nucleic acid integrity before storage.

[0405] 85a. The composition of any one of items 84, 84a and 85, wherein the nucleic acid integrity of the composition after storage for at least four weeks, preferably at a temperature of about 2°C to about 8°C, is at least 90% compared to the nucleic acid integrity before storage.

[0406] 85b. The composition of any one of items 84, 84a, 85 and 85a, wherein the nucleic acid integrity of the composition after storage for at least three months, preferably at a temperature of about 2°C to about 8°C for at least three months, is at least 90% compared to the nucleic acid integrity before storage.

[0407] 86. The composition of any one of items 84, 84a, 85, 85a and 85b, wherein the size of the nucleic acid particles (Z 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size of the nucleic acid particles before storage (Z 平均值 ) and / or size distribution and / or PDI.

[0408] 87. A method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a frozen composition prepared by the method of any one of items 43 to 74b, 76 and 77, and thawing the frozen composition to obtain the ready-to-use pharmaceutical composition.

[0409] 88. A method for preparing a ready-to-use pharmaceutical composition, comprising the steps of providing a liquid composition prepared by the method of any one of items 43 to 73 and 75 to 77, thereby obtaining the ready-to-use pharmaceutical composition.

[0410] 89. A ready-to-use pharmaceutical composition that can be prepared by the method of item 87 or 88.

[0411] 90. The composition of any one of items 1 to 42a, 80 to 86 and 89 for use in therapy.

[0412] 91. The composition of any one of items 1 to 42a, 80 to 86 and 89, for use in inducing an immune response in a subject.

[0413] 92. A method of transfecting a cell, comprising adding the composition of any one of items 1 to 42a, 80 to 86, and 89 to the cell; and incubating the mixture of the composition and the cell for a sufficient amount of time.

[0414] 93. The method of claim 92, wherein incubating the mixture of the composition and cells is performed in the presence of serum.

[0415] 93a. The method of claim 93, wherein the serum is human serum.

[0416] 94. The method of item 92, 93 or 93a, wherein the lipids that can ionize to release cations account for about 40 mol% to about 70 mol% of the total lipids present in the composition, for example, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%; the steroids account for about 15 mol% to about 40 mol% of the total lipids present in the composition, for example, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%; and the neutral lipids account for about 15 mol% to about 25 mol% of the total lipids present in the composition, for example, about 17 mol% to about 21 mol%.

[0417] 94a. The method of any one of items 92 to 94, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0418] 95. The method of any one of items 92 to 94a, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.5.

[0419] 96. The method of claim 92, wherein the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% of the total lipids present in the composition, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%; the steroids account for about 35 mol% to about 60 mol% of the total lipids present in the composition, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%; and the neutral lipids account for about 5 mol% to about 15 mol% of the total lipids present in the composition, for example, about 7 mol% to about 14 mol%.

[0420] 96a. The method of claim 96, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0421] 97. The method of any one of items 92, 96 and 96a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0.

[0422] 98. Use of the composition of any one of items 1 to 42a, 80 to 86 and 89 for transfecting cells.

[0423] 99. The use according to claim 98, wherein the transfection of cells is carried out in the presence of serum.

[0424] 99a. The use of claim 99, wherein the serum is human serum.

[0425] 100. The use described in item 98, 99 or 99a, wherein the lipid that can ionize to release cations accounts for about 40 mol% to about 70 mol% of the total lipids present in the composition, for example, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%; the steroid accounts for about 15 mol% to about 40 mol% of the total lipids present in the composition, for example, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%; and the neutral lipid accounts for about 15 mol% to about 25 mol% of the total lipids present in the composition, for example, about 17 mol% to about 21 mol%.

[0426] 100a. The use of any one of items 98 to 100, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0427] 101. The use according to any one of items 98 to 100a, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.5.

[0428] 102. The use described in item 98, wherein the lipids that can ionize to release cations account for about 20 mol% to about 40 mol% of the total lipids present in the composition, for example, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%; the steroids account for about 35 mol% to about 60 mol% of the total lipids present in the composition, for example, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol%; and the neutral lipids account for about 5 mol% to about 15 mol% of the total lipids present in the composition, for example, about 7 mol% to about 14 mol%.

[0429] 102a. The use of claim 102, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0430] 103. The use of any one of items 98, 102 and 102a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0.

[0431] Additional aspects of the disclosure are also disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0432] Figure 1: Schematic flow chart illustrating certain steps of the method according to the second aspect.

[0433] Figure 2 : Aggregation of non-PEG lipid particle compositions vs. stable polyphosphate lipid particle compositions. A: Lipid particle compositions comprising ionizable cationic lipids, steroids, and neutral lipids but without PEG and inorganic polyphosphate were prepared and their size (diameter, Z ave. (Z 平均值 B: Lipid particle compositions containing different concentrations (0 to 10 mM) of ionizable lipids, steroids, neutral lipids, and inorganic polyphosphate (triphosphate (3P)) but no PEG were prepared, and their sizes (diameter (Z)) were measured 16 hours after their preparation. ave. ), in nm).

[0434] Figure 3 Polyphosphate lipid granule compositions is all stable under various conditions.Prepared lipid granule compositions, it comprises RNA (with two different concentrations: 10 or 70mg / l), can ionize cationic lipid, steroid, neutral lipid, inorganic polyphosphate (triphosphate (3P), after particle formation, adds with the concentration of 2.5mM, optionally also is present in final filtration (dialysis) step) and has PEG lipid (+PEG) or does not have PEG lipid (-PEG).After storing under various conditions (5 ℃,-20 ℃ or-70 ℃), measure the colloidal parameters (diameter and polydispersity index (PDI) in nm) of particle compositions.

[0435] Figure 4:In the absence or presence of serum, the ionizable cationic lipid, steroid and neutral lipid of different mol ratios are on the impact of expression level.Use the composition with the DSPC molar percentage shown in the figure (the remainder of the lipid composition is ionizable lipid (ION) and cholesterol (CHOL) under the mol ratio shown in the figure) to prepare the lipid particle composition comprising RNA (encoding luciferase), ionizable cationic lipid (A: lipid XIV-3; B: lipid XIV-1; C: lipid XIV-2; D: lipid G (DPL-14)), steroid (cholesterol), neutral lipid (DSPC) and inorganic polyphosphate (triphosphate (3P)).In the presence of serum (+ serum (+ S)) or in the absence of serum (- serum (- S)), use any one of the compositions transfected cells.Determine luciferase expression, and based on the ratio (ratio + S / - S) of the luciferase expression in the presence of serum (+ S) and the luciferase expression in the absence of serum (- S) calculate serum stimulation. Figure 4 The results of luciferase expression and serum stimulation are shown (in log 10 (Ratio +S / -S) values ​​are expressed).

[0436] Fig. 5: the stabilization of lipid particle compositions using multivalent anions.Alkaline original colloid (raw colloid) uses (A) DODAP, (B) lipid D or (C) lipid XIV-2 to produce as ionizable lipid component, and does not contain stealth lipid.Material is dialyzed for buffer A (HEPES pH 7.4), buffer B (for buffer A plus 5mM sodium triphosphate), buffer C (for buffer A plus 5mM sodium diphosphate), buffer D (for buffer A plus 5mM sodium phosphate) or buffer E (for buffer A plus 5mM sodium citrate), obtain mature colloid (matured colloid).Handle the control material comprising PEG lipid in the same manner.Monitor particle size and the polydispersity of mature colloid in 48 hours.

[0437] Figure 6 Figure 5: Activity of lipid particle compositions using multivalent anions. The mature colloids described in Figure 5 were tested for their ability to transfect mRNA into HEK cells. The ionizable lipids were (A) DODAP, (B) lipid D, or (C) lipid XIV-2.

[0438] DETAILED DESCRIPTION

[0439] Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing certain specific embodiments only and are not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0440] Hereinafter, the key elements of the present disclosure will be described in more detail. These key elements are listed together with some specific embodiments, however, it should be understood that they can be combined in any way and in any quantity to produce other embodiments. The examples and preferred embodiments of the multiple descriptions should not be construed as limiting the present disclosure to only some embodiments clearly described. This specification sheet should be understood as supporting and encompassing the embodiments that will clearly describe the embodiment and any number of disclosed and / or preferred key element combinations. In addition, unless the context indicates otherwise, any arrangement and combination of all described key elements in this application should be considered to be disclosed by the specification sheet of the application.

[0441] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0442] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques, which are explained in the art (see, e.g.,

[0443] Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuchder Organischen Chemie", S.Hirzel VerlagStuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0444] Throughout this specification and the appended claims, unless the context requires otherwise, the words "comprises / comprising" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated members, integers or steps or groups of members, integers or steps, but not the exclusion of any other members, integers or steps or groups of members, integers or steps. The term "consisting essentially of is meant to exclude any other members, integers or steps in any substantial sense. The term "comprises / comprising" encompasses the term "consisting essentially of", which in turn encompasses the term "consisting of". Therefore, the term "comprises / comprising" may be replaced with the term "consisting essentially of" or "consisting of" at each occurrence in this application. Similarly, the term "consisting essentially of" may be replaced with the term "consisting of" at each occurrence in this application.

[0445] Unless otherwise indicated herein or clearly contradicted by context, unmodified nouns and similar referents used in the context of describing the disclosure (especially in the context of the claims) are understood to cover both the singular and the plural.

