Nucleic acid compositions containing polyvalent 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, particularly mRNA or inhibitory RNA, such as siRNA) compositions comprising polyvalent anions (e.g., inorganic polyphosphates), methods for making and storing such compositions, and the use of such compositions in therapy.

Description

[Technical Field]

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

[0002] background The use of recombinant nucleic acids (e.g., DNA or RNA) to deliver exogenous genetic information to target cells is well known. Recombinant nucleic acids can be administered to a subject in naked form, but they are typically administered in a composition. For example, nucleic acids such as RNA can be delivered to a subject using a variety of delivery vehicles, most of which are based on cationic polymers or lipids that form nanoparticles with the nucleic acid. Nanoparticles are intended to protect nucleic acids such as RNA from degradation, enable delivery of nucleic acids such as RNA to the target site, and facilitate cellular uptake and processing by the target cell. The efficiency of nucleic acid delivery depends in part on the molecular composition of the nanoparticle and can be affected by numerous parameters, including particle size, formulation, and charge or grafting with molecular moieties such as polyethylene glycol (PEG) or other ligands. The fate of such nanoparticle formulations is controlled by a variety of key factors, such as nanoparticle size and size distribution. These factors have been cited, for example, in the FDA's "Liposome Drug Products Guidance" since 2018 as specific attributes that should be analyzed and identified. Advantages of using RNA include transient expression and non-transforming characteristics. Furthermore, RNA does not need to enter the nucleus to be expressed and cannot integrate into the host genome, eliminating various risks such as carcinogenesis.

[0003] Grafting with PEG is thought to reduce serum interactions, increase serum stability, and prolong circulation time, which may be useful for certain targeting approaches. Ligands that bind to target site receptors can help improve targeting efficacy. Furthermore, PEGylation can be utilized in particle engineering. For example, when preparing lipid nanoparticles (LNPs) by mixing an aqueous phase of nucleic acid, such as RNA, with an organic phase of lipids, a specific ratio of PEG-conjugated lipids in the lipid mixture is required; otherwise, particles will aggregate during or after the mixing process. It has been shown that particle size can be tuned by varying the molar fraction of PEG-lipids containing various molar masses of PEG. Similarly, particle size can be adjusted by varying the molar mass of the PEG moiety in PEGylated lipids. Typical sizes available range from 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles thus formed further have the advantage of less interaction with serum components due to the PEG fraction and a long circulation half-life, which is desirable in many drug delivery approaches. Without PEG-lipids, discrete particle sizes cannot be formed; the particles form large aggregates and precipitate. Therefore, one of the primary roles of PEG-lipids is to promote particle self-assembly by providing a steric barrier on the surface of nascent particles, which form when nucleic acids, such as RNA, are rapidly mixed in an ethanol solution containing nucleic acid-binding lipids. PEG's steric hindrance prevents particle-to-particle fusion and promotes the formation of a homogeneous population of LNPs with diameters <100 nm.

[0004] PEG is the most widely used and gold-standard "stealth" polymer in drug delivery. PEG lipids are commonly incorporated into systems to prepare uniform, colloidally stable nanoparticle populations due to their hydrophilic steric hindrance properties (the PEG shell prevents electrostatic or van der Waals attractions that lead to aggregation). PEGylation can attract a shell of water around the polymer, protecting the particles from opsonization with serum proteins, thereby increasing serum half-life and resulting in improved pharmacokinetics. The acyl chain length (C) of the lipids can be adjusted to suit the specific application. 18 , C 16 or C 14 Varying the C (C ) alters the stability of the PEG-lipid incorporation into the particles, leading to modulation of pharmacokinetics. The C (C ) dissociates from LNPs in vivo with a short half-life of <30 min. 14 The use of PEG-lipids containing acyl chains results in optimal hepatocyte gene silencing efficacy (Chen et al., 2014, J. Control Release 196:106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163). Furthermore, by varying the PEG-lipid parameters, particle size can be tightly controlled; the higher the molecular weight of the PEG or the higher the mole fraction of PEG-lipid in the particle, the smaller the particle.

[0005] Despite these advantages, PEGylation of nanoparticles may also have several adverse effects that are detrimental to their intended use for drug delivery. PEGylation of liposomes and LNPs is known to reduce cellular uptake and endosomal escape, ultimately lowering overall transfection efficiency. Indeed, the PEG shell provides a steric barrier for efficient particle binding to cells and also prevents endosomal release by interfering with liposome and endosomal membrane fusion. Therefore, the type and amount of PEG lipid used must always be carefully adjusted. On the one hand, they must exert sufficient stealth effects in terms of in vivo and stabilization, while on the other hand, they must not interfere with transfection. This phenomenon is known as the "PEG dilemma."

[0006] PEGylation not only reduces transfection efficiency but is also associated with 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 into rats can significantly alter the pharmacokinetic behavior of a second dose when the second dose is administered several days apart (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 appears to be related to the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma increases particle clearance by the mononuclear phagocyte system (MPS), ultimately resulting in reduced drug efficacy.

[0007] PEG is also thought to induce complement activation, potentially leading to a hypersensitivity reaction also known as complement activation-associated pseudoallergy (CARPA). It is not yet clear from the literature whether complement activation is due to nanoparticles in general or the presence of PEG specifically.

[0008] The inclusion of PEG in other lipid particles can also elicit 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 elicited a strong immune response, resulting in rapid blood clearance after administration to mice. The magnitude of this response was sufficient to cause significant morbidity and, in some cases, mortality. The use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG-lipids abolished the response, suggesting that the rapid clearance of liposomally encapsulated oligodeoxynucleotides from the blood depended on the presence of PEG-lipids in the membrane. The generation of anti-PEG antibodies and presumed complement activation explained the rapid clearance of the vesicles from the blood.

[0009] PEG can induce immune responses and should be avoided in applications requiring multiple injections. Examples include nucleic acid (e.g., RNA, particularly mRNA)-based therapies, such as protein replacement therapy, where the risk may be particularly high due to the inherent immunogenic potential of nucleic acids (especially RNA). Other examples include protein knockdown therapies using inhibitory RNA (e.g., siRNA), antisense oligonucleotides, or DNA-based therapies.

[0010] Thus, there remains a need in the art for efficient compositions and methods for introducing nucleic acids, such as RNA, into cells that avoid the drawbacks associated with the use of PEG. Ideally, these compositions and methods should be such that (i) the compositions are stable and can be stored in liquid form at temperatures consistent with common pharmaceutical practice, particularly at temperatures of about -20°C, or even at temperatures between +2 and +20°C; (ii) the compositions can be repeatedly frozen and thawed; (iii) the compositions are ready to use; (iv) PEG-free compositions maintain high biological potency; and / or (v) the nucleic acids contained in the compositions are in a stable form and are not significantly degraded upon storage. The present disclosure addresses these and other needs.

[0011] The present inventors have surprisingly found that the compositions and methods described herein meet the above-mentioned requirements.In particular, it has been demonstrated that by using polyvalent anions such as inorganic polyphosphate, inorganic phosphate or citrate, it is possible to produce compositions that are stable (particularly with respect to the colloidal size of the particles contained in said compositions), can be stored in liquid form, can be repeatedly frozen and thawed, contain nucleic acid in a stable form, and maintain high biological potency even when compositions / particles contain / do not contain PEG-lipid or any other stealth lipid. Summary of the Invention

[0012] overview In a first aspect, the present disclosure provides a composition comprising: (i) a nucleic acid; (ii) a cationically ionizable lipid; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion, such as inorganic polyphosphate.

[0013] As shown in the present application, aggregation of particles (e.g., lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), or mixtures thereof) formed from nucleic acids (e.g., RNA, such as mRNA) in compositions (e.g., aqueous compositions) can be prevented by adding polyanions such as inorganic polyphosphate to the compositions, even when the compositions / particles contain or do not contain PEG-lipids or any other stealth lipids. Furthermore, the present application surprisingly demonstrates that by adjusting the amounts of lipids relative to one another in the presence of polyanions such as inorganic polyphosphate, it is possible to obtain nucleic acid (e.g., RNA) compositions that exhibit different biological performance: when used for cell transfection, nucleic acid compositions with higher relative amounts of ionizable lipids and neutral lipids and lower relative amounts of steroids result in higher expression of the nucleic acid in transfected cells when transfection is performed in the presence of serum compared to expression obtained when transfection is performed in the absence of serum (this effect is referred to herein as "serum stimulation" and is similar to the biological performance of standard nucleic acid compositions containing PEG-lipids). In contrast, nucleic acid compositions used to transfect cells that contain low relative amounts of ionizable lipids and neutral lipids and high relative amounts of steroids exhibit comparable or reduced nucleic acid expression in transfected cells when transfected in the presence of serum (i.e., these compositions exhibit some or no serum inhibition, but no serum stimulation). Thus, the claimed compositions are stable and can be stored at temperatures consistent with conventional pharmaceutical practice, providing ready-to-use compositions and maintaining high biological potency even when the compositions / particles contain or do not contain PEG-lipids or any other stealth lipids. Furthermore, depending on the relative amounts of lipids relative to one another, the claimed compositions may exhibit different biological performance characteristics, i.e., they may or may not be irritating to serum.

[0014] The term "polyvalent anion" as used herein may be understood to refer to an ion having multiple (i.e., one or more) negative charges. For example, a polyvalent anion may be a dianion, i.e., an anion having a 2-charge, or an anion having two negative charges. In another example, a polyvalent anion may be a trianion, i.e., an anion having a 3-charge, or an anion having three negative charges. In yet another example, a polyvalent anion may be a tetraanion, i.e., an anion having a 4-charge, or an anion having four negative charges. In a further example, a polyvalent anion may have multiple negative charges. Typically, a polyvalent anion is not or does not contain a nucleic acid such as DNA or RNA. In certain embodiments, a polyvalent anion has no more than 20 negative charges (i.e., 20-charges), preferably no more than 10 negative charges (i.e., 10-charges), or most preferably no more than 5 negative charges (i.e., 5-charges). 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] Polyvalent anions include inorganic polyphosphates (as further defined herein), inorganic phosphates (e.g., PO 3-), sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate, or tetrathionate, 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, mesoxalic 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. Typically, polyvalent anions in water are molecularly dissolved as solutes and do not form supramolecular assemblies such as micelles. Structurally, polyanions are not or do not include negatively charged amphiphiles having hydrophilic and lipophilic portions (e.g., polyanions are not negatively charged lipids).

[0016] In some embodiments, the polyvalent 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 (such as a linear inorganic triphosphate).

[0017] In certain embodiments of the first aspect, the inorganic polyphosphate has the formula [P x O (3x+1) ] y where x is an integer and is at least 2, preferably at least 3; and y is the anionic charge. For example, when x is 3, the inorganic polyphosphate has the formula [PO 10 ] 5- Similarly, when x is 4, the inorganic polyphosphate has the formula [PO 13 ] 6- is a linear or branched inorganic tetraphosphate containing

[0018] In certain embodiments of the first aspect, the inorganic polyphosphate is selected from the group consisting of diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof, e.g., selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof. In some preferred embodiments of the first aspect, the inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof. In some preferred embodiments of the first aspect, the inorganic polyphosphate is triphosphate.

[0019] In some embodiments, the polyvalent anion is inorganic phosphate (e.g., PO 3- ), sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate, or tetrathionate. In some embodiments, the polyvalent anion is inorganic phosphate (e.g., PO 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, mesoxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid, or saccharinic acid).

[0021] In some embodiments, the polyvalent 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 inorganic polyphosphate (as defined herein), inorganic phosphate (e.g., PO 3-), sulfate, succinate, glutarate, tartarate, malate, citric acid, or mixtures thereof. In certain embodiments, the polyvalent anion is inorganic polyphosphate (as defined herein), inorganic phosphate, or citric acid. In some most preferred embodiments, the polyvalent anion is inorganic polyphosphate (as defined herein).

[0023] In some embodiments of the first aspect, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 1: 2. For example, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is 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) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 2: 3. In some preferred embodiments of the first aspect, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 4: 3.

[0024] In certain embodiments of the first aspect, the composition is substantially free of lipids comprising polyethylene glycol (PEG). In certain embodiments, the composition is substantially free of compounds comprising PEG. In certain embodiments, the composition is substantially free of PEG. Accordingly, in certain embodiments of the first aspect, the composition comprises (i) a nucleic acid; (ii) a cationically ionizable lipid; (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 the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartaric acid, malate, citric acid, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, and citric acid. In some of these embodiments, the polyvalent anion is inorganic polyphosphate.

[0025] In certain embodiments of the first aspect, the composition is also substantially free of another polymer-conjugated lipid. In certain embodiments, the other polymer-conjugated lipid is a polysarcosine-conjugated lipid. In certain embodiments, the composition is substantially free of any polymer-conjugated lipid (including PEG-lipids and polysarcosine-conjugated lipids). Thus, in certain embodiments of the first aspect, the composition comprises (i) a nucleic acid; (ii) a cationically ionizable lipid; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion, wherein the composition is substantially free of any polymer-conjugated lipid (including PEG-lipids and polysarcosine-conjugated lipids). In some of these embodiments, the polyvalent anion is inorganic polyphosphate, inorganic phosphate (e.g., PO4 3-), sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithioate, or tetrathioate, 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, mesoxalic 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 the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, and citric acid. 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 about 4.0 to about 8.0. In some embodiments of the first aspect, the pH of the composition is about 4.5 to about 8.0, e.g., about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 6.8 to about 7.9, or about 7.0 to about 7.8.

[0027] In certain embodiments of the first aspect, 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 1.0% (v / v), e.g., less than about 0.5% (v / v). For example, the amount of water contained in the composition can 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), at least 90% (w / w), or at least 95% (w / w). In particular, if the composition includes a cryoprotectant, the amount of water included in the composition can 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 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 non-water solvents in the composition may be less than about 0.5% (v / v), e.g., 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 standard conditions should be considered a cryoprotectant, not a non-water solvent. In other words, the above-mentioned optimal limit that the total amount of non-water solvents in the composition may be less than about 0.5% (v / v), e.g., less than about 0.4% (v / v), does not apply to a cryoprotectant that is liquid under standard conditions.

[0028] In certain embodiments of the first aspect, the composition has an osmolality of up to about 1000×10 -3 In certain embodiments of the first aspect, the osmolality of the composition is up to about 1000×10 -3 In some embodiments, the osmolality of the composition is up to about 500×10 -3 osmol / kg, e.g., up to approximately 490 × 10-3 osmol / kg, maximum approximately 480×10 -3 osmol / kg, maximum approximately 470×10 -3 osmol / kg, maximum approximately 460×10 -3 osmol / kg, maximum approximately 450×10 -3 osmol / kg, maximum approximately 440×10 -3 osmol / kg, maximum approximately 430×10 -3 osmol / kg, maximum approximately 420×10 -3 osmol / kg, maximum approximately 410×10 -3 osmol / kg, maximum approximately 400×10 -3 osmol / kg, maximum approximately 390×10 -3 osmol / kg, maximum approximately 380×10 -3 osmol / kg, maximum approximately 370×10 -3 osmol / kg, maximum approximately 360×10 -3 osmol / kg, maximum approximately 350×10 -3 osmol / kg, maximum approximately 340×10 -3 osmol / kg, maximum approximately 330×10 -3 osmol / kg, maximum approximately 320×10 -3 osmol / kg, maximum approximately 310×10 -3 osmol / kg, or up to approximately 300 × 10 -3 In certain embodiments of the first aspect, the osmolality of the composition is about 100×10 -3 osmol / kg ~ approx. 500 x 10 -3 osmol / kg, e.g., approximately 300 × 10 -3 If the composition does not contain a cryoprotectant, the osmolality of the composition is 300 x 10 -3 Less than osmol / kg, e.g., up to about 250 × 10 -3 osmol / kg, maximum approximately 200×10 -3 osmol / kg, maximum approximately 150×10 -3 osmol / kg, maximum approximately 100×10 -3 osmol / kg, maximum approximately 50×10 -3 osmol / kg, maximum approximately 40×10 -3 osmol / kg, or up to approximately 30 × 10 -3The osmol / kg of the composition may be 100 osmol / kg. If the composition includes a cryoprotectant, it is preferred that the majority of the osmolality of the composition be 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 one embodiment of the first aspect, the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 500 mg / L. In one embodiment, the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 100 mg / L. In one embodiment, the concentration of nucleic acid (particularly RNA) in the composition is about 5 mg / L to about 500 mg / L, for example, about 10 mg / L to about 400 mg / L, about 10 mg / L to about 300 mg / L, about 10 mg / L to about 200 mg / L, about 10 mg / L to about 150 mg / L, or about 10 mg / L to about 100 mg / L, preferably about 10 mg / L to about 140 mg / L, more preferably about 20 mg / L to about 130 mg / L, and even more preferably about 30 mg / L to about 120 mg / L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is about 5 mg / L to about 150 mg / L, e.g., about 10 mg / L to about 140 mg / L, about 20 mg / L to about 130 mg / L, about 25 mg / L to about 125 mg / L, about 30 mg / L to about 120 mg / L, about 35 mg / L to about 115 mg / L, about 40 mg / L to about 110 mg / L, about 45 mg / L to about 105 mg / L, or about 50 mg / L to about 100 mg / L. In some embodiments of the first aspect (particularly embodiments in which the composition is in a frozen form), the concentration of nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 50 mg / L. In certain embodiments of the first aspect (particularly those in which 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 certain embodiments of the first aspect, the composition comprises a cryoprotectant. In certain embodiments of the first aspect, the composition is substantially free of cryoprotectants.

[0031] In certain embodiments of the first aspect, the molar ratio of polyvalent anion to cationically ionizable lipid 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 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 certain embodiments of the first aspect (particularly those in which the composition is in frozen form), the molar ratio of polyvalent anion to cationically ionizable lipid 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 nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 50 mg / L. In certain embodiments of the first aspect (particularly those in which the composition is in liquid form), the molar ratio of polyvalent anion to cationically ionizable lipid 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 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 the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartaric acid, malate, citrate, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, and citrate.

[0032] In some embodiments of the first aspect, the polyvalent anion is a linear inorganic polyphosphate, particularly triphosphate, and the molar ratio of inorganic polyphosphate to cationically ionizable lipid 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. In some preferred embodiments of the first aspect, the inorganic polyphosphate is a linear inorganic polyphosphate (particularly triphosphate), the molar ratio of inorganic polyphosphate to cationically ionizable lipid 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 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 inorganic polyphosphate is linear inorganic polyphosphate (particularly triphosphate), the molar ratio of inorganic polyphosphate to cationically ionizable lipid 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 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 in which the composition is in liquid form), the inorganic polyphosphate is linear inorganic polyphosphate (particularly triphosphate), the molar ratio of inorganic polyphosphate to cationically ionizable lipid 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 nucleic acid (particularly RNA) in the composition is about 10 mg / L to about 100 mg / L.

[0033] In certain embodiments of the first aspect, the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

[0034] In certain embodiments of the first aspect, the cationically ionizable lipid has the formula (X): [ka] 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 37 are as defined herein. In some embodiments, the cationically ionizable lipid is selected from structures X-1 to X-36 (shown herein); structures A to G (shown herein); or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14). In some embodiments, the cationically ionizable lipid is a lipid having structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., a lipid having structure G). In certain embodiments, the cationically ionizable lipid is a lipid having structure D.

[0035] In certain embodiments of the first aspect, the cationically ionizable lipid has formula (XI): [ka] wherein R1, R2, R3, R4, L2, G2 and m are as defined herein. In some embodiments, the cationically ionizable lipid is selected from structures (XIV-1), (XIV-2) and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-1. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-2. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-3.

[0036] In some embodiments of the first aspect, the cationically ionizable lipid is fully or partially replaced with a cationic lipid. In some embodiments, the cationic lipid is selected from structures XV-1 to XV-6 (shown herein). In these embodiments in which the cationically ionizable lipid is fully replaced with a cationic lipid, the molar ratio of the polyvalent anion (such as inorganic polyphosphate) to the cationically ionizable lipid is replaced with the molar ratio of the polyvalent anion (such as inorganic polyphosphate) to the cationic lipid. In some embodiments, the molar ratio of polyvalent anion (such as inorganic polyphosphate) to 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 polyvalent anion (such as inorganic polyphosphate) to cationic lipid is at least about 2:3, e.g., at least about 4:3). In those embodiments in which the cationically ionizable lipid is partially replaced with a cationic lipid, the molar ratio of polyvalent anion (such as inorganic polyphosphate) to cationically ionizable lipid is replaced with the molar ratio of polyvalent anion (such as inorganic polyphosphate) to the sum of cationic lipid and cationically ionizable lipid.Thus, in some embodiments, the molar ratio of the polyvalent anion (such as inorganic polyphosphate) to the sum of the cationic lipid and the cationically ionizable 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 (such as inorganic polyphosphate) to the sum of the cationic lipid and the cationically ionizable lipid is at least about 2:3, e.g., at least about 4:3).

[0037] In certain embodiments of the first aspect, the cationically ionizable lipids comprise about 20 mol% to about 75 mol%, e.g., about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% of the total lipids present in the composition (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high). In these embodiments where some or all of the cationically ionizable lipids are substituted with cationic lipids, the same ranges (e.g., about 20 mol% to about 75 mol%) as specified above for the total cationically ionizable lipids and cationic lipids preferably apply.

[0038] In certain embodiments of the first aspect, the steroid comprises a sterol. In some preferred embodiments of the first aspect, the steroid comprises or is cholesterol.

[0039] In certain embodiments of the first aspect, the steroid comprises about 15 mol% to about 60 mol%, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% of the total lipids present in the composition (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high).

[0040] In certain embodiments of the first aspect, the neutral lipid is a phospholipid. In certain embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. In certain embodiments, the phospholipid has a T above 30°C. 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.

