Compositions and methods

Abstract

An aqueous dispersion having an aqueous mobile phase and a dispersed phase is described; wherein the dispersed phase comprises a lipid mixture comprising a cationically ionizable lipid; and the aqueous mobile phase comprises anions of an aqueous acid; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA. Methods of making the aqueous dispersions, nucleic acid-lipid particles and methods of making them using the aqueous dispersions, and their use in medicine are disclosed.

Description

Technical Field

[0001] The present disclosure generally relates to lipid-based formulations suitable for carrying nucleic acids, particularly RNA, lipid particles comprising such nucleic acids, aqueous lipid dispersions capable of receiving nucleic acids, and methods for preparing the same, particularly such methods that do not involve the use of organic solvents. Background of the Invention

[0003] The traditional preparation approach of preparing nucleic acid-containing lipid nanoparticles is carried out by a one-step method in which a nucleic acid (such as RNA) in an aqueous buffer is mixed with three parts of lipid mixtures dissolved in an organic solvent. Although great success has been achieved with this approach, established technology is usually affected by manufacturing process complexity, such as requiring subsequent tangential flow filtration (TFF) steps to remove the organic solvent for dissolving lipids. This further processing increases the complexity of technology, and ultimately increases manufacturing lead time (manufacturing lead time) and associated costs. In addition, this scheme is usually unfavorable for individualized patient treatment, because a large number of batches need to be produced within a very short turnaround time. When using a compound with limited chemical stability, the use of an organic solvent in preparing RNA lipid nanoparticle preparations (wherein requiring longer processing time) may also be a shortcoming.

[0004] WO 2018 / 089801 describes a method for preparing empty lipid nanoparticles, wherein a lipid dissolved in ethanol is mixed with a citrate buffer of pH 4.5, followed by purification using tangential flow filtration, wherein exchange buffer occurs. These preformed empty lipid nanoparticles can then be mixed with mRNA to produce loaded lipid nanoparticles, wherein the mRNA is encapsulated. WO 2020 / 047061 similarly describes a method for preparing empty lipid nanoparticles, wherein a lipid dissolved in ethanol is mixed with a citrate buffer of pH 4.5, followed by buffer exchange (e.g., by tangential flow filtration) to produce empty lipid nanoparticles in a 10% weight / volume trehalose buffer. These preformed empty lipid nanoparticles can then be mixed with mRNA to produce loaded lipid nanoparticles, wherein the mRNA is encapsulated.

[0005] WO2022 / 032087 describes a method for preparing an empty lipid nanoparticle solution (empty-LNP solution), which comprises: i) a nanoprecipitation step comprising: ia) mixing a lipid solution comprising ionizable lipids, structural lipids and phospholipids with an aqueous buffer solution comprising a first buffer, thereby forming an intermediate empty lipid nanoparticle solution (intermediate empty-LNP solution) comprising an intermediate empty nanoparticle (intermediate empty LNP); ib) the intermediate empty-LNP solution is maintained for a residence time; and ic) a diluent solution is added to the intermediate empty-LNP solution, thereby forming the empty-LNP solution. The empty-LNP solution can be further processed to produce an empty-LNP preparation. Also described is a method for preparing the LNP of a load by mixing an empty-LNP solution or an empty-LNP preparation with a nucleic acid.

[0006] However, although ethanol, citrate buffer and other destabilizing agents may not be present during the addition of mRNA, the formation of empty lipid nanoparticles in all of these documents uses a buffer, particularly a citrate buffer or an acetate buffer. It is believed that the presence of inorganic ions in such buffers (e.g., citrate buffer) can destroy the colloidal properties of the lipid nanoparticle formulation and thus be detrimental to the formulation stability.

[0007] WO2022 / 069632 describes a method for preparing RNA lipid complex particles for delivering RNA to target tissues. The method described in this document only uses cationic lipids as defined herein. Therefore, the document does not disclose a method for preparing aqueous dispersions (such as pre-LNP or nucleic acid-lipid particles), wherein the lipid is a cationic ionizable lipid as defined herein.

[0008] WO2011 / 144745 describes a kind of method for the preparation of liposome, and described liposome can load pharmacy and / or diagnostic active agent and / or cosmetic agent, and they are dissolved by liposomal membrane basically.The method described in this file only adopts the cationic lipid defined herein.Therefore, this file does not disclose the method for preparing aqueous dispersion (such as pre-LNP or nucleic acid-lipid granule), and wherein said lipid is the ionizable lipid of cation as defined herein.

[0009] WO 2022 / 101471 and WO 2022 / 101486 describe pharmaceutical compositions comprising lipid nanoparticles and mRNA and methods for their preparation and storage. Specifically, these documents describe a mixture of DODMA, DOPE, cholesterol, and C in a molar ratio of 40:10:48:2 in ethanol. 16 -PEG 2000-A lipid mixture of ceramide. These documents describe mixing liposomes consisting of this lipid mixture in 5mM acetic acid aqueous solution with RNA (10mM HEPES, 0.1mM EDTA, pH 7.0) to produce RNA-lipid complexes, which can then be diluted in a sucrose-containing buffer to a final sucrose concentration of 10%. These documents do not describe lipid mixtures in aqueous solutions containing cryoprotectants such as sucrose in the absence of RNA. Furthermore, the document does not teach or suggest the steps of freezing or lyophilizing the resulting aqueous dispersion and the resulting advantages of producing personalized therapies.

[0010] WO2021 / 155274 describes a method for preparing an empty lipid nanoparticle (empty-LNP) solution comprising empty lipid nanoparticles, wherein the method comprises mixing a lipid solution with a solution comprising a first buffer, thereby forming an empty-LNP solution comprising empty LNP, wherein the empty-LNP solution comprises acetate buffer and has a pH in the range of about 4.6 to about 6.0. The empty LNP solution can then be mixed with RNA or other nucleic acids to produce the LNP of load, wherein the lipid encapsulates the RNA. However, due to similar reasons outlined above about WO 2020 / 047061 and WO 2018 / 089801, it is found that the inorganic ions present in the acetate buffer used in the method described in this document are harmful to the stability of the lipid nanoparticle formulation. SUMMARY OF THE INVENTION

[0012] In a first aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase;

[0013] in:

[0014] The dispersed phase comprises a lipid mixture including cationic lipids or cationic ionizable lipids; and

[0015] The aqueous mobile phase comprises an anion of an aqueous acid;

[0016] wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA, and

[0017] The aqueous mobile phase contains a cryoprotectant.

[0018] In one embodiment of this aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase;

[0019] in:

[0020] The dispersed phase comprises a lipid mixture including cationic ionizable lipids; and

[0021] The aqueous mobile phase comprises an anion of an aqueous acid;

[0022] wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA, and

[0023] The aqueous mobile phase contains a cryoprotectant.

[0024] In a second aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase; wherein:

[0025] The dispersed phase comprises a cationic lipid or a cationic ionizable lipid; and

[0026] The aqueous mobile phase comprises an anion of an aqueous acid;

[0027] in:

[0028] The concentration of the aqueous acid is at least 6 mM; and

[0029] The aqueous mobile phase is substantially free of inorganic cations, organic solvents, and RNA.

[0030] In a third aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase; wherein:

[0031] The dispersed phase comprises a cationic lipid or a cationic ionizable lipid; and

[0032] The aqueous mobile phase comprises malate anion or succinate anion;

[0033] The aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA.

[0034] In a fourth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, the method comprising mixing:

[0035] (i) a lipid mixture comprising cationic lipids or cationic ionizable lipids;

[0036] (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant;

[0037] to produce an aqueous dispersion comprising the anion of the aqueous acid.

[0038] In one embodiment of the fourth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, the method comprising mixing:

[0039] (i) a lipid mixture comprising cationic ionizable lipids;

[0040] (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant;

[0041] to produce an aqueous dispersion comprising the anion of the aqueous acid.

[0042] In a fifth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, the method comprising:

[0043] (a) Mixing:

[0044] (i) a lipid mixture comprising a cationic lipid or a cationic ionizable lipid; and

[0045] (ii) an aqueous phase comprising an aqueous acid;

[0046] to produce a first intermediate aqueous dispersion comprising an anion of said aqueous acid; and

[0047] (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce the aqueous dispersion.

[0048] In one embodiment of the fifth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, the method comprising:

[0049] (a) Mixing:

[0050] (i) a lipid mixture comprising cationically ionizable lipids; and

[0051] (ii) an aqueous phase comprising an aqueous acid;

[0052] to produce a first intermediate aqueous dispersion comprising an anion of said aqueous acid; and

[0053] (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce the aqueous dispersion.

[0054] In a sixth aspect, the present disclosure provides a method of forming an aqueous dispersion comprising an anion of an aqueous acid, the method comprising:

[0055] (a) Mixing:

[0056] (i) a lipid mixture comprising a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent; and

[0057] (ii) aqueous phase;

[0058] The lipid mixture and / or the aqueous phase comprises the aqueous acid;

[0059] to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid;

[0060] (b) subjecting the first intermediate acidified aqueous lipid dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5 to remove the organic solvent and produce a second intermediate aqueous dispersion; and

[0061] (c) adding a cryoprotectant to the second intermediate aqueous dispersion;

[0062] to produce an aqueous dispersion comprising the anion of the aqueous acid.

[0063] In a seventh aspect, the present disclosure provides a method of forming an aqueous dispersion comprising an anion of an aqueous acid, the method comprising:

[0064] i) mixing a lipid mixture and an aqueous phase, wherein the lipid mixture comprises a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent,

[0065] wherein the lipid solution and / or the aqueous phase comprises an aqueous acid,

[0066] to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid;

[0067] ii) subjecting the first intermediate acidified aqueous lipid dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5, or at a pH of 6.5 to 8.5, to remove the organic solvent and produce a second intermediate aqueous dispersion; and

[0068] iii) adding a cryoprotectant to the second intermediate aqueous dispersion;

[0069] to produce the aqueous dispersion;

[0070] The aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA.

[0071] In an eighth aspect, the present disclosure provides a method of forming the aqueous dispersion of the second aspect, the method comprising mixing:

[0072] (i) a lipid mixture comprising cationic lipids or cationic ionizable lipids;

[0073] (ii) an aqueous phase comprising an aqueous acid;

[0074] wherein the concentration of the aqueous acid is at least 6 mM;

[0075] to produce an aqueous dispersion comprising the anion of the aqueous acid.

[0076] In a ninth aspect, the present disclosure provides a method of forming the aqueous dispersion of the third aspect, the method comprising mixing:

[0077] (i) a lipid mixture comprising cationic lipids or cationic ionizable lipids;

[0078] (ii) an aqueous phase comprising malic acid or succinic acid;

[0079] to produce an aqueous dispersion comprising malate anions or succinate anions.

[0080] In a tenth aspect, the present disclosure provides a method of forming a lipid particle containing a nucleic acid (e.g., RNA, such as mRNA), the method comprising:

[0081] i) preparing an aqueous dispersion according to the method of any one of the first to ninth aspects; and

[0082] ii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid to produce lipid particles containing the nucleic acid.

[0083] In an eleventh aspect, the present disclosure provides a method of forming a lipid particle containing a nucleic acid (e.g., RNA, such as mRNA), the method comprising:

[0084] i) mixing a lipid mixture and an aqueous phase, wherein the lipid mixture comprises a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent,

[0085] wherein the lipid solution and / or the aqueous phase comprises an aqueous acid,

[0086] to produce a first intermediate dispersion comprising an anion of the aqueous acid;

[0087] ii) subjecting the first intermediate dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5, or at a pH of 6.5 to 8.5, to remove the organic solvent and produce a second intermediate aqueous dispersion,

[0088] iii) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion, wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and nucleic acids, and

[0089] iv) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid to produce lipid particles containing the nucleic acid.

[0090] In a twelfth aspect, the present disclosure provides a lipid-nucleic acid particle (e.g., a lipid-RNA particle), such as a lipid nanoparticle, obtained or obtainable by the method of the tenth or eleventh aspect.

[0091] In a thirteenth aspect, the present disclosure provides a pharmaceutical composition comprising the lipid particle of the twelfth aspect and a drug carrier.

[0092] In a fourteenth aspect, the present disclosure provides the lipid particle of the twelfth aspect for medical use.

[0093] In a fifteenth aspect, the present disclosure provides the lipid particle of the twelfth aspect for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for inducing an immune response.

[0094] In a sixteenth aspect, the present disclosure provides the lipid particle of the twelfth aspect for use in treating cancer.

[0095] In a seventeenth aspect, the present disclosure provides use of the lipid particle of the twelfth aspect in the preparation of a medicament for the prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for inducing an immune response.

[0096] In an eighteenth aspect, the present disclosure provides use of the lipid particle of the twelfth aspect in the preparation of a medicament for treating cancer.

[0097] In a nineteenth aspect, the present disclosure provides a method for prophylactically and / or therapeutically treating a disease involving an antigen and / or inducing an immune response in a subject in need thereof, the method comprising administering to the subject the lipid particle of the twelfth aspect.

[0098] In a twentieth aspect, the present disclosure provides a method of prophylactically and / or therapeutically treating cancer in a subject in need thereof, the method comprising administering to the subject the lipid particle of the twelfth aspect.

[0099] In a twenty-first aspect, the present disclosure provides a lyophilized composition comprising the aqueous dispersion of any one of the first to third aspects.

[0100] In a twenty-second aspect, the present disclosure provides a frozen composition comprising the aqueous dispersion of any one of the first to third aspects, wherein the frozen composition is at a temperature between -15°C and -90°C.

[0101] Advantages and surprising discoveries

[0102] The inventors of the present invention have unexpectedly discovered that the methods described herein can prepare aqueous dispersion compositions (without nucleic acid) and nucleic acid-lipid particles without the use of organic solvents. This avoids the risk of organic solvents degrading chemically unstable lipids and the need for complex purification processes to remove organic solvents. A large number of preformed aqueous dispersions can be used to prepare multiple batches of nucleic acid-lipid particles (e.g., patient-specific mRNA lipid nanoparticles) preparations, and are particularly useful in areas such as personalized immunotherapy platforms where small batches of final products are required.

[0103] In addition, the methods disclosed herein provide excellent manufacturing advantages and compatibility with Class D manufacturing environments. In addition, compared to other classical lipid nanoparticle preparation approaches, the nucleic acid-lipid particles formed using this new process have been shown to have improved colloidal stability, lipid stability, and RNA integrity under frozen and liquid conditions while maintaining biological efficacy. In addition, the methods disclosed herein have great flexibility to change the properties of the formulation, such as particle size, surface charge, and functionalization, without affecting the robustness of the process. In addition, the nucleic acid-lipid particles manufactured by the disclosed methods can also be functionalized with ligands to target specific cells, organs, etc.

[0104] In addition, compared with the method described in WO 2020 / 047061, WO 2018 / 089801, WO2021 / 155274 and WO2022 / 032087, method described herein avoids the use of inorganic ions present in citrate and acetate buffer, and thus avoids the deleterious effects of inorganic ions on nucleic acid-lipid granule preparations. Specifically, it is believed that the use of malic acid or succinic acid can further improve the maintenance of colloidal stability and / or RNA integrity, such as in RNA-lipid granule formation and subsequent storage process. It is also surprisingly found that higher concentrations of acid (such as acetic acid with a concentration of 6mM or higher) can provide improved colloidal properties and / or can not negatively impact particle stability and / or RNA integrity. Adding a cryoprotectant such as sucrose to aqueous dispersions can enhance the long-term stability of lipid granules, and is conducive to storing under freezing conditions.

[0105] Furthermore, the present inventors have discovered that adding a cryoprotectant during the initial mixing step in the process of forming the aqueous dispersion has the advantage that an HPLC step between the tangential flow filtration (TFF) and dilution steps is not required, as it is no longer necessary to measure the lipid concentration to determine the amount of cryoprotectant to add by dilution. This simplifies and shortens the manufacturing process.

[0106] In addition, and compared with the method described in WO 2022 / 101486 and WO 2022 / 101471, the inventors of the present invention have found that adding a cryoprotectant in the initial mixing step in the method for forming an aqueous dispersion has the advantage of enhancing colloidal stability. Without wishing to be bound by theory, it is believed that including a cryoprotectant in the initial mixing step avoids changes in osmotic pressure in filtration (e.g., TFF) / dialysis or dilution steps, thereby enhancing colloidal stability. Surprisingly, it has also been found that loading nucleic acids (such as RNA) into aqueous dispersions containing cryoprotectants can produce available nucleic acid-lipid particle compositions: this was considered impossible in the past because loading nucleic acids into more viscous aqueous dispersions is expected to be challenging. In particular, it has been surprisingly found that nucleic acids (e.g., RNA) may be loaded into aqueous dispersions comprising higher sucrose concentrations (e.g., 20% sucrose), which allows nucleic acid-lipid particles to be prepared in a one-step procedure, thereby avoiding further dilution steps to add cryoprotectants. This allows the second step of the preparation process to be simplified and shortened, which is conducive to RNA integrity and particle stability. Furthermore, at the date of publication of WO 2022 / 101486 and WO 2022 / 101471, it had not been foreseen that aqueous dispersions could be frozen or lyophilized, and the advantages of using a two-step process to produce personalized therapies had not been suggested.