[0446] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0447] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the present disclosure and does not limit the scope of the present disclosure otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0448] As used herein, "and / or" is considered specific disclosure that each of the two specified features or components has or does not have the other. For example, "X and / or Y" is considered specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually listed herein.

[0449] In the context of this disclosure, the term "approximately" represents an accuracy interval that a person of ordinary skill in the art will understand, to still ensure the technical effect of the feature in question. The term generally represents a deviation from the indicated numerical value of ±10%, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±10%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±5%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±4%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±3%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±2%. In some embodiments, "approximately" represents a deviation from the indicated numerical value of ±1%. In some embodiments, "about / approximately" means a deviation of ±0.9% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.8% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.7% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.6% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.5% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.4% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.3% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.2% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.1% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.05% from the indicated numerical value. In some embodiments, "about / approximately" means a deviation of ±0.01% from the indicated numerical value. As will be understood by one of ordinary skill in the art, the specific such deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have greater such deviations than anthropogenic or engineered technological effects.

[0450] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0451] Several documents are cited throughout the text of this specification. Each document cited herein, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's instructions, guidelines, etc., is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0452] definition

[0453] The following definitions are provided for all aspects of this disclosure. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0454] As used herein, terms such as "reduce / reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, such as a decrease of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

[0455] As used herein, terms such as "enhance" or "increase" refer to the ability to cause an overall increase in levels or enhance, for example, at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more. In some embodiments, these terms relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0456] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is between 7.3 and 7.5. In some embodiments, the physiological pH is between 7.35 and 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0457] As used herein, "physiological conditions" refer to conditions (particularly pH and temperature) in a living subject (particularly a human). Preferably, physiological conditions mean physiological pH and / or a temperature of about 37°C.

[0458] As used herein, "% (w / v)" (or "% w / v") refers to weight to volume percentage, which is a unit of concentration measuring the amount of solute in grams (g) as a percentage of the total volume of a solution in milliliters (ml).

[0459] As used herein, "volume %" or "% (v / v)" (or "% v / v") refers to volume percent, which is a unit of concentration measuring the amount of a liquid substance (in milliliters (ml)) expressed as a percentage of the total volume of a solution (in milliliters (ml)).

[0460] As used herein, "weight %" or "% (w / w)" (or "% w / w") refers to weight percent, which is a unit of concentration measuring the amount of a substance (in grams (g)) expressed as a percentage of the total weight (in grams (g)) of the total composition.

[0461] "Mol %" as used in this disclosure is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0462] "Mol % of total lipids" as used in this disclosure is defined as the ratio of the moles of one lipid component to the total moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0463] As used herein, the term "relative amount" refers to the total molar number of a component (e.g., lipid component) relative to all components (e.g., all lipid components). For example, if the composition comprises 3 lipid components (lipid 1, lipid 2, and lipid 3) with an amount of 45 mol, 45 mol, and 10 mol, the relative amount is 45 mol% (for lipid 1), 45 mol% (for lipid 2), and 10 mol% (for lipid 3). Improving the relative amount of a component requires reducing the relative amount of at least one other component (making the absolute amount of lipid remain substantially constant), and vice versa. For example, the relative amount of lipid 1 and 3 increases (to, for example, 58 mol% (for lipid 1) and 20 mol% (for lipid 3)) and the relative amount of lipid 2 will need to be reduced (to a lower relative amount, for example, 22 mol%). In addition, the relative amount of lipid 1 and 3 decreases (to, for example, 39 mol% (for lipid 1) and 5 mol% (for lipid 3)) and the relative amount of lipid 2 will need to be increased (to a higher relative amount, for example, 56 mol%).

[0464] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Therefore, the ionic strength, I, is mathematically represented by the following formula:

[0465]

[0466] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is taken over all the different ions (i) in the solution.

[0467] According to the present disclosure, in some embodiments, the term "ionic strength" refers to the presence of monovalent ions.

[0468] With respect to the presence of divalent inorganic ions, particularly divalent inorganic cations, in one embodiment, the concentration or effective concentration (presence of free ions) is sufficiently low (presence of free ions) due to the presence of the chelating agent to prevent RNA degradation. In one embodiment, the concentration or effective concentration of the divalent inorganic ions is below the catalytic level for hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent inorganic ions is 20 μM or less. In one embodiment, free divalent inorganic ions are absent or substantially absent.

[0469] As used herein, "molar ratio" refers to the ratio between the molar amounts of any two substances. For example, if a first substance is present in a composition in an amount of 1 millimole (mmol) and a second substance is present in the composition in an amount of 2 millimoles (mmol), then the molar ratio of the first substance to the second substance is 1:2 or 0.5.

[0470] "Osmolarity" refers to the concentration of a specific solute, expressed as osmoles of solute per kilogram of solvent.

[0471] The term "lyophilization" and variations thereof refer to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the freezing medium in the substance to sublime directly from the solid phase to the gas phase. Thus, the terms "lyophilization" and "freeze drying" are used interchangeably herein.

[0472] The term "spray drying" refers to the spray drying of a substance by mixing a (heated) gas with an atomizing (spray-like) fluid in a vessel (spray dryer), wherein the solvent from the formed droplets evaporates to produce a dry powder.

[0473] The term "reconstitution" relates to the addition of a solvent (eg, water) to a dried product to return it to a liquid state, eg, its original liquid state.

[0474] The term "freezing" refers to the solidification of a liquid, usually with the removal of heat. In some embodiments, freezing is the opposite action of thawing.

[0475] The term "thawing" refers to the liquefaction of a solid, typically by the addition of heat. In some embodiments, thawing is the reverse of freezing.

[0476] As used herein, the term "aqueous phase" in relation to compositions / formulations comprising particles (particularly LNPs, liposomes and / or lipoplexes) refers to the mobile or liquid phase, i.e., the continuous aqueous phase that includes all components dissolved therein but (formally) does not include the particles. Thus, if the particles (e.g., LNPs) are dispersed in the aqueous phase, and the aqueous phase is substantially free of compound X, then the aqueous phase is free of X in such a manner that it is practically feasible and practicable, for example, the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is possible that the particles dispersed in the aqueous phase may contain compound X in an amount exceeding 1% by weight.

[0477] The term “recombinant” in the context of this disclosure means “prepared by genetic engineering.” In some embodiments, a “recombinant object” in the context of this disclosure does not occur in nature.

[0478] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in a laboratory is naturally occurring. The term "found in nature" means "occurring in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0479] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15° C., preferably about 15° C. to about 35° C., about 15° C. to about 30° C., about 15° C. to about 25° C., or about 17° C. to about 22° C. Such temperatures would include 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., and 22° C.

[0480] The term "alkyl" refers to a monovalent group of a saturated straight or branched hydrocarbon. Preferably, the alkyl group contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, abbreviated as C 1-12 Alkyl, (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, abbreviated as C 1-10alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethyl-propyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. "Substituted alkyl" means an alkylene group in which one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms are replaced with a substituent other than hydrogen (when replacing more than one hydrogen atom, the substituents may be the same or different). Preferably, the substituents other than hydrogen are primary substituents as specified herein.Some examples of substituted alkyl groups include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0481] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Some exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1 -isopentylene, 1,1-sec-pentylene, 1,1-neopentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1 ,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1-isoheptylene), octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene), etc. A straight-chain alkylene moiety having at least 3 carbon atoms and one free valence at each end may also be referred to as a multiple methylene group (e.g., 1,4-butylene may also be referred to as a tetramethylene group). Generally speaking, as an alternative to using the ending "ylene" of the above alkylene moieties, the ending "diyl" may also be used (e.g., 1,2-butylene may also be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkylene, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent as specified herein.

[0482] The term "alkenyl" refers to a monovalent group of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, and if the number of carbon atoms in the alkenyl group is an odd number, the division result is rounded to the next integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. Preferably, an alkenyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group contains 2 to 12 carbon atoms (abbreviated as C 2-12The alkylene group comprises an alkylene group (e.g., 2 to 10) carbon atoms, and 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, 4 or 5) carbon-carbon double bonds, more preferably, 2 to 8 carbon atoms and 1, 2, 3 or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in a cis (Z) or trans (E) configuration. Some exemplary alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonen ...9-nonenyl, 10-nonenyl, 11-nonenyl, 12-nonenyl, 13-nonenyl, 14-nonenyl, 15-nonenyl, 16-nonenyl, 17-nonenyl, 18-nonenyl, 19-nonenyl, 20-nonenyl, 21-nonenyl, 22-nonenyl, 23-nonenyl, 24-nonenyl, 25-nonenyl, 26-nonenyl, 27-nonenyl, 28-nonenyl, 29-nonenyl, 30-nonenyl, 31-non The double bond may be alkyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, etc. If the alkenyl group is attached to a nitrogen atom, the double bond may not be alpha to the nitrogen atom. "Substituted alkenyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent as specified herein.

[0483] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total carbon atoms can be six to thirty, typically six to twenty, typically six to eighteen. An alkynyl group may optionally have one or more carbon-carbon double bonds. Generally speaking, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is an odd number, the division result is rounded to the next integer. For example, for an alkynyl group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, an alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6, more preferably 1 or 2 carbon-carbon triple bonds.