[0041] In certain embodiments of the first aspect, the neutral lipids comprise from about 5 mol% to about 25 mol%, e.g., from about 15 mol% to about 25 mol%, or from about 17 mol% to about 21 mol%, of the total lipids present in the composition (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or from about 5 mol% to about 15 mol%, or from about 7 mol% to about 14 mol%, (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high).

[0042] In certain embodiments of the first aspect, the cationically ionizable lipid comprises about 20 mol% to about 70 mol% of the total lipid present in the composition; the steroid comprises about 15 mol% to about 60 mol% of the total lipid present in the composition; and the neutral lipid (e.g., phospholipid) comprises about 5 mol% to about 25 mol% of the total lipid present in the composition.

[0043] In certain embodiments of the first aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 15 mol% to about 25 mol%, e.g., about 17 mol% to about 21 mol%, of the total lipid present in the composition. These embodiments of the compositions of the first aspect, i.e., compositions with high relative amounts of ionizable lipid and neutral lipid and low relative amounts of steroid, are particularly suitable for transfecting cells in the presence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0044] In certain embodiments of the first aspect, in which the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low, the molar ratio of steroids to neutral lipids is up to 2.5, preferably 1 to 2.5. Thus, in certain embodiments of the first aspect, the cationically ionizable lipids comprise 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 comprise 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 comprise 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 up to 2.5, preferably 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0045] In some alternative embodiments of the first aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 5 mol% to about 15 mol%, e.g., about 7 mol% to about 14 mol% of the total lipid present in the composition. These alternative embodiments of the compositions of the first aspect, i.e., compositions having low relative amounts of ionizable lipid and neutral lipid and high relative amounts of steroid, are particularly suitable for transfecting cells in the absence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0046] In certain embodiments of the first aspect, in which the relative amounts of ionizable lipid and neutral lipid are low and the relative amount of steroid is high, the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is between 3.0 and 10.0, for example between 5.0 and 7.0. Thus, in certain embodiments of the first aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid comprises 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 lipid present in the composition; the neutral lipid comprises about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipid present in the composition; and the molar ratio of steroid to neutral lipid is at least 3.0, preferably, the ratio is 3.0 to 10.0, e.g., 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0047] In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids can include cationic lipids. In these embodiments, when cationic lipids are present, the sum of the amount of (1) cationically ionizable lipid and the amount of (2) cationic lipid is used in the calculation. For example, if the amount of cationically ionizable lipid in the composition is about 20 mol% to about 70 mol% and the composition also contains cationic lipid, the sum of the amount of (1) cationically ionizable lipid and the amount of (2) cationic lipid is about 20 mol% to about 70 mol%.

[0048] In certain embodiments of the first aspect, the lipids included in the composition are only cationically ionizable lipids, steroids and neutral lipids, particularly only cationically ionizable lipids, steroids and phospholipids.

[0049] 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 a nucleic acid, at least a portion of a cationically ionizable lipid, at least a portion of a steroid, and at least a portion of a neutral lipid; and wherein at least a portion of a polyvalent anion (such as inorganic polyphosphate) is associated with the particles. In some embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In some embodiments, the particles comprise or are LNPs. In some embodiments, the particles comprise or are liposomes. In some embodiments, the particles comprise or are LPXs. In some embodiments, the particles comprise or are a mixture of LNPs and liposomes. In some embodiments, the particles comprise or are a mixture of LNPs and LPXs. In some embodiments, the particles comprise or are a mixture of liposomes and LPXs. In some embodiments, the particles comprise or are a mixture of LNPs, liposomes, and LPXs.

[0050] In certain embodiments of the first aspect in which the composition comprises particles dispersed in an aqueous phase, the particles comprise essentially all of the lipids present in the composition (particularly all of the cationically ionizable lipids, steroids, and neutral lipids).

[0051] In certain embodiments of the first aspect, in which the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of cationically ionizable lipids, steroids, and neutral lipids (e.g., substantially free of lipids).

[0052] In certain 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 acid (particularly RNA) present in the composition. In certain embodiments, the particles comprise at least 75%, preferably at least 85%, of the nucleic acid (particularly RNA) present in the composition.

[0053] In certain embodiments of the first aspect, in which the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of nucleic acids.

[0054] In certain embodiments of the first aspect, in which the composition comprises particles dispersed in an aqueous phase, the nucleic acid (such as RNA) is encapsulated within or associated with the particles.

[0055] In some embodiments of the first aspect, where 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%, more preferably at least 50%, of the polyvalent anions (e.g., polyphosphate) present in the composition are associated with the particles.

[0056] In certain embodiments of the first aspect, in which the composition comprises particles dispersed in an aqueous phase, the particles have a size of from about 30 nm to about 500 nm, hi certain embodiments, the particles have a size of from about 50 nm to about 150 nm.

[0057] In certain embodiments of the first aspect, the nucleic acid is DNA.

[0058] In certain embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA, (eg, siRNA).

[0059] In certain embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) that (i) contains modified nucleosides in place of uridine; (ii) has a coding sequence that is codon-optimized; and / or (iii) has a coding sequence that has an increased G / C content compared to the wild-type coding sequence. In certain embodiments, the modified nucleosides are selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0060] 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 polyA 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 polyA sequence. In some embodiments, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably an interrupted sequence of A nucleotides. In some embodiments, the 5' cap is a Cap 1 or Cap 2 structure.

[0061] In certain embodiments of the first aspect, the nucleic acid is RNA (such as mRNA) and encodes one or more polypeptides. In certain 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.

[0062] In certain 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 the protein, or an immunogenic fragment of the protein or its immunogenic variant. In certain embodiments, the pathogen is a pathogen that causes an infectious disease.

[0063] In certain 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 the SARS-CoV-2 S protein or its immunogenic variant. In certain embodiments, the RNA (such as an mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant.

[0064] In some embodiments of the first aspect, nucleic acid is inhibitory RNA (such as siRNA), and selectively hybridizes and / or is specific to target mRNA.In some embodiments, target mRNA comprises ORF that encodes the pharmaceutically active peptide or polypeptide whose expression (particularly, for example, compared with the expression in healthy subjects, increased expression) is associated with disease.In some embodiments, target mRNA comprises ORF that encodes the pharmaceutically active peptide or polypeptide whose expression (particularly, for example, compared with the expression in healthy subjects, increased expression) is associated with cancer.

[0065] In certain embodiments of the first aspect, the composition is in liquid form, preferably at a temperature of about 2°C to about 10°C.

[0066] In one embodiment of the first aspect, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least one week, preferably at a temperature above 0°C, e.g., about 2°C to about 8°C, is such that the desired effect, e.g., induction of an immune response, can be achieved. In one embodiment, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage 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), preferably at a temperature above 0°C, e.g., about 2°C to about 8°C, is at least 90% of the nucleic acid integrity before storage. In one embodiment, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least four weeks, preferably at a temperature above 0°C, e.g., about 2°C to about 8°C, is at least 90% of the nucleic acid integrity before storage. In one embodiment, the nucleic acid integrity (e.g., RNA integrity) of the composition after storage for at least three months, preferably at a temperature above 0°C, e.g., about 2°C to about 8°C, is at least 90% of the nucleic acid integrity before storage.

[0067] In certain embodiments of the first aspect, the initial nucleic acid integrity (e.g., initial RNA integrity) of the composition (i.e., after manufacture but before storage) is at least 50%, and preferably after storage at a temperature above 0°C, e.g., from about 2°C to about 8°C, 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, or at least 3 months), the nucleic acid integrity (e.g., RNA integrity) of the composition is at least 90% of the initial RNA integrity.

[0068] Additionally or alternatively, in certain embodiments of the first aspect, the size (Z) of nucleic acid particles (such as RNA particles) in the liquid composition is preferably measured after storage (e.g., at least 1 week) at a temperature above 0°C, e.g., from about 2°C to about 8°C. 平均) (and / or size distribution and / or polydispersity index (PDI)) are such that a desired effect, for example, for inducing an immune response, can be achieved. In one embodiment, the size (Z) of the nucleic acid particles (such as RNA particles) in the liquid composition after storage (e.g., at least one week) preferably at a temperature of 0°C or higher, for example, about 2°C to about 8°C, is 平均 ) (and / or size distribution and / or polydispersity index (PDI)) is used to determine the size (Z) of the initial composition, i.e., nucleic acid particles (such as RNA particles) before storage. 平均 ) (and / or size distribution and / or PDI) are essentially equal to the size (Z) of nucleic acid particles (such as RNA particles) after storage at a temperature preferably above 0°C, e.g., from about 2°C to about 8°C, for at least 1 week (e.g., at least 4 weeks, or at least 3 months). 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm. In one embodiment, the PDI of nucleic acid particles (such as RNA particles) after storage at a temperature of preferably 0°C or higher, for example, about 2°C to about 8°C, for at least one week (e.g., at least four weeks, or at least three months) is less than 0.3, preferably less than 0.2, and more preferably less than 0.1. In one embodiment, the size (Z) of nucleic acid particles (such as RNA particles) after storage at a temperature of preferably 0°C or higher, for example, about 2°C to about 8°C, for at least one week (e.g., at least four weeks, or at least three months) is less than 0.3, preferably less than 0.2, and more preferably less than 0.1. 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and is the size (Z) of nucleic acid particles (such as RNA particles) after storage of the liquid composition at a temperature of preferably 0°C or higher, for example, about 2°C to about 8°C, for at least 1 week (for example, at least 4 weeks, or at least 3 months). 平均 ) (and / or size distribution and / or PDI) are determined by the size (Z) of nucleic acid particles (such as RNA particles) before storage. 平均) (and / or size distribution and / or PDI) of nucleic acid particles (such as RNA particles) after storage of the liquid composition for at least 1 week (e.g., at least 4 weeks, or at least 3 months), preferably at a temperature of 0°C or higher, for example, from about 2°C to about 8°C. 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the PDI of the nucleic acid particles (such as RNA particles) after storage of the liquid composition at a temperature of preferably 0°C or higher, for example, about 2°C to about 8°C, for example, at 0°C or higher, for at least 1 week (for example, at least 4 weeks, or at least 3 months) is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0069] In some embodiments of the first aspect, the composition is in a frozen form (e.g., at -20°C). In some embodiments, the nucleic acid integrity (such as 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 (such as RNA integrity) before freezing the composition. In some embodiments, the size (Z) of nucleic acid particles (such as RNA particles), particularly LNPs, after thawing the frozen composition is 平均 ) and / or size distribution and / or polydispersity index (PDI) are used to measure the size (Z) of nucleic acid particles (such as RNA particles) in the composition prior to freezing. 平均 ) and / or size distribution and / or PDI.

[0070] In certain embodiments of the first aspect, the initial nucleic acid integrity (such as initial RNA integrity) of the composition (i.e., after manufacture but before freezing) is at least 50%, and the nucleic acid integrity (such as RNA integrity) of the composition after thawing the frozen composition is at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and even more preferably substantially 100% of the initial nucleic acid integrity (such as initial RNA integrity).

[0071] Additionally or alternatively, in certain embodiments of the first aspect, the size (Z) of nucleic acid particles (such as RNA particles) after thawing the frozen composition is 平均 ) (and / or size distribution and / or polydispersity index (PDI)) refers to the size (Z) of the nucleic acid particles (such as RNA particles) before freezing the composition. 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid particles (such as RNA particles) after thawing the frozen composition is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm. In one embodiment, the PDI of nucleic acid particles (such as RNA particles) after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In one embodiment, the size (Z 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the size (Z 平均 ) (and / or size distribution and / or PDI) are determined by the size (Z 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid particles (such as RNA particles) after thawing the frozen composition is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, and more preferably about 40 nm to about 120 nm, and the PDI of the nucleic acid particles (such as RNA particles) after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0072] In certain embodiments of the first aspect, the size (e.g., RNA particles) and nucleic acid integrity (e.g., RNA integrity) of the composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, even more preferably after four freeze / thaw cycles, and even more preferably after five or more freeze / thaw cycles, are essentially equal to the size (e.g., RNA particles) and nucleic acid integrity (e.g., RNA integrity) of the nucleic acid particles in the initial composition (i.e., before the composition was first frozen).

[0073] In a second aspect, the present disclosure provides a method for making a composition comprising particles dispersed in a final aqueous phase, wherein the composition comprises: (i) nucleic acid; (ii) cationically ionizable lipid; (iii) steroid; (iv) neutral lipid; and (v) polyvalent anion (such as inorganic polyphosphate); the particles comprise at least a portion of the nucleic acid, at least a portion of the cationically ionizable lipid, and at least a portion of the steroid; at least a portion of the polyvalent anion (such as inorganic polyphosphate) is associated with the particles; and the final aqueous phase comprises a final buffer system; wherein the method comprises: (I) preparing a formulation comprising particles dispersed in a final aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationically ionizable lipid, at least a portion of a steroid, and at least a portion of a neutral lipid, and at least a portion of a polyvalent anion (such as inorganic polyphosphate) is associated with the particles; and (II) optionally freezing the formulation to about −10° C. or below; thereby obtaining a composition, Here, step (I) is (a) providing (e.g., preparing) a nucleic acid solution comprising water and a first buffer system; (b) preparing (e.g., preparing) an organic solution containing a cationically ionizable lipid, a steroid, and a neutral lipid; (c) combining the nucleic acid solution provided under (a) with the organic solution provided under (b), thereby producing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system; (d) mixing the first intermediate formulation produced under (c) with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby producing 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 (e) filtering (e.g., by dialysis, tangential flow filtration, or diafiltration) and / or diluting the second intermediate formulation produced under (d) with a final aqueous buffer comprising a final buffer system; This involves producing a formulation comprising particles dispersed in a final aqueous phase.

[0074] As demonstrated in the present application, the methods of the present application can be used to produce compositions comprising particles dispersed in a final aqueous phase, wherein the composition comprises (i) nucleic acid; (ii) cationically ionizable lipid; (iii) steroid; (iv) neutral lipid; and (v) polyvalent anion (such as inorganic polyphosphate); and wherein the particles comprise at least a portion of the nucleic acid, at least a portion of the cationically ionizable lipid, and at least a portion of the steroid, wherein the presence of the polyvalent anion (such as inorganic polyphosphate) can prevent aggregation of the particles even when the composition / particles contain / do not contain PEG-lipids or any other stealth lipids (polymer-conjugated lipids). Furthermore, the present application surprisingly demonstrates that, by using the present method, it is possible to obtain nucleic acid (e.g., RNA) compositions that exhibit various biological properties, particularly by adjusting the amounts of lipids relative to one another: when used to transfect cells, nucleic acid compositions with high relative amounts of ionizable and neutral lipids and low relative amounts of steroids result in higher nucleic acid expression in transfected cells when transfected in the presence of serum compared to expression obtained when transfected in the absence of serum (these compositions are similar to standard nucleic acid compositions containing PEG-lipids). In contrast, nucleic acid compositions with low relative amounts of ionizable and neutral lipids and high relative amounts of steroids used to transfect cells result in equivalent or reduced nucleic acid expression in transfected cells when transfected in the presence of serum (i.e., these compositions show some or no serum inhibition, but no serum stimulation).

[0075] An exemplary flowchart of steps (c) through (e) of the method of the second embodiment is shown in Figure 1A. First, an aqueous nucleic acid solution (containing a first buffer system, e.g., a buffer system having a pH of less than about 6.0, e.g., about 3.5 to about 5.9) is mixed with an organic (e.g., ethanol) lipid solution ("lipid in organic solution" in Figure 1A), thereby forming a first intermediate formulation containing particles dispersed in a first aqueous phase containing a first buffer system. The first intermediate formulation is then mixed with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby producing a second intermediate formulation containing particles dispersed in a second aqueous phase containing a second buffer system, wherein at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is bound to the particles. The second intermediate formulation is then filtered (e.g., using dialysis, tangential flow filtration, and / or diafiltration) and / or diluted with a final aqueous buffer containing a final buffer system, thereby producing a formulation containing particles dispersed in a final aqueous phase. For example, the second intermediate formulation is filtered to remove undesired compounds (e.g., organic solvents (such as ethanol), and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally their respective countercations used to maintain the pH below 6.0) from the second intermediate formulation, and / or to increase the nucleic acid (such as RNA) concentration, and / or to change the pH, and / or to change the buffer system to the final buffer system.

[0076] In certain embodiments of the second aspect, step (d) is performed up to about 20 minutes after step (c). In certain embodiments, step (d) is performed up to about 19 minutes (e.g., up to about 18 minutes, up to about 17 minutes, up to about 16 minutes, up to about 15 minutes, up to about 14 minutes, up to about 13 minutes, up to about 12 minutes, up to about 11 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6 minutes, or up to about 5 minutes) after step (c).

[0077] In some embodiments of the second aspect, step (I) further comprises one or more optional processing steps selected from dilution and filtration, such as dialysis, tangential flow filtration, and diafiltration, after step (c) and / or step (d) and / or step (e) (preferably only after step (d) and / or step (e)). For example, the dilution step comprises adding a dilution solution (e.g., water) to the intermediate formulation. Such a dilution solution may contain one or more additional compounds (e.g., cryoprotectants) and optionally a buffer system (e.g., a final buffer system). The dilution step may be performed to dilute undesired compounds in the intermediate formulation (e.g., organic solvents (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, and / or to add one or more additional compounds (e.g., cryoprotectants). One or more filtration steps (including steps (e), (h'), (i'), and (j') identified herein) can be used to remove undesired compounds (e.g., organic solvents (such as ethanol) and / or one or more dibasic and / or polybasic organic acids) from the intermediate formulation, and / or to increase the nucleic acid (such as RNA) concentration of the intermediate formulation, and / or to change the pH and / or buffer system of the intermediate formulation. For this purpose, an aqueous buffer can be used that is free of undesired compounds (allowing the undesired compounds to be filtered or washed out of the intermediate formulation into the aqueous buffer), and / or that is hypertonic relative to the aqueous buffer (allowing water to flow from the intermediate formulation into the aqueous buffer), and / or has a different pH and / or buffer system than the pH and / or buffer system of the intermediate formulation.

[0078] In certain embodiments of the second aspect, step (a) comprises: (a') preparing an aqueous nucleic acid solution; (b') preparing a first aqueous buffer comprising a first buffer system; and (c') mixing the aqueous nucleic acid solution prepared under (a') with the first aqueous buffer prepared under (b'), thereby preparing (e.g., producing) a nucleic acid solution comprising water and the first buffer system.

[0079] In some embodiments of the second aspect, the organic solution comprises an organic solvent selected from lower alcohols, such as alcohols having up to 6 carbon atoms (especially aliphatic alcohols), and mixtures of two or more of these alcohols. In preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from the group consisting of ethanol, propanol, isopropanol, 1,2-propanediol, and mixtures of two or more of these alcohols.

[0080] In certain embodiments of the second aspect, step (I) comprises: (a') preparing an aqueous nucleic acid solution; (b') providing a first aqueous buffer comprising a first buffer system; (c') mixing the aqueous nucleic acid solution prepared under (a') with the first aqueous buffer prepared under (b'), thereby producing a nucleic acid solution comprising water and a first buffer system; (d') providing (e.g., preparing) an organic solution containing a cationically ionizable lipid, a steroid, and a neutral lipid; (e') mixing the nucleic acid solution prepared under (c') with the organic solution prepared (e.g., prepared) under (d'), thereby producing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system; (f') optionally diluting the first intermediate formulation produced under (e') with water or an additional aqueous buffer comprising an additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in an additional aqueous phase comprising the first buffer system or an additional buffer system, wherein the additional aqueous buffer can be the same as or different from the first aqueous buffer; (g') mixing the first intermediate formulation obtained in step (e') if step (f') is not present, or the further intermediate formulation obtained in step (f') if step (f') is present, with a polyvalent anion (such as an inorganic polyphosphate) or a salt thereof (as specified herein), thereby producing a second intermediate formulation comprising particles dispersed in a second aqueous phase, wherein at least a portion of the polyvalent anion (such as an inorganic polyphosphate) is associated with the particles; (h') optionally filtering the second first intermediate formulation produced under (g') with an additional aqueous buffer comprising an additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system, wherein the additional aqueous buffer can be the same as or different from the first and / or second aqueous buffer; (i') optionally repeating step (h') one, two or more times, wherein a further intermediate formulation comprising particles dispersed in a further aqueous phase comprising a further buffer system obtained after one cycle of step (h') is used as a second intermediate formulation in a next cycle, wherein in each cycle, the further aqueous buffer can be the same as or different from the first and / or second aqueous buffer; (j') filtering the second intermediate formulation obtained in step (g') if step (h') is not present, or the further intermediate formulation obtained in step (h') if step (h') is present but step (i') is not present, or the further intermediate formulation obtained after step (i') if steps (h') and (i') are present, with a final aqueous buffer comprising a final buffer system; and (k') optionally diluting the formulation obtained in step (j') with a diluent solution; This involves producing a formulation comprising particles dispersed in a final aqueous phase.