[0107] BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG1 (a) shows a general manufacturing scheme for aqueous dispersions of the present invention (also described herein as "preformed lipid nanoparticles" ("pre-LNPs")); (b) shows a preparation scheme for exemplary aqueous dispersions of the present invention (also described herein as "preformed lipid nanoparticles," i.e., prior to introduction of nucleic acids) (where IPA refers to isopropyl alcohol);

[0109] Figure 2 An exemplary preparation scheme of RNA-lipid particles according to the present invention is shown;

[0110] Figure 3 The stability of aqueous dispersions according to the invention (Example 1) with grafted lipids when stored under liquid and frozen conditions is shown, expressed as particle size (a) and polydispersity index (b);

[0111] Figure 4 Shown are the long-term stability of RNA-lipid particles according to the invention (Example 1) prepared according to the two-step method described herein at -80°C and -20°C as a function of particle size (a), polydispersity index (b) and RNA integrity (c);

[0112] Figure 5The INF-γ ELISpot shown shows a higher T-cell response for the drug product prepared according to the method of the present invention (designated LNP2) (Example 2), all formulations tested had similar lipids and N / P ratios and were administered at the same dose;

[0113] Figure 6 The stability of an aqueous dispersion of the invention comprising DODMA and C14-Psar(23)-Ac (Example 2) when stored under liquid (4°C and 25°C) conditions is shown, expressed in terms of particle size (a) and polydispersity index (b);

[0114] Figure 7 The stability of aqueous dispersions according to the present invention (Example 3) containing DODMA and DMG-PEG2k at different pH values ​​is shown for 5 mM acetic acid (a), 40 mM acetate buffer (b), and 10 mM HEPES buffer (c);

[0115] Figure 8 The colloidal stability of grafted free RNA-lipid particles according to the present invention (Example 4) (formulated with an N / P ratio of 6, pH 5.5, an RNA content of 0.1 mg / mL, and stored in HEPES buffer containing 10% (w / v) sucrose; the lipid mixture consisted of the ionizable lipid HY-501, cholesterol, and DSPC in a molar ratio of 47.5:42.5:10) monitored under frozen and liquid conditions over a period of 3 months is shown, with particle size (a) and polydispersity (b) both within the specifications for the duration of the experiment;

[0116] Figure 9 The stability of aqueous dispersions according to the invention (Example 6) containing α-tagged lipids when stored under liquid conditions (4°C and 25°C) is shown, expressed as particle size (a) and polydispersity index (b);

[0117] Figure 10 shows the particle size (a) and polydispersity index (b) analysis of functionalized RNA-lipid particles according to the present invention (Example 6) after two freeze-thaw (FT) cycles from -20°C to room temperature and from -80°C to room temperature;

[0118] Figure 11 Particle size and polydispersity index (PDI) of crude RNA-lipid particles, α-labeled RNA-lipid particles with post-insertion protocol and functionalized RNA-lipid particles according to the present invention (Example 7) are shown;

[0119] Figure 12The freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 10A) with different concentrations (1.25, 2.5, and 5 mM) of acetic acid is shown;

[0120] FIG13 shows the freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 10B) using 5 mM acetic acid and diluted and stored in (A) 8% w / v sucrose and (B) 12% w / v sucrose;

[0121] Figure 14 The freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 10C) with 5 mM acetic acid and diluted and stored in 10% trehalose is shown;

[0122] Figure 15 The freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 10D) with 5 mM acetic acid and diluted and stored in 5% w / v glucose is shown;

[0123] FIG. 16 shows the results of the present invention (Example 11) using 2.5 mM acetic acid ( Figure 16A ) or 5 mM acetic acid ( Figure 16B ) Freeze-thaw stability of the prepared pre-LNP;

[0124] Figure 17 The freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 12) with different concentrations (2.5, 5 and 10 mM) of malic acid is shown;

[0125] Figure 18 Shown are the freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 12) using a mixture of 5 mM acetic acid + different concentrations of malic acid as indicated;

[0126] FIG19 shows the freeze-thaw stability of pre-LNPs prepared and purified according to the present invention (Example 14) using a) 5 mM acetic acid, 5% w / v sucrose or b) 5 mM acetic acid, 10% w / v sucrose mixture;

[0127] Figure 20 (Comparative) shows the freeze-thaw stability of pre-LNPs prepared and purified according to Example 15 (Comparative) using the following buffers: (A) 5 mM acetate buffer, pH 5.0 or 5.5 mM, (B) citrate buffer with different concentrations (2.5, 5, 10, and 20 mM); (C) 30 mM succinate buffer, approximately pH 4, (D) 30 mM malate buffer, approximately pH 4;

[0128] Figure 21 The colloidal stability of RNA-LNPs according to the present invention (Example 17) during five freeze-thaw cycles is shown;

[0129] Figure 22 The long-term stability of RNA-LNP according to the present invention (Example 17) is shown;

[0130] Figure 23 Particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 18) are shown;

[0131] Figure 24 The long-term stability of RNA-LNP according to the present invention (Example 18) is shown;

[0132] Figure 25 shows the particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 19);

[0133] Figure 26 Particle size and PDI liquid states of RNA-LNPs according to the present invention (Example 20) are shown - for 2.5 mM acetic acid, different storage matrices are indicated: (a) 60 mM HEPES, 3 mM Tris, 30% sucrose, pH 6.3; (b) 50 mM Tris, 30% sucrose, pH 8.5;

[0134] Figure 27 Particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 21) are shown;

[0135] Figure 28 shows the particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 22);

[0136] Figure 29 The long-term stability of concentrated LNPs according to the present invention (Example 23) is shown (RNA concentration (mg / mL): Group A = 0.1; Group B = 0.2; Group C = 0.3; Group D = 0.6; Group D = 1.3);

[0137] Figure 30 Particle size and PDI of RNA-LNPs according to the present invention (Example 25) prepared from lyophilized and reconstituted pre-LNPs are shown;

[0138] Figure 31 Particle size and PDI of freeze-thawed LNPs according to the present invention (Example 26) are shown;

[0139] Figure 32 shows an exemplary simplified preparation scheme of RNA-LNP according to the present invention, as described in Example 27; and

[0140] Figure 33Particle size and PDI of RNA-LNPs according to the present invention (Example 27) are shown.

[0141] Detailed description

[0142] Hereinafter, the elements of the present disclosure will be described in more detail. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any way and in any number to produce other embodiments. The various described embodiments and preferred embodiments should not be interpreted as limiting the present disclosure to only the embodiments clearly described. This description should be understood to support and include such embodiments: it will clearly describe the embodiment and any number of disclosed and / or preferred element combinations. In addition, any arrangement and combination of all described elements in this application should be considered as disclosed by the description of the application, unless the context otherwise indicates.

[0143] Preferably, the terms used herein are in accordance with the terms in "A multilingual glossary of biotechnological terms: (IUPAC recommendations)", HGW Leuenberger, B. Nagel, and H. eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, which are explained in the literature in the field (see, for example, 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; ChemieLexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory Manual, 2nd Edition, edited by J.Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0144] All methods described herein can be performed in any suitable order, unless otherwise noted herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure as otherwise claimed. Any language in the specification should not be construed as indicating any unclaimed element essential to the practice of the present disclosure.

[0145] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0146] Several documents are cited in the text of this application. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is disqualified from antedating such disclosure by virtue of prior invention.

[0147] definition

[0148] In the following, definitions applicable to all aspects of the present disclosure are provided. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its meaning generally accepted in the art.

[0149] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variations such as "include" 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 is meant to exclude other members, integers or steps in any substantive sense. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, the term "comprising" may be replaced by the term "consisting essentially of" or "consisting of" at each occurrence in this application. Likewise, the term "consisting essentially of" may be replaced by the term "consisting of" at each occurrence in this application.

[0150] The use of the terms "a," "an," and "the" and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0151] As used herein, "and / or" should be understood as specific disclosure of each of the two specified features or components, whether or not with the other. For example, "X and / or Y" should be considered specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually listed herein.

[0152] In the context of this disclosure, the term "about" represents an accuracy interval that will be understood by those of ordinary skill in the art to still ensure the technical effect of the feature in question. The term generally indicates a deviation from the numerical value shown of ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05% and, for example, ±0.01%. For example, with respect to pH values, the term "about" may preferably indicate a deviation from the numerical value shown of up to 0.3. Those of ordinary skill in the art will understand that this specific deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have this type of deviation greater than artificial or engineered technical effects.

[0153] As used herein, the expression "substantially free of X" means that the compositions described herein are free of X in a practical and practicable 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 %, or less than 0.001 wt %) based on the total weight of the mixture. The specific meaning of the term "substantially free" with respect to certain components of the compositions is defined herein.

[0154] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In certain embodiments, the physiological pH is from 7.3 to 7.5. In certain embodiments, the physiological pH is from 7.35 to 7.45. In certain embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0155] As used herein, "physiological conditions" refers to conditions (particularly 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.

[0156] As used in this disclosure, "mol %" is defined as the ratio of the number of moles of a component to the total number of moles of all components multiplied by 100.

[0157] As used in this disclosure, "mol % of a lipid mixture" is defined as the ratio of the moles of that particular lipid component to the total moles of all lipids in the lipid mixture multiplied by 100. In this context, in certain embodiments, the terms "total lipids" and / or "total lipid mixture" include lipids and lipid-like substances.

[0158] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In certain embodiments, the hydrocarbyl group is non-cyclic, for example, linear (straight chain) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Specific examples of hydrocarbyl groups are C 1-40 Alkyl (such as C 6-40 Alkyl, C 6-30 Alkyl, C 6-20 Alkyl or C 10-20 alkyl), C having 1, 2 or 3 double bonds 2-40 Alkenyl (such as C 6-40Alkenyl, C 6-30 Alkenyl or C 6-20 alkenyl), aryl and aryl (C 1-6 In certain embodiments, the hydrocarbyl group is optionally substituted with one or more (such as 1, 2, or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0159] The term "heteroalkyl" refers to an alkyl group as defined above, wherein 1, 2, 3 or 4 carbon atoms in the alkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus or sulfur (preferably O, S or N). In one embodiment, the heteroalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0160] The term "alkyl" refers to a monovalent residue of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 40 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-eicosyl, n-triacontyl, n-tetracontyl, and the like. "Substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A. Examples of substituted alkyl groups include chloromethyl, dichloromethyl, fluoromethyl and difluoromethyl.

[0161] The term "alkylene" refers to a divalent residue of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises 1 to 40 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-iso-butylene, 1,2-iso-butylene, and 1,3-iso-butylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,6-pentylene, 1,7-pentylene, 1,8-pentylene, 1,9-pentylene, 2,10-pentylene), 1,1-iso-butylene, 1,2-iso-butylene, and 1,3-iso-butylene), ,1-isopentylene, 1,1-sec-pentylene, 1,1-neopentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene and 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1-isoheptylene), octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene), etc. A straight-chain alkylene moiety having at least 3 carbon atoms and one free valence at each end may also be referred to as a plurality of methylene groups (e.g., 1,4-butylene may also be referred to as tetramethylene). Generally, for alkylene moieties, in addition to using the ending "alkenylene" as described above, the ending "diyl" may also be used (e.g., 1,2-butylene may also be referred to as butane-1,2-diyl)."Substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkylene group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylene group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0162] Term " alkenyl " represents the monovalent residue of the unsaturated straight chain or branched hydrocarbon with at least one carbon-carbon double bond.Usually, the maximum number of carbon-carbon double bonds in alkenyl group can equal the integer that carbon atom number in alkenyl group is calculated divided by 2, and if carbon atom number in alkenyl group is an odd number, then the division result is rounded down to the nearest integer.For example, for alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4.Preferably, alkenyl group has 1 to 6 (such as 1 to 4) individual (that is, 1,2,3,4,5 or 6) carbon-carbon double bonds.Preferably, alkenyl group comprises 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Therefore, in a preferred embodiment, alkenyl group comprises 2 to 40 (such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10) carbon atoms and 1, 2, 3, 4, 5 or 6 (for example, 1, 2, 3, 4 or 5) individual carbon-carbon double bonds, such as comprising 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. Carbon-carbon double bonds can be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 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-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-decenyl, 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."Substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkenyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkenyl group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkenyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0163] The term "alkenylene" refers to a divalent residue of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Typically, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, and if the number of carbon atoms in the alkenylene group is an odd number, the division result is rounded down to the nearest integer. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4) (i.e., 1, 2, 3, 4, 5 or 6) carbon-carbon double bonds. Preferably, alkenylene group comprises 2 to 12 (such as 2 to 10) carbon atoms, that is, 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, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.Therefore, in a preferred embodiment, described alkenylene group comprises 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5 or 6 (such as 1, 2, 3, 4 or 5) individual carbon-carbon double bond, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3 or 4 carbon-carbon double bond, such as 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon double bond, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bond.Carbon-carbon double bond can be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propylene-1,2-diyl, 1-propylene-1,3-diyl, 1-propylene-2,3-diyl, allylene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, and the like. "Substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkenylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 15 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkenylene group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkenylene group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0164] The term "alkynyl" refers to a straight or branched monovalent hydrocarbon moiety with at least one carbon-carbon triple bond, wherein the total carbon atoms can be 6 to 40, such as 6 to 30, typically 6 to 20, such as 6 to 18. Alkynyl groups can optionally have one or more carbon-carbon triple bonds. Typically, the maximum number of carbon-carbon triple bonds in the alkynyl group can equal the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and if the number of carbon atoms in the alkynyl group is an odd number, the division result is rounded down to the nearest integer. For example, for an alkynyl group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4) individual (that is, 1, 2, 3, 4, 5 or 6), more preferably 1 or 2 carbon-carbon triple bonds. "Substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkynyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkynyl group are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkynyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0165] The term "cycloalkyl" and "cycloalkenyl" represent the cyclic non-aromatic forms of "alkyl" and "alkenyl", which preferably have 3 to 40 (such as 3 to 30, such as 3 to 20, such as 3 to 14) carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, that is, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and adamantyl. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl and cyclodecenyl. A cycloalkyl or cycloalkenyl group may be composed of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). "Substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the cycloalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the cycloalkyl or cycloalkenyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0166] The terms "cycloalkylene" and "cycloalkenylene" represent cyclic non-aromatic forms of "alkylene" and "alkenylene" that preferably have 3 to 40 (such as 3 to 30, such as 3 to 20, such as 3 to 14) carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, that is, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene and cycloheptylene. Exemplary cycloalkenylene groups include cyclopentenylene and cyclohexenylene.

[0167] The term "aryl" refers to a monovalent residue of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9 or 10, such as 5, 6 or 10) carbon atoms, which can be arranged in a ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthracenyl and phenanthrenyl. Preferably, "aryl" refers to a monocycle containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Fullerenes are not encompassed by aryl. "Substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the aryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 5 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the aryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the aryl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A. Examples of substituted aryl groups include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilino, 4-hydroxyphenyl and methoxyphenyl (i.e., 2-, 3 or 4-methoxyphenyl).

[0168] The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group as defined above, wherein one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S or N. Preferably, heteroaryl represents a 5- or 6-membered aromatic monocyclic ring in which 1, 2 or 3 carbon atoms are replaced by heteroatoms of the same or different O, N or S. Alternatively, it refers to an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4 or 5 carbon atoms are replaced by heteroatoms of the same or different O, N or S. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl , benzisothiazolyl, benzotriazolyl, quinolyl, isoquinolyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perylene-1,1-diazolinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the heteroaryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the heteroaryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heteroaryl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0169] The term "heterocyclyl" or "heterocycle" refers to a cycloalkyl group as defined above, wherein 1, 2, 3 or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus or sulfur (preferably O, S or N). The heterocyclyl group preferably has 1 or 2 rings containing 3 to 10 (such as 3, 4, 5, 6 or 7) ring atoms. Preferably, in each ring of the heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also intended to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also known as piperidinyl), piperazinyl, di- and tetra-hydrofuranyl, di- and tetra-hydrothienyl, di- and tetra-hydropyranyl, cyclohexamethylenetetramine, lactones, lactams, cyclic imides, and cyclic anhydrides. "Substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocyclyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heterocyclyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0170] The term "alkylcycloalkyl" refers to a cycloalkyl group as defined above, substituted with an alkyl group as defined above, the cycloalkyl portion being attached to the remainder of the molecule. Each cycloalkyl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted alkylcycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylcycloalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylcycloalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0171] The term "cycloalkylalkyl" refers to an alkyl group as defined above, substituted with a cycloalkyl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each cycloalkyl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted cycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the cycloalkylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the cycloalkylalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0172] The term "alkylcycloalkylalkyl" refers to an alkyl group, as defined above, substituted with a cycloalkyl group, as defined above, wherein the alkyl portion is attached to the rest of the molecule and the cycloalkyl portion is substituted with another alkyl group. Each cycloalkyl and alkyl portion of the group may have any of the broadest or preferred meanings described above. "Substituted alkylcycloalkylalkyl" means that one or more (such as from 1 to the maximum number of hydrogen atoms bound to the alkylcycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylcycloalkylalkyl group is substituted with one or more (such as 1, 2, or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0173] The term "alkylaryl" refers to an aryl group as defined above, substituted with an alkyl group as defined above, the aryl moiety being attached to the remainder of the molecule. Each aryl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted alkylaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylaryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or aryl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylaryl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0174] The term "arylalkyl" refers to an alkyl group as defined above, substituted with an aryl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each aryl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted arylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the arylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or aryl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the arylalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0175] The term "alkylheteroaryl" refers to a heteroaryl group as defined above, substituted with an alkyl group as defined above, the heteroaryl moiety being attached to the remainder of the molecule. Each heteroaryl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted alkylheteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylheteroaryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or heteroaryl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylheteroaryl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0176] The term "heteroarylalkyl" refers to an alkyl group as defined above, substituted with a heteroaryl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each aryl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted heteroarylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the heteroarylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or heteroaryl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heteroarylalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0177] The term "alkylheterocyclyl" refers to a heterocyclyl group as defined above, substituted with an alkyl group as defined above, the heteroaryl moiety being attached to the remainder of the molecule. Each heterocyclyl and alkyl moiety of the group may take any of the broadest or preferred meanings described above. "Substituted alkylheterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkylheterocyclyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or heteroaryl moiety of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylheterocyclyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0178] The term "heterocyclylalkyl" refers to an alkyl group as defined above, substituted with a heterocyclyl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each heterocyclyl and alkyl portion of the group may take any of the broadest or preferred meanings described above. "Substituted heterocyclylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to the heterocyclylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms of the alkyl or heterocyclyl portion of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heterocyclylalkyl group is substituted with one or more (such as 1, 2 or 3, such as 1 or 2, such as 1) substituents selected from List A.