[0484] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon with at least one carbon-carbon double bond. Generally speaking, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, and if the number of carbon atoms in the alkenylene group is an odd number, the division result is rounded to the next integer. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. Preferably, an alkenylene group comprises 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, 4 or 5) carbon-carbon double bonds, more preferably, it comprises 2 to 8 carbon atoms and 1, 2, 3 or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in cis (Z) or trans (E) configuration. Some exemplary alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. If the alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent as specified herein.

[0485] The term "cycloalkyl" refers to a cyclic non-aromatic form of an alkyl group having preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyls include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl and adamantyl. Cycloalkyls can be composed of one ring (monocycle), two rings (bicycle) or more than two rings (polycycle).

[0486] The term "cycloalkylene" refers to a cyclic, non-aromatic form of an "alkylene" group, and is a pair, ortho or separated diradical. In certain embodiments, the cycloalkylene group (i) is monocyclic or polycyclic (e.g., bicyclic or tricyclic) and / or (ii) is 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14-membered, e.g., 3 to 12-membered or 3 to 10-membered). In one embodiment, the cycloalkylene group is a mono-, bi- or tricyclic 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14-membered, e.g., 3 to 12-membered or 3 to 10-membered) cycloalkylene group. Generally speaking, as an alternative to using the ending "sub-" of the above-mentioned cycloalkylene moiety, the ending "di-" can also be used (e.g., 1,2-cyclopropylene can also be referred to as cyclopropane-1,2-diyl). Some exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantylene (e.g., tricyclo[3.3.1.1 3,7 ] decane-2,2-diyl). "Substituted cycloalkylene" means an alkylene group in which one or more (e.g., from 1 to the maximum number of hydrogen atoms bound to the cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., from 1 to 5, from 1 to 4, or from 1 to 3, or from 1 to 2) hydrogen atoms are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a primary substituent as specified herein.

[0487] The term "cycloalkenylene" represents a cyclic non-aromatic form of an alkenylene group, and is a paired, ortho or separated diradical. Generally speaking, the maximum number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2, and if the number of carbon atoms in the cycloalkenylene group is an odd number, the division result is rounded to the next integer. For example, for the cycloalkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5 or 6 carbon-carbon double bonds. In certain embodiments, the cycloalkenylene group (i) is a monocycle or polycycle (e.g., a bicycle or tricycle) and / or (ii) is 3 to 14 yuan (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 yuan, such as 3 to 12 yuan or 3 to 10 yuan). In one embodiment, cycloalkenylene is a mono-, bi- or tricyclic 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14-membered, such as 3 to 12-membered or 3 to 10-membered) cycloalkenylene. Some exemplary cycloalkenylenes include cyclohexene, cycloheptene, cyclopropene, cyclobutene, cyclopentene and cyclooctenene. "Substituted cycloalkenylene" means that one or more (e.g., 1 to the maximum number of hydrogen atoms bound to the cycloalkenylene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituents other than hydrogen are first-level substituents as specified herein.

[0488] The term "aryl" refers to a monovalent group of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9 or 10, such as 5, 6 or 10) carbon atoms, which may be arranged in one ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropane, The term "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Some preferred examples are phenyl and naphthyl. Aryl does not include fullerenes.

[0489] The term "aromatic" as used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aromatic group is hydrogenated (partially or fully), the resulting hydrogenated cyclic structure is classified as a cycloalkyl for the purposes of this disclosure. Similarly, if a bicyclic or polycyclic aromatic group (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for the purposes of this disclosure (even though one ring (e.g., in 1,2-dihydronaphthyl)) is still aromatic.

[0490] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, the hydrocarbyl group is non-cyclic, such as linear (straight chain) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Some specific examples of hydrocarbyl groups are C 1-6 Alkyl, aryl and aryl (C 1-6 In some embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more primary substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0491] Usually the first-stage substituent is preferably selected from: C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1 or 2, and C 1-3 Alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred first-stage substituents are selected from: methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl or Br) and -CF3, such as halogen (such as F, Cl or Br) and -CF3.

[0492] As used herein, the term "tertiary amine moiety" refers to a moiety comprising a nitrogen atom substituted with three organic substituents (wherein the substituents may be the same or different from each other). In some embodiments, the organic substituents are selected from optionally substituted (e.g., substituted with one or more primary substituents as defined herein) hydrocarbon groups (e.g., alkyl groups, particularly C 1-6 alkyl).

[0493] As used herein, the term "filtration" refers to any process involving the removal or separation of at least one component (e.g., permeable molecules such as salts, small proteins, solvents, etc.) of a liquid composition based on the molecular size of the component. Such separations can use micromolecularly permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., for dialysis). Thus, examples of filtration include dialysis, tangential flow filtration, and diafiltration.

[0494] The expression "after thawing the frozen composition" as used herein in the context of a frozen composition means that the frozen composition must be thawed in terms of characteristics (e.g., nucleic acid integrity (e.g., RNA integrity) and / or size (Z) of particles (e.g., LNPs) contained in the composition. 平均值 ) and / or size distribution and / or PDI) can be measured before thawing.

[0495] A "monovalent" compound refers to a compound having only one functional group of interest. For example, a monovalent acid refers to a compound having only one acid group (e.g., one carboxyl (-COOH) group). For example, a monovalent cation refers to a compound having only one cationic group, such as an alkaline cation (e.g., Na + , K + 、Li + ), ammonium cation (NH4 + ) or an organic compound having a primary, secondary or tertiary amine group (such as a protonated form of triethylamine, trimethylamine, etc.), or an organic compound having a quaternary amine group.

[0496] A "divalent" or "dibasic" compound refers to a compound having two functional groups of interest. For example, a dibasic organic acid has two carboxyl groups.

[0497] "Polyvalent," "multivalent," "polybasic," or "multibasic" compounds refer to compounds having two or more functional groups of interest, preferably three or more functional groups of interest. For example, a polybasic organic acid has two or more, preferably three or more, acidic carboxyl groups.

[0498] "Polyphosphate" refers to a compound containing two or more consecutive phosphate groups, preferably three or more consecutive phosphate groups. Examples of polyphosphate include inorganic polyphosphates and esters of polyphosphoric acid (e.g., triphosphate) and one or more organic alcohols (e.g., nucleotides, oligonucleotides, or polynucleotides having at least three consecutive phosphate groups).

[0499] The expression " inorganic polyphosphate " used in this article means to comprise two or more continuous phosphate groups, preferably three or more continuous phosphate groups, and does not comprise the compound of any organic part covalently bound thereto (for example, inorganic polyphosphate lacks covalent bond between any oxygen or phosphorus atom comprised in carbon atom and polyphosphate anion part).Therefore, the term " inorganic polyphosphate " does not comprise esters of polyphosphoric acid and one or more organic alcohols.Therefore, the expression " inorganic polyphosphate " does not comprise nucleotides, oligonucleotides or polynucleotides, even if they comprise at least two, preferably at least three continuous phosphate groups.Inorganic polyphosphate can be linear (that is, all phosphate moieties of polyphosphate are arranged in chain), branched or cyclic.In some embodiments, polyphosphate comprises formula [P x O (3x+1) ] y , wherein x is an integer and is at least 2, preferably at least 3; and y is an anionic charge. The example of polyphosphate includes diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, higher homologues and mixtures thereof (particularly the linear form of these polyphosphates), particularly triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, higher homologues and mixtures thereof (particularly the linear form of these polyphosphates). Preferred polyphosphate includes diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate and mixtures thereof (particularly the linear form of these polyphosphates), for example triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate and mixtures thereof (particularly the linear form of these polyphosphates), for example triphosphate, tetraphosphate, pentaphosphate and mixtures thereof (particularly the linear form of these polyphosphates). Particularly preferred polyphosphate is triphosphate.

[0500] Similarly, as used herein, the expression "inorganic phosphate" means a compound comprising only one phosphate group and not any organic moiety covalently bound thereto (e.g., an inorganic phosphate lacks a covalent bond between a carbon atom and any oxygen or phosphorus atom contained in the phosphate anion moiety). Thus, the expression "inorganic phosphate" does not include esters of phosphoric acid with one or more organic alcohols. Thus, the expression "inorganic phosphate" does not include nucleotides, oligonucleotides, or polynucleotides, even if they contain one phosphate group. Inorganic phosphates may be linear or cyclic. In some embodiments, an inorganic phosphate comprises the formula [PO4] 3- .

[0501] As used herein, the expression "substantially free of X" means that a mixture (e.g., the aqueous phase of a composition or formulation described herein) is free of X in such a manner that it is practically possible and practicable to do so. For example, if the mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the total weight of the mixture.

[0502] For example, as used herein, "substantially free of lipids containing polyethylene glycol (PEG)" means that a mixture (e.g., the aqueous phase of a composition or formulation described herein) is free of lipids containing PEG in a manner that is practically feasible and practicable. For example, if the mixture is substantially free of lipids containing PEG, the amount of lipids containing PEG in the mixture can be less than 1 wt % (e.g., less than 0.5 wt %, less than 0.4 wt %, less than 0.3 wt %, less than 0.2 wt %, less than 0.1 wt %, less than 0.09 wt %, less than 0.08 wt %, less than 0.07 wt %, less than 0.06 wt %, less than 0.05 wt %, less than 0.04 wt %, less than 0.03 wt %, less than 0.02 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.001 wt %) based on the gross weight of the mixture. Similar considerations apply to the expressions "substantially free of any PEG-containing compounds", "substantially free of PEG" and "substantially free of lipids, steroids and neutral lipids that can ionize into cations".