[0081] An exemplary flow chart of steps (e'), (g'), and (j') according to these embodiments of the second aspect is shown in Figure 1B (also showing optional processing steps (f'), (h'), (i'), and (k')). First, in step (e'), an aqueous nucleic acid solution (comprising a first buffer system, e.g., a buffer system having a pH less than about 6.0, e.g., a pH of about 3.5 to about 5.9) is mixed with an organic lipid solution, thereby forming a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising the first buffer system. Optionally, the first intermediate formulation is then (f') diluted with water or an additional aqueous buffer comprising the additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in a further aqueous phase comprising the first or additional buffer system, where the additional aqueous buffer can be the same as or different from the first aqueous buffer. Optional step (f') can be performed to dilute undesired compounds in the first intermediate formulation (e.g., organic solvents (e.g., ethanol) and / or one or more dibasic and / or polybasic organic acids), change the pH, and / or change the buffer system. In a specific embodiment, step (f') is used to change the pH of the first intermediate formulation to 7 to 9, preferably 7.5 to 8.5, and more preferably 7.5 to 8.0. Then, in step (g'), the first intermediate formulation (if step (f') is not present) or each further intermediate formulation (if step (f') is present) is mixed with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby producing a second intermediate formulation comprising particles dispersed in a second aqueous phase containing a second buffer system, wherein at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is bound to the particles. Optionally, the second intermediate formulation is then (h') filtered (e.g., by dialysis, tangential flow filtration, or diafiltration) using an additional aqueous buffer comprising an additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system, where the additional aqueous buffer can be the same as or different from the first and / or second aqueous buffer; and optionally, step (h') under (i') is repeated once, twice, or more times.Optional steps (h') and (i') can be performed to remove undesired compounds (e.g., organic solvents (such as ethanol) and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally their respective countercations) from the second intermediate formulation, and / or to increase the nucleic acid (such as RNA) concentration, and / or to change the pH, and / or to change the buffer system. Then, in step (j'), the second intermediate formulation (if step (h') is present) or each further intermediate formulation (if step (h') or both steps (h') and (i') are present) is filtered (e.g., by dialysis, tangential flow filtration, or diafiltration) using a final aqueous buffer containing the final buffer system. Step (j') is performed to remove undesired compounds (e.g., organic solvents (such as ethanol), and / or one or more monobasic, dibasic, and / or polybasic organic acids, and optionally their respective countercations) from the intermediate formulation, and / or to increase the nucleic acid (such as RNA) concentration, 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 (e.g., to add a cryoprotectant).

[0082] In one embodiment 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 more preferably 7.5 to 8.0, and is performed within 12 hours, preferably 4 hours, and more preferably 30 minutes after step (e'). In a specific embodiment 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 step (g') of adding a polyvalent anion, and the combined duration of steps (f') and (g') is 12 hours or less, preferably 4 hours or less, and more preferably 30 minutes or less after step (e').

[0083] In certain embodiments of the second aspect, step (g') is performed up to about 20 minutes after step (e'). In certain embodiments, step (g') is performed up to about 19 minutes (e.g., up to about 18 minutes, up to about 17 minutes, up to about 16 minutes, up to about 15 minutes, up to about 14 minutes, up to about 13 minutes, up to about 12 minutes, up to about 11 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6 minutes, or up to about 5 minutes) after step (e').

[0084] In certain embodiments of the second aspect, step (f') is absent, and step (g') is performed up to about 20 minutes after step (e'). In certain embodiments, step (g') is performed up to about 19 minutes (e.g., up to about 18 minutes, up to about 17 minutes, up to about 16 minutes, up to about 15 minutes, up to about 14 minutes, up to about 13 minutes, up to about 12 minutes, up to about 11 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6 minutes, or up to about 5 minutes) after step (e').

[0085] In certain embodiments of the second aspect, step (f') is absent. An exemplary flow chart of steps (e'), (g'), and (j') according to these embodiments of the second aspect (i.e., step (f') is absent) is shown in Figure 1C (also showing optional processing steps (h'), (i'), and (k')). These embodiments are particularly preferred when step (g') is performed up to about 20 minutes after step (e').

[0086] In certain embodiments of the second aspect, step (f') is present.

[0087] In some embodiments of the second aspect, the organic solution provided in step (e') comprises an organic solvent selected from lower alcohols, e.g., alcohols having up to 6 carbon atoms (especially aliphatic alcohols), and mixtures thereof (e.g., mixtures of two or more of these alcohols). In preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from the group consisting of ethanol, propanol, isopropanol, 1,2-propanediol, and mixtures of two or more of these alcohols.

[0088] In certain embodiments of the second aspect, the polyvalent anion or salt thereof is inorganic phosphate, sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate, tetrathionate, or a salt thereof. In certain embodiments, the polyvalent anion is inorganic phosphate (e.g., PO 3- )

[0089] In certain 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, mesoxalic acid, malic acid, tartaric acid, aspartic acid, glutamic acid, hydroxyglutaric acid, or saccharinic acid), or a salt thereof.

[0090] In certain embodiments, the polyvalent 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.

[0091] In some preferred embodiments of the second aspect, the polyvalent anion or salt thereof is an inorganic polyphosphate, inorganic phosphate (e.g., PO 3-), sulfuric acid, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, or mixtures thereof. In certain embodiments, the polyvalent anion or salt thereof is inorganic polyphosphate, inorganic phosphate, citric acid, or a salt thereof.

[0092] In a most preferred embodiment of the second aspect, the polyvalent anion or salt thereof is an inorganic polyphosphate or salt thereof.

[0093] In certain embodiments of the second aspect where the polyvalent anion or salt thereof is an inorganic polyphosphate or salt thereof, the inorganic polyphosphate or salt thereof can be any linear, cyclic, or branched inorganic polyphosphate or salt thereof. In certain embodiments of the second aspect, the inorganic polyphosphate or salt thereof is a linear inorganic polyphosphate (such as a linear inorganic triphosphate) or salt thereof.

[0094] In certain embodiments of the second aspect wherein the polyvalent anion or salt thereof is an inorganic polyphosphate or salt thereof, the inorganic polyphosphate or salt thereof 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 balancing. M contains or has only one type (e.g., Na + only), or two or more types (e.g., Na + and K. + a mixture of, or Na + and H + A mixture of Na + K + and H + In some embodiments, each M can be H + , alkali cations (e.g., Li + , Na + , K. + ), ammonium (i.e., NH4 +), and monovalent organic cations (e.g., monovalent organic amines such as trimethylamine, triethylamine, etc.). In certain embodiments, each M is independently selected from the group consisting of H + , Na + , K. + , Li + , and NH4 + For example, if x is 3, then the inorganic polyphosphate or salt thereof is a linear inorganic triphosphate or salt of the formula [PO 10 ]M y’ (e.g., [P 10 Similarly, if x is 4, the inorganic polyphosphate or salt thereof is a linear or branched inorganic tetraphosphate or salt of the formula [PO 13 ]M y (e.g., [P4O 13 ]Na6 or [P4O 13 ]Na4K2) or its salts.

[0095] In certain embodiments of the second aspect where the polyvalent anion or salt thereof is an inorganic polyphosphate or salt thereof, the inorganic polyphosphate or salt thereof is selected from the group consisting of diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, salts thereof, and mixtures thereof, e.g., selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof. In some preferred embodiments of the second aspect, the inorganic polyphosphate or salt thereof is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, salts thereof, and mixtures thereof. In some preferred embodiments of the second aspect, the inorganic polyphosphate or salt thereof is triphosphate or salt thereof.

[0096] In some embodiments of the second aspect, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 1: 2. For example, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid 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) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 2: 3. In some preferred embodiments of the second aspect, the molar ratio of (v) polyvalent anion (such as inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 4: 3.

[0097] In certain embodiments of the second aspect, particularly if it is desired to produce the composition in frozen form, the method of the second aspect includes freezing the (II) formulation to below about −10° C. Thus, in these embodiments, performing the method of the second aspect results in the composition in frozen form.

[0098] In some alternative embodiments, particularly if it is desired to produce a composition in liquid form, the method of the second aspect does not include step (II). Thus, in these embodiments, performing the method of the second aspect results in a composition in liquid form.

[0099] In certain embodiments of the second aspect, the organic solution prepared (e.g., produced) under (b) or (d'), respectively, further comprises an acid. In certain 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 organic acid, a dibasic organic acid, or a 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, trimesic acid, etc.)). 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).

[0100] In certain embodiments of the second aspect, the composition is substantially free of lipids comprising polyethylene glycol (PEG). In certain embodiments, the composition is substantially free of compounds comprising PEG. In certain embodiments, the composition is substantially free of PEG. In some of these embodiments, the polyvalent anion or salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion or salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof. In some of these embodiments, the polyvalent anion or salt thereof is inorganic polyphosphate or salt thereof.

[0101] In some embodiments of the second aspect, the composition is also substantially free of another polymer-conjugated lipid. In some embodiments, the other polymer-conjugated lipid is a polysarcosine-conjugated lipid. Thus, 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 salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion or salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof. In some of these embodiments, the polyvalent anion or salt thereof is inorganic polyphosphate or salt thereof.

[0102] In some embodiments of the second aspect, the final pH of the buffer system (and the pH of the composition) is about 4.0 to about 8.0. For example, the final pH of the buffer system (and the pH of the composition) can be about 4.5 to about 8.0, e.g., about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 6.8 to about 7.9, about 7.0 to about 7.8, or about 7.5.

[0103] In certain embodiments of the second aspect, the first buffer system (and the pH of the nucleic acid (e.g., RNA) solution prepared / obtained in step (a) or (c')) has a pH of 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 buffer system (and the pH of the nucleic acid (e.g., RNA) solution prepared / obtained in step (a) or (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. For this purpose, the nucleic acid (e.g., RNA) solution prepared / obtained in step (a) or (c') contains 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 (e.g., hydrochloric acid) or a monobasic organic acid (e.g., acetic acid). In certain embodiments, step (e) is preferably carried out under conditions that remove one or more undesired substances (e.g., organic solvents (such as ethanol) and / or one or more acids), resulting in a formulation comprising particles dispersed in a final aqueous phase, which is substantially free of such one or more undesired substances. For example, such conditions may include subjecting the second intermediate formulation comprising particles dispersed in an intermediate aqueous phase obtained in step (d) to at least one step of filtration, such as dialysis, tangential flow filtration, or diafiltration, using a final buffer comprising a final buffer system (i.e., a final buffer substance), wherein the final buffer is free of one or more undesired substances.Alternatively, such conditions may include: (1) subjecting the intermediate formulation (i.e., the second intermediate formulation) obtained in step (d) comprising particles dispersed in a second intermediate aqueous phase to at least one dilution step using water or a further aqueous buffer comprising a further buffer system, thereby producing a further intermediate formulation comprising particles dispersed in a further aqueous phase comprising the first buffer system or a further buffer system (wherein the further buffer system of the further aqueous buffer may be the same as or different from the buffer system used in step (a)); and (2) subjecting the further intermediate formulation obtained in step (1) to at least one filtration step, such as dialysis, tangential flow filtration, or diafiltration, using a final aqueous buffer (wherein at least the final aqueous buffer (preferably the intermediate aqueous buffer and the final aqueous buffer) are free of one or more undesired substances).

[0104] Similarly, in certain embodiments of the second aspect, when step (I) comprises steps (a') to (e'), (g'), and (j') (and optionally one or more of steps (f'), (h'), (i'), and (k')), the first aqueous buffer solution (and the pH of the nucleic acid (e.g., RNA) solution obtained under step (c')) has a pH of 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 under 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 first aqueous phase) provided under (b') may further comprise one or more acids (e.g., one or more monobasic, dibasic, or polybasic acids). In these embodiments, at least one of steps (f') through (j') is preferably carried out under conditions that remove one or more undesired substances (e.g., organic solvents (such as ethanol) and / or one or more monobasic, dibasic, or polybasic acids) from the first intermediate formulation and / or the second intermediate formulation and / or the further intermediate formulation, resulting in a further intermediate formulation comprising particles dispersed in the further aqueous phase or final aqueous phase, wherein the further aqueous phase and / or final aqueous phase are substantially free of the one or more undesired substances. For example, such conditions may include using 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 all of the further aqueous buffer solution and the final buffer solution) is free of the one or more undesired substances. In certain embodiments, the filtration step may be independently selected from dialysis, tangential flow filtration and diafiltration, preferably selected from dialysis and tangential flow filtration.

[0105] 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 and pH of the first buffer system used in step (a) are the same as the buffer system and pH of the final aqueous buffer used in step (e). For example, in this embodiment of the second aspect, only one aqueous buffer is used. In such cases, the pH of the second intermediate formulation can be adjusted by adding a polyvalent anion (such as inorganic polyphosphate) in combination with its respective countercation. For example, the pH of the second intermediate formulation can be achieved by adding a solution of pentasodium triphosphate, tetrasodium diphosphate, disodium hydrogen phosphate, or trisodium citrate.

[0106] Similarly, in certain embodiments of the second aspect, when step (I) comprises steps (a') through (e'), (g'), and (j') (and optionally one or more of steps (f'), (h'), (i'), and (k')), the first buffer system and all further buffer systems used in steps (b'), (f'), (h'), and (i') each comprise the final buffer substance used in step (j'), and preferably the buffer system and pH of the first aqueous buffer and all further aqueous buffers used in steps (b'), (f'), (h'), and (i') each are the same as the buffer system and pH of the final aqueous buffer. In one embodiment, for example, the homogeneous buffer system (i.e., each of the first and additional buffer systems used in the method) can comprise acetic acid and tris-hydroxymethylaminomethane, where acetic acid is preferred in steps (a')-(d'), and tris-hydroxymethylaminomethane is added in (e') in an amount to achieve a pH of 7-9, preferably 7.5-8.5, prior to the addition of the polyvalent anion in step (f'). In another example, a polyvalent anion can be a component of the buffer system, such as in the case of a buffer consisting of citric acid and tris-hydroxymethylaminomethane, where citric acid is added in steps (a')-(d') in combination with 0.1-2, preferably 0.2-1 equivalents of tris-hydroxymethylaminomethane, and tris-hydroxymethylaminomethane is added in (e') in an amount to achieve a pH of 7-9, preferably 7.5-8.5, and step (f') is used to add water or is not present. In this example, the materials used in steps (e') and (f') may be reversed, and the first intermediate formulation may be diluted with water before adjusting the pH with tris-hydroxymethylaminomethane solution in step (f'). In certain embodiments of the second aspect, the formulation and / or composition obtained in step (I) comprises a cryoprotectant. In certain embodiments of the second aspect, the formulation and / or composition obtained in step (I) is substantially free of a cryoprotectant.

[0107] In certain embodiments of the second aspect, the formulation and / or composition comprises water as a major component and / or the total amount of solvents other than water in the composition is less than about 1.0% (v / v), e.g., less than about 0.5% (v / v). For example, the amount of water in the formulation and / or composition can 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), at least 90% (w / w), or at least 95% (w / w). When the formulation and / or composition includes a cryoprotectant, the amount of water included in the formulation and / or composition can 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). When the formulation and / or composition is substantially free of a cryoprotectant, the amount of water included in the formulation and / or composition can be at least 95% (w / w). Additionally or alternatively, the total amount of non-aqueous solvents in the composition may be less than about 1.0% (v / v), e.g., 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 standard conditions should be considered a cryoprotectant, rather than a non-aqueous solvent. In other words, the total amount of solvents other than water contained in the composition may be less than about 1.0% (v / v), e.g., less than about 0.5% (v / v). The above optimum limits do not apply to cryoprotectants that are liquid under standard conditions.

[0108] In certain embodiments of the second aspect, the osmolality of the composition is at most about 1000×10 -3In some embodiments, the osmolality of the composition is up to about 500×10 -3 osmol / kg, e.g., up to approximately 490 × 10 -3 osmol / kg, maximum approximately 480×10 -3 osmol / kg, maximum approximately 470×10 -3 osmol / kg, maximum approximately 460×10 -3 osmol / kg, maximum approximately 450×10 -3 osmol / kg, maximum approximately 440×10 -3 osmol / kg, maximum approximately 430×10 -3 osmol / kg, maximum approximately 420×10 -3 osmol / kg, maximum approximately 410×10 -3 osmol / kg, maximum approximately 400×10 -3 osmol / kg, maximum approximately 390×10 -3 osmol / kg, maximum approximately 380×10 -3 osmol / kg, maximum approximately 370×10 -3 osmol / kg, maximum approximately 360×10 -3 osmol / kg, maximum approximately 350×10 -3 osmol / kg, maximum approximately 340×10 -3 osmol / kg, maximum approximately 330×10 -3 osmol / kg, maximum approximately 320×10 -3 osmol / kg, maximum approximately 310×10 -3 osmol / kg or up to approximately 300 × 10 -3 If the composition does not contain a cryoprotectant, the osmolality of the composition is 300 x 10 -3 Less than osmol / kg, e.g., up to about 250 × 10 -3 osmol / kg, maximum approximately 200×10 -3 osmol / kg, maximum approximately 150×10 -3 osmol / kg, maximum approximately 100×10 -3 osmol / kg, maximum approximately 50×10 -3 osmol / kg, maximum approximately 40×10 -3 osmol / kg or up to approximately 30 × 10 -3The osmol / kg of the composition may be 100 osmol / kg. If the composition includes a cryoprotectant, it is preferred that the majority of the osmolality of the composition be 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.

[0109] In one embodiment of the second aspect, the concentration of nucleic acid (e.g., RNA) in the composition is about 1 mg / L to about 500 mg / L, for example, about 1 mg / L to about 100 mg / L. In another embodiment, the concentration of nucleic acid (e.g., RNA) in the composition is about 5 mg / L to about 500 mg / L, for example, about 10 mg / L to about 400 mg / L, about 10 mg / L to about 300 mg / L, about 10 mg / L to about 200 mg / L, about 10 mg / L to about 150 mg / L, or about 10 mg / L to about 100 mg / L, preferably about 10 mg / L to about 140 mg / L, more preferably about 20 mg / L to about 130 mg / L, and more preferably about 30 mg / L to about 120 mg / L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is about 5 mg / L to about 150 mg / L, e.g., about 10 mg / L to about 140 mg / L, about 20 mg / L to about 130 mg / L, about 25 mg / L to about 125 mg / L, about 30 mg / L to about 120 mg / L, about 35 mg / L to about 115 mg / L, about 40 mg / L to about 110 mg / L, about 45 mg / L to about 105 mg / L, or about 50 mg / L to about 100 mg / L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is 1 mg / L to about 50 mg / L, or about 10 mg / L to about 100 mg / L.

[0110] In certain embodiments of the second aspect (particularly those embodiments in which the method comprises freezing the (II) formulation at or below about -10°C, and thus the composition produced 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.

[0111] In certain embodiments of the second aspect (particularly those embodiments in which the method does not include step (II) and therefore 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.

[0112] In some embodiments of the second aspect, the molar ratio of the polyvalent anion or salt thereof to the cationically ionizable lipid 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 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 in which the method includes freezing the (II) formulation below about -10°C, and the composition produced thereby is in frozen form), the molar ratio of the polyvalent anion or salt thereof to the cationically ionizable lipid 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 nucleic acid (particularly RNA) in the composition is about 1 mg / L to about 50 mg / L. In certain embodiments of the second aspect (particularly those in which the method does not include step (II) and the composition produced by the method is therefore in liquid form), the molar ratio of polyvalent anion or salt thereof to cationically ionizable lipid 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 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 salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartarate, malate, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof.

[0113] In some embodiments of the second aspect, the polyvalent anion is 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 (e.g., a linear triphosphate or a salt thereof) as defined herein, and the molar ratio of the inorganic polyphosphate or a salt thereof to the cationically ionizable lipid 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. 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 cationically ionizable lipid 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 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 certain embodiments of the second aspect (particularly those embodiments in which the method (II) comprises freezing the formulation to about −10° C. or below, and therefore the composition produced by the method is in frozen form), the inorganic polyphosphate is a linear inorganic polyphosphate (particularly a triphosphate) or a salt thereof, the molar ratio of inorganic polyphosphate or its salt to cationically ionizable lipid 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 nucleic acid (particularly RNA) in the composition is from about 1 mg / L to about 50 mg / L. In certain embodiments of the second aspect (particularly those embodiments in which the method does not include step (II) and therefore the composition produced by the method is in liquid form), the inorganic polyphosphate or salt thereof is a linear inorganic polyphosphate (particularly a triphosphate) or salt thereof, the molar ratio of inorganic polyphosphate or salt thereof to cationically ionizable lipid 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 nucleic acid (particularly RNA) in the composition is from about 10 mg / L to about 100 mg / L.

[0114] In certain embodiments of the second aspect, the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

[0115] In certain embodiments of the second aspect, the cationically ionizable lipid has the formula (X): [ka] 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 37 is as defined herein. In some embodiments, the cationically ionizable lipid 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-dioleyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14). In some embodiments, the cationically ionizable lipid is a lipid having structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., a lipid having structure G). In certain embodiments, the cationically ionizable lipid is a lipid having structure D.

[0116] In certain embodiments of the second aspect, the cationically ionizable lipid has formula (XI): [ka] wherein R1, R2, R3, R4, L2, G2 and m are as defined herein. In some embodiments, the cationically ionizable lipid is selected from structures (XIV-1), (XIV-2) and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-1. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-2. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-3.

[0117] In some embodiments of the second aspect, the cationically ionizable lipid is completely or partially replaced with a cationic lipid. In some embodiments, the cationically ionizable lipid is selected from structures XV-1 to XV-6 (shown herein). In these embodiments in which the cationically ionizable lipid is completely replaced with a cationic lipid, the molar ratio of the polyvalent anion (such as inorganic polyphosphate) or a salt thereof to the cationically ionizable lipid is replaced with the molar ratio of the polyvalent anion (such as inorganic polyphosphate) or a salt thereof to the cationic lipid. In some embodiments, the molar ratio of the polyvalent anion (such as inorganic polyphosphate) or its salt 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 (such as inorganic polyphosphate) or its salt to the cationic lipid is at least about 2:3, e.g., at least about 4:3). In those embodiments in which the cationically ionizable lipid is partially replaced with a cationic lipid, the molar ratio of polyvalent anion (such as inorganic polyphosphate) to cationically ionizable lipid is replaced with the molar ratio of polyvalent anion (such as inorganic polyphosphate) to the sum of cationic lipid and cationically ionizable lipid.In some embodiments, the molar ratio of the polyvalent anion (such as inorganic polyphosphate) to the sum of the cationic lipid and the cationically ionizable 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 (such as inorganic polyphosphate) to the sum of the cationic lipid and the cationically ionizable lipid is at least about 2:3, e.g., at least about 4:3).