[0179] The term "organosulfate" or "sulfate ester" refers to a compound of the formula R-OSO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). When the group is deprotonated, the term "sulfate" is used. Depending on the pH, the sulfate group can be protonated or deprotonated (in anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0180] The term "sulfonic acid" or "sulfonate" refers to a compound of the formula R-SO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). When the group is deprotonated, the term "sulfonate" is used. Depending on the pH, the sulfonic acid group can be protonated or deprotonated (in anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0181] The term "carboxylic acid" or "carboxylate" refers to a compound of the formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). When the group is deprotonated, the term "carboxylate" is used. Depending on the pH, the carboxylic acid can be protonated or deprotonated (in anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0182] The term "dicarboxylic acid" or "dicarboxylate" refers to a compound of the formula HO2C-R'-CO2H, wherein R' is an alkylene or alkenylene group (both as defined above, both in the broadest and preferred aspects). When the group is deprotonated, the term "dicarboxylate" is used. Depending on the pH, the dicarboxylic acid can be protonated or deprotonated (in anionic amphiphiles as defined below, the dicarboxylic acid group is typically protonated at acidic or neutral pH and deprotonated at alkaline pH).

[0183] The term "hydroxycarboxylic acid" or "hydroxycarboxylate" refers to a compound of the formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), substituted with one or more (preferably 1 to 5, such as 1, 2 or 3) hydroxyl groups. When the group is deprotonated, the term "hydroxycarboxylate" is used. Depending on the pH, the hydroxycarboxylic acid may be protonated or deprotonated (in anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0184] As used herein, the term "ester" refers to a compound having the structure RC(O)O-R' (including isomeric arrangements thereof, R-OC(O)-R', unless otherwise indicated), wherein R and R' are each independently a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). When the term refers to a substituent that is attached to the rest of the molecule, the ester portion may have the structure RC(O)O or R-OC(O)-, wherein R is as defined above. In one embodiment, the two ends of the ester structure are each covalently attached to a C atom of the same organic group or two separate organic groups (e.g., an alkylene group as another component of the linker).

[0185] The term "half-ester" as used herein with respect to a functional moiety relates to an ester of a dicarboxylic acid as defined above, wherein one of the carboxylic acid groups forms an ester bond with the remainder of the molecule and the other carboxylic acid group is free. Depending on the pH, the free carboxylic acid group may be protonated or deprotonated (in anionic amphiphiles as defined below, the free carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0186] The term "phosphate" or "organophosphate" refers to a compound of the formula RO-P(=O)(OH)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). Depending on the pH, the phosphate group may be protonated or deprotonated (in anionic amphiphiles as defined below, the phosphate group is typically deprotonated at physiological pH).

[0187] The term "phosphonate" or "organophosphonic acid" refers to a compound of formula RP(=O)(OH)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects). Depending on the pH, the phosphonate group may be protonated or deprotonated (in anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0188] "Halogen" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0189] "Amine" refers to the group -NR2, where each R is a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and is preferably an alkyl group such as C 1-6 Alkyl group. When both R groups are hydrogen, the amine group is a primary amine group. When one R group is hydrogen and the other R group is not hydrogen, the amine group is a secondary amine group. When both R groups are not hydrogen, the amine group is a tertiary amine group.

[0190] "Quaternary ammonium" salts are salts containing the group -N + wherein each R is a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and is preferably an alkyl group such as C 1-6 Alkyl groups. Unlike certain amines as defined above, which are protonated only at certain pHs, quaternary ammonium salts carry a constitutive positive charge (as defined herein) at all pHs.

[0191] "Hydroxy" refers to the group -OH. "Mercapto" refers to the group -SH. "Nitro" refers to the group -NO2.

[0192] "Ether" refers to an oxygen atom to which two hydrocarbyl or heterohydrocarbyl groups, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, both in the broadest and preferred aspects) are linked. The ether may be a cyclic ether, in which the two hydrocarbyl groups together form a ring, and may include a dioxolane group.

[0193] "Thioether" refers to a sulfur atom linked to two hydrocarbyl or heterohydrocarbyl groups such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, both in the broadest and preferred aspects). The ether may be a cyclic thioether in which the two hydrocarbyl groups together form a ring, and may include a dithiane group.

[0194] "Amide" refers to the group -C(=O)NR(R') where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and preferably an alkyl group such as C 1-6 Alkyl group.

[0195] "Hydroxyamide" refers to the group -C(=O)O-NR(R') where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects).

[0196] "Sulfonamide" refers to the group -S(=O)2NRR', where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and preferably an alkyl group such as C 1-6 Alkyl group.

[0197] "Carbamate" refers to the group -OC(=O)NRR', where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and preferably an alkyl group such as C 1-6 Alkyl group.

[0198] "Amidine" refers to the group -C(=NR)NR'R", wherein R, R' and R" are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and is preferably an alkyl group such as C 1-6 Alkyl group.

[0199] "Guandinium" refers to the group -NR-C(=NR')NR"R"' or =NC(NRR')(NR"R"') where R, R', R" and R'" are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl or heterocyclylalkyl group (all as defined above, both in the broadest and preferred aspects), and is preferably an alkyl group such as C 1-6 Alkyl group.

[0200] The above definitions may be modified according to normal chemical nomenclature by replacing the suffix "-ium" when referring to any protonated basic nitrogen atom. For example, a guanidinium group is a protonated guanidine, an ammonium group is a protonated ammonia or a protonated primary, secondary, or tertiary amine, an imidazolium group is a protonated imidazole, a pyridinium group is a protonated pyridine, an amidinium group is a protonated amidine, and a piperazinium group is a protonated piperazine.

[0201] "Carbohydrate" means a carbohydrate having the empirical formula C m (H2O) n Compound, wherein m can be identical or different from n. The term "carbohydrate residue" or "carbohydrate moiety" defines a residue connected to another atom, wherein one hydrogen atom of the carbohydrate is replaced by a bond connected to the rest of the molecule. The carbohydrate moiety can be a monosaccharide moiety. The monosaccharide moiety can have a D or L- configuration. In addition, the monosaccharide moiety can be an aldose or ketose moiety. Suitably, the monosaccharide moiety can have 3 to 8, preferably 4 to 6, more preferably 5 or 6 carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (i.e., it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose, and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0202] In another embodiment, the monosaccharide moiety is a pentose moiety (ie it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0203] In another embodiment, the carbohydrate can be a higher sugar (i.e., a disaccharide or oligosaccharide) comprising more than one monosaccharide moiety linked together by a glycosidic bond. When the monosaccharide moiety is a hexose moiety, the glycosidic bond can be a 1-α, 1'-α glycosidic bond, a 1,2'-glycosidic bond (which can be a 1-α2' or 1'-β-2' glycosidic bond), a 1,3'-glycosidic bond (which can be a 1-α-3' or 1-β-3'-glycosidic bond), a 1,4'-glycosidic bond (which can be a 1-α-4' or 1-β-4'-glycosidic bond), a 1,6'-glycosidic bond (which can be a 1-α-6' or 1-β-6'-glycosidic bond), or any combination thereof. In one embodiment, the higher sugar comprises two monosaccharide units (i.e., a disaccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose, and trehalulose. In another embodiment, the higher sugar comprises 3 to 10 monosaccharide units (i.e., oligosaccharides) in the chain, which can be branched or unbranched. Preferably, the oligosaccharide comprises 3 to 8, more preferably 3 to 6 monosaccharide units. The example of suitable oligosaccharide includes maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose.

[0204] "List A" substituents are selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5- or 6-membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR', -N(R')2, -S(O) 0-2 R', -S(O) 1-2 OR', -OS(O) 1-2 R', -OS(O) 1-2 OR', -S(O) 1-2 N(R')2、-OS(O) 1-2 N(R')2、-N(R')S(O) 1-2 R', -N(R')S(O) 1-2 OR', -C(=X 1 )R'、-C(=X 1 )X 1 R', -X 1 C(=X1 )R' and -X 1 C(=X 1 )X 1 R', where X 1 independently selected from O, S, NH and N(CH3); and each R' is independently selected from H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 alkynyl, 5 or 6 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 5 or 6 membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from C 1-3 Alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(C 1-3 Alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 Alkyl), -N(C 1-3 Alkyl)2, -NHS(O)2(C 1-3 Alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z 、-C(=O)OH、-C(=O)O(C1 -3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z 、-NHC(=O)(C1-3 alkyl), -NHC(=NH)NH z-2 (C1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z , wherein each z is independently 0, 1 or 2, and each C 1-3 Alkyl is independently methyl, ethyl, 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , -C(=O)OH and -C(=O)OCH3, wherein z is 0, 1 or 2 and C 1-3 Alkyl is methyl, ethyl, propyl or isopropyl. In certain embodiments, the List A substituent is selected from List A2 consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl or Br) and -CF3.

[0205] amino acids

[0206] In certain embodiments, the methods and compositions of the present invention, particularly further processing steps such as dialysis or filtration steps, steps of dilution or addition of storage media, and storage steps, use amino acids.

[0207] "Amino acid" is used in its broadest sense as it is normally understood in the art, i.e., a compound containing an amine group (as defined and exemplified above, both in its broadest and preferred aspects) and a carboxylic acid group (as defined and exemplified above, both in its broadest and preferred aspects). The amino acid may contain other functional groups as defined and exemplified herein.

[0208] It is well known to those skilled in the art that, according to pH, amino acid can exist in various forms. In one embodiment, amino acid is zwitterionic form (i.e., wherein the proton from the carboxylic acid group is transferred to the amino group, thereby leaving a negative carboxylic acid group and a positive ammonium group). In one embodiment, amino acid is neutral form (i.e., wherein the amino group and the carboxylic acid group are uncharged). In one embodiment, usually at acidic pH, amino acid is cationic form (i.e., wherein only the amine group is protonated, thereby having an uncharged carboxylic acid group and a positive ammonium group). In one embodiment, usually at alkaline pH, amino acid is anionic form (i.e., wherein only the carboxylic acid group is deprotonated, thereby leaving a negative carboxylic acid group and an uncharged amine group). Amino acid in this manual is named according to its neutral structure according to the general method of this area. The use of any specific amino acid name is not meant to be limited to neutral structure, but includes all neutral, protonated, deprotonated and zwitterionic structures.

[0209] In one embodiment, the amino acid is an alpha amino acid (i.e., wherein the amino group is present on a carbon adjacent to the carbon forming the carboxylic acid group). Typically, such alpha amino acids have the general formula (neutral structure) H2N-CH(R)-CO2H, where the group R is referred to as the side chain. Proline and its derivatives differ from this structure in that the nitrogen atom forms part of the pyrrolidine ring.

[0210] In one embodiment, the amino acid is a protein-forming amino acid. Examples of protein-forming amino acids include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0211] In one embodiment, the amino acid is a substituted proteomic amino acid, i.e., a proteomic amino acid selected from those listed above, substituted with one or more substituents selected from List A. Examples of such substituted proteomic amino acids include 3-hydroxyglutamate, 2-methyl-L-serine, and O-methyl-L-serine.

[0212] In one embodiment, the amino acid is a non-protein-forming amino acid. Examples of protein-forming amino acids include α-aminoadipic acid, β-alanine, α-aminoisobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid, dehydroalanine, norvaline, alloisoleucine, allothreonine, homocysteine, homoserine, isoserine, citrulline, ornithine, homophenylalanine, 7-azatryptophan, norleucine, homoserine, sarcosine, L-β-homoleucine, and substituted derivatives thereof wherein the substituent is selected from any one of List A.

[0213] In one embodiment, the amino acid is an acidic amino acid. In one embodiment, the acidic amino acid has an isoelectric point (pI) lower than 4, i.e., the pH at which the molecule has no net charge. In one embodiment, the acidic amino acid is an amino acid with an acidic side chain. Examples of acidic side chains include carboxylic acids, sulfonic acids, organosulfuric acids, phosphonic acids, and phosphates, as defined and illustrated above. Preferably, the acidic amino acid is an amino acid with a carboxylic acid side chain. Examples of acidic amino acids include aspartic acid, glutamic acid, and substituted derivatives thereof wherein the substituent is selected from List A. More preferably, the acidic amino acid is selected from aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and α-aminoadipic acid.

[0214] In one embodiment, the amino acid is a neutral amino acid. In one embodiment, the neutral amino acid has an isoelectric point (pi) of 4 to 7.8. In one embodiment, the neutral amino acid is an amino acid lacking an acidic or basic side chain. Examples of neutral amino acids include serine, threonine, asparagine, glutamine, cysteine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, and substituted derivatives thereof wherein the substituent is selected from any of List A. More preferably, the neutral amino acid is selected from leucine and isoleucine.

[0215] In one embodiment, the amino acid is a basic amino acid. In one embodiment, the basic amino acid has an isoelectric point (pI) greater than 7.8, preferably greater than 8.5. In one embodiment, the basic amino acid is an amino acid with a basic side chain. Examples of basic side chains include amines, amidines and guanidines and nitrogen-containing heteroaryl and heterocyclic groups, all as defined and illustrated above. Examples of basic amino acids include arginine, histidine, lysine, and substituted derivatives thereof wherein the substituents are selected from any of List A. More preferably, the basic amino acid is selected from arginine, histidine and lysine.

[0216] Nucleic Acids

[0217] The lipid particle composition of the present application contains an active ingredient. The active ingredient is a nucleic acid. Preferably, the lipid particle composition of the present application contains RNA, such as mRNA. Generally, the lipid particle composition described herein comprises lipid particles that encapsulate nucleic acid. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA.

[0218] Nucleic acids can exist as single-stranded or double-stranded and linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to the present disclosure, the term "isolated nucleic acid" means that the nucleic acid (i) is amplified in vitro, for example, by polymerase chain reaction (PCR) of DNA or in vitro transcription of RNA (using, for example, RNA polymerase), (ii) is produced by cloning and recombination, (iii) is purified, for example, by cleavage and separation by gel electrophoresis, or (iv) is synthesized, for example, by chemical synthesis.

[0219] The term "nucleoside" refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a core base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. A nucleic acid can include one or more modified nucleosides or nucleotides. Examples of modified nucleosides or nucleotides that can be incorporated into nucleic acids include N7-alkylguanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N7-C1-4alkylguanine, N6-C1-4alkyl-adenine, 5-C1-4alkyl-cytosine, 5-C1-4alkyl-uracil and N(1)-C1-4alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (ψ) and N1-methyl-pseudouridine (m1Ψ).

[0220] RNA

[0221] In certain embodiments of all aspects of the present disclosure, the nucleic acid is RNA. According to the present disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acid adenosine (A), uridine (U), cytidine (C) and guanosine (G). In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. "Ribonucleotide" as used herein means a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. RNA encompasses but is not limited to double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides. Such changes may point to internal RNA nucleotides or RNA ends to add non-nucleotide substances. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For purposes of the present disclosure, these altered / modified nucleotides (or modified nucleosides) may be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides (i.e., altered / modified RNAs) may be referred to as analogs of naturally occurring RNAs. A molecule contains "mostly ribonucleotide residues" if the content of ribonucleotide residues in the molecule exceeds 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) of the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplified RNA (saRNA), trans-amplified RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA) or microRNA (miRNA)), activated RNA (such as small activated RNA) and immunostimulatory RNA (isRNA). In certain embodiments, "RNA" means mRNA. The active ingredient can be mRNA, saRNA, taRNA or a mixture thereof. The active ingredient is preferably mRNA. In some cases, the active ingredient is not siRNA.

[0222] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. The RNA may be capable of or be configured to express a coded peptide, polypeptide or protein. For example, the RNA may be encoding and may be configured to express a pharmaceutically active peptide or protein. In certain embodiments, the RNA may interact with the cell translation mechanism, thereby realizing the translation of the peptide or protein. The cell may produce the coded peptide or protein in the cell (for example, in the cytoplasm), may secrete the coded peptide or protein, or may produce it on the surface. Alternatively, the RNA may be a non-coding RNA such as antisense-RNA, microRNA (miRNA) or siRNA.

[0223] mRNA

[0224] In preferred embodiments of all aspects of the present disclosure, the nucleic acid is mRNA. According to the present disclosure, the term "mRNA" refers to "messenger-RNA" and includes "transcripts" that can be generated using a DNA template. Typically, mRNA encodes a peptide, polypeptide, or protein. As known in the art, RNA (such as mRNA) typically contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR).

[0225] mRNA is single-stranded but may contain self-complementary sequences that allow portions of the mRNA to fold and pair with themselves to form a double helix.

[0226] According to the present disclosure, "dsRNA" refers to double-stranded RNA, and is RNA having two partially or completely complementary strands.