[0503] The expression "nucleic acid integrity" refers to the percentage of full-length (i.e., unfragmented) nucleic acids contained in a sample relative to the total amount of nucleic acids (i.e., unfragmented nucleic acids plus fragmented nucleic acids). Nucleic acid integrity can be determined by chromatographic separation of nucleic acids (e.g., separation using capillary electrophoresis), determining the peak area of ​​the main nucleic acid peak (i.e., the peak area of ​​the full-length (i.e., unfragmented) nucleic acids), determining the peak area of ​​the total nucleic acids, and dividing the peak area of ​​the main nucleic acid peak by the peak area of ​​the total nucleic acids. Similarly, the expression "RNA integrity" refers to the percentage of full-length (i.e., unfragmented) RNA contained in a sample relative to the total amount of RNA (i.e., unfragmented RNA plus fragmented RNA). RNA integrity can be determined by chromatographic separation of RNA (e.g., separation using capillary electrophoresis), determining the peak area of ​​the main RNA peak (i.e., the peak area of ​​the full-length (i.e., unfragmented) RNA), determining the peak area of ​​the total RNA, and dividing the peak area of ​​the main RNA peak by the peak area of ​​the total RNA.

[0504] The term "cryoprotectant" relates to a substance that is added to a preparation (eg a formulation or composition) to protect the active ingredients in the formulation during the freezing phase.

[0505] The term "lyoprotectant" relates to substances added to the formulation to protect the active ingredient during the drying phase.

[0506] The expression "glass transition temperature" (abbreviation: T g ) refers to the temperature range over which the glass transition occurs. The term "glass transition" refers to the gradual and reversible transformation of an amorphous substance (or an amorphous region in a semi-crystalline substance) from a hard (e.g., relatively brittle "glass-like") amorphous state to a viscous (e.g., rubbery) molten state as the temperature rises. Methods for determining the glass transition temperature are known to those skilled in the art and include dilatometry, dielectric methods, dynamic mechanical (DTMA) methods, calorimetry (DSC) or refractometry or NMR spectroscopy. Suitable standardized methods include DIN 53765: 1994-03 and ISO 11357-2: 1999-03.

[0507] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150 consecutive amino acids connected to each other by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules, but the terms "protein" and "polypeptide" are often used synonymously herein.

[0508] "Therapeutic proteins" when provided to a subject in a therapeutically effective amount, have a positive or beneficial effect on the subject's condition or disease state. In some embodiments, therapeutic proteins have curative or palliative properties and can be administered to improve, alleviate, relieve, reverse a disease or condition, delay the onset of a disease or condition, or alleviate the severity of one or more symptoms of a disease or condition. Therapeutic proteins can have preventive properties and can be used to delay the onset of a disease or alleviate the severity of such a disease or pathological condition. The term "therapeutic protein" includes complete proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens used for vaccination and immunostimulants (e.g., cytokines). The terms "therapeutic protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

[0509] According to various embodiments of the present disclosure, nucleic acids encoding peptides, polypeptides, or proteins, such as RNA (e.g., mRNA), are taken up or introduced, i.e., transfected or transduced, into cells, which may be present in vitro or in a subject, resulting in expression of the peptides, polypeptides, or proteins. The cells may express the encoded peptides, polypeptides, or proteins intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptides, polypeptides, or proteins, and / or may express the peptides, polypeptides, or proteins on their surfaces.

[0510] According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and mean, with respect to a particular peptide, polypeptide or protein, that a nucleic acid can be expressed to produce the peptide, polypeptide or protein if present in a suitable environment, preferably within a cell.

[0511] The term "portion" refers to a fraction. With respect to a specific structure, such as an amino acid sequence or a protein, the term "portion" thereof may refer to a continuous or discontinuous fraction of the structure.

[0512] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure (e.g., an amino acid sequence or protein) refers to a continuous element of the structure. When used in the context of a composition, the term "portion" means a portion of the composition. For example, a portion of a composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0513] With respect to an amino acid sequence (peptide, polypeptide or protein), a "fragment" refers to a portion of an amino acid sequence, i.e., a sequence representing an amino acid sequence that is shortened at the N-terminus and / or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame comprises a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence comprise, for example, sequences of, for example, at most 8, in particular at most 10, at most 12, at most 15, at most 20, at most 30 or at most 55 consecutive amino acids of an amino acid sequence.

[0514] According to the present disclosure, a part or fragment of a peptide, polypeptide or protein preferably has at least one functional property of the peptide, polypeptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides, polypeptides or proteins, enzymatic activity, interaction with antibodies and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide, polypeptide or protein has at least one pharmacological activity of the peptide, polypeptide or protein from which the fragment is derived. A part or fragment of a peptide, polypeptide or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 continuous amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably comprises a sequence of at most 8, particularly at most 10, at most 12, at most 15, at most 20, at most 30 or at most 55 continuous amino acids of the peptide or protein.

[0515] As used herein, "variant" with respect to an amino acid sequence (peptide, polypeptide, or protein) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified form of a wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid differences.

[0516] "Wild-type" or "WT" or "native" with respect to an amino acid sequence means an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, polypeptide, or protein has an amino acid sequence that has not been intentionally modified. Similarly, "wild-type" or "WT" or "native" with respect to a nucleic acid sequence means a nucleic acid sequence found in nature, including allelic variations. For example, a wild-type coding sequence means a coding sequence that is found in nature and has not been intentionally modified.

[0517] As used herein, "coding sequence" means the portion of a nucleic acid (eg, the DNA or RNA of a gene) that encodes a protein.

[0518] The expression "guanosine / cytosine (G / C) content" or "G / C content" refers to the percentage of bases in a DNA or RNA molecule that are guanine (G) or cytosine (C). The G / C content can be given for a specific portion of the DNA or RNA, or for the entire genome. When the G / C content refers to a portion, it can refer to the G / C content of a single gene or a portion (domain) of a gene, a group of genes or gene clusters, a noncoding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0519] For the purposes of this disclosure, "variants" of an amino acid sequence (peptide, protein, or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformers, isoforms, allelic variants, species variants, and species homologs, particularly those occurring in nature. The term "variant" particularly includes fragments of an amino acid sequence.

[0520] Amino acid insertion variants include insertions of single or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product is also possible. Amino acid addition variants include one or more amino acids, such as amino and / or carboxyl terminal fusions of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its place. Priority is given to modifications in positions in the amino acid sequence that are non-conserved between homologous proteins or peptides and / or replacing amino acids with other amino acids having similar properties. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of the families of amino acids to which their side chains are associated. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In one embodiment, conservative amino acid replacements include replacements within the following groups:

[0521] -Glycine, alanine;

[0522] -valine, isoleucine, leucine;

[0523] -Aspartic acid, glutamic acid;

[0524] -Asparagine, glutamine;

[0525] -serine, threonine;

[0526] - lysine, arginine; and

[0527] -Phenylalanine, tyrosine.

[0528] In some embodiments, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and the amino acid sequences of a variant of the given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids (in some embodiments, contiguous amino acids). In some embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignments for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0529] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences.

[0530] The terms "% identical" and "% identity" or similar terms are particularly intended to refer to the percentage of nucleotides or amino acids that are identical between the sequences to be compared in an optimal alignment. The percentages are purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the search similarity algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) the expectation threshold is set to 10; (ii) the word length is set to 28; (iii) the maximum match within the query range is set to 0; (iv) the match / mismatch score is set to 1, -2; (v) the gap cost is set to linear; and (vi) a filter for low complexity regions is being used. In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10; (ii) word length set to 3; (iii) maximum match within query range set to 0; (iv) matrix set to BLOSUM62; (v) gap cost set to existence: 11 extension: 1; and (vi) condition composition scoring matrix adjustment.

[0531] Percent identity is calculated by determining the number of identical positions in the sequences being compared, dividing that number by the number of positions being compared (eg, the number of positions in the reference sequence), and multiplying the result by 100.

[0532] In some embodiments, the degree of similarity or identity is given for a region of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some embodiments, contiguous nucleotides). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0533] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0534] The amino acid sequence variants described herein can be readily prepared by a skilled artisan, for example, by recombinant DNA manipulation. DNA sequence manipulations for preparing peptides or proteins with substitutions, additions, insertions, or deletions are described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, as by solid phase synthesis and similar methods.

[0535] In some embodiments, a fragment or variant of an amino acid sequence (peptide, polypeptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to the functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent). With respect to antigens or antigenic sequences, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence (immunogenic fragment) comprising an amino acid sequence that has one or more amino acids changed compared to the amino acid sequence of the parent molecule or sequence and is still capable of achieving one or more functions of the parent molecule or sequence (e.g., inducing an immune response). In one embodiment, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the properties of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant can be reduced but still significantly present, for example, the immunogenicity of the functional variant can be at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant can be enhanced compared to the parent molecule or sequence.