[0118] In certain embodiments of the second aspect, the cationically ionizable lipids comprise from about 20 mol% to about 75 mol%, e.g., from about 40 mol% to about 70 mol%, from about 45 mol% to about 65 mol%, or from about 50 mol% to about 60 mol% of the total lipids present in the organic solution (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or from about 20 mol% to about 40 mol%, from about 25 mol% to about 40 mol%, or from about 25 mol% to about 35 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high). In certain embodiments of the second aspect, the cationically ionizable lipid comprises about 20 mol% to about 75 mol%, e.g., about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, or about 50 mol% to about 60 mol% of the total lipid present in the composition (particularly in embodiments where the relative amounts of ionizable lipid and neutral lipid are high and the relative amount of steroid is low); or about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol% (particularly in embodiments where the relative amounts of ionizable lipid and neutral lipid are low and the relative amount of steroid is high). In those embodiments where the cationically ionizable lipid is fully or partially substituted with cationic lipid, the same ranges (e.g., about 20 mol% to about 75 mol%) as defined above for the total of cationically ionizable lipid and cationic lipid preferably apply.

[0119] In certain embodiments of the second aspect, the steroid comprises a sterol. In some preferred embodiments of the second aspect, the steroid comprises or is cholesterol.

[0120] In certain embodiments of the second aspect, the steroid constitutes about 15 mol% to about 60 mol%, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% of the total lipids present in the organic solution (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high). In certain embodiments of the second aspect, the steroid constitutes about 15 mol% to about 60 mol%, e.g., about 15 mol% to about 40 mol%, about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol% of the total lipids present in the composition (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low); or about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, or about 45 mol% to about 60 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high).

[0121] In some embodiments of the second aspect, the neutral lipid is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. In some embodiments, the phospholipid has a T greater than 30°C. 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.

[0122] In certain embodiments of the second aspect, the neutral lipids constitute from about 5 mol% to about 25 mol% of the total lipids present in the organic solution (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low), e.g., from about 15 mol% to about 25 mol% or from about 17 mol% to about 21 mol%; or from about 5 mol% to about 15 mol% or from about 7 mol% to about 14 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high). In certain embodiments of the second aspect, the neutral lipids constitute from about 5 mol% to about 25 mol% of the total lipids present in the composition (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are high and the relative amount of steroids is low), e.g., from about 15 mol% to about 25 mol% or from about 17 mol% to about 21 mol%; or from about 5 mol% to about 15 mol% or from about 7 mol% to about 14 mol% (particularly in embodiments where the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high).

[0123] In certain embodiments of the second aspect, the cationically ionizable lipids comprise from about 20 mol% to about 70 mol% of the total lipids present in the organic solution; the steroids comprise from about 15 mol% to about 60 mol% of the total lipids present in the organic solution; and the neutral lipids (e.g., phospholipids) comprise from about 5 mol% to about 25 mol% of the total lipids present in the organic solution.

[0124] In certain embodiments of the second aspect, the cationically ionizable lipids comprise 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; the steroids (preferably cholesterol) comprise 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; and the neutral lipids (preferably phospholipids) comprise 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. In certain embodiments of the second aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 15 mol% to about 25 mol%, e.g., about 17 mol% to about 21 mol%, of the total lipid present in the composition. These embodiments of the second aspect, i.e., embodiments for producing nucleic acid compositions with high relative amounts of ionizable lipid and neutral lipid and low relative amounts of steroid, are particularly suitable for transfecting cells in the presence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0125] In certain embodiments of the second aspect, in which the relative amounts of ionizable lipids and neutral lipids are higher and the relative amount of steroids is lower, the molar ratio of steroids to neutral lipids is up to 2.5, preferably 1 to 2.5. Thus, in certain embodiments of the second aspect, the cationically ionizable lipids comprise 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 composition); the steroids comprise 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 composition); the neutral lipids comprise 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 composition); and the molar ratio of steroids to neutral lipids is up to 2.5, preferably 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0126] In some alternative embodiments of the second aspect, the cationically ionizable lipid comprises 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 lipid present in the organic solution; the steroid (preferably cholesterol) comprises 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 lipid present in the organic solution; and the neutral lipid (preferably a phospholipid) comprises about 5 mol% to about 15 mol%, e.g., about 7 mol% to about 14 mol%, of the total lipid present in the organic solution. In certain embodiments of the second aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 5 mol% to about 15 mol%, e.g., about 7 mol% to about 14 mol%, of the total lipid present in the composition. These embodiments of the second aspect, i.e., those for producing nucleic acid compositions with low relative amounts of ionizable lipid and neutral lipid and high relative amounts of steroid, are particularly suitable for transfecting cells in the absence of serum. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0127] In certain embodiments of the second aspect, in which the relative amounts of ionizable lipids and neutral lipids are low and the relative amount of steroids is high, the molar ratio of steroid to neutral lipids is at least 3.0, preferably said ratio is between 3.0 and 10.0, for example between 5.0 and 7.0. Thus, in certain embodiments of the second aspect, the cationically ionizable lipid comprises 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 lipid present in the organic solution (or composition); the steroid comprises 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 lipid present in the organic solution (or composition); the neutral lipid comprises about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipid present in the organic solution (or composition); and the molar ratio of steroid to neutral lipid is at least 3.0, preferably 3.0 to 10.0, e.g., 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0128] In certain embodiments of the second aspect, the organic solution / composition further comprises one or more additional lipids. In these embodiments in which cationic lipids are present, the sum of the amount of (1) cationically ionizable lipid and the amount of (2) cationic lipid is used in the calculation. For example, if the amount of cationically ionizable lipid in the organic solution / composition is about 20 mol% to about 70 mol%, and the organic solution / composition also contains cationic lipid, then the sum of the amount of (1) cationically ionizable lipid and the amount of (2) cationic lipid should be about 20 mol% to about 70 mol%.

[0129] In certain embodiments of the second aspect, the organic solution, the composition, or both, are substantially free of lipids containing polyethylene glycol (PEG). In certain embodiments of the second aspect, the organic solution, the composition, or both, are substantially free of compounds containing PEG. In certain embodiments of the second aspect, the organic solution, the composition, or both, are substantially free of PEG. In some of these embodiments, the polyvalent anion or salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof. In some of these embodiments, the polyvalent anion is inorganic polyphosphate or a salt thereof.

[0130] In some embodiments of the second aspect, the organic solution is also substantially free of another polymer-conjugated lipid. In some embodiments, the other polymer-conjugated lipid is a polysarcosine-conjugated lipid. Thus, 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 polymer-conjugated lipids (including PEG-lipids and polysarcosine-conjugated lipids). In some of these embodiments, the polyvalent anion or a salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof. In some of these embodiments, the polyvalent anion is inorganic polyphosphate or a salt thereof.

[0131] In some embodiments of the second aspect, the lipids contained in the organic solution are exclusively cationically ionizable lipids, steroids, and neutral lipids, particularly cationically ionizable lipids, steroids, and phospholipids. 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 lipids contained in the composition are exclusively cationically ionizable lipids, steroids, and neutral lipids, particularly cationically ionizable lipids, steroids, and phospholipids. In some of these embodiments, the polyvalent anion or a salt thereof is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinic acid, glutaric acid, tartaric acid, malic acid, citric acid, salts thereof, and mixtures thereof. In some of these embodiments, the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, citric acid, and salts thereof. In some of these embodiments, the polyvalent anion is inorganic polyphosphate or a salt thereof.

[0132] In certain embodiments of the second aspect, the particle comprises or is selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPX), and mixtures thereof. In certain embodiments, the particle comprises or is LNPs. In certain embodiments, the particle comprises or is liposomes. In certain embodiments, the particle comprises or is LPX. In certain embodiments, the particle comprises or is a mixture of LNPs and liposomes. In certain embodiments, the particle comprises or is a mixture of LNPs and LPX. In certain embodiments, the particle comprises or is a mixture of liposomes and LPX. In certain embodiments, the particle comprises or is a mixture of LNPs, liposomes, and LPX.

[0133] In certain embodiments of the second aspect, the particles comprise essentially all of the lipids present in the composition (particularly all of the cationically ionizable lipids, steroids and neutral lipids).

[0134] In certain embodiments of the second aspect, the aqueous phase is substantially free of cationically ionizable lipids, steroids, and neutral lipids (eg, substantially free of lipids).

[0135] In certain 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 acid (particularly RNA) present in the composition. In certain embodiments, the particles comprise at least 75%, preferably at least 85%, of the nucleic acid (particularly RNA) present in the composition.

[0136] In certain embodiments of the second aspect, the aqueous phase is substantially free of nucleic acids.

[0137] In certain embodiments of the second aspect, the nucleic acid (such as RNA) is encapsulated within or associated with the particle.

[0138] 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 polyvalent anions (e.g., inorganic polyphosphates) present in the composition are associated with particles. In some embodiments, at least 20%, more preferably at least 50%, of the polyvalent anions (e.g., inorganic polyphosphates) present in the composition are associated with particles.

[0139] In some embodiments of the second aspect, the particles have a size of about 30 nm to about 500 nm, hi some embodiments, the particles have a size of about 50 nm to about 150 nm.

[0140] In certain embodiments of the second aspect, the nucleic acid is DNA.

[0141] In certain embodiments of the second aspect, the nucleic acid is RNA (eg, mRNA or inhibitory RNA, such as siRNA).

[0142] In certain embodiments of the second aspect, the RNA (e.g., mRNA) (i) contains modified nucleosides in place of uridine; (ii) has a coding sequence that is codon-optimized; and / or (iii) has a coding sequence that has an increased G / C content compared to the wild-type coding sequence. In certain embodiments, the modified nucleosides are selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0143] In certain embodiments of the second aspect, the RNA (e.g., mRNA) comprises 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 polyA sequence. In certain embodiments, the RNA (e.g., mRNA) comprises all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In certain embodiments, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

[0144] In certain embodiments of the second aspect, the RNA (such as mRNA) encodes one or more polypeptides. In certain 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.

[0145] In certain 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 the protein, or an immunogenic fragment of the protein or its immunogenic variant. In certain embodiments, the pathogen is a pathogen that causes an infectious disease.

[0146] In certain embodiments of the second aspect, the pharmaceutically active polypeptide and / or 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 its immunogenic variant. In certain embodiments, the RNA (such as an mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant.

[0147] In some embodiments of the second aspect, nucleic acid is inhibitory RNA (such as siRNA), and selectively hybridizes with target mRNA and / or is specific to target mRNA.In some embodiments, target mRNA comprises ORF encoding pharmaceutically active peptide or polypeptide, and particularly comprises ORF encoding pharmaceutically active peptide or polypeptide, whose expression (especially, for example, compared with the expression in healthy subjects, increased expression) is associated with disease.In some embodiments, target mRNA comprises ORF encoding pharmaceutically active peptide or polypeptide, whose expression (especially, for example, compared with the expression in healthy subjects, increased expression) is associated with cancer.

[0148] It will be understood that any embodiment described herein in the context of the first aspect may be applied to any embodiment of the second aspect.

[0149] In a third aspect, the present disclosure provides a method for storing a composition, comprising preparing a composition according to the method of the second aspect and storing the composition at a temperature ranging from about −90° C. to about −10° C., e.g., from about −90° C. to about −40° C., from about −40° C. to about −25° C., from 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, 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, 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, and more preferably at least 6 months. In some embodiments of the third aspect, the composition can be stored at −70° C.

[0150] In certain embodiments of the third aspect, the composition comprises a cryoprotectant. In certain embodiments of the third aspect, the composition is substantially free of cryoprotectants.

[0151] In certain embodiments of the third aspect, the method of storing the composition includes producing the composition according to the method of the second aspect, including step (II) (i.e., freezing the formulation to about -10°C or below); storing the frozen composition at a temperature in the range of about -90°C to about -10°C for a period of time (e.g., at least 1 week); and storing the frozen composition at a temperature in the range of about 0°C to about 20°C for a period of time (e.g., at least 4 weeks).

[0152] It will be understood that any embodiment described herein in the context of the first or second aspect may be applied to any embodiment of the third aspect.

[0153] In a fourth aspect, the present disclosure provides a method for storing a liquid composition, comprising preparing a liquid composition according to the method of the second aspect and storing the liquid composition at a temperature ranging from about 0°C to about 20°C, e.g., from about 1°C to about 15°C, from about 2°C to about 10°C, or from 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, and more preferably at least 6 months.

[0154] In certain embodiments of the fourth aspect, the composition comprises a cryoprotectant. In some preferred embodiments of the fourth aspect, the composition is substantially free of cryoprotectants.

[0155] It will be understood that any embodiment described herein in the context of the first or second aspect may be applied to any embodiment of the fourth aspect.

[0156] In a fifth aspect, the present disclosure provides a composition producible by the method of the second, third, or fourth aspect. In some embodiments of the fifth aspect, the composition may be in a frozen form, which may be stored at a temperature of about -90°C or higher, for example, about -90°C to about -10°C. For example, the frozen composition of the fifth aspect may be stored at a temperature ranging from about -90°C to about -40°C, or from about -40°C to about -25°C, or from about -25°C to about -10°C, or at a temperature of about -20°C. In some embodiments of the fifth aspect, the composition may be 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. For example, the frozen composition may 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, and more preferably at least 6 months.

[0157] In certain embodiments of the fifth aspect, the composition comprises a cryoprotectant. In certain embodiments of the fifth aspect, the composition is substantially free of cryoprotectants.

[0158] In certain embodiments of the fifth aspect, wherein the composition is in frozen form, the nucleic acid integrity (particularly RNA integrity) of the frozen composition after thawing is at least 90%, at least 95%, at least 97%, at least 98% or substantially 100% compared to the nucleic acid integrity (particularly RNA integrity) of the composition before the composition was frozen, e.g., after thawing a frozen composition stored at -20°C.

[0159] In one embodiment, the initial nucleic acid integrity (particularly the initial RNA integrity) of the composition (i.e., after manufacture but before freezing) is at least 50%, and the nucleic acid integrity (particularly the 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%, and more preferably substantially 100% of the initial nucleic acid integrity (particularly the initial RNA integrity).

[0160] Additionally or alternatively, in certain embodiments of the fifth aspect, when the composition is in frozen form, the size (Z) of nucleic acid (such as RNA) particles after thawing the frozen composition is 平均 ) (and / or size distribution and / or polydispersity index (PDI)) refers to the size (Z) of nucleic acid (e.g., RNA) particles in the composition prior to freezing. 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid (such as RNA) particles after thawing the frozen composition is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm. In one embodiment, the PDI of nucleic acid (such as RNA) particles after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In one embodiment, the size (Z 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the size (Z) of nucleic acid (RNA, etc.) particles after thawing the frozen composition 平均 ) (and / or size distribution and / or PDI) are the size (Z 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid (such as RNA) particles after thawing the frozen composition is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the PDI of the nucleic acid (RNA, etc.) particles after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0161] In certain embodiments, the size and nucleic acid integrity (e.g., RNA) particles of the composition 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, more preferably after five or more freeze / thaw cycles, are essentially equal to the size and nucleic acid integrity (e.g., RNA) particles of the initial composition (i.e., before the composition was first frozen).

[0162] In another embodiment of the fifth aspect, the composition is in liquid form.

[0163] In some embodiments of the fifth aspect, in which the composition is in liquid form, the nucleic acid integrity (especially RNA integrity) of the liquid composition is such that it can achieve a desired effect, such as inducing an immune response, when stored, for example, at 0°C or above for at least one week. For example, when stored at 0°C or above 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 (especially RNA integrity) of the liquid composition can be at least 90%, at least 95%, at least 97%, or at least 98% of the nucleic acid integrity (especially RNA integrity) before storage. In some embodiments, the nucleic acid integrity (especially RNA integrity) of the composition after storage for at least four weeks, preferably at 0°C or above, for example, at a temperature of about 2°C to about 8°C, is at least 90%, at least 95%, at least 97%, or at least 98% of the nucleic acid integrity (especially RNA integrity) before storage.

[0164] In certain embodiments, the initial nucleic acid integrity (particularly the initial RNA integrity) of the liquid composition (i.e., after manufacture but before storage) is at least 50%, and preferably after storage at a temperature of 0°C or above, e.g., about 2°C to about 8°C, 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, or at least 3 months), the nucleic acid integrity (particularly the RNA integrity) of the liquid composition is at least 90% of the initial nucleic acid integrity (particularly the initial RNA integrity).

[0165] Additionally or alternatively, in certain embodiments of the fifth aspect wherein the composition is in liquid form, the size (Z) of nucleic acid (e.g., RNA) particles in the liquid composition is determined to be 0.05 or less when stored, e.g., at a temperature of 0°C or higher, for at least 1 week. 平均 ) (and / or size distribution and / or polydispersity index (PDI)) are such that a desired effect, such as the effect of inducing an immune response, can be achieved. For example, when stored at a temperature of 0° C. or higher for at least one week, the size (Z 平均 ) (and / or size distribution and / or polydispersity index (PDI)) is used to determine the initial composition, i.e., the size (Z 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid (e.g., RNA) particles after storage of the liquid composition, e.g., at 0°C or above for at least 1 week, is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm. In one embodiment, the PDI of the nucleic acid (e.g., RNA) particles after storing 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. In one embodiment, the size (Z) of the nucleic acid (e.g., RNA) particles in the nucleic acid liquid composition after storing the liquid composition, for example, at 0°C or higher for at least one week, is 平均) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the size (Z 平均 ) (and / or size distribution and / or PDI) are the size (Z 平均 ) (and / or size distribution and / or PDI). In some embodiments, the size (Z) of nucleic acid (e.g., RNA) particles after storage of the liquid composition, e.g., at 0°C or above for at least 1 week, is essentially equal to 平均 ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, and more preferably about 40 nm to about 120 nm, and the PDI of the nucleic acid (e.g., RNA) particles after storing 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).

[0166] It will be understood that any embodiment described herein in the context of the first, second, third, or fourth aspect may be applied to any embodiment of the fifth aspect.

[0167] In a sixth aspect, the present disclosure provides a method of producing a ready-to-use pharmaceutical composition, comprising the steps of providing a frozen composition produced by the method of the second, third or fourth aspect, and thawing the frozen composition, thereby obtaining a ready-to-use pharmaceutical composition.

[0168] It will be understood that any embodiment described herein in the context of the first, second, third, fourth or fifth aspect may be applied to any embodiment of the sixth aspect.

[0169] In a seventh aspect, the present disclosure provides a method of making a ready-to-use pharmaceutical composition, comprising providing a liquid composition made by the method of the second, third or fourth aspect, thereby obtaining a ready-to-use pharmaceutical composition.

[0170] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspect may be applied to any embodiment of the seventh aspect.

[0171] In an eighth aspect, the present disclosure provides a ready-to-use pharmaceutical composition producible by the method of the sixth or seventh aspect.

[0172] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth or seventh aspect may be applied to any embodiment of the eighth aspect.

[0173] In a ninth aspect, the present disclosure provides a composition of any one of the first, fifth and eighth aspects for use in therapy.

[0174] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect may be applied to any embodiment of the ninth aspect.

[0175] In a tenth aspect, the present disclosure provides a composition of any one of the first, fifth, eighth, and ninth aspects for use in inducing an immune response.

[0176] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspects may be applied to any embodiment of the tenth aspect.

[0177] In an eleventh aspect, the present disclosure provides a method for transfecting cells, comprising adding a composition according to any one of the first, fifth, or eighth aspects to cells and incubating the mixture of the composition and the cells for a sufficient period of time. In some embodiments, particularly in those in which the nucleic acid is DNA or RNA (e.g., mRNA) and encodes a pharmaceutically active protein, the mixture of the composition and the cells is incubated for a sufficient period of time to allow expression of the pharmaceutically active protein. In some embodiments, particularly in those in which the nucleic acid is an inhibitory RNA (e.g., siRNA) against a target mRNA, the mixture of the composition and the cells is incubated for a sufficient period of time to allow inhibition of transcription and / or translation of the target mRNA. In some embodiments, the amount of time sufficient is at least 1 hour (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 (e.g., up to about 36 hours or up to about 24 hours). In some embodiments of the eleventh aspect, the mixture of the composition and the cells is incubated in the presence of serum (e.g., human serum).

[0178] In certain embodiments of the eleventh aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 15 mol% to about 25 mol%, e.g., about 17 mol% to about 21 mol%, of the total lipid present in the composition. In these embodiments (i.e., compositions with high relative amounts of ionizable lipid and neutral lipid and low relative amounts of steroid), incubation of the composition and cell mixture is preferably performed 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.

[0179] In certain embodiments of the eleventh aspect relating to compositions containing a higher relative amount of ionizable lipids and neutral lipids and a lower relative amount of steroids, the molar ratio of steroid to neutral lipids is up to 2.5, preferably 1 to 2.5. Thus, in certain embodiments of the eleventh aspect, the cationically ionizable lipids comprise 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 comprise 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 comprise 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 steroid to neutral lipids is up to 2.5, preferably 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0180] In some alternative embodiments of the eleventh aspect, the cationically ionizable lipids comprise 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 steroids (preferably cholesterol) comprise 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 lipids (preferably phospholipids) comprise about 5 mol% to about 15 mol%, e.g., about 7 mol% to about 14 mol%, of the total lipids present in the composition. In these embodiments (i.e., compositions with low relative amounts of ionizable lipids and neutral lipids and high relative amounts of steroids), the incubation of the composition and cell mixture can be performed in the presence or absence of serum, e.g., in the absence of serum.