[0227] In a preferred embodiment of the present disclosure, the mRNA relates to an RNA transcript encoding a peptide, polypeptide or protein.

[0228] In certain embodiments, preferably the RNA encoding the peptide, polypeptide or protein has a length of at least 45 nucleotides, such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides, preferably at most 15,000, such as at most 14,000, at most 13,000, at most 12,000 nucleotides, at most 11,000 nucleotides or at most 10,000 nucleotides.

[0229] In certain embodiments, RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, RNA (such as mRNA) is produced by in vitro transcription using a DNA template. As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., the generation of RNA) is performed in a cell-free manner. That is, IVT does not use living / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysate or isolated components thereof, including RNA polymerase (preferably T7, T3 or SP6 polymerase)). In vitro transcription methods are known to those of skill in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, a variety of in vitro transcription kits are commercially available, for example, from ThermoFisher Scientific (such as TranscriptAid TM T7 test kit, T7 test kit, ), New England BioLabs Inc. (such as HiScribe TM T7 kit, HiScribe TM T7ARCA mRNA kit), Promega (such as RiboMAX TM 、 systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe TM ).

[0230] In order to provide a modified RNA (such as mRNA), corresponding modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during the synthesis (preferably in vitro transcription), or modifications can be made in the mRNA after transcription and / or the modifications can be added to the mRNA. RNA (such as mRNA) can be modified. RNA (such as mRNA) can contain modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (ψ) or N(1)-methyl-pseudouridine (m1ψ). One or more uridines in the RNA described herein can be replaced by modified nucleosides. The modified nucleosides can be modified uridines. RNA can contain modified nucleosides that replace at least one uridine. Preferably, RNA can contain modified nucleosides that replace each uridine (for example, all uridines in the RNA are replaced by modified nucleosides). Modified nucleosides can be independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ).

[0231] In certain embodiments, the RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from an appropriate DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. DNA templates for in vitro transcription can be obtained by cloning a nucleic acid (particularly cDNA) and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0232] In certain embodiments of the present disclosure, the RNA (such as mRNA) is a "replicon RNA" (such as "replicon mRNA") or "replicon" for short, particularly a "self-replicating RNA" (such as "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). The lipid particles containing RNA as described herein can contain mRNA, saRNA, taRNA or a mixture thereof. The lipid particles containing RNA as described herein can contain an mRNA encoding a replicase protein, and one or more RNA molecules that can be copied or amplified by a replicase.

[0233] Inhibitory RNA

[0234] In certain embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

[0235] The term "inhibitory RNA" as used herein refers to an RNA that selectively hybridizes with a target mRNA and / or has specificity thereto to inhibit (e.g., reduce) its transcription and / or translation. Inhibitory RNA includes an RNA molecule with an antisense orientation sequence relative to the target mRNA. Suitable inhibitory oligonucleotides are typically 5 to several hundred nucleotides in length, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. The example of inhibitory RNA includes antisense RNA, ribozymes, iRNA, siRNA and miRNA. In certain embodiments of all aspects of the present disclosure, the inhibitory RNA is siRNA.

[0236] The term "antisense RNA" as used herein refers to an RNA that hybridizes under physiological conditions with the DNA comprising a specific gene or with the mRNA of the gene to inhibit the transcription of the gene and / or the translation of the mRNA. The size of the antisense RNA can be 15 nucleotides to 15,000 nucleotides, preferably 20 to 12,000 nucleotides, particularly 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150 or 65 to 100 nucleotides.

[0237] As used herein, "small interfering RNA" or "siRNA" refers to an RNA molecule that is capable of specifically binding to a portion of a target mRNA, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. This binding induces a process in which the portion of the target mRNA is cut or degraded, and thereby inhibits gene expression of the target mRNA. The range of 19 to 25 nucleotides is the most preferred size for siRNA. Typically, siRNA comprises a single molecule in which two complementary portions are base paired and covalently linked by a single-stranded "hairpin" region. Without wishing to be bound by any theory, it is believed that the hairpin region of the siRNA molecule is cut by the "Dicer" protein (or its equivalent) within the cell to form two separate base-paired RNA molecules of the siRNA.

[0238] As used herein, "target mRNA" refers to an RNA molecule that is targeted for downregulation. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide specified herein. In certain embodiments, the pharmaceutically active peptide or polypeptide is a peptide or polypeptide whose expression (particularly increased expression, e.g., compared to expression in healthy subjects) is associated with a disease. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, the expression of which (particularly increased expression, e.g., compared to expression in healthy subjects) is associated with cancer.

[0239] According to the present disclosure, siRNA can be targeted to any segment of about 19 to 25 consecutive nucleotides in any target mRNA sequence ("target sequence"). For example, techniques for selecting target sequences for siRNA are provided in TuschlT. et al., "The siRNA User Guide" (revised October 11, 2002) (the entire disclosure of which is incorporated herein by reference). Further guidance on selecting target sequences and / or designing siRNA can be found on the webpage of Protocol Online (www.protocol-online.com) using the keyword "siRNA". Therefore, in certain embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any consecutive segment of about 19 to about 25 nucleotides in the target mRNA.

[0240] siRNA can be obtained using a variety of techniques known to those skilled in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter. The selection of other suitable promoters is within the skill of the art. The selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing siRNA into the plasmids, and in vitro transcription (IVT) methods for the siRNA are within the skill of the art.

[0241] The term "miRNA" (microRNA) used herein refers to non-coding RNA having a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides, and which induces the degradation of target mRNA and / or prevents its translation. MiRNA is typically found in plants, animals, and certain viruses, where they are encoded by eukaryotic nuclear DNA (in plants and animals) and viral DNA (in viruses whose genomes are based on DNA), respectively. MiRNA is a post-transcriptional regulatory factor that binds to a complementary sequence on a target messenger RNA transcript (mRNA), typically leading to translational repression or target degradation and gene silencing.

[0242] miRNA can be obtained using a variety of techniques known to those skilled in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as miRNA cDNA synthesis kits sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter.

[0243] DNA

[0244] In certain embodiments of all aspects of the present disclosure, the nucleic acid is DNA. In this article, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" stands for "deoxy"). In preferred embodiments, the DNA contains all or most of the deoxyribonucleotide residues. "Deoxyribonucleotides" as used herein refer to nucleotides lacking a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA (such as partially purified DNA), substantially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by adding, deleting, replacing and / or changing one or more nucleotides. Such changes can be directed to the addition of non-nucleotide substances to internal DNA nucleotides or to the ends of DNA. It is also contemplated herein that the nucleotides in the DNA can be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered to be analogs of naturally occurring DNA. A molecule contains "mostly deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule exceeds 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) of the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). The DNA may be recombinant DNA and may be obtained by cloning nucleic acids (particularly cDNA). cDNA may be obtained by reverse transcription of RNA.

[0245] Pharmaceutically active peptides or polypeptides

[0246] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or RNA (preferably mRNA)) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if transcription and translation of the RNA (preferably mRNA) corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes the protein. Similarly, if translation of an RNA (such as an mRNA) (e.g., in a cell) produces a protein, then the RNA encodes the protein.

[0247] In certain embodiments, the active ingredient is an RNA (preferably mRNA) described in the present disclosure, which comprises a nucleic acid sequence (e.g., ORF) encoding one or more polypeptides (e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins). In certain embodiments, the RNA (preferably mRNA) described in the present disclosure is capable of expressing the peptides or proteins, particularly if transferred into a cell or subject. Therefore, in certain embodiments, the RNA (preferably mRNA) described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a continuous codon segment starting from a start codon and ending at a stop codon. Such RNA (preferably mRNA) encoding a pharmaceutically active peptide or protein is also referred to herein as "pharmaceutically active RNA" (or "pharmaceutically active mRNA"). In certain embodiments, the RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, such as two, three, four or more peptides or polypeptides. In certain embodiments, the RNA (preferably mRNA) described in the present disclosure includes a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5 or more) patient-specific antigens suitable for personalized cancer therapy. In certain embodiments, the lipid particle composition comprising RNA can include one or more RNAs, wherein each RNA encodes different peptides or proteins.

[0248] Preferably, the RNA (i) contains structural elements (5' cap, 5' UTR, 3' UTR, poly A sequence) optimized for maximum efficacy of the RNA in terms of stability and translation efficiency; (ii) is modified to optimize the efficacy of the RNA (e.g., increase translation efficacy, reduce immunogenicity and / or reduce cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (particularly cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine); and / or codon optimization), or (iii) both (i) and (ii).

[0249] The term "pharmaceutically active peptide or protein" may be understood to refer to a peptide or protein that can be used to treat an individual in which expression of the peptide or protein would be beneficial, e.g., to ameliorate the symptoms of a disease or disorder. Preferably, the pharmaceutically active peptide or protein has curative or palliative properties and can be administered to ameliorate, relieve, alleviate, reverse, delay the onset of, or lessen the severity of one or more symptoms of a disease or disorder. The pharmaceutically active peptide or protein may have prophylactic properties and can be used to delay the onset of a disease or disorder or to lessen the severity of such a disease or disorder.

[0250] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, interferons such as interferon-α (IFN-α), interferon-β (IFNβ) or interferon-γ (IFN-γ), interleukins such as interleukin 2 (IL2), IL-4, IL7, IL-10, IL-11, IL12, IL15, IL-21 and IL23, colony stimulating factors such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), erythropoiesis-stimulating factor (EGF), erythropoiesis-stimulating factor (EGF), cytokine receptor antagonist (CRPT ... epoxin (EPO) and bone morphogenetic protein (BMP); members of the immunoglobulin superfamily, including antibodies (e.g., IgG), T cell receptors (TCR), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds such as tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic or viral antigens), allergens and self-antigens.

[0251] Aqueous dispersion

[0252] The present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase. In this specification, the term "dispersion" is used in its broadest sense as it is commonly used in chemistry, i.e., a system in which distributed particles of one material (the "dispersed phase") are dispersed in a phase of another material (the "continuous phase" or "mobile phase").

[0253] In one embodiment, the dispersion is a solid-liquid dispersion, wherein the dispersed phase is solid and the mobile phase is liquid. In one embodiment, the dispersion is a liquid-liquid dispersion, wherein both the dispersed phase and the mobile phase are liquids.

[0254] In one embodiment, the dispersion is a colloid. As used herein, the term "colloid" describes a stable mixture in which the dispersed particles do not settle out. Typically, the dispersed particles have a size of approximately 1 nm to 1 μm in at least one direction, or in such a system, discontinuities are found over distances of this magnitude.

[0255] In one embodiment, the dispersion is a suspension. As used herein, the term "suspension" refers to a non-uniform dispersion of relatively large particles in a medium. Unlike solutions and colloids, suspended particles may settle out of the mixture if left undisturbed for extended periods of time. The terms "colloid" and "suspension" are sometimes used interchangeably or synonymously, and in some cases, colloids are considered a subtype of suspension.

[0256] In one embodiment, the mobile phase is a solution. As used herein, the term "solution" is a homogeneous mixture comprising a solvent (which is typically water) and a solute (which can be a salt, a buffer, a tonicity agent, etc.), as long as these substances are molecularly distributed in the solvent. The mobile phase may comprise a solute, as further described herein.

[0257] Described dispersed phase comprises the lipid mixture that comprises cationic lipid or cation ionizable lipid as defined herein.In one embodiment, described dispersed phase comprises the lipid mixture that comprises cationic ionizable lipid as defined herein.In one embodiment, described dispersed phase comprises the lipid mixture that comprises cationic ionizable lipid as defined herein.In one embodiment, described dispersed phase comprises the lipid mixture that contains cationic ionizable lipid as defined herein, wherein said lipid mixture does not comprise cationic lipid as defined herein.

[0258] In one embodiment, the aqueous dispersion is substantially free of inorganic cations.

[0259] In one embodiment, the aqueous dispersion is substantially free of organic solvents.

[0260] In one embodiment, the aqueous dispersion is substantially free of nucleic acids. In one embodiment, the aqueous dispersion is substantially free of RNA.

[0261] In the present disclosure, it is preferred that the aqueous dispersion comprises preformed lipid nanoparticles (pre-LNP). In the present disclosure, such preformed LNP can be understood as an oil-in-water emulsion, wherein the pre-LNP core material is preferably in a liquid state, and therefore has a fusing point lower than body temperature. Therefore, the preformed LNP generally comprises a central complex disordered non-lamellar phase made from lipid, but does not contain nucleic acid substantially. This is in contrast to the structure of the liposome comprising a single layer or multilamellar vesicle particle, in which the thin layer comprises a lipid bilayer around the aqueous cavity of the encapsulation. The lipid for forming the LNP generally does not form lamellar (double-layer) phase in water under physiological conditions. The LNP generally does not comprise or encapsulate an aqueous core. The LNP generally comprises a lipid (or oily) core.

[0262] In some cases, the pre-LNP described herein is not a liposome.In some cases, the pre-LNP described herein or the nucleic acid lipid particle formed from the pre-LNP is not a lipoplex.

[0263] In some embodiments, the preformed LNP of the present invention is a nucleic acid-containing LNP. The ...

[0264] In certain embodiments, pre-LNP described herein has an average diameter in the range of about 40nm to about 1000nm, about 40nm to about 800nm, about 40nm to about 700nm, about 40nm to about 600nm, about 40nm to about 500nm, about 40nm to about 450nm, about 40nm to about 400nm, about 40nm to about 350nm, about 40nm to about 300nm, about 40nm to about 250nm, about 40nm to about 200nm, about 40nm to about 150nm, about 40nm to about 100nm, about 40nm to about 90nm, about 40nm to about 80nm, about 40nm to about 70nm. In certain embodiments, pre-LNP described herein has an average diameter less than 100nm. In certain embodiments, pre-LNP described herein has an average diameter of about 30nm to about 100nm. In certain embodiments, pre-LNP described herein has an average diameter of about 40nm to about 100nm. In certain embodiments, the pre-LNPs described herein have an average diameter of about 40 nm to about 70 nm.

[0265] In some cases, the aqueous dispersion comprises a dispersed phase comprising pre-LNPs having a size (i.e., diameter) of about 20 nm to about 500 nm, about 20 nm to about 200 nm, about 30 nm to about 100 nm, or preferably about 60 nm to about 100 nm.

[0266] In one embodiment, the aqueous mobile phase comprises a cryoprotectant, as described in more detail below. This may be introduced into the mixing step or into further processing steps, as described in more detail below.

[0267] In one embodiment, the dispersed phase comprises a lipid mixture comprising a cationic lipid or a cationic ionizable lipid; and the aqueous mobile phase comprises an anion of an aqueous acid;

[0268] wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA, and

[0269] The aqueous mobile phase contains a cryoprotectant.

[0270] In one embodiment, the dispersed phase comprises a cationic lipid or a cationic ionizable lipid; and the aqueous mobile phase comprises an anion of an aqueous acid;

[0271] in:

[0272] The concentration of the aqueous acid is at least 6 mM; and

[0273] The aqueous mobile phase is substantially free of inorganic cations, organic solvents, and RNA.

[0274] In one embodiment, the dispersed phase comprises a cationic lipid or a cationic ionizable lipid; and the aqueous mobile phase comprises a malate anion or a succinate anion;

[0275] The aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA.

[0276] In one embodiment, the aqueous dispersion has a maximum pH of about 4.5, such as, for example, a maximum pH between 4.2 and 4.8. In one embodiment, the aqueous dispersion has a maximum pH of 4.5. The aqueous dispersion may have a pH of less than 4.5.

[0277] In one embodiment, the aqueous dispersion has a pH of about 2.5 to about 4.5. The aqueous dispersion may have a pH of 2.5 to 4.5. The aqueous dispersion may have a pH of 2.5 to 3.5 or 3.5 to 4.5. The aqueous dispersion may have a pH of 4.0 to 4.5. The aqueous dispersion may have a pH of about 4.5.

[0278] In some embodiments, the aqueous mobile phase comprises an anion of an aqueous acid. The acid can be any inorganic acid or organic acid, which is at least partially miscible with water and can be at least partially deprotonated in water to produce an anion (i.e., conjugate base) of the acid. Therefore, it will be appreciated by those skilled in the art that, according to pH and acid intensity, the aqueous mobile phase can contain different proportions of undissociated acid and its corresponding anion. Strong acid is completely or mostly deprotonated in water, so the material in the aqueous solution is mainly (completely in certain embodiments) an anion of the acid. On the contrary, weak acid can not be completely deprotonated in water, so the material in the aqueous solution will comprise a mixture of undissociated acid and its conjugate base, and the relative amount of each substance depends on pH.

[0279] In one embodiment, the anion is an acetate anion. In one embodiment, the anion is a malate anion. In one embodiment, the anion is a succinate anion.

[0280] In addition, aqueous acid can react with cationic ionizable lipid to produce cationic ionizable lipid in its charged form and acid in its anionic form. The extent to which such reaction occurs depends on factors such as the alkalinity and pH of the cationic ionizable lipid (when present in neutral form).

[0281] In addition, the negative ion of aqueous acid can interact with the charged head group of the composition of cationic lipid to form lipid salt. It is anticipated that the interaction between the negative ion of aqueous acid and cationic lipid or cationic ionizable lipid will promote the formation of stable lipid particles. For example, the formation of lipid salt between the negative ion of aqueous acid and cationic lipid may affect the shape factor kappa (κ) (i.e., the volume ratio between the polar part and the non-polar part of the lipid; κ=molecular volume (head, polar) / molecular volume (tail, non-polar)) of cationic lipid and promote the formation of lipid nanoparticle structure (see, for example, WO2008 / 043575, WO2009 / 047006, Siepi et al., Biophys J.2011, 100, 2412-2421). For example, lipid salt can have a shape factor κ less than 0.25, optionally less than 0.15.