[0536] An amino acid sequence (peptide, protein or polypeptide) that is "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. In some embodiments, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence can be a variant of the specific sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that antigens suitable for use herein can be altered so that their sequences are different from the naturally occurring or natural sequences from which they are derived while retaining the desired activity of the native sequence.

[0537] In some embodiments, "isolated" means changed or removed (e.g., purified) from its natural state or from an artificial composition (e.g., a composition from a production process). For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from coexisting substances in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment, such as a host cell. In some embodiments, the RNA (e.g., mRNA) used in the present disclosure is in a substantially purified form. In some embodiments, a solution (preferably an aqueous solution) of RNA (e.g., mRNA) in substantially purified form comprises a first buffer system.

[0538] The term "genetic modification" or simply "modification" includes transfecting cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids (particularly RNA) into cells. For the purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, wherein the cells may be present in a subject, such as a patient. Thus, according to the present disclosure, cells used to transfect nucleic acids described herein may be present in vitro (e.g., in cell culture) or in vivo, where, for example, the cells may form part of an organ, tissue, and / or body of a patient. According to the present disclosure, transfection may be transient or stable. For some applications of transfection, it is sufficient if only the transfected genetic material is transiently expressed. RNA can be transfected into cells to transiently express the protein it encodes. Since nucleic acids introduced during transfection generally do not integrate into the nuclear genome, the exogenous nucleic acids will be diluted or degraded by mitosis. Cells that allow for free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection. Generally speaking, nucleic acid encoding an antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its encoded protein.

[0539] The present disclosure includes analogs of peptides, polypeptides or proteins. According to the present disclosure, analogs of peptides, polypeptides or proteins are modified forms of the peptides, polypeptides or proteins from which they are derived, and have at least one functional property of the peptides, polypeptides or proteins. For example, a pharmacologically active analog of a peptide, polypeptide or protein has at least one pharmacological activity of the peptide, polypeptide or protein from which the analog is derived. Such modifications include any chemical modification and include single or multiple replacements, deletions and / or additions of any molecule (e.g., carbohydrates, lipids and / or proteins or peptides) associated with the protein, polypeptide or peptide. In one embodiment, "analogs" of proteins, polypeptides or peptides include those modified forms obtained by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protection / blocking groups, proteolytic cleavage or binding to antibodies or other cellular ligands. The term "analog" also extends to all functional chemical equivalents of the proteins, polypeptides and peptides.

[0540] As used herein, "activation" or "stimulation" refers to the state of cells (e.g., immune effector cells such as T cells) that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" particularly refers to immune effector cells that are undergoing cell division.

[0541] The term "sensitization" refers to the process in which immune effector cells (eg, T cells) first come into contact with their specific antigen and result in differentiation into effector cells (eg, effector T cells).

[0542] The term "clonal expansion" or "amplification" refers to a process in which a specific entity multiplies. In some embodiments, the term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize the antigen are expanded. In some embodiments, the expansion results in the differentiation of the immune effector cells.

[0543] According to the present disclosure, "antigen" encompasses any substance that will cause an immune response and / or any substance to which an immune response or immune mechanism (e.g., a cellular response) is directed. This also includes situations in which the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present disclosure, the term "antigen" includes any molecule comprising at least one epitope (e.g., a T cell epitope). Preferably, the antigen in the context of the present disclosure is a molecule that, optionally after processing, induces an immune response that is preferably specific for the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0544] According to the present disclosure, any suitable antigen as a candidate for an immune response can be used, wherein the immune response can be a humoral immune response or a cellular immune response or the two thereof. In the case of some embodiments of the present disclosure, the antigen is presented by a cell, preferably by an antigen presenting cell (in the case of an MHC molecule), which causes an immune response against the antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or can be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein, or a part thereof.

[0545] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains an epitope that will stimulate the host's immune system to produce an antigen-specific cellular immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with an infection caused by a microorganism, typically a microbial antigen (e.g., a bacterial or viral antigen), or antigens associated with a cancer (typically a tumor), such as a tumor antigen.

[0546] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular, one that is primarily present intracellularly or as a surface antigen of a tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen from a virus, bacteria, unicellular organism, or parasite, for example, a viral antigen such as a viral ribonucleoprotein or envelope protein. In particular, the antigen should be presented by an MHC molecule, which leads to modulation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular activation of cells of the immune system (preferably CD4+ and CD8+ lymphocytes), in particular by modulation of the activity of the T cell receptor.

[0547] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to those antigens produced intracellularly or produced as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetosulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer and that is characteristic of a tumor or cancer cell in terms of type and / or expression level.

[0548] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0549] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. Bacterial antigens may be derived from the bacterial cell wall or cytoplasmic membrane.

[0550] The term "epitope" refers to an antigenic determinant in a molecule (e.g., an antigen), i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by a T cell or B cell, particularly when presented in the context of an MHC molecule. The epitope of a protein can comprise a continuous or discontinuous portion of the protein and can be, for example, about 5 to about 100 amino acids, about 5 to about 50, about 8 to about 0, about 10 to about 25 amino acids in length, e.g., an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, the epitope in the context of the present disclosure is a T cell epitope.

[0551] Terms such as "epitope", "antigenic fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and, for example, may relate to an incomplete representation of an antigen that is, for example, capable of eliciting an immune response against the antigen or eliciting a cell that expresses or contains and presents the antigen. In some embodiments, the term relates to an immunogenic portion of an antigen. Preferably, it is a portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen that is recognized by the immune system). For example, an epitope can be recognized by a T cell, a B cell or an antibody. The epitope of an antigen can include a continuous or discontinuous portion of the antigen, and can be about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, most preferably about 8 to about 25 amino acids in length, such as an epitope length can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T cell epitopes.

[0552] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when present in the presence of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind to peptide epitopes and present them to be recognized by the T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invasive microorganisms) to T cells. In the case of class I MHC / peptide complexes, binding peptides are typically about 8 to about 10 amino acids long, but longer or shorter peptides may also be effective. In the case of class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, but longer and shorter peptides may also be effective.

[0553] The length of peptide and protein antigens can be 2 to 100 amino acids, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0554] The peptide or protein antigen can be any peptide or protein that can induce or enhance the ability of the immune system to produce antibodies and T cell responses against the peptide or protein.

[0555] In some embodiments, vaccine antigens (i.e., antigens that have induced an immune response in the subject by inoculation) are recognized by immune effector cells. In some embodiments, if vaccine antigens are recognized by immune effector cells, then the vaccine antigens can induce stimulation, sensitization and / or amplification of immune effector cells carrying antigen receptors that recognize the vaccine antigens in the presence of appropriate co-stimulatory signals. In the case of the embodiments of the present disclosure, vaccine antigens are preferably presented or present on the surface of cells (preferably antigen presenting cells). In some embodiments, antigens are presented by diseased cells (such as tumor cells or infected cells). In some embodiments, antigen receptors are TCRs that are combined with the epitope of the antigen presented in the case of MHC. In some embodiments, when expressed by T cells and / or present on T cells, the combination of TCR and the antigen presented by cells (such as antigen presenting cells) causes stimulation, sensitization and / or amplification of the T cells. In some embodiments, when expressed by and / or present on a T cell, binding of the TCR to an antigen present on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0556] In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B cell receptor binds to a native epitope of the antigen.

[0557] The term "expressed on the surface of a cell" or "associated with the cell surface" means that a molecule, such as an antigen, is associated with and located at the plasma membrane of a cell, wherein at least a portion of the molecule is oriented toward the extracellular space of the cell and is accessible from outside the cell, for example, by an antibody located outside the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can occur through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the cell surface can be a transmembrane protein with an extracellular portion, or can be a protein that is associated with the cell surface through interaction with another protein that is a transmembrane protein.

[0558] "Cell surface" or "surface of a cell" is used according to its normal meaning in the art and therefore includes the outside of the cell that can be bound by proteins and other molecules. An antigen is expressed on the cell surface if it is located on the surface of the cell and can be bound by, for example, an antigen-specific antibody added to the cell. In some embodiments, the antigen expressed on the cell surface is an integral membrane protein with an extracellular portion that can be recognized by a CAR.

[0559] In the context of the present disclosure, the term "extracellular portion" or "extracellular domain" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, for example, by a binding molecule such as an antibody (which is located outside the cell). In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0560] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL) (CD8+ T cells) including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen targeted by the T cell (particularly when presented on the surface of antigen-presenting cells or diseased cells such as cancer cells in the context of MHC molecules) and preferably exerts the effector function of the T cell. If the T cell kills a target cell expressing an antigen, it is considered that the cell is specific for the antigen. T cell specificity can be evaluated using any of a variety of standard techniques (e.g., in a chromium release assay or a proliferation assay). Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the present disclosure, RNA (particularly mRNA) encodes at least one epitope.

[0561] The term "target" shall mean a substance, such as a cell or tissue, that is the target of an immune response (e.g., a cellular immune response). Targets include cells that present an antigen or an antigenic epitope (i.e., a peptide fragment derived from an antigen). In one embodiment, a target cell is a cell that expresses an antigen and preferably presents class I MHC to the antigen.

[0562] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association of one or more of these fragments with an MHC molecule (e.g., association by binding) for presentation by a cell (preferably an antigen-presenting cell) to a specific T cell. Antigen-presenting cells can be divided into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0563] "Antigen-responsive CTL" refers to CD8 + T cells, the CD8 + T cells respond to antigens, or peptides derived from the antigens, which are presented using MHC class I on the surface of antigen-presenting cells.