[0181] In certain embodiments of the eleventh aspect relating to a composition having a low relative amount of ionizable lipid and neutral lipid and a high relative amount of steroid, the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is between 3.0 and 10.0, for example between 5.0 and 7.0. Thus, in certain embodiments of the eleventh aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid comprises 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 lipid present in the composition; the neutral lipid comprises about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipid present in the composition; and the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, e.g., 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0182] In certain embodiments of the eleventh aspect, the method is performed in vivo (i.e., the cells form part of an organ, tissue, and / or organism of a subject). In certain embodiments of the eleventh aspect, the method is performed in vitro (i.e., the cells do not form part of an organ, tissue, and / or organism of a subject, e.g., the cells are in ex vivo cell culture).

[0183] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspects may be applied to any embodiment of the eleventh aspect.

[0184] In a twelfth aspect, the present disclosure provides a use of the composition of any one of the first, fifth, or eighth aspects for transfecting a cell. In certain embodiments of the twelfth aspect, the transfection of the cell is performed in the presence of serum (such as human serum).

[0185] In certain embodiments of the twelfth aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises about 20 mol% to about 40 mol% of the total lipid present in the composition; and the neutral lipid (preferably phospholipid) comprises about 15 mol% to about 25 mol% of the total lipid present in the composition. In these embodiments (i.e., compositions with high relative amounts of ionizable lipid and neutral lipid and low relative amounts of steroid), incubation of the composition and cell mixture is preferably performed 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.

[0186] In certain embodiments of the twelfth aspect, which relates to a composition having a high relative amount of ionizable lipid and neutral lipid and a low relative amount of steroid, the molar ratio of steroid to neutral lipid is up to 2.5, preferably 1 to 2.5. Thus, in certain embodiments of the twelfth aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid comprises 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 lipid present in the composition; the neutral lipid comprises about 15 mol% to about 25 mol% (e.g., about 17 mol% to about 21 mol%) of the total lipid present in the composition; and the molar ratio of steroid to neutral lipid is up to 2.5, preferably 1 to 2.5. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0187] In some other embodiments of the twelfth aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid (preferably cholesterol) comprises 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 lipid present in the composition; and the neutral lipid (preferably a phospholipid) comprises about 5 mol% to about 15 mol%, e.g., about 7 mol% to about 14 mol%, of the total lipid present in the composition. In these embodiments (i.e., compositions with low relative amounts of ionizable lipid and neutral lipid and high relative amounts of steroid), the incubation of the composition and cell mixture can be performed in the presence or absence of serum, e.g., in the absence of serum.

[0188] In certain embodiments of the twelfth aspect relating to a composition having a low relative amount of ionizable lipid and neutral lipid and a high relative amount of steroid, the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is between 3.0 and 10.0, for example, between 5.0 and 7.0. Thus, in certain embodiments of the twelfth aspect, the cationically ionizable lipid comprises 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 lipid present in the composition; the steroid comprises 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 lipid present in the composition; the neutral lipid comprises about 5 mol% to about 15 mol% (e.g., about 7 mol% to about 14 mol%) of the total lipid present in the composition; and the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, e.g., 5.0 to 7.0. In some of these embodiments, the steroid is cholesterol and the neutral lipid is a phospholipid.

[0189] In certain embodiments of the twelfth aspect, the use is an in vivo use (i.e., the cells form part of an organ, tissue, and / or organism of a subject). In certain embodiments of the twelfth aspect, the use is an in vitro use (i.e., the cells do not form part of an organ, tissue, and / or organism of a subject, e.g., the cells are in ex vivo cell culture).

[0190] It will be understood that any embodiment described herein in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspects may be applied to any embodiment of the twelfth aspect.

[0191] In a further 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, such as for inducing immune response.In some embodiments, the kit is used for inducing immune response against pathogens, such as for treating or preventing infectious diseases.

[0192] Further itemized embodiments are as follows: 1. A composition comprising: (i) a nucleic acid; (ii) a cationically ionizable lipid; (iii) a steroid; (iv) a neutral lipid; and (v) a polyvalent anion.

[0193] 2. The composition according to item 1, wherein the polyvalent anion comprises 2 to 10 negative charges, optionally 2 to 5 negative charges.

[0194] 3. The composition of claim 1 or 2, wherein the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartarate, malate, citric acid, or a mixture thereof.

[0195] 4. The composition according to any one of items 1 to 3, wherein the polyvalent anion is inorganic polyphosphate.

[0196] 4a. Inorganic polyphosphate is represented by the formula [Px O (3x+1) ] y Item 5. The composition of item 4, comprising: wherein x is an integer and is at least 3; and y is an anionic charge.

[0197] 4b. The composition of claim 4 or 4a, wherein the inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof, preferably selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof, and more preferably the inorganic polyphosphate is triphosphate.

[0198] 4c. The composition according to any one of items 4 to 4b, wherein the inorganic polyphosphate is a linear inorganic polyphosphate, for example, a linear inorganic triphosphate.

[0199] 5. The composition according to any one of paragraphs 1 to 4c, wherein the molar ratio of (v) polyvalent anion (e.g., inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 1:2, preferably at least about 2:3, for example at least about 4:3.

[0200] 6. The composition according to any one of items 1 to 5, which is substantially free of lipids containing polyethylene glycol (PEG), preferably substantially free of any compound containing PEG, and more preferably substantially free of PEG.

[0201] 6a. The composition according to any one of items 1 to 6, which is substantially free of polymer-conjugated lipids.

[0202] 7. The composition according to any one of items 1 to 6a, wherein the pH of the composition is about 4.0 to about 8.0, preferably about 4.5 to about 8.0, for example, about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 6.8 to about 7.9, or about 7.0 to about 7.8.

[0203] 8. The composition according to any one of items 1 to 7, wherein water is the main 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).

[0204] 9. The osmotic pressure of the composition is at most about 1000 x 10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg ~ approx. 500 x 10 -3 osmol / kg, more preferably about 300×10 -3 Item 9. The composition according to any one of items 1 to 8, wherein the concentration is osmol / kg.

[0205] 10. The composition according to any one of Items 1 to 9, 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.

[0206] 11. The composition of any one of paragraphs 1 to 10, wherein the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

[0207] 12. The cationically ionizable lipid is represented by the formula (X): [ka] [During the ceremony, L 10 and L 20 One of the following 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 20The other 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- or a direct bond; G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C 2-12 alkenylene; G 3 is C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 is cycloalkenylene; R a is H or C 1-12 is alkyl; R 35 and R 36 are each independently C 6-24 Alkyl or C 6-24 is alkenyl; R 37 H, OR 50 , CN, -C(=O)OR 40 , -OC(=O)R 40 or -NR 50 C(=O)R 40 and; R 40 is C 1-12 is alkyl; R 50 is H or C 1-6 is alkyl; and x is 0, 1, or 2. Item 12. The composition according to any one of Items 1 to 11, which has the structure: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0208] 13. The cationic or cationically ionizable lipid has the formula (XI): [ka] [During the ceremony, each of R1 and R2 is independently R5 or -G1-L1-R6, where at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is independently C 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 cycloalkyl; Each of R5 and R6 is independently an acyclic hydrocarbyl 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 independently represents -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x selected from the group consisting of -, -SS-, -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 is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2. Item 12. The composition according to any one of items 1 to 11, having the structure:

[0209] 14. The composition according to any one of items 1 to 13, wherein the cationically ionizable lipid constitutes about 20 mol% to about 75 mol%, 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% of the total lipid present in the composition.

[0210] 15. The composition according to any one of items 1 to 14, wherein the steroid comprises a sterol such as cholesterol.

[0211] 16. The composition according to any one of items 1 to 15, wherein the steroid constitutes about 15 mol% to about 60 mol%, 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% of the total lipids present in the composition.

[0212] 17. The composition according to any one of items 1 to 16, wherein the neutral lipid is a phospholipid, preferably selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, and more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE), and dipalmitoyl-phosphatidylethanolamine (DPPE).

[0213] 18. The composition according to any one of items 1 to 17, wherein the neutral lipid constitutes about 5 mol% to about 25 mol%, 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% of the total lipid present in the composition.

[0214] 19. The composition of any one of paragraphs 1 to 18, wherein the cationically ionizable lipids constitute about 20 mol% to about 70 mol% of the total lipids present in the composition; the steroids constitute about 15 mol% to about 60 mol% of the total lipids present in the composition; and the neutral lipids (e.g., phospholipids) constitute about 5 mol% to about 25 mol% of the total lipids present in the composition.

[0215] 20. The composition according to any one of items 1 to 19, wherein the cationically ionizable lipid constitutes 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 total lipid present in the composition; the steroid constitutes 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 total lipid present in the composition; and the neutral lipid constitutes about 15 mol% to about 25 mol%, for example, about 17 mol% to about 21 mol%, of the total lipid present in the composition.

[0216] 20a. The composition according to any one of items 1 to 20, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0217] 21. The composition according to any one of items 1 to 20a, wherein the molar ratio of steroid to neutral lipid is up to 2.5, preferably the ratio is 1 to 2.5.

[0218] 22. The composition according to any one of items 1 to 19, wherein the cationically ionizable lipid constitutes 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 lipid present in the composition; the steroid constitutes 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 lipid present in the composition; and the neutral lipid constitutes about 5 mol% to about 15 mol%, for example, about 7 mol% to about 14 mol%, of the total lipid present in the composition.

[0219] 22a. The composition according to item 22, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0220] 23. The composition according to any one of paragraphs 1 to 19, 22 and 22a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, for example 5.0 to 7.0.

[0221] 24. The composition of any one of paragraphs 1 to 23, wherein the composition comprises particles dispersed in an aqueous phase, the particles comprising at least a portion of a nucleic acid, at least a portion of a cationically ionizable lipid, at least a portion of a steroid, and at least a portion of a neutral lipid; and at least a portion of a polyvalent anion (e.g., inorganic polyphosphate) is associated with the particles.

[0222] 25. The composition according to item 24, wherein the particle is selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.

[0223] 25a. The composition of paragraph 24 or paragraph 25, wherein the particles comprise essentially all of the cationically ionizable lipids, steroids, and neutral lipids present in the composition.

[0224] 25b. The composition of any one of paragraphs 24 to 25a, wherein the aqueous phase is substantially free of cationically ionizable lipids, steroids, and neutral lipids.

[0225] 26. The composition of any one of paragraphs 24 to 25b, wherein the particles comprise at least 50%, preferably at least 75%, and more preferably at least 85% of the nucleic acids present in the composition.

[0226] 26a. The composition according to any one of items 24 to 26, wherein the aqueous phase is substantially free of nucleic acids.

[0227] 27. The composition of any one of paragraphs 24 to 26a, wherein at least 10%, preferably at least 20%, and more preferably at least 50% of the polyvalent anions (e.g., inorganic polyphosphates) present in the composition are associated with particles.

[0228] 28. The composition according to any one of items 24 to 27, wherein the particles have a size of about 30 nm to about 500 nm, for example, about 50 nm to about 150 nm.

[0229] 29. The composition of any one of items 1 to 28, wherein the nucleic acid is RNA, preferably mRNA.

[0230] 29a. The composition of any one of items 1 to 28, wherein the nucleic acid is DNA.

[0231] 29b. The composition of any one of paragraphs 1 to 28, wherein the nucleic acid is an inhibitory RNA such as an siRNA.

[0232] 30. The composition of paragraph 29, wherein the RNA (1) contains modified nucleosides in place of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (2) has a coding sequence that is codon-optimized; and / or (3) has a coding sequence that has an increased G / C content compared to the wild-type coding sequence.

[0233] 31. The composition of paragraph 29 or 30, wherein the RNA comprises at least one, preferably all, of the following components: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

[0234] 32. The composition of paragraph 31, wherein the polyA sequence comprises at least 100 A nucleotides, and wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

[0235] 33. The composition of item 31 or 32, wherein the 5' cap is a Cap 1 structure or a Cap 2 structure.

[0236] 34. The composition of any one of paragraphs 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.

[0237] 35. The composition of paragraph 34, wherein the pharmaceutically active polypeptide and / or 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.

[0238] 36. The composition of clause 34 or clause 35, wherein 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 the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0239] 37. The composition according to any one of items 1 to 36, wherein the composition is in liquid form and preferably at a temperature of about 2°C to about 10°C.

[0240] 37a. The composition according to Item 37, wherein the concentration of the nucleic acid in the composition is about 10 mg / L to about 100 mg / L.

[0241] 38. The composition of any one of paragraphs 1 to 37a, 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, is at least 90% compared to the nucleic acid integrity before storage.

[0242] 38a. The composition of any one of paragraphs 1 to 38, wherein the nucleic acid integrity of the composition after storage for at least 4 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.

[0243] 38b. The composition of any one of paragraphs 1 to 38a, wherein the nucleic acid integrity of the composition after storage for at least 3 months, preferably at a temperature of about 2°C to about 8°C, is at least 90% compared to the nucleic acid integrity before storage.

[0244] 39. The size of the nucleic acid particles (Z) of the composition after storage 平均 ) and / or size distribution and / or polydispersity index (PDI) of the nucleic acid particles before storage (Z平均 ) and / or size distribution and / or PDI essentially equal to the composition according to any one of items 24 to 38b.

[0245] 40. The composition of any one of paragraphs 1 to 36, wherein the composition is in frozen form.

[0246] 40a. The composition according to Item 40, wherein the concentration of the nucleic acid in the composition is about 1 mg / L to about 50 mg / L.

[0247] 40b. The composition of paragraph 40 or paragraph 40a, wherein the nucleic acid integrity after thawing the frozen composition is at least 50% compared to the nucleic acid integrity before freezing the composition.

[0248] 41. The composition of paragraph 40, 40a, or 40b, wherein the nucleic acid integrity after thawing the frozen composition is at least 90% or substantially 100% compared to the nucleic acid integrity before freezing the composition.

[0249] 42. Size of nucleic acid particles after thawing of frozen composition (Z 平均 ) and / or size distribution and / or polydispersity index (PDI) of the nucleic acid particles before freezing the composition (Z 平均 42. The composition according to any one of items 40 to 41, wherein the particle size distribution and / or PDI are essentially equal.

[0250] 42a. The composition of any one of paragraphs 40 to 42, wherein the particle size and nucleic acid integrity of the composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, is essentially equal to the particle size and nucleic acid integrity before the composition was first frozen.

[0251] 43. A method for producing a composition comprising particles dispersed in a final aqueous phase, wherein the composition comprises (i) nucleic acid; (ii) cationically ionizable lipid; (iii) steroid; (iv) neutral lipid; and (v) polyvalent anion (e.g., inorganic polyphosphate); the particles comprise at least a portion of the nucleic acid, at least a portion of the cationically ionizable lipid, and at least a portion of the steroid; at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is associated with the particles; and the final aqueous phase comprises a final buffer system; wherein the method comprises: (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 cationically ionizable lipid, at least a portion of the steroid, and at least a portion of the neutral lipid, and at least a portion of the polyvalent anion (e.g., inorganic polyphosphate) is associated with the particles; and (II) optionally freezing the formulation to about −10° C. or below; thereby obtaining a composition, Here, step (I) is (a) providing a nucleic acid solution comprising water and a first buffer system; (b) preparing an organic solution containing a cationically ionizable lipid, a steroid, and a neutral lipid; (c) combining the nucleic acid solution provided under (a) with the organic solution provided under (b), thereby producing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system; (d) mixing the first intermediate formulation produced under (c) with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby producing 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 (e) filtering and / or diluting the second intermediate formulation prepared under (d) with a final aqueous buffer comprising a final buffer system; thereby producing a formulation comprising particles dispersed in a final aqueous phase.

[0252] 43a. The method of paragraph 43, wherein step (a) comprises: (a') preparing an aqueous nucleic acid solution; (b') preparing a first aqueous buffer solution comprising a first buffer system; and (c') mixing the aqueous nucleic acid solution prepared under (a') with the first aqueous buffer solution prepared under (b'), thereby preparing a nucleic acid solution comprising water and the first buffer system.

[0253] 43b. The method of claim 43 or 43a, wherein the organic solution comprises an organic solvent selected from the group consisting of alcohols having up to 6 carbon atoms and mixtures of two or more of these alcohols, preferably an organic solvent selected from the group consisting of ethanol, propanol, isopropanol, 1,2-propanediol and mixtures of two or more of these alcohols.

[0254] 44. The method of any one of paragraphs 43 to 43b, wherein step (I) further comprises one or more steps selected from dilution and filtration.

[0255] 45. Step (I) is (a') preparing an aqueous nucleic acid solution; (b') providing a first aqueous buffer comprising a first buffer system; (c') mixing the aqueous nucleic acid solution prepared under (a') with the first aqueous buffer prepared under (b'), thereby producing a nucleic acid solution comprising water and a first buffer system; (d') providing (e.g., preparing) an organic solution containing a cationically ionizable lipid, a steroid, and a neutral lipid; (e') mixing the nucleic acid solution prepared under (c') with the organic solution prepared (e.g., prepared) under (d'), thereby producing a first intermediate formulation comprising particles dispersed in a first aqueous phase comprising a first buffer system; (f') optionally diluting the first intermediate formulation produced under (e') with water or an additional aqueous buffer comprising an additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in an additional aqueous phase comprising the first buffer system or an additional buffer system, wherein the additional aqueous buffer can be the same as or different from the first aqueous buffer; (g') mixing the first intermediate formulation obtained in step (e') if step (f') is not present, or the further intermediate formulation obtained in step (f') if step (f') is present, with a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof, thereby producing 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; (h') optionally filtering the second first intermediate formulation produced under (g') with an additional aqueous buffer comprising an additional buffer system, thereby producing a further intermediate formulation comprising particles dispersed in an additional aqueous phase comprising an additional buffer system, wherein the additional aqueous buffer can be the same as or different from the first and / or second aqueous buffer; (i') optionally repeating step (h') one, two or more times, wherein a further intermediate formulation comprising particles dispersed in a further aqueous phase comprising a further buffer system obtained after one cycle of step (h') is used as a second intermediate formulation in a next cycle, wherein in each cycle, the further aqueous buffer can be the same as or different from the first and / or second aqueous buffer; (j') filtering the second intermediate formulation obtained in step (g') if step (h') is not present, or the further intermediate formulation obtained in step (h') if step (h') is present but step (i') is not present, or the further intermediate formulation obtained after step (i') if steps (h') and (i') are present, with a final aqueous buffer comprising a final buffer system; and (k') optionally diluting the formulation obtained in step (j') with a diluent solution; Item 45. The method according to any one of Items 43 to 44, thereby producing a formulation comprising particles dispersed in a final aqueous phase.

[0256] 45a. The method of claim 45, wherein the organic solution comprises an organic solvent selected from the group consisting of alcohols having up to 6 carbon atoms and mixtures of two or more of these alcohols, preferably an organic solvent selected from the group consisting of ethanol, propanol, isopropanol, 1,2-propanediol and mixtures of two or more of these alcohols.

[0257] 45b. The method of paragraph 45 or paragraph 45a, wherein step (f') is not present.

[0258] 45c. The method according to paragraph 45 or paragraph 45a, wherein step (f') is present.

[0259] 45d. The method according to any one of paragraphs 45 to 45c, wherein step (g') is carried out at most about 20 minutes after step (e').

[0260] 45e. The method according to any one of paragraphs 43 to 45d, wherein the organic solution prepared under (b) or (d'), respectively, further comprises an acid.

[0261] 45f. The method according to any one of items 43 to 45e, wherein step (f') comprises changing the pH of the first intermediate formulation to about pH 7.0 to about 9.0, optionally about pH 7.5 to about 8.5, preferably about pH 7.5 to about 8.0.

[0262] 45g. The method according to paragraph 45f, wherein step (f') is carried out within 12 hours, preferably within 4 hours, more preferably within 30 minutes after step (e').

[0263] 45h. The method according to item 45f or 45g, wherein step (f') is used to change the pH of the first intermediate formulation to pH 7 to 9, and then a polyvalent anion is added in step (g'), and the combined duration of steps (f') and (g') is 12 hours or less, preferably 4 hours or less, more preferably 30 minutes or less after step (e').

[0264] 46. ​​The method according to any one of items 43 to 45h, wherein the filtration is dialysis, tangential flow filtration or diafiltration, preferably dialysis or tangential flow filtration.

[0265] 47. The method of any one of paragraphs 43 to 46, wherein the polyvalent anion comprises 2 to 10 negative charges, optionally 2 to 5 negative charges.

[0266] 47a. The method of any one of paragraphs 43 to 47, wherein the polyvalent anion is selected from the group consisting of inorganic polyphosphate, inorganic phosphate, sulfate, succinate, glutarate, tartarate, malate, citrate, or a mixture thereof.

[0267] 47b. The method according to any one of items 43 to 46, wherein the polyvalent anion is an inorganic polyphosphate or a salt thereof.

[0268] 47c. Inorganic polyphosphate or its salt is represented by 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 balancing.

[0269] 48. Each M is H + , an alkali cation, an ammonium, and a monovalent organic cation, and preferably each M is H + , Na + , K. + , Li + , and NH4 + The method of claim 47c, wherein the compound is independently selected from the group consisting of:

[0270] 49. The method according to any one of paragraphs 47b to 48, wherein the inorganic polyphosphate or salt thereof is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, salts thereof and mixtures thereof, preferably selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, salts thereof and mixtures thereof, more preferably the inorganic polyphosphate or salt thereof is triphosphate or a salt thereof.

[0271] 50. The method of any one of paragraphs 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.

[0272] 51. The method of any one of paragraphs 43 to 50, wherein the molar ratio of (v) polyvalent anion (e.g., inorganic polyphosphate) to (ii) cationically ionizable lipid is at least about 1:2, preferably at least about 2:3, for example at least about 4:3.