[0282] In one embodiment, the aqueous acid is an inorganic acid. Examples of suitable inorganic acids include hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0283] In one embodiment, the aqueous acid is a water-soluble organic acid.Examples of suitable inorganic acids include sulfonic acids, carboxylic acids, dicarboxylic acids, hydroxycarboxylic acids (all as defined herein) or amino acids.

[0284] In one embodiment, the water-soluble organic acid is selected from acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid and citric acid or a combination thereof. The water-soluble organic acid may be selected from acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid and citric acid. The water-soluble organic acid may be selected from acetic acid, malic acid and succinic acid. The water-soluble organic acid may be selected from acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid and oxalic acid. The water-soluble organic acid may be selected from malic acid, maleic acid, succinic acid, ascorbic acid and oxalic acid and citric acid. The water-soluble organic acid may be selected from malic acid, maleic acid, succinic acid, ascorbic acid and oxalic acid.

[0285] In one embodiment, the water-soluble weak organic acid is acetic acid. In one embodiment, the water-soluble weak organic acid is malic acid. In one embodiment, the water-soluble weak organic acid is succinic acid.

[0286] In one embodiment, the aqueous mobile phase further comprises a cryoprotectant as described and illustrated herein. In one embodiment, the aqueous dispersion comprises a cryoprotectant as described and illustrated herein. In one embodiment, the cryoprotectant is a carbohydrate, such as a monosaccharide or a disaccharide. In one embodiment, the cryoprotectant is selected from sucrose, trehalose, and glucose, or any mixture thereof. Preferably, the cryoprotectant is sucrose.

[0287] In one embodiment, the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 1% to about 30% (w / v), about 2% to about 20% (w / v) or about 5% to about 15% (w / v). In one embodiment, the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 8% to about 12% (w / v), optionally about 10% (w / v). In one embodiment, the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 15% to about 25% (w / v), optionally about 18% to about 22% (w / v). In one embodiment, the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 20% (w / v). In one embodiment, the cryoprotectant is glucose and is present in the aqueous dispersion at a concentration of about 1% to about 15% (w / v), optionally about 2% to about 10% (w / v). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 4% to about 8% (w / v), optionally about 5% (w / v). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 8% to about 12% (w / v), optionally about 10% (w / v).

[0288] In one embodiment, the aqueous dispersion is substantially free of acetate buffer and citrate buffer. The aqueous dispersion may be substantially free of acetate buffer. The aqueous dispersion may be substantially free of citrate buffer. The aqueous mobile phase may be substantially free of citrate buffer. The aqueous dispersion and / or the aqueous mobile phase may be substantially free of citrate buffer comprising about 10 mM citrate, about 150 mM NaCl, and a pH of about 4.5. The aqueous dispersion may be substantially free of buffering agents. The aqueous dispersion may be substantially free of acetate buffer, citrate buffer, phosphate buffer, and / or tris buffer. The aqueous dispersion may be substantially free of buffering agents selected from ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. The aqueous dispersion may be substantially free of buffering agents selected from ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, tris(hydroxymethyl)aminomethane (Tris), sodium phosphate, and HEPES.

[0289] In one embodiment, the aqueous dispersion is substantially free of inorganic cations. Such inorganic cations are considered to affect the colloidal stability of the lipid dispersion and reduce the stability of the preparation. In one embodiment, the aqueous dispersion is substantially free of (as defined herein) alkali metal ions. In one embodiment, the aqueous dispersion is substantially free of inorganic cations such as ammonium, sodium and / or potassium ions.

[0290] In one embodiment, the aqueous dispersion is substantially free of (as defined herein) organic solvents. In one embodiment, the term "substantially free of organic solvents" means that the aqueous dispersion contains less than about 5,000 ppm by weight of organic solvent, such as less than about 4,000 ppm, such as less than about 3,000 ppm, such as less than about 2,000 ppm, such as less than about 1,000 ppm, such as less than about 900 ppm, such as less than about 800 ppm, such as less than about 700 ppm, such as less than about 600 ppm, such as less than about 500 ppm, such as less than about 400 ppm, such as less than about 300 ppm, such as less than about 200 ppm, such as less than about 100 ppm, as a proportion of the total weight of the aqueous dispersion.

[0291] For example, the aqueous dispersion can be substantially free of water-soluble organic solvents such as C1-4 alcohols (e.g., isopropanol or ethanol), ketones (e.g., acetone), or mixtures thereof; and / or non-polar organic solvents such as hydrocarbons such as pentane or hexane; chlorinated hydrocarbons such as dichloromethane or chloroform; or mixtures thereof. In one embodiment, the aqueous dispersion is substantially free of organic solvents comprising isopropanol, ethanol, and / or acetone.

[0292] In one embodiment, the concentration of the aqueous acid is at least 6 mM. In one embodiment, the concentration of the aqueous acid is in the range of 1 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 2.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 10 mM. In this context, it should be understood that this concentration includes undissociated acid and its conjugate base.

[0293] In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 20: 1 to about 1: 20. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 10: 1 to about 1: 10. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 5: 1 to about 1: 5. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 3: 1 to about 1: 3. In this context, it should be understood that the moles of the cationic lipid or cation ionizable lipid include the undissociated lipid and its conjugate acid, and the moles of the aqueous acid include the undissociated acid and its conjugate base.

[0294] When the acid is a strong acid, in one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:10 and 10:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:5 and 5:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:3 and 3:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:2 and 2:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:1.5 and 1.5:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:1.2 and 1.2:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:1.

[0295] When the acid is a weak acid, in one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:20 and 5:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:10 and 2.5:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:6 and 1.5:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:4 and 1.25:1. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of between 1:3 and 1:1.33. In one embodiment, the cationic lipid or cation ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:2.

[0296] In some cases, the aqueous dispersion comprises the dispersed phase that contains lipid granule.In some cases, the lipid granule of the dispersed phase is lipid nanoparticle.In some cases, the lipid granule of the dispersed phase is not liposome.In some cases, the aqueous dispersion comprises the dispersed phase, and the dispersed phase comprises the lipid granule with the size (that is, diameter) of about 20nm to about 500nm, about 20nm to about 200nm, about 30nm to about 180nm, about 40nm to about 120nm or preferably about 40nm to about 80nm.In some cases, the aqueous dispersion comprises the dispersed phase, and the dispersed phase comprises the lipid granule with the size (that is, diameter) that is no more than about 200nm.

[0297] Storage matrix - cryoprotectants and other components

[0298] In one embodiment, the aqueous dispersion (typically containing pre-LNP) also contains a storage matrix. In this specification, the term "storage matrix" when used in its broadest sense generally encompasses any substance typically used to aid storage and increase the shelf life of an aqueous dispersion. The storage matrix is ​​typically added to the aqueous dispersion after a filtration (e.g., TFF) / dialysis step.

[0299] In one embodiment, the storage matrix comprises a cryoprotectant. In this specification, the term "cryoprotectant" when used in its broadest sense refers to any substance capable of protecting a composition from damage caused by freezing and / or ice formation. Examples of cryoprotectants include glycols (i.e., alcohols containing at least two hydroxyl groups, such as glycerol and propylene glycol) and carbohydrates, as defined and illustrated herein.

[0300] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or a disaccharide. In one embodiment, the cryoprotectant is selected from sucrose, trehalose, lactose and glucose or any mixture thereof. In one embodiment, the cryoprotectant is selected from sucrose, trehalose and glucose or any mixture thereof. Preferably, the cryoprotectant is sucrose.

[0301] When the aqueous dispersion also contains a carbohydrate storage matrix, typically, it is present at a concentration of about 1% to about 30% (w / v). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 10% (w / v).

[0302] In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 1% to about 30% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 10% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 15% to about 25% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 18% to about 22% (w / v). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 20% (w / v).

[0303] In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 1% to about 15% (w / v). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 2% to about 10% (w / v). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 4% to about 8% (w / v). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 5% (w / v). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 10% (w / v).

[0304] Method for forming aqueous dispersion

[0305] In another aspect, the present disclosure provides methods of preparing the aqueous dispersions of the present invention.

[0306] Many methods of preparing dispersions are known in the art, and the skilled artisan can readily select an appropriate method and apply that method to prepare the dispersion compositions of the present invention.

[0307] In one embodiment, the method comprises mixing:

[0308] (i) a lipid mixture comprising cationic lipids or cationic ionizable lipids;

[0309] (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant;

[0310] to produce an aqueous dispersion comprising the anion of the aqueous acid.

[0311] In one embodiment, the method comprises:

[0312] (a) Mixing:

[0313] (i) a lipid mixture comprising a cationic lipid or a cationic ionizable lipid; and

[0314] (ii) an aqueous phase comprising an aqueous acid;

[0315] to produce a first intermediate aqueous dispersion comprising an anion of said aqueous acid; and

[0316] (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce the aqueous dispersion.

[0317] In one embodiment, the method comprises:

[0318] (a) Mixing:

[0319] (i) a lipid mixture comprising a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent; and

[0320] (ii) aqueous phase;

[0321] The lipid mixture and / or the aqueous phase comprises the aqueous acid;

[0322] to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid;

[0323] (b) subjecting the first intermediate acidified aqueous lipid dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5 to remove the organic solvent and produce a second intermediate aqueous dispersion; and

[0324] (c) adding a cryoprotectant to the second intermediate aqueous dispersion;

[0325] To produce an aqueous dispersion comprising an anion of the aqueous acid. The addition of the cryoprotectant typically does not affect the pH, such that the pH of the aqueous dispersion comprising an anion of the aqueous acid is substantially the same as the pH of the second intermediate aqueous dispersion (i.e., about 2.5 to about 5.5).

[0326] In one embodiment, the method comprises:

[0327] i) mixing a lipid mixture and an aqueous phase, wherein the lipid mixture comprises a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent,

[0328] wherein the lipid solution and / or the aqueous phase comprises an aqueous acid,

[0329] to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid;

[0330] ii) subjecting the first intermediate acidified aqueous lipid dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5 or at a pH of about 6.5 to about 8.5 to remove the organic solvent and produce a second intermediate aqueous dispersion; and

[0331] iii) adding a cryoprotectant to the second intermediate aqueous dispersion;

[0332] to produce the aqueous dispersion;

[0333] wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA. The addition of the cryoprotectant typically does not affect the pH, such that the pH of the aqueous dispersion is substantially the same as the pH of the second intermediate aqueous dispersion (i.e., about 2.5 to about 5.5 or about 6.5 to about 8.5).

[0334] In one embodiment, the method comprises:

[0335] i) preparing a solution of a lipid mixture comprising a cationic lipid or a cationic ionizable lipid dissolved in a water-soluble and / or non-polar organic solvent (preferably wherein such solvent is volatile);

[0336] ii) evaporating the organic solvent below atmospheric pressure to provide the lipid mixture in the form of a lipid film (or layer), optionally a thin film, typically a homogeneous film;

[0337] iii) adding aqueous acid to a thin film (e.g., a film) of the lipid mixture,

[0338] to produce said aqueous dispersion; and

[0339] iv) diluting the aqueous dispersion with a cryoprotectant;

[0340] wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA. An exemplary suitable solvent for dissolving the lipid mixture in step i) may be, for example, a 1:1 mixture of methanol and dichloromethane.

[0341] This method is referred to herein as the "thin film method."

[0342] In one embodiment of the above thin film method, the method further comprises the following step ii') after step ii):

[0343] ii') reducing the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably aperture extrusion.

[0344] In one embodiment, the method comprises:

[0345] i) preparing a solution of a lipid mixture comprising a cationic lipid or a cationic ionizable lipid dissolved in a non-polar, water-immiscible organic solvent (or a mixture of a non-polar, water-immiscible organic solvent and a polar organic solvent if lipid solubility requires);

[0346] ii) adding an aqueous phase to produce a first intermediate composition comprising said lipid mixture,

[0347] wherein the solution of the lipid mixture and / or the aqueous phase is acidified;

[0348] iv) removing the organic solvent by standard unit operations such as evaporation or filtration (preferably by evaporation) at subatmospheric pressure to produce a second intermediate composition comprising the lipid mixture;

[0349] v) sonicating the second intermediate composition to produce the aqueous dispersion; and

[0350] vi) diluting the aqueous dispersion with a cryoprotectant;

[0351] The aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA.

[0352] This method is referred to herein as the "emulsification method."

[0353] In one embodiment, the emulsification method further comprises the following step v') after step v):

[0354] v') reducing the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably aperture extrusion.

[0355] In one embodiment, the method further comprises storing the aqueous dispersion at a pH between 2.5 and 5.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH between 3.0 and 5.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH between 3.5 and 5.0. In one embodiment, the method further comprises storing the aqueous dispersion at a pH between 3.5 and 4.5.

[0356] In one embodiment, the method of preparing the aqueous dispersion of the present invention is carried out at about 0° C. to about 25° C., optionally about 4° C. to about 25° C., preferably about 15° C. to about 25° C. In one embodiment, the method of preparing the aqueous dispersion of the present invention is carried out at about room temperature (e.g., 18-25° C.).

[0357] Mixing step

[0358] The mixing step of the method of forming an aqueous dispersion of the present invention comprises mixing an organic phase comprising a lipid mixture comprising (i) a cationically ionizable lipid dissolved in a water-miscible organic solvent; and (ii) an aqueous phase comprising an anion of an aqueous acid, wherein the aqueous dispersion is substantially free of inorganic cations, organic solvent, and RNA.

[0359] In the method of the present invention, the organic solvent (e.g., the water-soluble organic solvent) can be selected from the list of Class 2 and Class 3 solvents, as described in the FDA's "Q3C-Tables and List Guidance for Industry", June 2017, 3rd Revision (see, for example, https: / / www.fda.gov / media / 71737 / download). When the organic solvent is a water-soluble organic solvent, examples include C1-4 alcohols (e.g., isopropanol or ethanol), ketones (e.g., acetone), or mixtures thereof. When the organic solvent is a non-polar organic solvent, examples include hydrocarbons such as pentane or hexane; chlorinated hydrocarbons such as dichloromethane or chloroform; or mixtures thereof. The organic solvent (e.g., water-soluble organic solvent) is preferably ethanol or isopropanol.

[0360] In one embodiment, the lipid mixture does not comprise phosphatidylserine.

[0361] In a preferred embodiment, the acid is a water-soluble organic acid as generally defined above.Examples of suitable organic acids include sulfonic acid, phosphoric acid, phosphonic acid, carboxylic acid, dicarboxylic acid or hydroxycarboxylic acid (all as defined herein).

[0362] In one embodiment, the water-soluble organic acid is selected from acetic acid, malic acid, succinic acid and citric acid or a combination thereof. In one embodiment, the water-soluble organic acid may be selected from acetic acid and malic acid or a combination thereof.

[0363] In one embodiment, the water-soluble organic acid is acetic acid. In one embodiment, the water-soluble organic acid is malic acid. In one embodiment, the water-soluble organic acid is succinic acid. In one embodiment, the water-soluble organic acid is citric acid.

[0364] In one embodiment, the concentration of the acid is in the range of about 0.1 to about 20mM. In one embodiment, the concentration of the acid is in the range of about 0.2 to about 15mM. In one embodiment, the concentration of the acid is in the range of about 0.5 to about 10mM. In one embodiment, the concentration of the acid is in the range of about 1 to about 5mM. In one embodiment, the concentration of the acid is in the range of about 2 to about 10mM. In one embodiment, the concentration of the acid is in the range of about 0.5 to about 5mM. In one embodiment, the concentration of the acid is in the range of about 3 to about 15mM. In one embodiment, the concentration of the acid is in the range of about 5 to about 8mM. In one embodiment, the concentration of the acid is in the range of about 8 to about 12mM. In this context, it should be understood that this concentration includes undissociated acid and its conjugate base.

[0365] In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.2 to about 20 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.5 to about 10 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.5 to about 2 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 1.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 1.25 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.5 to about 4 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 3.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 2.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration in the range of about 1 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration in the range of about 2 to about 7 mM. In one embodiment, the acid is acetic acid and is present at a concentration in the range of about 4 to about 6 mM. In one embodiment, the acid is acetic acid and is present at a concentration in the range of about 4.5 to about 5.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 5 mM.

[0366] In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.1 to about 5 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.4 to about 4 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.8 to about 2 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 1.5 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 1.25 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.5 to about 4 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 3.5 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 2 to about 3 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 2.5 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 8 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 8 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 2 to about 7 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 4 to about 6 mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 4.5 to about 5.5 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 5 mM.

[0367] In one embodiment, the acid is citric acid and the concentration of the acid is greater than 0.3mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 0.2 to about 15mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 0.5 to about 10mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 1 to about 8mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 2 to about 7mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 4 to about 6mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 4.5 to about 5.5mM. In one embodiment, the acid is citric acid and exists at a concentration of about 5mM.

[0368] In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 0.2 to about 10mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 0.4 to about 5mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 1 to about 3.5mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 2 to about 3mM. In one embodiment, the acid is succinic acid and exists with a concentration of about 2.5mM.

[0369] In one embodiment, mixing is performed using a T-blender or a Y-blender.

[0370] In one embodiment, the flow rate during mixing is at least 50 mL / min. The flow rate during mixing can be about 50 mL / min to about 400 mL / min, optionally about 100 mL / min to about 300 mL / min, optionally about 150 mL / min to about 250 mL / min. The volume ratio of the organic solvent to the aqueous phase can be about 1:6 to about 6:1, optionally about 1:2 to about 1:6, optionally about 1:4.