[0564] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines (e.g., IFN-γ and TNF-α), upregulation of activation markers (e.g., CD44 and CD69), and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately indicate CTL responsiveness.

[0565] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the overall body response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against" with respect to a substance (e.g., an antigen, cell, or tissue) refer to an immune response, such as a cellular response, against the substance. An immune response may include one or more reactions selected from the group consisting of: the development of antibodies against one or more antigens, and the expansion of antigen-specific T lymphocytes, such as CD4 T lymphocytes. + and CD8 + T lymphocytes, such as CD8 + T lymphocytes, which can be detected in various in vitro proliferation or cytokine production assays.

[0566] In the context of the present disclosure, the terms "induce an immune response" and "cause an immune response" and similar terms refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / preventative / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B or C), CMV or RSV)). In this context, "induce" can mean that there is no immune response to a particular antigen or pathogen before induction, but it can also mean that there is a certain level of immune response to a particular antigen or pathogen before induction, and that after induction, the immune response is enhanced. Therefore, in this context, "induce an immune response" also includes "enhance an immune response." In some embodiments, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease condition is improved by inducing an immune response.

[0567] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are intended to include cellular responses directed against cells characterized by expression of antigen and / or presentation of antigen by class I and / or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (also known as CD4 +T cells) play a key role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, such as diseased cells.

[0568] The term "humoral immune response" refers to the process in living organisms in which antibodies are produced in response to a substance or organism, which ultimately neutralize and / or eliminate it. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-associated receptors that bind a single specific antigen. After binding to the appropriate antigen and receiving a variety of other activation signals, B lymphocytes divide, which produces memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies to the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. When re-exposed to the specific antigen, these lymphocytes provide the cellular basis of memory and lead to a gradual escalation of the antibody response.

[0569] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions known as complementarity determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2 and HCDR3, and the CDRs of VL are called LCDR1, LCDR2 and LCDR3. The heavy and light chain variable regions contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein CH can be further subdivided into a constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxyl terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be immunologically active portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0570] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, for example to antigen receptors such as antibodies or B cell receptors (B cell receptors, BCRs). Immunoglobulins are characterized by structural domains (i.e., immunoglobulin domains) that have a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are typically part of the BCR. Soluble immunoglobulins are often called antibodies. Immunoglobulins typically comprise several chains, typically two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V and L chains. L (variable light chain) domain, CL (constant light chain) domain, V H (variable heavy chain) domain and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which make up the different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl termini. In mammals, there are two types of light chains, namely λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved among different immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0571] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the administration to the individual of one or more immunogens or antigens as described herein, or derivatives thereof, particularly in the form of RNA (especially mRNA) encoding the same, and the stimulation of an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens.

[0572] By "cell characterized by presenting an antigen" or "cell presenting an antigen" or "MHC molecules presenting an antigen on the surface of an antigen-presenting cell" or similar expressions is meant a cell, such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen-presenting cell, which presents an antigen or an antigenic peptide directly or after processing in the presence of an MHC molecule (preferably MHC class I and / or MHC class II molecule, most preferably MHC class I molecule).

[0573] In the context of this disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA). Subsequently, the RNA (particularly mRNA) can be translated into a peptide, polypeptide or protein.

[0574] With respect to RNA, the terms "expression" or "translation" refer to the process in the cell's ribosomes by which a strand of mRNA directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0575] The term "serum" as used herein, particularly with respect to incubating cells in the presence of serum, means the fluid obtained by removing cells and clotting factors from whole blood, such as whole blood obtained from humans or mice. In some embodiments, the serum is human serum or mouse serum.

[0576] The pharmaceutical preparations described herein, particularly kits...

Claims

1. A composition comprising (i) a nucleic acid; (ii) a lipid that can ionize into a cation; (iii) a steroid; (iv) a neutral lipid; and (v) an inorganic polyphosphate.

2. The composition of claim 1, wherein the inorganic polyphosphate comprises the formula [P x O (3x+1) ] y , wherein x is an integer and is at least 3; and y is the anionic charge.

3. The composition of claim 1 or 2, wherein the inorganic polyphosphate is selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof, preferably selected from triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof, more preferably, the inorganic polyphosphate is triphosphate.

4. The composition of any one of claims 1 to 3, wherein the inorganic polyphosphate is a linear inorganic polyphosphate, such as a linear inorganic triphosphate.

5. The composition of any one of claims 1 to 4, wherein the molar ratio of (v) inorganic polyphosphate to (ii) lipid that can ionize into a cation is at least about 1:2, preferably at least about 2:3, such as at least about 4:

3.

6. The composition of any one of claims 1 to 5, which is substantially free of lipids comprising polyethylene glycol (PEG), preferably substantially free of any PEG-containing compound, more preferably substantially free of PEG.

7. The composition of any one of claims 1 to 6, wherein the pH of the composition is from about 4.0 to about 8.0, preferably from about 4.5 to about 8.0, such as from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, or from about 7.0 to about 7.

8.

8. The composition of any one of claims 1 to 7, wherein water is the major component of the composition and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

9. The composition of any one of claims 1 to 8, wherein the composition has an osmotic concentration of at most about 1000×10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg to about 500×10 -3 osmol / kg, more preferably about 300×10 - 3 osmol / kg.

10. The composition of any one of claims 1 to 9, wherein the concentration of the nucleic acid in the composition is from about 1 mg / l to about 500 mg / l, such as from about 1 mg / l to about 100 mg / l, from about 5 mg / l to about 100 mg / l, or from about 10 mg / l to about 100 mg / l.

11. The composition of any one of claims 1 to 10, wherein the lipid that can ionize into a cation comprises a head group comprising at least one tertiary amine moiety.

12. The composition of any one of claims 1 to 11, wherein the lipid that can ionize into a cation has a structure of formula (X) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L 10 and L 20 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-,-C(=O)NR a -, NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O-, and L 10 and L 20 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a , NR a C(=O)NR a , -OC(=O)NR a -, -OC(=O)NR a C(=O)O- or a direct bond; G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene or C 2-12 alkenylene; G 3 It is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 Cycloalkenylene; R a Is H or C 1-12 alkyl; R 35 and R 36 Each independently is C 6-24 Alkyl or C 6-24 alkenyl; R 37 is H, OR 50 , CN, -C(=O)OR 40 , -OC(=O)R 40 or -NR 50 C(=O)R 40 ; R 40 It is C 1-12 alkyl; R 50 Is H or C 1-6 Alkyl; and x is 0, 1, or 2.

13. The composition of any one of claims 1 to 11, wherein the cationic lipid or the lipid that can ionize cations has the structure of formula (XI): in Each of R1 and R2 is independently R5 or -G1-L1-R6, wherein at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 Cycloalkyl; Each of R5 and R6 is independently a non-cyclic hydrocarbon group having at least 10 carbon atoms; Each of G1 and G2 is independently an unsubstituted C 1-12 Alkylene or C 2-12 alkenylene; Each of L1 and L2 is independently selected from -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-; Ra is H or C 1-12 alkyl; m is 0, 1, 2, 3 or 4; and x is 0, 1, or 2.

14. The composition of any one of claims 1 to 13, wherein the lipid that can ionize out a cation accounts for about 20 mol % to about 75 mol % of the total lipid present in the composition, such as about 40 mol % to about 70 mol %, about 45 mol % to about 65 mol %, about 50 mol % to about 60 mol %, about 20 mol % to about 40 mol %, about 25 mol % to about 40 mol %, or about 25 mol % to about 35 mol %.

15. The composition of any one of claims 1 to 14, wherein the steroid comprises a sterol, such as cholesterol.

16. The composition of any one of claims 1 to 15, wherein the steroid accounts for about 15 mol % to about 60 mol % of the total lipids present in the composition, e.g., about 15 mol % to about 40 mol %, about 20 mol % to about 35 mol %, about 20 mol % to about 30 mol %, about 35 mol % to about 60 mol %, about 40 mol % to about 60 mol %, or about 45 mol % to about 60 mol %.

17. The composition of any one of claims 1 to 16, wherein the neutral lipid is a phospholipid, preferably selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE) and dipalmitoyl-phosphatidylethanolamine (DPPE).

18. The composition of any one of claims 1 to 17, wherein the neutral lipid accounts for about 5 mol % to about 25 mol %, e.g., about 10 mol % to about 25 mol %, about 15 mol % to about 25 mol %, about 17 mol % to about 21 mol %, about 5 mol % to about 15 mol %, or about 7 mol % to about 14 mol % of the total lipids present in the composition.

19. The composition of any one of claims 1 to 18, wherein the lipid that can ionize out cations accounts for about 20 mol % to about 70 mol % of the total lipids present in the composition; the steroid accounts for about 15 mol % to about 60 mol % of the total lipids present in the composition; and the neutral lipid (e.g., phospholipid) accounts for about 5 mol % to about 25 mol % of the total lipids present in the composition.