[0273] 52. The method of any one of paragraphs 43 to 51, wherein the composition is substantially free of lipids containing PEG, preferably substantially free of any compounds containing PEG, and more preferably substantially free of PEG.

[0274] 52a. The method of any one of paragraphs 43 to 52, wherein the composition is substantially free of any polymer-conjugated lipid.

[0275] 53. The method of any one of paragraphs 43 to 52a, wherein (1) the nucleic acid solution obtained in step (a) has a pH of less than 6.0, preferably at most about 5.0, more preferably at most about 4.5; or (2) the first aqueous buffer has a pH of less than 6.0, preferably at most about 5.0, more preferably at most about 4.5.

[0276] 54. The method according to any one of items 43 to 53, wherein the pH of the composition is about 4.0 to about 8.0, preferably about 4.5 to about 8.0, for example, about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 6.8 to about 7.9, or about 7.0 to about 7.8.

[0277] 55. The method of any one of paragraphs 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).

[0278] 56. The osmolality of the composition is up to about 1000 x 10 -3 osmol / kg, preferably about 100×10 -3 osmol / kg ~ approx. 500 x 10 -3 osmol / kg, more preferably about 300×10 -3Item 56. The method according to any one of Items 43 to 55, wherein the concentration is osmol / kg.

[0279] 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.

[0280] 58. The method of any one of paragraphs 43 to 57, wherein the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

[0281] 59. The method of any one of paragraphs 43 to 58, wherein the cationically ionizable lipid, steroid, and neutral lipid are present in the organic solution in a molar ratio of about 20 mol% to about 70 mol% cationically ionizable lipid; about 15 mol% to about 60 mol% steroid; and about 5 mol% to about 25 mol% neutral lipid (e.g., phospholipid).

[0282] 60. The method of any one of paragraphs 43 to 59, wherein the cationically ionizable lipid, steroid, and neutral lipid are present in the organic solution in a molar ratio of 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 cationically ionizable lipid; 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.

[0283] 60a. The method according to any one of items 43 to 60, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0284] 61. The method according to any one of paragraphs 43 to 60a, wherein the molar ratio of steroid to neutral lipid in the organic solution is up to 2.5, preferably said ratio is 1 to 2.5.

[0285] 62. The method of any one of paragraphs 43 to 59, wherein the cationically ionizable lipid, steroid, and neutral lipid are present in the organic solution in a molar ratio of 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 cationically ionizable lipid; 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.

[0286] 62a. The method according to paragraph 62, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0287] 63. The method of any one of paragraphs 43 to 59, 62 and 62a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, for example 5.0 to 7.0.

[0288] 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.

[0289] 65. The method of any one of paragraphs 43 to 64, wherein the particle is selected from the group consisting of a lipid nanoparticle (LNP), a liposome, a lipoplex (LPX), and a mixture of two or more thereof.

[0290] 65a. The method of any one of paragraphs 43 to 65, wherein the particles comprise essentially all of the cationically ionizable lipids, steroids, and neutral lipids present in the composition.

[0291] 65b. The method of any one of paragraphs 43 to 65a, wherein the final aqueous phase is substantially free of cationically ionizable lipids, steroids, and neutral lipids.

[0292] 65c. The method of any one of paragraphs 43 to 65b, wherein the particles comprise at least 50%, preferably at least 75%, more preferably at least 85% of the nucleic acids present in the composition.

[0293] 65d. The method of any one of paragraphs 43 to 65c, wherein the final aqueous phase is substantially free of nucleic acids.

[0294] 65e. The method of any one of paragraphs 43 to 65d, wherein at least 10%, preferably at least 20%, and more preferably at least 50% of the polyvalent anions (e.g., inorganic polyphosphates) present in the composition are particle-associated.

[0295] 66. The method of any one of paragraphs 43 to 65e, wherein the nucleic acid is RNA, preferably mRNA.

[0296] 66a. The method of any one of paragraphs 43 to 65e, wherein the nucleic acid is DNA.

[0297] 66b. The method of any one of paragraphs 43 to 65e, wherein the nucleic acid is an inhibitory RNA such as an siRNA.

[0298] 67. The method of paragraph 66, wherein the RNA (i) contains modified nucleosides in place of uridine, preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (ii) has a coding sequence that is codon-optimized; and / or (iii) has a coding sequence that has an increased G / C content compared to the wild-type coding sequence.

[0299] 68. The method of paragraph 66 or paragraph 67, wherein the RNA comprises at least one, and preferably all, of a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

[0300] 69. The method of paragraph 68, wherein the polyA sequence comprises at least 100 A nucleotides, and wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

[0301] 70. The method of paragraph 68 or paragraph 69, wherein the 5' cap is a Cap 1 structure or a Cap 2 structure.

[0302] 71. The method of any one of paragraphs 66 and 67 to 70, wherein the RNA encodes one or more polypeptides, wherein preferably one or more polypeptides are pharmaceutically active polypeptides and / or comprise an epitope for inducing an immune response against an antigen in a subject.

[0303] 72. The method of paragraph 71, wherein the pharmaceutically active polypeptide and / or 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.

[0304] 73. The method of clause 71 or clause 72, wherein the pharmaceutically active polypeptide and / or 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.

[0305] 74. (II) The method according to any one of items 43 to 73, comprising a step of freezing the formulation to about -10°C or below.

[0306] 74a. The method according to Item 74, wherein the concentration of the nucleic acid in the composition is about 1 mg / L to about 50 mg / L.

[0307] 74b. The method of paragraph 74 or paragraph 74a, wherein the final aqueous buffer comprises a polyvalent anion (e.g., inorganic polyphosphate) or a salt thereof.

[0308] 75. The method according to any one of items 43 to 73, which does not include step (II).

[0309] 75a. The method according to Item 75, wherein the concentration of the nucleic acid in the composition is about 10 mg / L to about 100 mg / L.

[0310] 76. A method for storing a composition, comprising producing a composition according to the method of any one of items 43 to 74b, and storing the composition at a temperature in the range 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.

[0311] 77. The method of paragraph 76, wherein the composition is stored for at least 1 month, for example, 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.

[0312] 78. A method for storing a composition, comprising producing a composition according to the method of any one of items 43 to 75a, and storing the composition at a temperature in the range 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.

[0313] 79. The method of paragraph 78, wherein the 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, or at least 24 months.

[0314] 80. A composition producible by the method according to any one of paragraphs 43 to 79.

[0315] 81. The composition according to paragraph 80, which is in a frozen form.

[0316] 81a. The composition according to Item 81, wherein the concentration of the nucleic acid in the composition is about 1 mg / L to about 50 mg / L.

[0317] 81b. The composition of paragraph 81 or paragraph 81a, wherein the nucleic acid integrity after thawing the frozen composition is at least 50% compared to the nucleic acid integrity of the composition before freezing the composition.

[0318] 81c. The composition of any one of paragraphs 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 freezing the composition.

[0319] 82. The composition of paragraph 81, paragraph 81a, paragraph 81b and paragraph 81c, 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.

[0320] 83. Size of nucleic acid particles after thawing of frozen composition (Z 平均 ) and / or size distribution and / or polydispersity index (PDI) of the nucleic acid particles before freezing the composition (Z 平均 83. The composition according to any one of items 81 to 82, wherein the particle size distribution and / or PDI are essentially equal.

[0321] 83a. The composition of any one of paragraphs 81 to 83, wherein the particle size and nucleic acid integrity of the composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, is essentially equal to the particle size and nucleic acid integrity before the composition was first frozen.

[0322] 84. The composition according to paragraph 80, which is in liquid form.

[0323] 84a. The composition according to Item 84, wherein the concentration of the nucleic acid in the composition is about 10 mg / L to about 100 mg / L.

[0324] 85. The composition of paragraph 84 or paragraph 84a, wherein the nucleic acid integrity after storage of the composition for at least one week, preferably at a temperature of about 2°C to about 8°C, is at least 90% compared to the nucleic acid integrity before storage.

[0325] 85a. The composition of any one of paragraphs 84, 84a, and 85, wherein the nucleic acid integrity after storage of the composition for at least 4 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.

[0326] 85b. The composition of any one of paragraphs 84, 84a, 85 and 85a, wherein the nucleic acid integrity after storage of the composition for at least 3 months, preferably at a temperature of about 2°C to about 8°C, is at least 90% compared to the nucleic acid integrity before storage.

[0327] 86. The size (Z) of nucleic acid particles after storing the composition for at least one week 平均 ) and / or size distribution and / or polydispersity index (PDI) of the nucleic acid particles before storage (Z 平均 ) and / or size distribution and / or PDI essentially equal to the composition of any one of paragraphs 84, 84a, 85, 85a and 85b.

[0328] 87. A method for producing a ready-to-use pharmaceutical composition, comprising the steps of providing a frozen composition produced by the method of any one of items 43 to 74b, 76 and 77, and thawing the frozen composition, thereby obtaining a ready-to-use pharmaceutical composition.

[0329] 88. A method for producing a ready-to-use pharmaceutical composition, comprising the step of providing a liquid composition produced by the method of any one of paragraphs 43 to 73 and 75 to 77, thereby obtaining a ready-to-use pharmaceutical composition.

[0330] 89. A ready-to-use pharmaceutical composition that can be produced by the method according to paragraph 87 or 88.

[0331] 90. A composition described in any one of items 1 to 42a, items 80 to 86, and item 89 for use in treatment.

[0332] 91. The composition of any one of paragraphs 1 to 42a, 80 to 86, and 89 for use in inducing an immune response in a subject.

[0333] 92. A method for transfecting cells, comprising adding to cells the composition described in any one of paragraphs 1 to 42a, paragraphs 80 to 86, and paragraph 89; and incubating the mixture of the composition and the cells for a sufficient period of time.

[0334] 93. The method of paragraph 92, wherein the mixture of the composition and the cells is incubated in the presence of serum.

[0335] 93a The method of paragraph 93, wherein the serum is human serum.

[0336] 94. The method of paragraph 92, paragraph 93, or paragraph 93a, wherein the cationically ionizable lipid constitutes 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 total lipid present in the composition; the steroid constitutes 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 total lipid present in the composition; and the neutral lipid constitutes about 15 mol% to about 25 mol%, for example, about 17 mol% to about 21 mol%, of the total lipid present in the composition.

[0337] 94a. The method according to any one of items 92 to 94, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0338] 95. The method of any one of paragraphs 92 to 94a, wherein the molar ratio of steroid to neutral lipid is up to 2.5, preferably the ratio is 1 to 2.5.

[0339] 96. The method of claim 92, wherein the cationically ionizable lipid constitutes 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 lipid present in the composition; the steroid constitutes 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 lipid present in the composition; and the neutral lipid constitutes about 5 mol% to about 15 mol%, for example, about 7 mol% to about 14 mol%, of the total lipid present in the composition.

[0340] 96a. The method of paragraph 96, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0341] 97. The method of any one of paragraphs 92, 96 and 96a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, for example 5.0 to 7.0.

[0342] 98. Use of the composition of any one of paragraphs 1 to 42a, 80 to 86, and 89 to transfect a cell.

[0343] 99. The use of paragraph 98, wherein the transfection of cells is carried out in the presence of serum.

[0344] 99a. The use of paragraph 99, wherein the serum is human serum.

[0345] 100. The use of paragraph 98, 99, or 99a, wherein the cationically ionizable lipid constitutes 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 total lipid present in the composition; the steroid constitutes 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 total lipid present in the composition; and the neutral lipid constitutes about 15 mol% to about 25 mol%, for example about 17 mol% to about 21 mol%, of the total lipid present in the composition.

[0346] 100a. The use according to any one of items 98 to 100, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0347] 101. The use according to any one of paragraphs 98 to 100a, wherein the molar ratio of steroid to neutral lipid is up to 2.5, preferably said ratio is 1 to 2.5.

[0348] 102. The use according to item 98, wherein the cationically ionizable lipid constitutes 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 lipid present in the composition; the steroid constitutes 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 lipid present in the composition; and the neutral lipid constitutes about 5 mol% to about 15 mol%, for example, about 7 mol% to about 14 mol%, of the total lipid present in the composition.

[0349] 102a. The use according to paragraph 102, wherein the steroid is cholesterol and the neutral lipid is a phospholipid.

[0350] 103. The use of any one of paragraphs 98, 102 and 102a, wherein the molar ratio of steroid to neutral lipid is at least 3.0, preferably said ratio is 3.0 to 10.0, for example 5.0 to 7.0.

[0351] Further aspects of the present disclosure are disclosed herein. [Brief explanation of the drawings]

[0352] [Figure 1] 4 is an exemplary flow chart illustrating certain steps according to the method of the second aspect.

[0353] [Figure 2]Non-PEG lipid particle compositions versus aggregation of stable polyphosphate lipid particle compositions. A: Lipid particle compositions containing cationically ionizable lipids, steroids, and neutral lipids, but no PEG or inorganic polyphosphate, were prepared and their size (diameter (Z-average) (nm)) was measured over time (up to 30 minutes). B: Lipid particle compositions containing various concentrations (0-10 mM) of cationically ionizable lipids, steroids, neutral lipids, and inorganic polyphosphate (triphosphate (3P)), but no PEG, were prepared and their size (diameter (Z-average) (nm)) was measured 16 hours after preparation.

[0354] [Figure 3] Polyphosphate lipid particle compositions are stable under various conditions. Lipid particle compositions containing RNA (at two different concentrations: 10 or 70 mg / L), cationically ionizable lipids, steroids, neutral lipids, inorganic polyphosphate (triphosphate (3P), added at 2.5 mM after particle formation and optionally present in the final filtration (dialysis) step), and PEG-lipids (+PEG) or without PEG-lipids (-PEG) were prepared. After storage under various conditions (5°C, -20°C, or -70°C), the colloidal parameters of the particle compositions (diameter (nm) and polydispersity index (PDI)) were measured.

[0355] [Figure 4]The effect of different molar ratios of cationically ionizable lipids, steroids, and neutral lipids on expression levels in the absence or presence of serum. Luciferase-encoding RNA, cationically ionizable lipids (A: lipid XIV-3, B: lipid XIV-1, C: lipid XIV-2, D: lipid G (DPL-14)), steroids (cholesterol), neutral lipids (DSPC), and inorganic polyphosphate (triphosphate (3P)) were prepared using compositions with the molar percentage of DSPC indicated. The remainder of the lipid composition consisted of ionizable lipids (ION) and cholesterol (CHOL) at the molar percentages indicated. Cells were transfected with either compositions in the presence (+serum (+S)) or absence (-serum (-S)). Luciferase expression was measured, and serum stimulation was calculated based on the ratio of luciferase expression in the presence (+S) of serum to that in the absence (-S) of serum (ratio +S / -S). FIG. 4 shows the results of luciferase expression, serum stimulation (expressed as log10 (ratio +S / -S) values).

[0356] [Figure 5] Stabilization of lipid particle compositions using polyvalent anions. Stealth lipid-free alkaline raw colloids were prepared using (A) DODAP, (B) lipid D, or (C) lipid XIV-2 as the ionizable lipid component. The material was dialyzed against Buffer A (HEPES pH 7.4), Buffer B (Buffer A + 5 mM sodium triphosphate), Buffer C (Buffer A + 5 mM sodium diphosphate), Buffer D (Buffer A + 5 mM sodium phosphate), or Buffer E (Buffer A + 5 mM sodium citrate) to obtain mature colloids. Control material containing PEG-lipids was treated similarly. The particle size and polydispersity of the mature colloids were monitored over a 48-hour period.

[0357] [Figure 6]Activity of lipid particle compositions with multivalent anions. Colloids matured as described in Figure 5 were tested for their ability to transfect HEK cells with mRNA. The ionizable lipids were (A) DODAP, (B) lipid D, or (C) lipid XIV-2. DETAILED DESCRIPTION OF THE INVENTION

[0358] Array Description The following table lists the specific sequences referred to herein. [Table 1]

[0359] Detailed Description of the Invention Although the present disclosure is described in more detail below, it is understood that the disclosure is not limited to the particular methods, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0360] The elements of the present disclosure are described in more detail below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of all described elements herein should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0361] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0362] The practice of the present disclosure employs, unless otherwise indicated, conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as described in the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al., J. ... al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0363] Throughout this specification and the claims that follow, unless the context requires a contrary interpretation, the terms "comprise" and variations thereof, such as "comprises" and "comprising," should be understood to imply the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" means the exclusion of other members, integers, or steps of some essential significance. The term "comprises" includes the term "consisting essentially of," which in turn includes the term "consisting of." Thus, in each instance herein, the term "comprising" can be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each instance herein, the term "consisting essentially of" can be replaced with the term "consisting of."

[0364] As used in the context of describing this disclosure (particularly in the context of the claims), the singular and similar expressions should be construed to encompass both the singular and the plural unless otherwise stated herein or clearly contradicted by the context.

[0365] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0366] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to illustrate the disclosure and does not otherwise impose limitations on the scope of the claimed disclosure. No language herein should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0367] As used herein, "and / or" is to be construed as a specific disclosure of each of the two specified properties or components, with or without the other. For example, "X and / or Y" is to be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0368] In the context of the present disclosure, the term "about" refers to an interval of accuracy that is understood to allow a person skilled in the art to still be certain of the technical effect of the property in question. The term typically refers to a deviation of ±10% from the indicated numerical value, e.g., ±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%, e.g., ±0.01%. In some embodiments, "about" refers to a deviation of ±10% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±5% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±4% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±3% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±2% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.9% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.8% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.7% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.6% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.5% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.4% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.3% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.2% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.05% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.01% from the indicated numerical value. As will be recognized by one of ordinary skill in the art, the specific deviation from a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects generally exhibit greater deviations than man-made or engineered technical effects.

[0369] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range, and unless otherwise stated herein, each separate value is included herein as if it were individually referenced herein.

[0370]

[0001] Throughout this specification, several documents have been cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0371] definition Below are provided definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise specified: Any undefined terms have their art-recognized meanings.

[0372] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by 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 terms includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0373] As used herein, terms such as "enhancement" and "increase" refer to the ability to cause an overall increase or enhancement of, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% of the level. 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%.

[0374] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0375] As used herein, "physiological conditions" refers to the conditions (especially pH and temperature) in a living subject, particularly a human. Preferably, physiological conditions refer to physiological pH and / or a temperature of about 37°C.

[0376] As used in this disclosure, "%(w / v)" (or "% w / v") refers to weight / volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (ml).

[0377] As used in this disclosure, "volume percent" or "%(v / v)" (or "%v / v") refers to volume percent, a unit of concentration that measures the amount of a liquid substance in milliliters (ml), expressed as a percentage of the total volume of the solution in milliliters (ml).

[0378] As used in this disclosure, "% by weight" or "%(w / w)" (or "% w / w") refers to weight percent, a unit of concentration measuring the amount of a substance in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

[0379] As used in this disclosure, "mol %" is defined as the ratio of the number of moles of a component to the sum of the number of moles of all components multiplied by 100.

[0380] As used herein, "mol % of total lipids" is defined as the ratio of the number of moles of a lipid component to the sum of the number of moles of total lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0381] As used herein, the term "relative amount" refers to the moles of a component (e.g., lipid component) relative to the total moles of all components (e.g., total lipid components).For example, if a composition contains three lipid components (lipid 1, lipid 2 and lipid 3) in amounts of 45 mol, 45 mol and 10 mol, respectively, then the relative amounts are 45 mol% (lipid 1), 45 mol% (lipid 2) and 10 mol% (lipid 3).To increase the relative amount of a component, it is necessary to reduce the relative amount of at least one other component (so that the absolute amount of lipid remains substantially constant), and vice versa.For example, to increase the relative amount of lipid 1 and lipid 3 (e.g., to 58 mol% (lipid 1) and 20 mol% (lipid 3)), it is necessary to reduce the relative amount of lipid 2 (to a lower relative amount, for example, 22 mol%). Furthermore, reducing the relative amounts of lipid 1 and lipid 3 (e.g., to 39 mol% (lipid 1) and 5 mol% (lipid 3)) requires increasing the relative amount of lipid 2 (to a higher relative amount, e.g., 56 mol%).

[0382] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species and their respective charges in a particular solution. Thus, ionic strength, I, can be mathematically determined by the formula:

number

[0383] According to the present disclosure, in certain embodiments, the term "ionic strength" refers to the presence of monovalent ions.

[0384] With respect to the presence of divalent inorganic ions, particularly divalent inorganic cations, their concentration or effective concentration (the presence of free ions) due to the presence of chelating agent is low enough in some embodiments to prevent RNA degradation.In some embodiments, the concentration or effective concentration of divalent inorganic ions is below the catalytic level of the hydrolysis of the phosphodiester bond between RNA nucleotides.In some embodiments, the concentration of free divalent inorganic ions is below 20 μM.In some embodiments, free divalent inorganic ions are absent or essentially absent.

[0385] As used herein, "molar ratio" refers to the ratio of 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 a 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.

[0386] "Osmolality" refers to the concentration of a particular solute expressed as moles of solute per kilogram of solvent.

[0387] The term "lyophilize" or "freeze-drying" refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or less than 1 Pa or less) to allow the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilization" and "freeze-drying" are used interchangeably herein.

[0388] The term "spray drying" refers to the process of spray drying a substance by mixing a (heated) gas with a fluid in the form of an atomized atomization (atomization) in a vessel (spray dryer) where the solvent evaporates from the droplets formed, leaving a dry powder.

[0389] The term "reconstitution" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as the original liquid state.

[0390] The term "freezing" refers to the solidification of a liquid, usually by the removal of heat. In certain embodiments, freezing is the opposite process to thawing.

[0391] The term "thawing" refers to the liquefaction of a solid, usually by the addition of heat. In some embodiments, thawing is the opposite of freezing.