[0371] The pH of the aqueous dispersion prepared according to any of the above methods may be from about 2.5 to about 5.5, optionally from about 2.5 to about 4.5. The pH of the aqueous dispersion prepared according to any of the above methods may be from about 2.5 to about 3.5. The pH of the aqueous dispersion produced according to any of the above methods may be from about 3.5 to about 4.5. The pH of the aqueous dispersion produced according to any of the above methods may be from about 6.5 to about 8.5, optionally from 6.8 to 8.5, further optionally from about 7.0 to about 8.0.

[0372] Cryoprotectants during the mixing step

[0373] In one embodiment, the aqueous phase further comprises a cryoprotectant as defined and exemplified herein. In this embodiment, the aqueous phase thus comprises the anion of the aqueous acid as defined and exemplified above, and the cryoprotectant as defined and exemplified above.

[0374] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or a disaccharide. In one embodiment, the cryoprotectant is selected from sucrose, trehalose, lactose and glucose or any mixture thereof. In one embodiment, the cryoprotectant is selected from sucrose, trehalose and glucose or any mixture thereof. Preferably, the cryoprotectant is sucrose.

[0375] When the aqueous phase also contains a cryoprotectant that is a carbohydrate, typically, it is present at a concentration of about 1% to about 50% (w / v).

[0376] In one embodiment, the cryoprotectant is selected from sucrose, trehalose and lactose, and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the cryoprotectant is selected from sucrose, trehalose and lactose, and is present in a concentration of about 3% to about 25% (w / v). In one embodiment, the cryoprotectant is selected from sucrose, trehalose and lactose, and is present in a concentration of about 5% to about 20% (w / v). In one embodiment, the cryoprotectant is selected from sucrose, trehalose and lactose, and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the cryoprotectant is selected from sucrose, trehalose and lactose, and is present in a concentration of about 18% to about 22% (w / v).

[0377] In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1% to about 15% (w / v). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 3% to about 12% (w / v). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 5% to about 10% (w / v).

[0378] Additional processing steps

[0379] In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more additional processing steps. In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more additional dilution or purification steps.

[0380] Dialysis / filtration steps

[0381] In one embodiment, the purification step comprises a dialysis or filtration step, the purpose of which is generally to remove the organic solvent. In one embodiment, the dialysis or filtration step is performed at a pH of about 4.0 to about 5.0. In one embodiment, the dialysis or filtration step comprises tangential flow filtration.

[0382] In one embodiment, the dialysis or filtration step employs a composition comprising a compound selected from any one of the following classes (a) to (d):

[0383] (a) amino acids or mixtures thereof, preferably:

[0384] (i) an acidic amino acid, preferably selected from aspartic acid, glutamic acid, 3-hydroxyglutamic acid and α-aminoadipic acid; or a mixture thereof;

[0385] (ii) a basic amino acid, preferably selected from arginine, histidine and lysine or a mixture thereof;

[0386] or a mixture of (i) and (ii), or a mixture of either or both of (i) and (ii) with a neutral amino acid;

[0387] (b) an organic acid, preferably selected from acetic acid, malic acid, succinic acid, citric acid and methylmalonic acid; or a mixture thereof,

[0388] (c) a cryoprotectant, optionally wherein the cryoprotectant is a carbohydrate, such as a monosaccharide or disaccharide, preferably wherein the cryoprotectant is selected from sucrose, trehalose, lactose and glucose or any mixture thereof;

[0389] or any mixture thereof.

[0390] In one embodiment, the dialysis or filtration step is performed using one or more water-soluble weak organic acids. In one embodiment, the water-soluble weak organic acid is selected from acetic acid, malic acid, maleic acid, and succinic acid. In one embodiment, the water-soluble weak organic acid is acetic acid.

[0391] In one embodiment, the dialysis or filtration step is performed using an amino acid.

[0392] In one embodiment, the composition for dialysis or filtration is an acidic amino acid or a mixture thereof as defined and exemplified above. In one embodiment, the composition for dialysis or filtration is selected from aspartic acid, glutamic acid, 3-hydroxyglutamic acid and α-aminoadipic acid or a mixture thereof.

[0393] In one embodiment, the composition used for dialysis or filtration is a mixture of amino acids as defined and exemplified above, or a mixture thereof, and a water-soluble organic acid as defined and exemplified above, or a mixture thereof.

[0394] Dilution / addition of storage matrix / cryoprotectant

[0395] In one embodiment, the method may further comprise the step of adding a storage matrix to the aqueous dispersion. The method is preferably performed after the dialysis or filtration step. However, in an alternative, it may be performed immediately after the mixing step to form the aqueous dispersion.

[0396] The storage matrix used in this step can be any of those defined and illustrated above. In one embodiment, the storage matrix comprises a cryoprotectant so that the dilution step comprises adding a cryoprotectant. The cryoprotectant dilutes the aqueous dispersion and protects the pre-LNP from the damage caused by freezing. There is no particular restriction on cryoprotectant, as long as it can bring into play this function. In one embodiment, the cryoprotectant is selected from sucrose, trehalose, glucose, sorbitol, fructose, maltose, xylose and dextran or its arbitrary mixture. In one embodiment, the cryoprotectant is selected from sucrose, glycerol, trehalose, lactose, glucose and mannitol. In a preferred embodiment, the cryoprotectant is selected from sucrose, trehalose and glucose or its arbitrary mixture. In a more preferred embodiment, the cryoprotectant is selected from sucrose and trehalose or its mixture. In one embodiment, the cryoprotectant is sucrose.

[0397] In one embodiment, the storage matrix further comprises a compound selected from the following classes (a) to (c):

[0398] (a) amino acids as defined and exemplified above, such as

[0399] (i) an acidic amino acid as defined and exemplified above, preferably selected from aspartic acid, glutamic acid, 3-hydroxyglutamic acid and α-aminoadipic acid or a mixture thereof;

[0400] (ii) a basic amino acid as defined and exemplified above, preferably selected from arginine, histidine and lysine; or a mixture thereof;

[0401] or a mixture of (i) and (ii), optionally mixed with a neutral amino acid;

[0402] (b) an organic acid as defined and exemplified above, preferably selected from acetic acid, malic acid, succinic acid, citric acid and methylmalonic acid or mixtures thereof;

[0403] or any mixture thereof.

[0404] In one embodiment, the method further includes adding a peptide-conjugated lipid (as further described in this article) to the lipid granules contained in the dispersed phase of the aqueous dispersion. In some cases, the peptide-conjugated lipid can substitute (i.e., replace) the steroid (e.g., cholesterol) of the corresponding portion in the lipid granules contained in the dispersed phase of the aqueous dispersion.

[0405] Thus, the composition of the lipid particles contained in the dispersed phase of the aqueous dispersion prior to the addition of the peptide-conjugated lipid can comprise a cationic lipid or a cationic ionizable lipid as described herein, a neutral or zwitterionic phospholipid as described herein, a steroid as described herein, and an optional grafted lipid as described herein, in a molar ratio of 20-70 mol %: 5-15 mol %: 20-60 mol % and optionally 0.5-10 mol %; preferably 40-60 mol %: 8-12 mol %: 30-50 mol % and optionally 1.0-5 mol %. After the addition of the peptide-conjugated lipid, the peptide-conjugated lipid can comprise 0.05-1.0 mol %, optionally 0.1 to 0.5 mol %, preferably 0.1-0.3 mol % of the lipid particles contained in the dispersed phase of the aqueous dispersion, with a corresponding decrease in the mol % of the steroid.

[0406] In one embodiment, the purification is performed using an aqueous phase that is substantially free of buffer. In one embodiment, the purification is performed using an aqueous phase that is substantially free of buffer other than amino acids.

[0407] Additional optional steps

[0408] In one embodiment, the method further comprises the step of drying the aqueous dispersion. In one embodiment, the drying is lyophilization (freeze drying). In one embodiment, the drying is spray drying.

[0409] In one embodiment, the purification comprises sterile filtering the aqueous dispersion. Typically, the sterile filtration uses a 0.22 μm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0410] In one embodiment, the method further comprises storing the aqueous dispersion for 24 hours, 48 ​​hours, 72 hours, 5 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years or more. The aqueous dispersion can be stored at about 25°C, at about room temperature (e.g., 18-23°C), at about 4-8°C, at about 4°C, at about -20°C or at about -80°C. The aqueous dispersion can be stored at about 4°C or at about -20°C. In one embodiment, the method further comprises the step of freezing the aqueous dispersion, for example at a temperature between -15°C and -90°C, preferably at a temperature of about -18° to about -25°C. In one embodiment, the method further comprises the step of drying the aqueous dispersion. In one embodiment, the drying is freeze drying or spray drying.

[0411] In one embodiment, the aqueous dispersion is stable at 4°C for at least 3 months. In one embodiment, the aqueous dispersion is stable at -20°C for at least 6 months. Thus, in one embodiment, an aqueous dispersion is provided that is stable at 4°C for at least 3 months. In one embodiment, an aqueous dispersion is provided that is stable at -20°C for at least 6 months. In this context, "stable" is understood to mean that the size (Z) of the particles in the aqueous dispersion remains constant after storage at a specified temperature for a specified period of time. 平均 ) and / or size distribution and / or PDI are substantially equal to the size (Z) of the particles before storage and shortly after preparation. 平 For example, the size (Z) of the particles in the aqueous dispersion is 平均 ) and / or size distribution and / or PDI may vary by no more than 20%, optionally by no more than 10%, preferably by no more than 5% during a given storage.

[0412] Nucleic acid-lipid particles

[0413] The disclosure further provides a kind of lipid granule, it comprises lipid or lipid mixture and nucleic acid as defined herein.In one embodiment, provide by the lipid granule that the method for defining herein obtains or obtainable.Such granule is also referred to as " nucleic acid-lipid granule" in this article.When described nucleic acid is RNA, such granule is also referred to as " RNA-lipid granule" in this article.

[0414] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA, saRNA, taRNA or a mixture thereof. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA encoding one or more personalized cancer antigens.

[0415] In the present disclosure, it is preferred that the nucleic acid-lipid particle is a lipid nanoparticle (LNP). The function of LNP is to stabilize and encapsulate nucleic acid so that it can be delivered to cells while promoting its uptake by cells and being released into the cytosol. LNP and / or its lipid components can have adjuvant activity.

[0416] In the present disclosure, LNP can be understood as an oil-in-water emulsion, wherein the LNP core material is preferably in a liquid state and therefore has a melting point below body temperature. Therefore, LNP generally comprises a central complex of mRNA and lipids embedded in a disordered non-lamellar phase made of lipids. This is in contrast to the structure of liposomes comprising unilamellar or multilamellar vesicle particles, wherein the lamella comprises a lipid bilayer surrounding the encapsulated aqueous cavity. In some cases, nucleic acid-lipid particles described herein are not liposomes. In some cases, nucleic acid-lipid particles described herein are not lipoplexes.

[0417] Lipid nanoparticles (LNPs) can be obtained by combining nucleic acids with lipids. The lipids used to form LNPs do not typically form lamellar (double-layer) phases in water under physiological conditions. The LNPs typically do not include or encapsulate an aqueous core. The LNPs typically include a lipid (or oily) core.

[0418] In certain embodiments, the lipid nanoparticles described herein have a diameter of about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm. 100 nm to about 200 nm, about 150 nm to about 1000 nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 150 nm to about 500 nm, about 150 nm to about 450 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm to about In some embodiments, the lipid nanoparticles described herein have an average diameter in the range of about 60 nm to about 100 nm, about 200 nm to about 150 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, or about 200 nm to about 250 nm. In some embodiments, the lipid nanoparticles described herein have an average diameter in the range of about 60 nm to about 100 nm, in some embodiments.

[0419] In one embodiment, the nucleic acid-lipid particle is stable for at least 3 months at 4° C. In one embodiment, the nucleic acid-lipid particle is stable for at least 6 months at −20° C.

[0420] In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 4.0 and 6.5. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 4.5 and 6.0. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 4.6 and 5.8. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 5.0 and 5.5. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 5.1. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 5.2. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 5.3. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 5.4.

[0421] In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 7.0 and 9.0. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 7.0 and 8.5. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH between 7.5 and 8.1. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 7.8. In one embodiment, the nucleic acid-lipid particle is present in a composition having a pH of about 7.5.

[0422] Thus, in one embodiment, a nucleic acid-lipid particle is provided that is stable at 4°C for at least 3 months. In one embodiment, a nucleic acid-lipid particle is provided that is stable at -20°C for at least 6 months.

[0423] In one embodiment, the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particle does not decrease by more than 20% after the nucleic acid-lipid particle is stored at 4° C. for at least 3 months. In one embodiment, the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particle does not decrease by more than 20% after the nucleic acid-lipid particle is stored at −20° C. for at least 6 months.

[0424] Thus, in one embodiment, a nucleic acid-lipid particle (preferably an RNA-lipid particle) is provided, wherein the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particle is reduced by no more than 20% after the nucleic acid-lipid particle is stored at 4° C. for at least 3 months. In one embodiment, a nucleic acid-lipid particle (preferably an RNA-lipid particle) is provided, wherein the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particle is reduced by no more than 20% after the nucleic acid-lipid particle is stored at −20° C. for at least 6 months.

[0425] In one embodiment, the nucleic acid-lipid particles are capable of inducing a comparable or higher (e.g., 0.5-fold, 2-fold, 5-fold, 100-fold) antibody and / or T-cell response following in vivo administration compared to nucleic acid-lipid particles prepared using standard methods.

[0426] Thus, in one embodiment, a nucleic acid-lipid particle (preferably an RNA-lipid particle) is provided, wherein the nucleic acid-lipid particle is capable of inducing a comparable or higher (e.g., 0.5-fold, 2-fold, 5-fold, 100-fold) antibody and / or T-cell response following in vivo administration compared to nucleic acid-lipid particles prepared using standard methods.

[0427] Methods for forming nucleic acid-lipid particles

[0428] In yet another aspect, present disclosure provides the method for preparing nucleic acid-lipid granule disclosed herein.Usually, such method comprises adding an aqueous dispersion as described herein (usually containing pre-LNP) into the composition containing nucleic acid.In one embodiment, the composition containing nucleic acid is a solution containing nucleic acid.In one embodiment, the composition containing nucleic acid is an aqueous solution containing nucleic acid.

[0429] In one aspect, the method comprises:

[0430] i) preparing an aqueous dispersion as defined herein according to any of the methods defined herein; and

[0431] ii) mixing the aqueous dispersion with an aqueous solution containing nucleic acid to produce the nucleic acid-lipid particles.

[0432] In one embodiment, the method of forming RNA-lipid particles comprises:

[0433] i) preparing an aqueous dispersion as defined herein according to any of the methods defined herein; and

[0434] ii) mixing the aqueous dispersion with an aqueous solution containing RNA to produce the RNA-lipid particles.

[0435] In one embodiment, the method of forming nucleic acid-lipid particles comprises:

[0436] i) mixing a lipid mixture and an aqueous phase, wherein the lipid mixture comprises a cationic lipid or a cationic ionizable lipid dissolved in a water-miscible organic solvent,

[0437] wherein the lipid solution and / or the aqueous phase are acidified,

[0438] to produce an intermediate acidified aqueous lipid dispersion;

[0439] ii) subjecting the intermediate dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5 (preferably about 2.5 to about 4.5) or at a pH of about 6.5 to about 8.5 (preferably about 7.5 or about 8.5) to remove the organic solvent and produce an aqueous dispersion,

[0440] wherein the aqueous dispersion is substantially free of acetate buffer, citrate buffer, organic solvent, and RNA, and

[0441] wherein the aqueous dispersion comprises a cryoprotectant;

[0442] iii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid to produce the nucleic acid-lipid particles.

[0443] Mixing step

[0444] The mixing step of this aspect of the invention comprises mixing an aqueous dispersion as defined herein (typically containing pre-LNP) with an aqueous solution comprising nucleic acid as defined herein to produce said nucleic acid-lipid particles.

[0445] In one embodiment, the aqueous dispersion is provided at a neutral pH and the aqueous dispersion or the aqueous solution is acidified.

[0446] In one embodiment, the aqueous dispersion is provided at an acidic pH, and neither the aqueous dispersion nor the aqueous solution is acidified.

[0447] In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acids is 1.5:1 to 1:1.5. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acids is 1.2:1 to 1:1.2. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acids is 1.1:1 to 1:1.1. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acids is 1.05:1 to 1:1.05. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acids is 1:1.

[0448] In a preferred embodiment, the acid is a water-soluble organic acid as generally defined above.Examples of suitable organic acids include sulfonic acid, phosphoric acid, phosphonic acid, carboxylic acid, dicarboxylic acid or hydroxycarboxylic acid (all as defined herein).

[0449] In one embodiment, the water-soluble organic acid is selected from acetic acid, malic acid, succinic acid and citric acid or a combination thereof. In one embodiment, the water-soluble organic acid may be selected from acetic acid and malic acid or a combination thereof.

[0450] In one embodiment, the water-soluble organic acid is acetic acid. In one embodiment, the water-soluble organic acid is malic acid. In one embodiment, the water-soluble organic acid is succinic acid. In one embodiment, the water-soluble organic acid is citric acid.