20. The composition of any one of claims 1 to 19, wherein the lipid that can ionize out cations accounts for about 40 mol % to about 70 mol %, e.g., about 45 mol % to about 65 mol %, or about 50 mol % to about 60 mol % of the total lipids present in the composition; the steroid accounts for about 15 mol % to about 40 mol %, e.g., about 20 mol % to about 35 mol %, or about 20 mol % to about 30 mol % of the total lipids present in the composition; and the neutral lipid accounts for about 15 mol % to about 25 mol %, e.g., about 17 mol % to about 21 mol % of the total lipids present in the composition.

21. The composition of any one of claims 1 to 20, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.

5.

22. The composition of any one of claims 1 to 19, wherein the lipid that can ionize out cations accounts for about 20 mol % to about 40 mol %, e.g., about 25 mol % to about 40 mol %, or about 25 mol % to about 35 mol % of the total lipids present in the composition; the steroid accounts for about 35 mol % to about 60 mol %, e.g., about 40 mol % to about 60 mol %, or about 45 mol % to about 60 mol % of the total lipids present in the composition; and the neutral lipid accounts for about 5 mol % to about 15 mol %, e.g., about 7 mol % to about 14 mol % of the total lipids present in the composition.

23. The composition of any one of claims 1 to 19 and 22, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.

0.

24. The composition of any one of claims 1 to 23, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into a cation, at least a portion of the steroid, and at least a portion of the neutral lipid; and wherein at least a portion of the inorganic polyphosphate is associated with the particles.

25. The composition of claim 24, wherein the particle is selected from the group consisting of lipid nanoparticles (LNP), liposomes, lipid complexes (LPX), and mixtures thereof.

26. The composition of any one of claims 24 to 25, wherein the particles account for at least 50%, preferably at least 75%, more preferably at least 85% of the nucleic acid present in the composition.

27. The composition of any one of claims 24 to 26, wherein at least 10%, preferably at least 20%, and more preferably at least 50% of the polyphosphate present in the composition is associated with the particles.

28. The composition of any one of claims 24 to 27, wherein the particles have a size of about 30 nm to about 500 nm, such as about 50 nm to about 150 nm.

29. The composition of any one of claims 1 to 28, wherein the nucleic acid is RNA, preferably mRNA.

30. The composition of claim 29, wherein the RNA (1) comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U); (2) has a codon-optimized coding sequence; and / or (3) has a coding sequence with an increased G / C content compared to a wild-type coding sequence.

31. The composition of claim 29 or 30, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

32. The composition of claim 31, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted A nucleotide sequence.

33. The composition of claim 31 or 32, wherein the 5' cap is a cap 1 or cap 2 structure.

34. The composition of any one of claims 29 to 33, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and / or comprise an epitope for inducing an immune response to an antigen in a subject.

35. The composition of claim 34, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or its immunogenic variant.

36. The composition of claim 34 or 35, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

37. The composition of any one of claims 1 to 36, wherein the composition is in liquid form, preferably at a temperature of about 2°C to about 10°C.

38. The composition of any one of claims 1 to 37, wherein after storage for at least one week, preferably at a temperature of about 2°C to about 8°C for at least one week, the nucleic acid integrity of the composition is at least 90% compared to the nucleic acid integrity before storage.

39. The composition of any one of claims 24 to 38, wherein the size (Z) of the nucleic acid particles of the composition after storage is 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles prior to storage 平均值 ) and / or size distribution and / or PDI.

40. The composition of any one of claims 1 to 36, wherein the composition is in frozen form.

41. The composition of claim 40, wherein the nucleic acid integrity after thawing the frozen composition is at least 90% or substantially 100% compared to the nucleic acid integrity before the composition was frozen.

42. The composition of any one of claims 40 to 41, wherein the size (Z) of the nucleic acid particles after thawing the frozen composition 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles before the composition is frozen 平均值 ) and / or size distribution and / or PDI.

43. A method of preparing a composition comprising particles dispersed in a final aqueous phase, wherein the composition comprises (i) a nucleic acid; (ii) a lipid that can ionize to give off a cation; (iii) a steroid; (iv) a neutral lipid; and (v) an inorganic polyphosphate; wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize to give off a cation, and at least a portion of the steroid; wherein at least a portion of the inorganic polyphosphate is associated with the particles; and wherein the final aqueous phase comprises a final buffer system; The method comprises: (I) preparing a formulation comprising particles dispersed in the final aqueous phase, wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the lipid that can ionize into a cation, at least a portion of the steroid, and at least a portion of the neutral lipid, and wherein at least a portion of the inorganic polyphosphate is associated with the particles; and (II) optionally freezing the formulation to about -10°C or below, Thus, the composition is obtained, Wherein step (I) comprises: (a) providing a nucleic acid solution comprising water and a first buffer system; (b) providing an organic solution comprising the lipid that can ionize into cations, the steroid and the neutral lipid; (c) mixing the nucleic acid solution provided under (a) with the organic solution provided under (b), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system; (d) mixing the first intermediate preparation prepared under (c) with an inorganic polyphosphate or a salt thereof, thereby preparing a second intermediate preparation comprising particles dispersed in a second aqueous phase comprising a second buffer system, wherein at least a portion of the inorganic polyphosphate is associated with the particles; and (e) filtering and / or diluting the second intermediate preparation prepared under (d) using a final aqueous buffer solution comprising the final buffer system, The formulation comprising the particles dispersed in the final aqueous phase is thereby prepared.

44. The method of claim 43, wherein step (I) further comprises one or more steps selected from dilution and filtration.

45. The method of any one of claims 43 to 44, wherein step (I) comprises: (a') providing an aqueous nucleic acid solution; (b') providing a first aqueous buffer solution comprising a first buffer system; (c') mixing the aqueous nucleic acid solution provided under (a') with the first aqueous buffer solution provided under (b'), thereby preparing a nucleic acid solution comprising water and the first buffer system; (d') providing (e.g., preparing) an organic solution comprising the lipid that can ionize into cations, the steroid, and the neutral lipid; (e') mixing the nucleic acid solution prepared under (c') with the organic solution provided under (d'), thereby preparing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system; (f') optionally diluting the first intermediate formulation prepared under (e') with water or another aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising particles dispersed in an additional aqueous phase, the additional aqueous phase comprising the first buffer system or an additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution; (g') mixing said first intermediate preparation obtained in step (e') with an inorganic polyphosphate or a salt thereof if step (f') is absent, or mixing said further intermediate preparation obtained in step (f') with an inorganic polyphosphate or a salt thereof if step (f') is present, thereby preparing a second intermediate preparation comprising particles dispersed in a second aqueous phase, wherein at least a portion of said inorganic polyphosphate is associated with said particles; (h') optionally filtering the second first intermediate preparation prepared under (g') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate preparation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system, wherein the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution and / or the second aqueous buffer solution; (i') optionally repeating step (h') once or twice or more times, wherein the additional intermediate preparation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system obtained after one cycle is used as the second intermediate preparation for the next cycle, wherein in each cycle, the additional aqueous buffer solution may be the same as or different from the first aqueous buffer solution and / or the second aqueous buffer solution; (j') filtering the following using the final aqueous buffer solution comprising the final buffer system: if step (h') is absent, filtering the second intermediate preparation obtained in step (g'), or if step (h') is present and step (i') is absent, filtering the further intermediate preparation obtained in step (h'), or if steps (h') and (i') are present, filtering the further intermediate preparation obtained after step (i'); and (k') optionally diluting the preparation obtained in step (j') with a diluting solution; The formulation comprising the particles dispersed in the final aqueous phase is thereby prepared.

46. ​​The method of any one of claims 43 to 45, wherein filtration is dialysis, tangential flow filtration or diafiltration, preferably dialysis or tangential flow filtration.

47. The method of any one of claims 43 to 46, wherein the inorganic polyphosphate or salt thereof comprises or has the formula P x O (3x+1) M y’ , wherein x is an integer and is at least 3; each M is independently H + or cations; and y' is the number of cations required for charge balance.

48. The method of claim 47, wherein each M is independently selected from H + , alkaline cations, ammonium and monovalent organic cations, preferably each M is independently selected from H + 、Na + , K + , Li + and NH4 + .

49. The method of any one of claims 43 to 48, wherein the inorganic polyphosphate or its salt is selected from triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, salts thereof and mixtures thereof, preferably selected from triphosphate, tetraphosphate, pentaphosphate, salts thereof and mixtures thereof, more preferably, the inorganic polyphosphate or its salt is triphosphate or its salt.

50. The method of any one of claims 43 to 49, wherein the inorganic polyphosphate or salt thereof is a linear inorganic polyphosphate or salt thereof, such as a linear triphosphate or salt thereof.

51. The method of any one of claims 43 to 50, wherein the molar ratio of (v) inorganic polyphosphate to (ii) lipid that can ionize into a cation is at least about 1:2, preferably at least about 2:3, such as at least about 4:

3.

52. The method of any one of claims 43 to 51, wherein the composition is substantially free of PEG-containing lipids, preferably substantially free of any PEG-containing compound, more preferably substantially free of PEG.

53. The method of any one of claims 43 to 52, wherein (1) the pH of the nucleic acid solution obtained in step (a) is less than 6.0, preferably at most about 5.0, more preferably at most about 4.5; or (2) the pH of the first aqueous buffer solution is less than 6.0, preferably at most about 5.0, more preferably at most about 4.

5.