[0392] The term "aqueous phase," as used herein in connection with compositions / formulations comprising particles, particularly LNPs, liposomes, and / or lipoplexes, refers to a mobile or liquid phase, i.e., a continuous aqueous phase that (formally) contains all dissolved components except the particles. Thus, when particles such as LNPs are dispersed in an aqueous phase, and the aqueous phase is substantially free of compound X, the aqueous phase is substantially and as practically feasible as possible free of X, e.g., the concentration of compound X in the aqueous composition is less than 1% by weight. However, at the same time, it is possible that particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

[0393] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In certain embodiments, a "recombinant entity" in the context of this disclosure is not naturally occurring.

[0394] 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), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "naturally occurring" 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.

[0395] 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 include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0396] The term "alkyl" refers to a monoradical of a saturated straight or branched chain hydrocarbon. Preferably, the alkyl group has 1 to 12 (such as 1 to 10) carbon atoms, i.e., C 1-12 A radical of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., C 1-10 alkyl (abbreviated as alkyl) contains 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. Examples of alkyl groups include methyl, ethyl, propyl, iso-propyl (also known as 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. "Substituted alkyl" means that one or more of the hydrogen atoms (e.g., from 1 to the maximum number of hydrogen atoms bonded to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) of an alkylene group are replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents specified herein. Examples of substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0397] The term "alkylene" refers to a diradical of a saturated straight-chain or branched-chain hydrocarbon. Preferably, the alkylene contains 1 to 12 (such as 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. Examples of 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, 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- isomers of octylene (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 three carbon atoms and a free valence at each end may also be referred to as multiple methylenes (e.g., 1,4-butylene may also be referred to as tetramethylene).Generally, instead of using the suffix "ylene" as described above for the alkylene moiety, the suffix "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 of the hydrogen atoms of the alkylene group (e.g., from 1 to the maximum number of hydrogen atoms bonded to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) have been replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents as specified herein.

[0398] The term "alkenyl" refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Generally, 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 alkenyl group has an odd number of carbon atoms, rounding down the resulting fraction to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the 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, the alkenyl group comprises 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, for example 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group is 2-12Alkenyl groups contain 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 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 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 the cis (Z) or trans (E) configuration. Examples of alkenyl groups include vinyl, 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, and 5-nonenyl. , 6-nonenyl, 7-nonenyl, 8-nonenyl, 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, and the like. If the alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenyl" means that one or more of the hydrogen atoms (e.g., 1 to up to the maximum number of hydrogen atoms attached to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4 or 1-3 or 1 or 2) of the alkenyl group's hydrogen atoms are replaced with non-hydrogen substituents (when more than one hydrogen atom is replaced, the substituents can be the same or different).Preferably, the non-hydrogen substituents are first level substituents as specified herein.

[0399] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total number of carbon atoms ranging from 6 to 30, typically 6 to 20, and often 6 to 18. An alkynyl group may optionally have one or more carbon-carbon double bonds. Generally, 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 odd, rounding down the resulting fraction to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the 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.

[0400] The term "alkenylene" refers to an unsaturated straight-chain or branched hydrocarbon diradical having at least one carbon-carbon double bond. Generally, 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 odd, rounding down the resulting fraction to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the 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, the alkenylene group comprises 2 to 12 (such as 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, e.g. 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, the alkenylene group contains 2 to 12 (e.g., 2 to 10 carbon atoms) 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 the cis (Z) or trans (E) configuration. Examples of 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, allylidene, 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, and the like.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 of the hydrogen atoms of the alkenylene group (e.g., 1 to the maximum number of hydrogen atoms attached to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) are replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents as specified herein.

[0401] The term "cycloalkyl" refers to cyclic, non-aromatic versions of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 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. Examples of cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. A cycloalkyl group can consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).

[0402] The term "cycloalkylene" refers to a cyclic, non-aromatic version of "alkylene," and is a geminal, vicinal, or isolated diradical. In certain embodiments, the cycloalkylene is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and / or (ii) 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-, or 3-10-membered). In certain embodiments, the cycloalkylene is a monocyclic, bicyclic, 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-, or 3-10-membered) cycloalkylene. Generally, for cycloalkylene moieties, instead of using the suffix "ylene" as above, the suffix "diyl" may also be used (e.g., 1,2-cyclopropylene may also be referred to as cyclopropane-1,2-diyl). Examples of cycloalkylene groups are cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3.1.1]diyl). 3,7 ]decane-2,2-diyl). "Substituted cycloalkylene" means that one or more of the hydrogen atoms of the alkylene group (e.g., from 1 to the maximum number of hydrogen atoms attached to the cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4 or 1-3 or 1 or 2) have been replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents as specified herein.

[0403] The term "cycloalkenylene" refers to a cyclic, non-aromatic version of "alkenylene," and may be a geminal, vicinal, or isolated diradical. Generally, the maximum number of carbon-carbon double bonds in a cycloalkenylene group, if the number of carbon atoms in the cycloalkenylene group is odd, can be equal to the integer calculated by rounding down the fractional result to the next integer. For example, for a cycloalkenylene group having 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 is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and / or (ii) 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- or 3-10-membered). In certain embodiments, the cycloalkenylene is a monocyclic, bicyclic, 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- or 3-10-membered) cycloalkenylene. Examples of cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. "Substituted cycloalkenylene" means that one or more of the hydrogen atoms (e.g., 1 to the maximum number of hydrogen atoms bonded to the cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4 or 1-3 or 1 or 2) of the cycloalkenylene group are replaced with a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents as specified herein.

[0404] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, etc.) carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Examples of aryl groups include cyclopropenyl, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerene.

[0405] The term "aromatic" in reference to hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this disclosure. Similarly, if a bicyclic or polycyclic aryl (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this disclosure (even though one ring, as in 1,2-dihydronaphthyl, is still aromatic).

[0406] The term "hydrocarbyl," as used herein, refers to a monovalent organic group obtained by removing an H atom from a hydrocarbon molecule. In certain embodiments, the hydrocarbyl group is acyclic, e.g., linear (straight-chain) or branched. Representative examples of hydrocarbyl groups include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Specific examples of hydrocarbyl groups include C 1-6 Alkyl, aryl and aryl(C 1-6 In certain embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more first-level substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively non-polar.

[0407] Typical first level substituents are preferably C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , —C(═O)OH, and —C(═O)OCH3, where z is 0, 1, or 2; 1-3 Alkyl is methyl, ethyl, propyl, or isopropyl. Particularly preferred first level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF 3 .

[0408] As used herein, the term "tertiary amine moiety" refers to a moiety that includes a nitrogen atom substituted with three organic substituents, where the substituents can be the same or different from one another. In certain embodiments, the organic substituents are optionally substituted (e.g., with one or more first-level substituents as defined herein) hydrocarbyl groups (e.g., alkyl groups, particularly C 1-6 alkyl groups).

[0409] The term "filtration" as used herein refers to any process that involves the removal or separation of at least one component of a liquid composition (for example, permeable molecules such as salts, small proteins, solvents, etc.) based on the molecular size of the components contained in the composition. This separation can use a filter that is permeable to small molecules (for example, for diafiltration or tangential flow filtration) or a semipermeable membrane (for example, for dialysis). Thus, examples of filtration include dialysis, tangential flow filtration and diafiltration.

[0410] As used herein in reference to a frozen composition, the term "post-thaw frozen composition" refers to a frozen composition that has undergone thawing to determine whether the characteristics (e.g., nucleic acid integrity (e.g., RNA integrity) and / or size (Z) of particles (e.g., LNPs) contained in the composition have been altered. 平均 This means that the sample must be thawed before the particle size distribution and / or particle diameter distribution (PDI) can be measured.

[0411] 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). A monovalent cation refers to, for example, an alkali cation (e.g., Na + , K. + , Li + ), ammonium cation (NH4 + ), or organic compounds with one primary, secondary, or tertiary amine group (such as protonated forms of triethylamine, trimethylamine, etc.), or organic compounds with one quaternary amine group.

[0412] 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.

[0413] A "polyhydric" or "polybasic" compound refers to a compound 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, acid carboxyl groups.

[0414] "Polyphosphate" refers to a compound containing two or more consecutive phosphate groups, preferably three or more consecutive phosphate groups. Examples of polyphosphates include inorganic polyphosphates and esters of polyphosphates (such as triphosphates) with one or more organic alcohols, such as nucleotides, oligonucleotides, or polynucleotides having at least three consecutive phosphate groups.

[0415] As used herein, the phrase "inorganic polyphosphate" refers to a compound containing two or more consecutive phosphate groups, preferably three or more consecutive phosphate groups, without any organic moieties covalently bonded thereto (e.g., inorganic polyphosphate lacks a covalent bond between any oxygen or phosphorus atom and a carbon atom contained in the anionic portion of the polyphosphate). Thus, the phrase "inorganic polyphosphate" does not include esters of polyphosphate with one or more organic alcohols. Consequently, the phrase "inorganic polyphosphate" does not encompass nucleotides, oligonucleotides, or polynucleotides, even if they contain at least two, preferably at least three, consecutive phosphate groups. Inorganic polyphosphates can be linear (i.e., all phosphate moieties of the polyphosphate are arranged in a chain), branched, or cyclic. In certain embodiments, polyphosphates have the formula [P x O (3x+1) ] y where x is an integer and is at least 2, preferably at least 3; and y is the anionic charge. Examples of polyphosphates include diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, higher homologs, and mixtures thereof (especially the linear forms of these polyphosphates), particularly triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, higher homologs, and mixtures thereof (especially the linear forms of these polyphosphates). Preferred polyphosphates include diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof (especially the linear forms of these polyphosphates), for example, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof (especially the linear forms of these polyphosphates), for example, triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof (especially the linear forms of these polyphosphates). A particularly preferred polyphosphate is triphosphate.

[0416] Similarly, as used herein, the phrase "inorganic phosphate" refers to a compound containing only one phosphate group and no organic moiety covalently bonded thereto (e.g., inorganic phosphate lacks a covalent bond between any oxygen or phosphorus atom contained in the anion portion of the phosphate and a carbon atom). Thus, the phrase "inorganic phosphate" does not include esters of phosphoric acid with one or more organic alcohols. Accordingly, the phrase "inorganic phosphate" does not encompass nucleotides, oligonucleotides, or polynucleotides, even if they contain one phosphate group. Inorganic phosphates can be linear or cyclic. In certain embodiments, inorganic phosphates have the formula [PO4] 3- Includes:

[0417] As used herein, the phrase "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 a practical and feasible manner. For example, if a 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.

[0418] For example, as used herein, " substantially free of lipids comprising polyethylene glycol (PEG) " means that the mixture (for example, the aqueous phase of the compositions or preparations described herein) does not contain lipids comprising PEG in a practical and practically feasible manner.For example, if the mixture is substantially free of lipids comprising PEG, the amount of lipids comprising PEG in the mixture can be less than 1 wt % (for example, 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. The same applies to the expressions "substantially free of compounds containing PEG," "substantially free of PEG," and "substantially free of cationically ionizable lipids, steroids, and neutral lipids."

[0419] The term "nucleic acid integrity" refers to the percentage of full-length (i.e., unfragmented) nucleic acids relative to the total amount of nucleic acids contained in a sample (i.e., unfragmented + fragmented nucleic acids). Nucleic acid integrity can be determined by chromatographically separating nucleic acids (e.g., 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 acid), determining the peak area of ​​the total nucleic acid, and dividing the peak area of ​​the main nucleic acid peak by the peak area of ​​the total nucleic acid. Similarly, the expression "RNA integrity" refers to the percentage of full-length (i.e., unfragmented) RNA relative to the total amount of RNA contained in a sample (i.e., unfragmented + fragmented RNA). RNA integrity can be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of ​​the major RNA peak (i.e., the peak area of ​​full-length (i.e., unfragmented) RNA), determining the peak area of ​​total RNA, and dividing the peak area of ​​the major RNA peak by the peak area of ​​total RNA.

[0420] The term "cryoprotectant" relates to a substance added to a preparation (eg, a formulation or composition) to protect the active ingredients of the preparation during the freezing step.

[0421] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0422] The "glass transition temperature" (abbreviated as T g The term "glass transition" refers to the temperature range over which this glass transition occurs. The term "glass transition" refers to the gradual and reversible transition in an amorphous material (or amorphous region within a semicrystalline material) from a hard (e.g., relatively brittle "glassy") amorphous state to a viscous (e.g., rubbery) molten state as the temperature increases. Methods for measuring the glass transition temperature are known to those skilled in the art and include dilatometry, dielectric methods, dynamic mechanical analysis (DTMA), differential scanning calorimetry (DSC), refractometry, or NMR spectroscopy. Suitable standardized methods include DIN 53765: 1994-03 and ISO 11357-2: 1999-03.

[0423] According to this disclosure, the term "peptide" includes oligo- and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 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 linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used interchangeably herein.

[0424] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "therapeutic protein" includes whole proteins or peptides and can 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, vaccination antigens and immunostimulants such as cytokines. The terms "therapeutic protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

[0425] According to various embodiments of the present disclosure, a nucleic acid, such as an RNA (e.g., mRNA), encoding a peptide, polypeptide, or protein is taken up or introduced, i.e., transfected or transduced, into a cell, which may be in vitro or in a subject, resulting in expression of the peptide, polypeptide, or protein. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and / or nucleus), and may secrete and / or express the encoded peptide, polypeptide, or protein on its surface.

[0426] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein to mean that, with respect to a particular peptide, polypeptide, or protein, a nucleic acid can be expressed to produce the peptide, polypeptide, or protein when present in the appropriate environment, preferably within a cell.

[0427] The term "portion" refers to a fragment. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" can refer to a contiguous or discontinuous fragment of the structure.

[0428] The terms "portion" and "fragment" are used interchangeably herein and refer to continuous elements. For example, a portion of a structure, such as 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" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0429] A "fragment" of an amino acid sequence (peptide, polypeptide, or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (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 contains the start codon responsible for translation initiation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains 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 include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0430] According to the present disclosure, a portion or fragment of a peptide, polypeptide, or protein preferably possesses 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 possesses at least one of the pharmacological activities of the peptide, polypeptide, or protein from which it is derived. A portion 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 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, at least 12, at least 15, at least 20, at least 30, or at least 55 consecutive amino acids of the peptide or protein.

[0431] 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 version of the wild-type amino acid sequence. In certain 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, and preferably 1 to about 10 or 1 to about 5 amino acid differences compared to the parent.

[0432] "Wild-type" or "WT" or "native" with respect to an amino acid sequence refers to 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 refers to a nucleic acid sequence that has been found in nature, including allelic variations. For example, a wild-type coding sequence refers to a coding sequence that has been found in nature and has not been intentionally modified.

[0433] As used herein, "coding sequence" means a portion of a nucleic acid (eg, the DNA or RNA of a gene) that encodes a protein.

[0434] The phrase "guanosine / cytosine (G / C) content" or "G / C content" refers to the percentage of bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). The G / C content can be given for a specific portion of DNA or RNA, or for the entire genome. When G / C content refers to a portion, it can refer to the G / C content of an individual gene or part of a gene (domain), a group of genes or gene clusters, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

[0435] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes total variants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0436] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modification of positions of the amino acid sequence that are not conserved in homologous proteins or peptides and / or substitution of amino acids with others with similar properties are preferred. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitution of similarly charged or uncharged amino acids. Conservative amino acid changes are substitutions of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, tyrosine.

[0437] In some embodiments, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the amino acid sequence is 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 expressed over 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 entire 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 expressed over 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, consecutive amino acids. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0438] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0439] The terms "% identical" and "% identity" or similar terms refer specifically to the percentage of nucleotides or amino acids that are identical between the sequences being compared in optimal alignment. The percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences over a segment or "window of comparison" after optimal alignment 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 Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search 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 from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In one embodiment, the algorithm parameters used with the BLASTN algorithm on the NCBI website include (i) an expectation threshold setting of 10; (ii) a font size setting of 28; (iii) a maximum query range match setting of 0; (iv) a match / mismatch score setting of 1, -2; (v) a gap cost setting of linear; and (vi) a filter for low complexity regions. In one embodiment, the algorithm parameters used with the BLASTP algorithm on the NCBI website include (i) an expectation threshold setting of 10; (ii) a font size setting of 3; (iii) a maximum query range match setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap costs settings of presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0440] Percentage identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying this result by 100.

[0441] In some embodiments, the degree of similarity or identity is shown for a region that is 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, when the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is shown for at least about 100 nucleotides, at least about 120 nucleotides, at least about 140 nucleotides, at least about 160 nucleotides, at least about 180 nucleotides or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, the degree of similarity or identity is shown for the full length of the reference sequence.

[0442] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular 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% of the amino acid residues.

[0443] Amino acid sequence variants can be readily produced by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0444] 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 refers to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived, i.e., is functionally equivalent. For antigens or antigenic sequences, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids are modified, particularly compared to the amino acid sequence of the parent molecule or sequence, and can still perform one or more functions of the parent molecule or sequence, such as inducing an immune response (immunological fragment). In some embodiments, modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present, e.g., the immunogenicity of the functional variant may 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 may be enhanced compared to the parent molecule or sequence.

[0445] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. In certain embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence, or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence, or a fragment thereof. For example, it will be understood by those of skill in the art that antigens suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived, yet still retain the desired activity of the native sequence.

[0446] In some embodiments, "isolated" means altered or removed (e.g., purified) from a natural state or an artificial composition, such as a composition from a manufacturing 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 the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, 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 a substantially purified form comprises a first buffer system.

[0447] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For 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, where the cells may be present in a subject, e.g., a patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein may be present in vitro (e.g., in cell culture) or in vivo (e.g., the cells may form part of a patient's organ, tissue, and / or organism). According to this disclosure, transfection can be transient or stable. For some transfection applications, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during transfection are not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Episomal amplification of nucleic acids greatly reduces the rate of possible cellular dilution. If transfected nucleic acid is desired to 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, the nucleic acid encoding the antigen is transiently transfected into cells.RNA can be transfected into cells to transiently express its encoded protein.

[0448] The present disclosure includes analogs of peptides, polypeptides, or proteins. According to the present disclosure, a peptide, polypeptide, or protein analog is a modified form of the peptide, polypeptide, or protein from which it is derived, retaining at least one functional property of the peptide, polypeptide, or protein. For example, a pharmacologically active peptide, polypeptide, or protein analog retains at least one pharmacological activity of the peptide, polypeptide, or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecules associated with the protein, polypeptide, or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In certain embodiments, a "protein, polypeptide, or peptide" "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to antibodies or other cellular ligands. The term "analog" also extends to fully functional chemical equivalents of the proteins, polypeptides, and peptides.

[0449] As used herein, "activation" or "stimulation" refers to the state of a cell (e.g., an immune effector cell such as a T cell) that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of a signal transduction pathway, the induction of cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell that is undergoing cell division.

[0450] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0451] The term "clonal expansion" or "expansion" refers to the process by which a specific entity increases. In certain embodiments, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize the antigen are amplified. In certain embodiments, expansion leads to differentiation of the immune effector cells.

[0452] According to the present disclosure, "antigen" encompasses any substance that induces an immune response and / or any substance targeted by an immune response or immune mechanism, such as a cellular response. This also includes situations in which the antigen is processed into antigenic peptides and the immune response or immune mechanism targets one or more antigenic peptides, particularly if presented by MHC molecules. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present disclosure, the term "antigen" includes any molecule that contains at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that induces an immune response, optionally after processing, that is preferably specific to the antigen (including cells that express the antigen). In certain embodiments, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0453] According to the present disclosure, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a humoral or cellular immune response, or both. In the context of certain embodiments of the present disclosure, the antigen is presented by cells, preferably antigen-presenting cells, in the context of MHC molecules, which results in an immune response to the antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or 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, for example, a viral protein or a portion thereof.

[0454] 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 containing an epitope that stimulates the host's immune system to mount a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, such as tumor antigens, typically tumors.

[0455] In some embodiments, the antigen is a tumor antigen, i.e., a part of tumor cells, particularly those that occur mainly intracellularly or as surface antigens of tumor cells.In other embodiments, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as a virus, a bacterium, a single-cell organism or a parasite, for example, a viral antigen such as a viral ribonucleoprotein or coat protein.In particular, the antigen should be presented by MHC molecules, which leads to the regulation, particularly activation, of immune system cells, preferably CD4+ and CD8+ lymphocytes, particularly through the regulation of the activity of T cell receptors.

[0456] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. Specifically, it refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-iron protein, and γ-fetoprotein, as well as various viral tumor antigens. According to certain embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer and tumor or cancer cells with respect to type and / or expression level.

[0457] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0458] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0459] The term "epitope" refers to an antigenic determinant of a molecule, such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented by an MHC molecule. An epitope of a protein can include a continuous or discontinuous portion of the protein and can be, for example, about 5 to about 100, about 5 to about 50, about 8 to about 0, or about 10 to about 25 amino acids in length; for example, 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 certain embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0460] Terms such as "epitope," "antigenic fragment," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer to, for example, an incomplete representation of an antigen that can elicit an immune response, for example, against the antigen or a cell that expresses or contains and presents the antigen. In certain embodiments, the term relates to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented by an MHC molecule. Some preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length; for example, an epitope may 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, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0461] The term "T cell epitope" refers to a portion or fragment of a protein that, when presented by an MHC molecule, is recognized by a T cell. The terms "major histocompatibility complex" and abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may be effective.

[0462] Peptide and protein antigens can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length, and can range from 2 to 100 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0463] A peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to develop antibody and T cell responses against the peptide or protein.