[0451] In one embodiment, the concentration of the acid is in the range of about 0.1 to about 20mM. In one embodiment, the concentration of the acid is in the range of about 0.2 to about 15mM. In one embodiment, the concentration of the acid is in the range of about 0.5 to about 10mM. In one embodiment, the concentration of the acid is in the range of about 1 to about 5mM. In one embodiment, the concentration of the acid is in the range of about 2 to about 10mM. In one embodiment, the concentration of the acid is in the range of about 0.5 to about 5mM. In one embodiment, the concentration of the acid is in the range of about 3 to about 15mM. In one embodiment, the concentration of the acid is in the range of about 5 to about 8mM. In one embodiment, the concentration of the acid is in the range of about 8 to about 12mM. In this context, it should be understood that this concentration includes both the undissociated acid and its conjugate base.

[0452] In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.2 to about 20 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.5 to about 10 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 0.5 to about 4 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 3.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 2.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 1 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 2 to about 7 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 4 to about 6 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 4.5 to about 5.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 5.5 to about 9 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 6 to about 8.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 7 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 7.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 6 to about 14 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 7 to about 13 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 8 to about 12 mM. In one embodiment, the acid is acetic acid and is present at a concentration within the range of about 9 to about 11 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 10 mM.

[0453] In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.1 to about 5mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.4 to about 4mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.8 to about 2mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 1.5mM. In one embodiment, the acid is malic acid and is present at a concentration of about 1.25mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 0.5 to about 4mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 1 to about 3.5mM. In one embodiment, the acid is malic acid and is present at a concentration within the range of about 2 to about 3mM. In one embodiment, the acid is malic acid and is present at a concentration of about 2.5mM.

[0454] In one embodiment, the acid is citric acid and the concentration of the acid is greater than 0.3mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 0.2 to about 15mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 0.5 to about 10mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 1 to about 8mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 2 to about 7mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 4 to about 6mM. In one embodiment, the acid is citric acid and exists at a concentration in the range of about 4.5 to about 5.5mM. In one embodiment, the acid is citric acid and exists at a concentration of about 5mM.

[0455] In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 0.2 to about 10mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 0.4 to about 5mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 1 to about 3.5mM. In one embodiment, the acid is succinic acid and exists with a concentration in the range of about 2 to about 3mM. In one embodiment, the acid is succinic acid and exists with a concentration of about 2.5mM.

[0456] In a preferred embodiment of any of the above methods for forming nucleic acid-lipid particles, the aqueous dispersion comprises a cryoprotectant as defined and illustrated herein. In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or a disaccharide. In one embodiment, the cryoprotectant is selected from sucrose, trehalose, lactose and glucose or any mixture thereof. In one embodiment, the cryoprotectant is selected from sucrose, trehalose and glucose or any mixture thereof. Preferably, the cryoprotectant is sucrose or trehalose.

[0457] When the aqueous dispersion includes a cryoprotectant that is a carbohydrate, typically, it is present at a concentration of about 1% to about 30% (w / v).

[0458] In one embodiment, the cryoprotectant is sucrose or trehalose and is present in a concentration of about 1% to about 30% (w / v), optionally about 3% to about 25% (w / v), preferably about 5% to about 20% (w / v). In one embodiment, the cryoprotectant is sucrose or trehalose and is present in a concentration of about 8% to about 12% (w / v), such as about 10% (w / v). In one embodiment, the cryoprotectant is glucose and is present in a concentration of about 1% to about 15% (w / v), optionally about 3% to about 12% (w / v), preferably about 5% to about 10% (w / v).

[0459] In one embodiment, the cryoprotectant is sucrose or trehalose, and exists in a concentration of about 1% to about 30% (w / v), optionally about 10% to about 25% (w / v), preferably about 15% to about 25% (w / v). In one embodiment, the cryoprotectant is sucrose or trehalose, and exists in a concentration of about 18% to about 22% (w / v), such as about 20% (w / v). In one embodiment, the cryoprotectant is glucose and exists in a concentration of about 1% to about 15% (w / v), optionally about 5% to about 15% (w / v), preferably about 8% to about 12% (w / v). In such an embodiment, preferably, the nucleic acid-lipid particle is not further diluted and / or a cryoprotectant step is added. For example, the nucleic acid-lipid particle may not need any further processing steps.

[0460] In one embodiment, the method of forming nucleic acid-lipid particles of the present invention is carried out at about 0° C. to about 25° C., optionally about 4° C. to about 25° C., preferably about 15° C. to about 25° C. In one embodiment, the method of preparing the aqueous dispersion of the present invention is carried out at about room temperature (e.g., 18-25° C.).

[0461] In one embodiment, the aqueous solution containing nucleic acid also contains one or more buffers. In one embodiment, the buffer is 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (HEPES), optionally in combination with ethylenediaminetetraacetic acid (EDTA) or its acceptable salt. The aqueous solution containing nucleic acid can have a pH of about 6.5 to about 8.5, optionally a pH of about 6.8 to about 7.5. The aqueous solution containing nucleic acid can have a pH of about 7.0.

[0462] Optional further processing steps

[0463] In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle to one or more further processing steps.

[0464] In one embodiment, the method further includes adding a peptide-conjugated lipid (as further described in this article) to the nucleic acid-lipid particle. In some cases, the peptide-conjugated lipid can replace (i.e., replace) the steroid (e.g., cholesterol) of the corresponding portion in the nucleic acid-lipid particle. Before adding the peptide-conjugated lipid, the composition of the nucleic acid-lipid particle can include cationic lipids or cationic ionizable lipids as described herein, neutral or zwitterionic phospholipids as described herein, steroids as described herein, and optional grafted lipids as described herein, in a molar ratio of 20-70mol%: 5-15mol%: 20-60mol% and optional 0.5-10mol%; optionally, 40-60mol%: 8-12mol%: 30-50mol% and optional 1.0-5mol%. After addition of the peptide-conjugated lipid, the peptide-conjugated lipid may comprise 0.05-1.0 mol%, optionally 0.1 to 0.5 mol%, preferably 0.1-0.3 mol% of the lipid in the nucleic acid-lipid particle, with a corresponding decrease in the mol% of steroid.

[0465] In one embodiment, the method comprises further performing one or more purification steps on the nucleic acid-lipid particle. In one embodiment, the purification step comprises a dialysis or filtration step. In one embodiment, the dialysis or filtration step comprises a tangential flow filtration. In one embodiment, the method does not comprise filtering or dialysis steps on the nucleic acid-lipid particle. In one embodiment, the method does not comprise performing a tangential flow filtration step on the nucleic acid-lipid particle.

[0466] In one embodiment, the method includes further carrying out one or more dilution steps to the nucleic acid-lipid granule. In one embodiment, the one or more dilution steps include adding a cryoprotectant. In a preferred embodiment, the method does not include carrying out any of the following to the nucleic acid-lipid granule: (i) dialysis or filtration step (for example, TFF step), (ii) dilution step, and (iii) comprising the dilution step of adding a cryoprotectant. In one embodiment, the cryoprotectant is selected from sucrose, glycerol, trehalose, lactose, glucose and mannitol. In one embodiment, the cryoprotectant is sucrose.

[0467] In one embodiment, the purification step is performed using an aqueous phase that is substantially free of buffer.

[0468] In one embodiment, the method further comprises a sterile filtration step of the nucleic acid-lipid particles. Typically, the sterile filtration uses a 0.22 μm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0469] In one embodiment, the method further comprises the step of drying the nucleic acid-lipid particles. In one embodiment, the drying is freeze drying. In one embodiment, the drying is spray drying.

[0470] In one embodiment, the one or more purification steps of the nucleic acid-lipid particles do not include a tangential flow filtration step.

[0471] In one embodiment, the nucleic acid-lipid particle is not subjected to any further purification steps.

[0472] In one embodiment, the method further comprises the step of diluting the lipid granules with a storage matrix. In one embodiment, the storage matrix comprises one or more buffers. In one embodiment, the buffer or its mixture has a pH of 4.5 to 8.5. In one embodiment, the buffer is selected from 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris-(hydroxymethyl) aminomethane (Tris), histidine, triethanolamine or its arbitrary mixture. In one embodiment, the buffer is a mixture of HEPES and Tris. The preferred molar ratio of HEPES:Tris is between 100:1 and 1:100, preferably 10:1 to 1:10.

[0473] Lipids and amphiphiles

[0474] The compositions of the present invention also contain mixtures of lipids.The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules comprising one or more hydrophobic parts or groups and one or more hydrophilic parts or groups.

[0475] Lipids are generally insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature enables the molecules to self-assemble into organized structures and distinct phases.

[0476] Lipids can comprise a polar portion and a non-polar (or non-polar) portion. The term "amphiphile" as used in this specification is broadly defined herein as a molecule comprising a hydrophobic portion and a hydrophilic portion and / or a polar portion and a non-polar portion. Since both cationic and anionic lipids contain such groups, they are amphiphiles. Therefore, in this specification, the term "cationic lipid" is synonymous with "cationic amphiphile" and the term "anionic lipid" is synonymous with "anionic amphiphile".

[0477] Hydrophobicity can be imparted by the inclusion of non-polar groups, including but not limited to long chain saturated and unsaturated hydrocarbon groups (as defined and exemplified above), such as alkyl, alkenyl and / or alkynyl groups and such groups substituted with one or more aryl, heteroaryl or cycloalkyl groups (as defined and exemplified above). The hydrophilic groups can comprise polar and / or charged groups, and include at least one amine and optionally hydrophilic uncharged groups such as hydroxyl, carbohydrate, sulfhydryl, nitro or the like, and can further include anionic groups such as phosphate, phosphonate, carboxylate, sulfate, sulfonate (all as defined and exemplified above) and other similar groups.

[0478] As used herein, the term "hydrophobic" with respect to a compound, group, or moiety means that the compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, excludes water molecules. In certain embodiments, the term "hydrophobic" refers to any compound, group, or moiety that is substantially immiscible or insoluble in aqueous solution. In certain embodiments, a hydrophobic compound, group, or moiety is substantially non-polar.

[0479] Examples of hydrophobic groups are hydrocarbyl groups (as defined and illustrated above), such as alkyl, alkenyl and / or alkynyl groups, and such groups substituted with one or more aryl, heteroaryl or cycloalkyl groups (as defined and illustrated above). The hydrophobic group may have functional groups (e.g., ethers, thioethers, esters, dioxolanes, halides, amides, sulfonamides, carbamates, etc.) and atoms other than carbon and hydrogen, as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

[0480] The hydrophobic portion of the lipid can have 24 to 60 carbon atoms and can be a hydrocarbon group (as described and exemplified above, typically comprising an alkyl, alkenyl or alkynyl group as described and exemplified above). The 24 to 60 carbon atoms can be divided into two or more hydrophobic portions, each such portion typically having at least 6 carbon atoms. An example of a fragmented hydrophobic portion in which each segment is a hydrocarbon group is a lipid comprising a DACA portion as described in WO2011 / 003834, wherein each acyl or alkyl group comprises 12 to 20 carbon atoms. Another example is a lipid in which the hydrophobic portion comprises a steroid portion (such as a cholesteryl portion).

[0481] The hydrophobic portion of the lipid preferably has 24 to 60 carbon atoms and may also be a heteroalkyl group, wherein the heteroatoms are selected from N, O or S, thereby forming one, two, three or four uncharged ether, thioether, ester, amide, carbamate, sulfonamide or the like groups. The 24 to 60 carbon atoms may be divided into two or more hydrophobic portions, provided that each such portion has at least 6 carbon atoms. Examples of fragmented hydrophobic portions in which each segment is a hydrocarbyl group are lipids comprising a diacylglycerol or dialkylglycerol portion, wherein each acyl or alkyl group comprises 12 to 20 carbon atoms. Examples of hydrophobic portions in which each segment is a heteroalkyl group are ester branched moieties in lipids, such as SM-102 or ALC-315, as defined and exemplified below.

[0482] Cationic lipids and cationic ionizable lipids

[0483] The aqueous dispersions and lipid particles of the present invention also contain cationic lipids or cationic ionizable lipids, or any mixture thereof.In one embodiment, the aqueous dispersions and lipid particles of the present invention comprise cationic ionizable lipids, and preferably do not comprise cationic lipids.

[0484] The term "cationic lipid" as used herein refers to a lipid or lipid-like material as defined herein with a constituent positive charge. In this context, "constitutive charge" refers to that the cationic lipid carries a positive charge at all physiological pHs. The cationic lipid carrying a constituent charged cation portion is typically a salt of a quaternary ammonium salt (as defined above) or an organic base (such as a nitrogenous base). Typically, such an organic base is a strong base (that is, a completely protonated base when dissolved in a solvent (such as, but not limited to, an aqueous solvent) so that the concentration of the unprotonated substance is too low to measure).

[0485] In one embodiment, the cationic lipid is a monovalent cationic lipid.

[0486] In one embodiment, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

[0487] Examples of cationic lipids include, but are not limited to, 1,2-dialkoxy-3-dimethylammonium propanes and 1,2-dienyloxy-3-dimethylammonium propanes (each alkyl or alkenyl moiety is as defined and exemplified above and preferably has 12 to 20 carbon atoms), such as 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-diacyloxy-3-dimethylammonium propanes (the alkyl or alkenyl moiety of each acyl moiety is as defined and exemplified above and preferably has 12 to 20 carbon atoms), such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP ); dimethyldioctadecyl ammonium (DDAB); dioctadecyldimethylammonium chloride (DODAC); 2,3-ditetradecyloxypropyl-(2-hydroxyethyl)-dimethylazonium (DMRIE); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA).

[0488] The structures of DODMA and DODAP are shown below.

[0489]

[0490] The structures of DOTMA, DOTAP, and their analogs are shown below.

[0491]

[0492] The structures of DOTAP and other suitable homologues are shown below.

[0493]

[0494] The structures of DOTMA, DORIE, and other suitable homologs are shown below.

[0495]

[0496] Other suitable cationic lipids are described in Sun and Lu, Pharmaceutical Research, 2023, https: / / doi.org / 10.1007 / s11095-022-03460-2.

[0497] In one embodiment, the lipid is a cationically ionizable lipid. As used herein, "cationically ionizable lipid" refers to a lipid or lipid-like substance that has a net positive charge or is neutral, depending on whether it is protonated or deprotonated, i.e., a lipid that is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is dissolved, the cationically ionizable lipid is either positively charged or neutral.

[0498] In certain embodiments, the cationically ionizable lipid comprises a head group including at least one nitrogen atom (N) capable of being protonated, preferably under physiological or mildly acidic conditions.

[0499] In one embodiment, the cationic ionizable lipid is a compound represented by formula (TL-I):

[0500]

[0501] or a pharmaceutically acceptable salt thereof, wherein:

[0502] L 1 and L 2 are each independently an optionally substituted C1-C 30 aliphatic groups;

[0503] L 3 is a bond, an optionally substituted C1-C 10 an aliphatic group or an optionally substituted 2- to 10-membered heteroaliphatic group;

[0504] X 1 and X 2 Each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-, where X 1 or X 2 One or two of them are selected from -S(O)2N(R 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-;

[0505] Each R 1is independently at each occurrence an optionally substituted C1-C 20 aliphatic group or H;

[0506] T 1 and T 2 Each independently is optionally substituted C3-C 30 aliphatic groups;

[0507] G is -N(R 2 )C(S)N(R 2 )2, -N + (R 3 )3、-OH、-N(R 2 )2、-N(R 5 )C(O)R 3 、-N(R 5 )S(O)2R 3 、-N(R 5 )C(O)N(R 3 )2、-CH(NR 2 )or - R 4 ;

[0508] Each R 2 is independently selected at each occurrence from H, an optionally substituted C1-C6 aliphatic group or OR 3 ; or R 2 Two instances of are taken together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocyclic ring or an optionally substituted 4- to 12-membered heteroaryl ring;

[0509] Each R 3 is independently selected at each occurrence from H and optionally substituted C1-C 10 an aliphatic group; and

[0510] R 4 is an optionally substituted 4- to 12-membered heterocyclic ring, an optionally substituted 4- to 12-membered heteroaryl ring, replaced by -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 )2 in one or more substituted C6-C 12 Aryl, or oxo, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 )2 in one or more substituted C3-C 12 cycloaliphatic groups;

[0511] Each R 5 independently selected from H and optionally substituted C1-C6 aliphatic groups.

[0512] In certain embodiments of Formula (TL-I), L 1 and L 2 Each independently is -(CH2) 6-10 -.

[0513] In certain embodiments of Formula (TL-I), X 1 and X 2 Each independently selected from -S(O)2N(R 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-.

[0514] In certain embodiments of Formula (TL-I), X 1 and X 2 Each is -S(O)2N(R 1 )-, where each R 1 Independently C1-C 10 Aliphatic group.

[0515] In certain embodiments of Formula (TL-I), T 1 and T 2 Each independently selected from optionally substituted C3-C 20 alkyl.

[0516] In certain embodiments of Formula (TL-I), T 1 and T 2 Each independently selected from:

[0517]

[0518]

[0519] In certain embodiments of Formula (TL-I), G is -N(R 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 .

[0520] In certain embodiments of Formula (TL-I), G is -N(H)C(S)N(R 2 )2, where each R 2 is selected from optionally substituted C1-C6 aliphatic groups and OH.

[0521] In certain embodiments of Formula (TL-I), G is -OH.

[0522] In certain embodiments of Formula (TL-I), G is selected from:

[0523]

[0524] In certain embodiments of Formula (TL-I), -L 3 -G is selected from:

[0525]

[0526] In certain embodiments of Formula (TL-I), the compound is represented by Formula (TL-IIa):

[0527]

[0528] or a pharmaceutically acceptable salt thereof.