54. The method of any one of claims 43 to 53, wherein the pH of the composition is from about 4.0 to about 8.0, preferably from about 4.5 to about 8.0, for example from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 6.8 to about 7.9, or from about 7.0 to about 7.

8.

55. The method of any one of claims 43 to 54, wherein water is a major component of the formulation and / or composition, and / or the total amount of solvents other than water contained in the composition is less than about 0.5% (v / v).

56. The method of any one of claims 43 to 55, wherein the composition has an osmolarity of at most about 1000 x 10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg to about 500×10 -3 osmol / kg, more preferably about 300×10 - 3 osmol / kg.

57. The method of any one of claims 43 to 56, wherein the concentration of the nucleic acid in the composition is from about 1 mg / l to about 500 mg / l, e.g., from about 1 mg / l to about 100 mg / l, from about 5 mg / l to about 100 mg / l, or from about 10 mg / l to about 100 mg / l.

58. The method of any one of claims 43 to 57, wherein the lipid that can ionize into a cation comprises a head group comprising at least one tertiary amine moiety.

59. The method of any one of claims 43 to 58, wherein the lipid that can ionize to give a cation, the steroid, and the neutral lipid are present in the organic solution in the following molar ratios: about 20 mol % to about 70 mol % of the lipid that can ionize to give a cation; about 15 mol % to about 60 mol % of the steroid; and about 5 mol % to about 25 mol % of the neutral lipid (e.g., phospholipid).

60. The method of any one of claims 43 to 59, wherein the lipid that can ionize to give a cation, the steroid, and the neutral lipid are present in the organic solution in the following molar ratios: about 40 mol % to about 70 mol %, for example, about 45 mol % to about 65 mol %, or about 50 mol % to about 60 mol % of the lipid that can ionize to give a cation; about 15 mol % to about 40 mol %, for example, about 20 mol % to about 35 mol %, or about 20 mol % to about 30 mol % of the steroid; and about 15 mol % to about 25 mol %, for example, about 17 mol % to about 21 mol % of the neutral lipid.

61. The method of any one of claims 43 to 60, wherein the molar ratio of steroid to neutral lipid in the organic solution is at most 2.5, preferably the ratio is from 1 to 2.

5.

62. The method of any one of claims 43 to 59, wherein the lipid that can ionize to give a cation, the steroid, and the neutral lipid are present in the organic solution in the following molar ratios: about 20 mol % to about 40 mol %, for example, about 25 mol % to about 40 mol %, or about 25 mol % to about 35 mol % of the lipid that can ionize to give a cation; about 35 mol % to about 60 mol %, for example, about 40 mol % to about 60 mol %, or about 45 mol % to about 60 mol % of the steroid; and about 5 mol % to about 15 mol %, for example, about 7 mol % to about 14 mol % of the neutral lipid.

63. The method of any one of claims 43 to 59 and 62, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.

0.

64. The method of any one of claims 43 to 63, wherein the particles have a size of about 30 nm to about 500 nm, such as about 50 nm to about 150 nm.

65. The method of any one of claims 43 to 64, wherein the particle is selected from the group consisting of lipid nanoparticles (LNP), liposomes, lipid complexes (LPX), and mixtures of two or more thereof.

66. The method of any one of claims 43 to 65, wherein the nucleic acid is RNA, preferably mRNA.

67. The method of claim 66, wherein the RNA (i) comprises modified nucleosides instead of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U); (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with an increased G / C content compared to a wild-type coding sequence.

68. The method of claim 66 or 67, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

69. The method of claim 68, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted A nucleotide sequence.

70. The method of claim 68 or 69, wherein the 5' cap is a cap 1 or cap 2 structure.

71. The method of any one of claims 66 to 70, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and / or comprise epitopes for inducing an immune response to an antigen in a subject.

72. The method of claim 71, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or its immunogenic variant.

73. The method of claim 71 or 72, wherein the pharmaceutically active polypeptide and / or the antigen or epitope is derived from or is: a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

74. The method of any one of claims 43 to 73, comprising (II) freezing the formulation to about -10°C or below.

75. The method of any one of claims 43 to 73, excluding step (II).

76. A method of storing a composition, comprising preparing a composition according to the method of any one of claims 43 to 74, and storing the composition at a temperature of about -90°C to about -10°C, for example, about -90°C to about -40°C, or about -25°C to about -10°C.

77. The method of claim 76, wherein the composition is stored for at least 1 month, e.g., at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

78. A method for storing a composition, comprising preparing a composition according to the method described in any one of claims 43 to 75, and storing the composition at a temperature of about 0°C to about 20°C, such as about 1°C to about 15°C, about 2°C to about 10°C, or about 2°C to about 8°C, or at a temperature of about 5°C.

79. The method of claim 78, wherein the composition is stored for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months.

80. A composition preparable by the method of any one of claims 43 to 79.

81. The composition of claim 80 in frozen form.

82. The composition of claim 81, wherein the nucleic acid integrity after thawing the frozen composition is at least 90% or substantially 100% compared to the nucleic acid integrity before the composition was frozen.

83. The composition of any one of claims 81 to 82, wherein the size (Z) of the nucleic acid particles after thawing the frozen composition is 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles before the composition is frozen 平均值 ) and / or size distribution and / or PDI.

84. The composition of claim 80 in liquid form.

85. The composition of claim 84, wherein the nucleic acid integrity of the composition after storage for at least one week, preferably at a temperature of about 2°C to about 8°C for at least one week, is at least 90% compared to the nucleic acid integrity before storage.

86. The composition of claim 84 or 85, wherein the size (Z) of the nucleic acid particles of the composition after storage for at least one week is 平均值 ) and / or a size distribution and / or a polydispersity index (PDI) substantially equal to the size (Z) of the nucleic acid particles prior to storage 平均值 ) and / or size distribution and / or PDI.

87. A method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of: Providing a frozen composition prepared by the method of any one of claims 43 to 74, 76 and 77, and thawing the frozen composition to obtain the ready-to-use pharmaceutical composition.

88. A method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of: A liquid composition prepared by the method of any one of claims 43 to 73 and 75 to 77 is provided, thereby obtaining the ready-to-use pharmaceutical composition.

89. A ready-to-use pharmaceutical composition preparable by the method of claim 87 or 88.

90. The composition of any one of claims 1 to 42, 80 to 86 and 89 for use in therapy.

91. The composition of any one of claims 1 to 42, 80 to 86 and 89 for use in inducing an immune response in a subject.

92. A method of transfecting a cell, comprising adding the composition of any one of claims 1 to 42, 80 to 86 and 89 to the cell; and incubating the mixture of the composition and the cell for a sufficient amount of time.

93. The method of claim 92, wherein incubating the mixture of the composition and cells is performed in the presence of serum.

94. The method of claim 92 or 93, wherein the lipid that can ionize to give a cation accounts for about 40 mol % to about 70 mol %, e.g., about 45 mol % to about 65 mol %, or about 50 mol % to about 60 mol % of the total lipids present in the composition; the steroid accounts for about 15 mol % to about 40 mol %, e.g., about 20 mol % to about 35 mol %, or about 20 mol % to about 30 mol % of the total lipids present in the composition; and the neutral lipid accounts for about 15 mol % to about 25 mol %, e.g., about 17 mol % to about 21 mol % of the total lipids present in the composition.

95. The method of any one of claims 92 to 94, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.

5.

96. The method of claim 92, wherein the lipid that can ionize to give a cation accounts for about 20 mol % to about 40 mol %, e.g., about 25 mol % to about 40 mol %, or about 25 mol % to about 35 mol % of the total lipids present in the composition; the steroid accounts for about 35 mol % to about 60 mol %, e.g., about 40 mol % to about 60 mol %, or about 45 mol % to about 60 mol % of the total lipids present in the composition; and the neutral lipid accounts for about 5 mol % to about 15 mol %, e.g., about 7 mol % to about 14 mol % of the total lipids present in the composition.

97. The method of claim 92 or 96, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.

0.

98. Use of the composition of any one of claims 1 to 42, 80 to 86 and 89 for transfecting cells.

99. The use of claim 98, wherein the transfecting cells is performed in the presence of serum.

100. The use of claim 98 or 99, wherein the lipid that can ionize to release cations accounts for about 40 mol% to about 70 mol% of the total lipids present in the composition, such as about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol%; the steroid accounts for about 15 mol% to about 40 mol% of the total lipids present in the composition, such as about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%; and the neutral lipid accounts for about 15 mol% to about 25 mol% of the total lipids present in the composition, such as about 17 mol% to about 21 mol%.

101. The use of any one of claims 98 to 100, wherein the molar ratio of steroid to neutral lipid is at most 2.5, preferably the ratio is from 1 to 2.

5.

102. The use of claim 98, wherein the lipid that can ionize to release cations accounts for about 20 mol % to about 40 mol %, for example, about 25 mol % to about 40 mol %, or about 25 mol % to about 35 mol % of the total lipids present in the composition; the steroid accounts for about 35 mol % to about 60 mol %, for example, about 40 mol % to about 60 mol %, or about 45 mol % to about 60 mol % of the total lipids present in the composition; and the neutral lipid accounts for about 5 mol % to about 15 mol %, for example, about 7 mol % to about 14 mol % of the total lipids present in the composition.

103. The use of claim 98 or 102, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably the ratio is from 3.0 to 10.0, such as from 5.0 to 7.0.

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