[0464] In some embodiments, vaccine antigens, i.e., antigens that induce an immune response when inoculated into a subject, are recognized by immune effector cells. In some embodiments, if a vaccine antigen is recognized by an immune effector cell, it can be induced in the presence of an appropriate costimulatory signal, stimulating, priming, and / or expanding immune effector cells bearing an antigen receptor that recognizes the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell. In some embodiments, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of an antigen presented by MHC. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In one embodiment, engagement of a TCR expressed by and / or present on a T cell to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, e.g., perforin and granzymes.

[0465] In certain embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope of the antigen. In certain embodiments, the antibody or B cell receptor binds to a natural epitope of the antigen.

[0466] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, for example, by an antibody located outside the cell. In this context, the 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 be via one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule that associates with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0467] "Cell surface" or "surface of a cell" is used according to its usual meaning in the art, and thus includes the outside of a cell that is accessible to binding by proteins and other molecules.An antigen is expressed on the surface of a cell if it is located on the surface of the cell, and is accessible to binding by, for example, an antigen-specific antibody added to the cell.In some embodiments, the antigen expressed on the surface of a cell is an integral membrane protein with an extracellular portion that can be recognized by CAR.

[0468] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, e.g., by binding of a molecule, such as an antibody, that is located on the outside of the cell. In certain embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0469] 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 an antigen, particularly when presented by an MHC molecule on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, to which the T cell targets and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, such as 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, the RNA (particularly mRNA) encodes at least one epitope.

[0470] The term "target" refers to a factor, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present antigens or antigen epitopes, i.e., peptide fragments derived from antigens. In some embodiments, target cells are cells that express antigens and preferably present the antigens by class I MHC.

[0471] "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), association of one or more of these fragments with an MHC molecule (e.g., by binding), and presentation to specific T cells by a cell, preferably an antigen-presenting cell. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0472] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen presented by class I MHC on the surface of an antigen-presenting cell or a peptide derived from the antigen. + It means T cells.

[0473] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0474] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the body's integrated 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" in reference to an agent such as an antigen, cell, or tissue refers to an immune response such as a cellular response directed against the agent. An immune response may be characterized by the development of antibodies against one or more antigens and the production of CD4 + and CD8 + T lymphocytes, e.g., CD8 + The expansion of antigen-specific T lymphocytes, such as T lymphocytes, may include one or more responses selected from the group consisting of T lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.

[0475] In the context of the present disclosure, the terms "induction of an immune response" and "elicitation of 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 / prophylactic / preventative and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably 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, "induction" can mean not only that there is no immune response against a specific antigen or pathogen before induction, but also that there is a certain level of immune response against a specific antigen or pathogen before induction, and that the immune response is enhanced after induction. Thus, in this context, "induction of an immune response" also includes "enhancement of an immune response." In certain embodiments, after induction of 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 state is alleviated by the induction of an immune response.

[0476] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include a cellular response to cells characterized by antigen expression and / or antigen with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that can act as "helpers" or "killers." Helper T cells (CD4 + T cells (also called killer cells) play a more central role in regulating the immune response and are classified as cytotoxic T cells, cytolytic T cells, and CD8 + T cells or CTLs (also called T cells or CTLs) kill cells, such as diseased cells.

[0477] The term "humoral immune response" refers to the process in living organisms by which antibodies are produced in response to factors and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide and produce memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope as recognized by the antigen receptor. Memory B lymphocytes remain quiescent until subsequently activated by a specific antigen. These lymphocytes provide the cellular basis of memory and the eventual recruitment of antibody responses upon re-exposure to the specific antigen.

[0478] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an epitope of an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of 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 regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3, and the CDRs of the VL are referred to as 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), where the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxy 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 of the classical complement system (C1q). Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or may be immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may 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.

[0479] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, such as antibodies or antigen receptors, such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins and are generally part of the BCR. Soluble immunoglobulins are generally referred to as antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are mainly V L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domains 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 are responsible for 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 the carboxy terminus. In mammals, there are two types of light chains, namely, lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among various immunoglobulin isotypes, while the variable portion is highly diverse and is responsible for antigen recognition.

[0480] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons and involve the administration to an individual of one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding same, as described herein, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

[0481] "Cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions mean that diseased cells, in particular cells such as tumor cells or infected cells or antigen-presenting cells, present antigens or antigenic peptides, either directly or after processing, by MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0482] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA (especially mRNA), which can then be translated into peptides, polypeptides, or proteins.

[0483] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0484] As used herein, the term "serum" refers to the fluid resulting from the removal of cells or clotting factors from whole blood, such as whole blood obtained from a human or mouse, particularly with respect to the incubation of cells in the presence of serum. In certain embodiments, the serum is human serum or mouse serum.

[0485] The pharmaceutical preparations, particularly kits, described herein may include instructional materials or directions. As used herein, "instruction materials" or "directions" includes publications, recordings, diagrams, or any other medium of representation that can be used to communicate the usefulness of the disclosed compositions and methods. The instructional materials of the disclosed kits can, for example, be attached to a container containing the disclosed compositions or shipped together with the container containing the compositions. Alternatively, the instructional materials can be shipped separately from the container, with the intention that the instructional materials and the composition be used cooperatively by the recipient.

[0486] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and the description includes instances where the event, circumstance, or condition occurs and instances where it does not occur.

[0487] A prodrug of a specific compound described herein is a compound that, upon administration to an individual, undergoes chemical conversion under physiological conditions to yield the specific compound. Furthermore, a prodrug can be converted to the specific compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the specific compound when placed in a transdermal patch reservoir with, for example, an appropriate enzyme or chemical reagent. Examples of prodrugs are in vivo hydrolyzable esters (using alcohol or carboxy groups contained in the specific compound) or amides (using amino or carboxy groups contained in the specific compound). Specifically, any amino group contained in the specific compound that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0488] In this specification, the structural formula of a compound may represent a certain isomer of the compound. However, it is understood that the present disclosure includes all isomers and isomeric mixtures, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbon atoms, stereoisomers, tautomers, etc., and is not limited to the description of the formula. Furthermore, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it is understood that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0489] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) positions of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that are reversibly interconverted into each other, even if spontaneously and pure, by the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is a keto-enol-tautomeric isomer. "Conformational isomers" are stereoisomers that can formally be interconverted by simple rotation about a single bond, including - in particular - those that lead to different three-dimensional forms of (hetero)cyclic rings, such as the chair, half-chair, boat and twist-boat forms of cyclohexane.

[0490] As used herein, the term "solvate" refers to an addition complex of a dissolved substance in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), where the addition complex exists in crystalline or mixed crystalline form. The amount of solvent contained in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solve...

Claims

1. A composition comprising (i) nucleic acids; (ii) cationically ionizable lipids; (iii) steroids; (iv) neutral lipids; and (v) inorganic polyphosphate.

2. (i) The inorganic polyphosphate contains the formula [P x O (3x + 1)] y [wherein x is an integer and at least 3; y is an anionic charge]; (ii) The inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof; (iii) The inorganic polyphosphate is linear inorganic polyphosphate; or (iv) The molar ratio of (v) inorganic polyphosphate to (ii) cationically ionizable lipids is at least 1:2, at least 2:3, or at least 4:

3. The composition according to claim 1.

3. The composition according to claim 1, wherein the inorganic polyphosphate is triphosphate.

4. (i) The composition does not contain lipids containing polyethylene glycol (PEG) or any compound containing PEG or does not contain PEG, The composition contains less than 1% by weight of polyethylene glycol (PEG) in lipids or compounds containing PEG, or PEG itself, based on the total weight of the composition; (ii) The pH of the composition is 4.0 to 8.0 or 7.0 to 7.8; (iii) Water is the main component of the composition and / or the total amount of solvents other than water contained in the composition is less than 0.5% (v / v); (iv) The osmotic pressure of the composition is at most 1000 × 10⁻³ osmol / kg, or between 100 × 10⁻³ osmol / kg and 500 × 10⁻³ osmol / kg; or (v) The concentration of nucleic acid in the composition is 1 mg / l to 500 mg / l, or 10 mg / l to 100 mg / l. The composition according to claim 1.

5. (i) The cationically ionizable lipid comprises a head group containing at least one tertiary amine moiety; (ii) Lipids that can be cationically ionized are given by formula (X) 【Chemistry 1】 [During the ceremony, One of L10 and L20 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- or -NR a C(=O)O-, and the other of L10 and L20 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- or -NR a C(=O)O- or direct bond; G1 and G2 are, independently, unsubstituted C1-C12 alkylenes or C2-12 alkenylenes; G3 is C1-24 alkylene, C2-24 alkenylene, C3-8 cycloalkylene, or C3-8 cycloalkenylene; Ra is H or C1-12 alkyl; R35 and R36 are each independently a C6-24 alkyl or C6-24 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 is a C1-12 alkyl group; R 50 is H or C 1-6 alkyl; and x is 0, 1, or 2. Having the structure of or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof; (iii) A lipid that can be cationically ionized, given formula (XI): 【Chemistry 2】 [During the ceremony, Each of R1 and R2 is independently R5 or -G1-L1-R6, where at least one of R1 and R2 is -G1-L1-R6; Each of R3 and R4 is independently selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, aryl, and C3-10 cycloalkyl; Each of R5 and R6 is independently an acyclic hydrocarbyl group having at least 10 carbon atoms; Each of G1 and G2 is independently an unsubstituted C1-12 alkylene or C2-12 alkenylene; Each of L1 and L2 is independently selected from the group consisting of -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 C1-12 alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2. Does it have the structure of; (iv) Cationically ionizable lipids constitute 20 mol% to 75 mol%, 40 mol% to 70 mol%, or 25 mol% to 35 mol% of the total lipids present in the composition; (v) Whether the steroid contains sterols or cholesterol; (vi) The steroid constitutes 15 mol% to 60 mol%, 15 mol% to 40 mol%, 35 mol% to 60 mol%, or 45 mol% to 60 mol% of the total lipids present in the composition; (vii) The neutral lipid is a phospholipid, or a phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, or a phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylethanolamine (DSPE), and dipalmitoyl-phosphatidylethanolamine (DPPE); (viii) Neutral lipids constitute 5 mol% to 25 mol%, 15 mol% to 25 mol%, or 7 mol% to 14 mol% of the total lipids present in the composition; or (ix) Cationically ionizable lipids constitute 20 mol% to 70 mol% of the total lipids present in the composition; steroids constitute 15 mol% to 60 mol% of the total lipids present in the composition; and neutral lipids constitute 5 mol% to 25 mol% of the total lipids present in the composition. The composition according to claim 1.

6. The composition according to claim 1, wherein cationically ionizable lipids constitute 40 mol% to 70 mol%, 45 mol% to 65 mol%, or 50 mol% to 60 mol% of the total lipids present in the composition; steroids constitute 15 mol% to 40 mol%, 20 mol% to 35 mol%, or 20 mol% to 30 mol%, of the total lipids present in the composition; and neutral lipids constitute 15 mol% to 25 mol%, or 17 mol% to 21 mol%, of the total lipids present in the composition.

7. The composition according to claim 1, wherein the molar ratio of steroid to neutral lipids is a maximum of 2.5, or the ratio is between 1 and 2.

5.

8. The composition according to claim 6, wherein the molar ratio of steroid to neutral lipids is a maximum of 2.5, or the ratio is between 1 and 2.

5.

9. The composition according to claim 1, wherein cationically ionizable lipids constitute 20 mol% to 40 mol% of the total lipids present in the composition; steroids constitute 35 mol% to 60 mol% of the total lipids present in the composition; and neutral lipids constitute 5 mol% to 15 mol% of the total lipids present in the composition.

10. The composition according to claim 1, wherein the molar ratio of steroid to neutral lipids is at least 3.0, or the ratio is 3.0 to 10.0 or 5.0 to 7.

0.

11. The composition according to claim 9, wherein the molar ratio of steroid to neutral lipids is at least 3.0, or the ratio is 3.0 to 10.0 or 5.0 to 7.

0.

12. (i) The composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of nucleic acids, at least a portion of cationically ionizable lipids, at least a portion of steroids, and at least a portion of neutral lipids; and at least a portion of inorganic polyphosphate is bound to the particles, wherein (a) The particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof; (b) The particles contain at least 50% of the nucleic acids present in the composition; (c) At least 10% of the polyphosphate present in the composition is bound to the particles; or (d) The particles have a size of 30 nm to 500 nm or 50 nm to 150 nm; or (ii) The composition (a) In liquid form; or (b) Frozen form That is The composition according to claim 1.

13. The composition according to claim 1, wherein the nucleic acid is RNA or mRNA.

14. (i) RNA (1) comprises a modified nucleoside instead of uridine, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methylpseudridine (m1ψ), and 5-methyluridine (m5U); (2) has a codon-optimized coding sequence; and / or (3) has a coding sequence with increased G / C content compared to a wild-type coding sequence; (ii) The RNA contains at least one or all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence; or (iii) RNA encodes one or more polypeptides The composition according to claim 13.

15. One or more encoded polypeptides (i) comprising a pharmaceutically active polypeptide and / or epitope for inducing an immune response to an antigen in a subject; (ii) comprising a pharmaceutically active polypeptide and / or epitope for inducing an immune response to an antigen in a subject, wherein the pharmaceutically active polypeptide and / or antigen or epitope is derived from or from a pathogen protein, an immunogenic variant of a protein, or an immunogenic fragment of a protein or its immunogenic variant; (iii) comprising a pharmaceutically active polypeptide and / or epitope for inducing an immune response to an antigen in a subject, wherein the pharmaceutically active polypeptide and / or antigen or epitope is derived from or is derived from the SARS-CoV-2 spike (S) protein, its immunogenic variant, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. The composition according to claim 14.

16. A method for producing a composition containing particles dispersed in the final aqueous phase, Here, the composition comprises (i) nucleic acids; (ii) cationically ionizable lipids; (iii) steroids; (iv) neutral lipids; and (v) inorganic polyphosphates; the particles comprise at least a portion of nucleic acids, at least a portion of cationically ionizable lipids, and at least a portion of steroids; at least a portion of inorganic polyphosphates is bound to the particles; and the final aqueous phase comprises a final buffer system; Here, the method is (I) A preparation comprising particles dispersed in a final aqueous phase, wherein the particles comprise at least a portion of nucleic acids, at least a portion of cationically ionizable lipids, at least a portion of steroids, and at least a portion of neutral lipids, and at least a portion of inorganic polyphosphate is bound to the particles; and (II) Optionally, freeze the preparation to -10°C or below. A method comprising obtaining a composition, Here, process (I) is (a) Prepare a nucleic acid solution containing water and a first buffer system; (b) Prepare an organic solution containing cationically ionizable lipids, steroids, and neutral lipids; (c) Mix the nucleic acid solution prepared under (a) and the organic solution prepared under (b) to produce a first intermediate formulation containing particles dispersed in a first aqueous phase containing a first buffer system; (d)(c) is mixed with an inorganic polyphosphate or a salt thereof to produce a second intermediate formulation containing particles dispersed in a second aqueous phase containing a second buffer system, wherein at least a portion of the inorganic polyphosphate is bound to the particles; and (e) Filter and / or dilute the second intermediate formulation prepared under (d) using the final aqueous buffer containing the final buffer system. The method comprising producing a formulation containing particles dispersed in the final aqueous phase.

17. (i) step (I) further comprises one or more steps selected from dilution and filtration; (ii) Process (I) is (a') Prepare an aqueous nucleic acid solution; (b') Prepare the first aqueous buffer containing the first buffer system; Mix the aqueous nucleic acid solution prepared under (c')(a') with the first aqueous buffer prepared under (b') to produce a nucleic acid solution containing water and the first buffer system; (d') Prepare (e.g., prepare) an organic solution containing cationically ionizable lipids, steroids, and neutral lipids; Mix the nucleic acid solution prepared under (e')(c') with the organic solution prepared under (d') (for example, prepared), thereby producing a first intermediate formulation containing particles dispersed in a first aqueous phase containing a first buffer system; (f') Optionally, the first intermediate formulation prepared under (e') is diluted with water or a further aqueous buffer containing an additional buffer system, thereby producing a further intermediate formulation containing particles dispersed in the first buffer system or a further aqueous phase containing an additional buffer system, where the further aqueous buffer may be the same as or different from the first aqueous buffer; (g') If step (f') does not exist, the first intermediate formulation obtained in step (e'), or if step (f') exists, the further intermediate formulation obtained in step (f'), is mixed with inorganic polyphosphate or a salt thereof to produce a second intermediate formulation containing particles dispersed in a second aqueous phase, wherein at least a portion of the inorganic polyphosphate is bound to the particles; (h') Optionally, the second first intermediate formulation prepared under (g') is filtered using a further aqueous buffer containing a further buffer system, thereby producing a further intermediate formulation containing particles dispersed in a further aqueous phase containing a further buffer system, where the further aqueous buffer may be the same as or different from the first and / or second aqueous buffer; (i') Optionally, repeat step (h') once, twice or more times, wherein a further intermediate formulation containing particles dispersed in a further aqueous phase containing a further buffer system obtained after one cycle of step (h') is used as the second intermediate formulation for the next cycle, wherein in each cycle the further aqueous buffer may be the same as or different from the first and / or second aqueous buffer; (j') If step (h') does not exist, the second intermediate formulation obtained in step (g'), or if step (h') exists but step (i') does not exist, the further intermediate formulation obtained in step (h'), or if steps (h') and (i') exist, the further intermediate formulation obtained after step (i'), is filtered using the final aqueous buffer containing the final buffer system; and (k') Optionally, dilute the formulation obtained in step (j') with the dilution solution; This involves producing a formulation containing particles dispersed in the final aqueous phase; or (iii) Filtration is dialysis, tangential flow filtration, or diafiltration. The method according to claim 16.

18. (i) an inorganic polyphosphate or a salt thereof comprises or has the formula P x O (3x + 1) M y' [wherein x is an integer and at least 3; each M is independently H+ or a cation; and y' is the number of cations required for charge equilibrium]; or Inorganic polyphosphate or a salt thereof contains or has the formula P x O (3x + 1) My' [wherein x is an integer and at least 3; each M is independently selected from the group consisting of H+, alkali cations, ammonium, and monovalent organic cations, or each M is independently selected from the group consisting of H+, Na+, K+, Li+, and NH4+]; (ii) Inorganic polyphosphate or a salt thereof is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, salts thereof and mixtures thereof; (iii) Whether the inorganic polyphosphate or a salt thereof is linear inorganic polyphosphate or a salt thereof; (iv) The molar ratio of inorganic polyphosphate to cationically ionizable lipids is at least 1:2 or at least about 2:3 or at least about 4:3; (v) The composition does not contain lipids containing PEG or any compound containing PEG or does not contain PEG, The composition contains less than 1% by weight of polyethylene glycol (PEG) in lipids or compounds containing PEG, or PEG itself, based on the total weight of the composition; (vi) (1) The nucleic acid solution obtained in step (a) has a pH of less than 6.0 or a maximum of 5.0 or a maximum of 4.5; or (2) The first aqueous buffer has a pH of less than 6.0 or a maximum of 4.5; (vii) The pH of the composition is between 4.0 and 8.0; (viii) water is the main component of the formulation and / or composition and / or the total amount of solvents other than water contained in the composition is less than 0.5% (v / v); (ix) The osmotic pressure of the composition is at most 1000 × 10⁻³ osmol / kg; (x) The concentration of nucleic acid in the composition is between 1 mg / l and 500 mg / l; (xi) The cationically ionizable lipid contains a head group comprising at least one tertiary amine moiety; (xii) In an organic solution, cationically ionizable lipids, steroids, and neutral lipids are present in the following molar ratios: 20 mol% to 70 mol% cationically ionizable lipids; 15 mol% to 60 mol% steroids; and 5 mol% to 25 mol% neutral lipids; (xiii) Whether cationically ionizable lipids, steroids, and neutral lipids are present in the organic solution in the following molar ratios: 40 mol% to 70 mol% cationically ionizable lipids; 15 mol% to 40 mol% steroids; and 15 mol% to 25 mol% neutral lipids; or Cationically ionizable lipids, steroids, and neutral lipids are present in the organic solution in the following molar ratios: 20 mol% to 40 mol% cationically ionizable lipids; 35 mol% to 60 mol% steroids; and 5 mol% to 15 mol% neutral lipids. The method according to claim 16.

19. (i) The particles have a size of 30 nm to 500 nm; (ii) The particles are selected from the group consisting of lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures of two or more thereof; or (iii) The nucleic acid is RNA. The method according to claim 16.

20. (II) The method according to claim 16, comprising freezing the formulation to -10°C or below.

21. The method according to claim 16, wherein step (II) is not included.

22. A composition that can be produced by the method described in any one of claims 16 to 21, (i) It is in a frozen state; or (ii) It is in liquid form. The aforementioned composition.

23. A ready-to-use pharmaceutical composition, (i) A method comprising the steps of preparing a frozen composition manufactured by the method of claim 20, thawing the frozen composition, and thereby obtaining a ready-to-use pharmaceutical composition; or (ii) A method comprising the step of preparing a liquid composition manufactured by the method of claim 21, thereby obtaining a ready-to-use pharmaceutical composition. The ready-to-use pharmaceutical composition that can be manufactured by the above method.

24. A composition according to any one of claims 1 to 15 for use in treatment.

25. A composition according to any one of claims 1 to 15 for use in inducing an immune response in a subject.

26. Use of the composition according to any one of claims 1 to 15 for transfecting cells in vitro.

27. ​​(i) The mixture of the composition and the cells is incubated for a sufficient amount of time; (ii) Cell transfection is carried out in the presence of serum; or (iii) The mixture of the composition and cells is incubated in the presence of serum for a sufficient amount of time. The use described in claim 26.

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