[0529] In certain embodiments of Formula (TL-I), the compound is represented by Formula (TL-IIc):

[0530]

[0531] or a pharmaceutically acceptable salt thereof.

[0532] In certain embodiments of Formula (TL-I), the compound is represented by Formula (TL-IIIb):

[0533]

[0534] or a pharmaceutically acceptable salt thereof.

[0535] In certain embodiments of Formula (TL-I), the compound is represented by Formula (TL-IIIe):

[0536]

[0537] or a pharmaceutically acceptable salt thereof.

[0538] In certain embodiments of Formula (TL-I), the compound is 7,7'-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octyheptane-1-sulfonamide)

[0539]

[0540] or a pharmaceutically acceptable salt thereof.

[0541] In certain embodiments of Formula (TL-I), the compound is 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylmeptane-1-sulfonamide)

[0542]

[0543] or a pharmaceutically acceptable salt thereof.

[0544] In certain embodiments of Formula (TL-I), the compound is

[0545]

[0546] or a pharmaceutically acceptable salt thereof.

[0547] The thioester compound of formula (TL-I) can be prepared according to PCT / EP2023 / 071270 (the contents of which are incorporated herein by reference).

[0548] In one embodiment, the cationic lipid or cationic ionizable lipid is selected from:

[0549] [(4-Hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315);

[0550] 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA);

[0551] 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA);

[0552] Heptatriacontriacontac-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (D-Lin-MC3-DMA);

[0553] 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0554] Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319);

[0555] Di-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonaneamido)-nonadecanedioate (A9);

[0556] (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5);

[0557] Heptadec-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);

[0558] O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butanoate (HY501);

[0559] 2-(Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2);

[0560] 4-((Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-4-amine (HYAM-2);

[0561] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405);

[0562] (2-(4-(dimethylamino)butyryl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405);

[0563] Palmitoyl-oleoyl-nor-arginine (PONA);

[0564] Guanidine-di[(heptadecyl)methyl]carboxylic acid (GUADACA);

[0565] 4-Methylpyridinium-di(heptadecyl)methylcarboxylic acid (MPDACA);

[0566] 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP);

[0567] 1,2-dioleoyl-3-dimethylammonium propane (DODAP);

[0568] 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA);

[0569] or any mixture thereof.

[0570] In one embodiment, the cationic ionizable lipid is selected from:

[0571] 7,7'-((4-Hydroxybutyl)azanediyl)bis(N-hexyl-N-octyheptane-1-sulfonamide)

[0572]

[0573] 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octyheptane-1-sulfonamide)

[0574]

[0575] Compounds with the following structure

[0576]

[0577] [(4-Hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315);

[0578] 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA);

[0579] 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA);

[0580] Heptatriacontriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (D-Lin-MC3-DMA);

[0581] 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0582] Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319);

[0583] Di-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonaneamido)-nonadecanedioate (A9);

[0584] (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5);

[0585] Heptadec-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);

[0586] O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butanoate (HY501);

[0587] 2-(Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2);

[0588] 4-((Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-4-amine (HYAM-2);

[0589] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405);

[0590] (2-(4-(dimethylamino)butyryl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405);

[0591] diheptadecan-9-yl 3,3'-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate

[0592] (BHD-C2C2-PipZ, described in US2022 / 0218622A1);

[0593] Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate

[0594] (BODD-C2C2-1Me-Pyr, described in US2022 / 0218622A1);

[0595] or any mixture thereof.

[0596] In one embodiment, the cationic ionizable lipid is selected from:

[0597] 7,7'-((4-Hydroxybutyl)azanediyl)-bis(N-hexyl-N-octyheptane-1-sulfonamide) (BNT-51);

[0598] 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octyhleptane-1-sulfonamide) (BNT-52);

[0599] Compounds with the following structure

[0600]

[0601] [(4-Hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);

[0602] 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA);

[0603] 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA);

[0604] Heptatriacontriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (D-Lin-MC3-DMA);

[0605] 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0606] Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319);

[0607] Di-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonaneamido)-nonadecanedioate (A9);

[0608] (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5);

[0609] Heptadec-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);

[0610] O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butanoate (HY501);

[0611] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405);

[0612] (2-(4-(dimethylamino)butyryl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405);

[0613] Diheptadecan-9-yl 3,3′-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ);

[0614] Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr);

[0615] Bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr);

[0616] Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1MePyr);

[0617] Bis(2-octyldodecyl) 3,3'-(((1-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-1Me-3PipD);

[0618] Bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA);

[0619] Bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ);

[0620] Bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr);

[0621] Bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ);

[0622] Di(nonadecan-9-yl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (DND-C2-C4-PipZ);

[0623] or any mixture thereof.

[0624] In one embodiment, the cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA). In one embodiment, the cationic lipid is 4-methylpyridinium-di(heptadecanyl)-methylcarboxylic acid (MPDACA). In one embodiment, the cationic lipid is 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP). In one embodiment, the cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

[0625] In one embodiment, the cationic ionizable lipid is [(4-hydroxybutyl) azanediyl]-bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationic ionizable lipid is 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA). In one embodiment, the cationic ionizable lipid is 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA). In one embodiment, the cationic ionizable lipid is heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (D-Lin-MC3-DMA). In one embodiment, the cationic ionizable lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In one embodiment, the cationic ionizable lipid is di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319). In one embodiment, the cationic ionizable lipid is di-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)-nonaneamido)-nonadecanedioate (A9). In one embodiment, the cationic ionizable lipid is (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5). In one embodiment, the cationic ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102). In one embodiment, the cationic ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butanoate (HY501). In one embodiment, the cationic ionizable lipid is 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2). In one embodiment, the cationic ionizable lipid is 7,7'-((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylmeptane-1-sulfonamide) (BNT-51). In one embodiment, the cationic ionizable lipid is 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylmeptane-1-sulfonamide) (BNT-52). In one embodiment, the cationic ionizable lipid is a compound having the structure In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-1Me-Pyr.

[0626] In certain embodiments, the cationic ionizable lipids are selected from those generally and specifically described in WO 2018 / 087753.

[0627] In certain embodiments, the cationic ionizable lipid is selected from the group consisting of:

[0628]

[0629] In one embodiment, the cationic ionizable lipid is 4-((di-((9Z,12Z)-octadec-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-4-amine (HYAM-2). In one embodiment, the cationic ionizable lipid is ((2-(4-(dimethylamino)butanoyl)-oxy)ethyl)-azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405). In one embodiment, the cationic ionizable lipid is (2-(4-(dimethylamino)butanoyl)-oxy)azanediylbis-(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405). In one embodiment, the cationic ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butyrate (HY501).

[0630] In one embodiment, the cationic lipid or cation ionizable lipid exists in an amount of 20 to 70mol% of the total lipid present in the lipid mixture. In one embodiment, the cationic lipid or cation ionizable lipid exists in an amount of 30 to 60mol% of the total lipid present in the lipid mixture. In one embodiment, the cationic lipid or cation ionizable lipid exists in an amount of 40 to 50mol% of the total lipid present in the lipid mixture. The term "lipid mixture" is applicable to the lipid mixture components of both the aqueous dispersion and the nucleic acid-lipid particle in this context.

[0631] Other lipids

[0632] The lipid mixture in aqueous dispersions of the present invention and lipid granule can further comprise one or more other lipids.In one embodiment, described one or more other lipids comprise anionic amphiphiles as defined below and illustrated.In one embodiment, described one or more other lipids comprise neutral or zwitterionic lipids as defined below and illustrated.In one embodiment, described one or more other lipids comprise steroids as defined below and illustrated.In one embodiment, described one or more other lipids comprise neutral lipids as defined below and illustrated.In one embodiment, described one or more other lipids comprise neutral lipids (such as steroids) as defined below and illustrated.In one embodiment, described one or more other lipids comprise lipids that are puted together with peptides as defined below and illustrated.

[0633] neutral lipids

[0634] The composition may further comprise a neutral lipid. The neutral lipid is preferably a neutral phospholipid. In one embodiment, the phospholipid may be a zwitterionic ion (i.e., it carries both a positive and a negative charge such that it is neutral at a pH around neutral).

[0635] In certain embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. The alkyl part of the acyl part of the phospholipid is as defined above, but preferably an alkyl group (as defined above) with 6 to 40, preferably 8 to 24 carbon atoms or an alkenyl group (as defined above) with 6 to 40, preferably 14 to 22 carbon atoms and 1 to 6 carbon-carbon double bonds. The acyl part of the phospholipid can be the same or different. In one embodiment, the acyl part is a saturated fatty acid part with 8 to 24 carbon atoms (including acyl carbon), preferably selected from tetracosanoyl, behenoyl, arachidonic acid, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl parts. In a specific embodiment, the neutral phospholipid has a T of 30°C or higher. m and is selected from distearoyl or dipalmitoyl or stearoyl-palmitoyl moieties. In one embodiment, the acyl moiety is an unsaturated fatty acid moiety having 14 to 22 carbon atoms (including the acyl carbon), preferably selected from oleoyl, linoleoyl and linolenoyl moieties.

[0636] Examples of such phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diacidonoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), dioctanoylphosphatidylcholine (D ... Trialkanoylphosphatidylcholine (DTPC), ditetracosanoylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-10-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidyl-ethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphatidylcholine (SM) and other phosphatidyl-ethanolamine lipids with different hydrophobic chains.

[0637] In certain embodiments, the neutral lipid is selected from DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE and SM, or any mixture thereof.

[0638] Thus, in certain embodiments, the lipid nanoparticle compositions described herein comprise a cationic lipid or a cationic ionizable lipid (as defined herein) and a phospholipid. In certain embodiments, the lipid nanoparticle compositions described herein comprise a cationic lipid or a cationic ionizable lipid and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or any mixture thereof.

[0639] In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 1 mol % to about 40 mol % of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 2 mol % to about 25 mol % of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipid present in the lipid mixture.

[0640] In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipid present in the lipid mixture.

[0641] In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of from about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of from about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.

[0642] In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipid present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipid present in the lipid mixture.

[0643] In each of the above embodiments, the term "lipid mixture" applies in this context to both the lipid mixture component of the aqueous dispersion and the nucleic acid-lipid particle.

[0644] steroids

[0645] The lipid nanoparticle compositions of the present invention also comprise a steroid. In one embodiment, the steroid comprises a sterol. In one embodiment, the steroid is cholesterol.

[0646] Thus, in certain embodiments, the lipid nanoparticle compositions described herein comprise a cationic ionizable lipid (as defined herein) and cholesterol.

[0647] In one embodiment, the steroid is present in an amount in the range of about 10mol% to about 65mol% of the total lipid present in the lipid mixture. In one embodiment, the steroid is present in an amount in the range of about 20mol% to about 60mol% of the total lipid present in the lipid mixture. In one embodiment, the steroid is present in an amount in the range of about 30mol% to about 50mol% of the total lipid present in the lipid mixture.

[0648] In certain embodiments, the combined concentration of neutral lipids (particularly, one or more phospholipids, especially phosphatidylcholines such as DSPC) and steroids (particularly, cholesterol) can be from about 0 mol% to about 70 mol%, such as from about 2 mol% to about 60 mol%, from about 5 mol% to about 55 mol%, from about 5 mol% to about 50 mol% of the total lipids present in the lipid mixture.

[0649] In each of the above embodiments, the term "lipid mixture" applies in this context to both the lipid mixture component of the aqueous dispersion and the nucleic acid-lipid particle.

[0650] Anionic amphiphiles

[0651] In one embodiment, the compositions of the present disclosure also include negatively charged amphiphiles ("anionic amphiphiles"). In this specification, the term "amphiphile" is generally defined as a molecule having a hydrophilic portion and a hydrophobic portion (as defined above). The negative charge is located in the hydrophilic portion of the amphiphile. The negatively charged amphiphile can have one negatively charged group or multiple (e.g., 2, 3, 4, or 5) negatively charged groups. Anionic amphiphiles having a single negatively charged group are preferred.

[0652] In the present invention, anionic amphiphiles have a pH-sensitive charge. In this context, "pH-sensitive charge" means that the amphiphile has a negative charge at alkaline or neutral pH, but may be neutral at acidic pH. In certain embodiments, the pH-sensitive charge is combined with a constitutive charge, such as in organophosphates, where such amphiphiles have two negative charges at alkaline or neutral pH, but only one negative charge at acidic pH. Amphiphiles with constitutively charged anionic moieties are typically salts of weak organic acids (i.e., organic acids that are largely undissociated when dissolved in a solvent, so that protons are only partially transferred to solvent molecules).

[0653] In one embodiment, the anionic amphiphile has a charged polar moiety selected from carboxylate or phosphate.

[0654] In one embodiment, the negatively charged amphiphile is a carboxylic acid or a carboxylate (as defined above, both in its broadest and preferred aspects).

[0655] In one embodiment, the negatively charged amphiphile has a pH sensitive charge and the pH sensitive anionic moiety is a carboxylic acid. One or more charged groups may be present in the amphiphile, and in a preferred embodiment, a single charged moiety is present in the amphiphile.

[0656] The polar region of the negatively charged amphiphile may contain additional uncharged polar moieties. Preferred uncharged polar moieties are hydroxyl or amide groups, and one or more uncharged polar moieties may be present in the negatively charged amphiphile.

[0657] In one embodiment, the negatively charged amphiphile is a half ester of a dicarboxylic acid and diacylglycerol. The hydrocarbyl portion of the acyl moiety of the diacylglycerol moiety is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 14 to 22 carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22 carbon atoms. The acyl moieties of the diacylglycerol moieties may be the same or different. In one embodiment, the acyl moiety is a saturated fatty acid moiety, preferably selected from behenoyl, arachidoyl, stearoyl, palmitoyl and myristoyl moieties. In one embodiment, the acyl moiety is an unsaturated fatty acid moiety, preferably selected from oleoyl, linoleoyl and linolenoyl moieties. The dicarboxylic acid moiety is as defined above and preferably has 2 to 8 carbon atoms, more preferably 2 to 6, even more preferably 2 to 4 carbon atoms. Examples of the dicarboxylic acid moiety include oxalate, malonate, succinate, glutarate, adipate, pimelate and suberate. Typical examples of such negatively charged amphiphiles include dimyristoylglyceryl hemisuccinate (DMGS), dipalmitoylglyceryl hemisuccinate (DPGS), palmitoylstearoylglyceryl hemisuccinate (PSGS), distearoylglyceryl hemisuccinate (DSGS), dioleoylglyceryl hemisuccinate (DOGS), palmitoyloleoylglyceryl hemisuccinate (POGS) and homologues of any of the above substances, wherein the dicarboxylic acid moiety is oxalate, malonate, succinate, glutarate, adipate, pimelate or suberate. Dimyristoylglyceryl hemisuccinate (DMGS) or dioleoylglyceryl hemisuccinate (DOGS) is preferred.

[0658] In one embodiment, the negatively charged amphiphile is a half ester of a dicarboxylic acid and a steroid. The dicarboxylic acid portion is as defined and exemplified above and typically contains a total of 2 to 6 carbon atoms (including the acyl carbon), preferably 3 to 5, and most preferably 4 carbon atoms. The ester group can preferentially esterify the 3' hydroxyl group on the steroid molecule.

[0659] In one embodiment, the negatively charged amphiphile is a half ester of a dicarboxylic acid and cholesterol. The dicarboxylic acid moiety is as defined above and preferably has 2 to 6 carbon atoms, more preferably 3 to 5, even more preferably 4 carbon atoms. Examples of dicarboxylic acid moieties include oxalate, malonate, succinate, glutarate, and adipate, with succinate being preferred. Typical examples of such negatively charged amphiphiles include cholesterol hemisuccinate and cholesterol hemisuccinate, with cholesterol hemisuccinate being preferred.

[0660] In one embodiment, the negatively charged amphiphile is a monoester or diester of phosphoric acid, wherein one of the hydroxyl groups of the phosphoric acid is esterified with diacylglycerol. The hydrocarbyl portion of the acyl moiety of the diacylglycerol moiety is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 14 to 22 carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22 carbon atoms. The acyl moieties of the diacylglycerol moieties may be the same or different. In one embodiment, the acyl moiety is a saturated fatty acid moiety, preferably selected from behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl moieties. In one embodiment, the acyl moiety is an unsaturated fatty acid moiety, preferably selected from oleoyl, linoleoyl and lineoyl moieties.

[0661] In one embodiment, the anionic amphiphile is a carboxylic acid, preferably selected from hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, tricosanoic acid, 2-hydroxytetradecanoic acid, 2-methyloctadecanoic acid, 2-bromohexadecanoic acid, 2-propylpentanoic acid, 2-butyloctanoic acid, 2-hexyldecanoic acid, 9-hydroxy-stearic acid, trans-2-decenoic acid, (9Z)-9-hexadecenoic acid, linoleic acid, linolenic acid, oleic acid, elaidic acid, arachidonic acid, cyclododecanoic acid, adamantyl acetic acid, dicyclohexyl acetic acid, trans-4-pentylcyclohexane-carboxylic acid, 4-(decyloxy)benzoic acid, 4-octylbenzoic acid, cholic acid and lithocholic acid, or any mixture thereof.

[0662] In one embodiment, the anionic amphiphile is a half ester of a dicarboxylic acid and a diacylglycerol, preferably selected from dimyristoyl hemisuccinate and dioleoyl hemisuccinate or any mixture thereof.

[0663] In one embodiment, the anionic amphiphile is a half-ester of a dicarboxylic acid and cholesterol, preferably selected from the group consisting of cholesterol hemisuccinate, cholesterol heimalonate and cholesterol hemiadipate or any mixture thereof.

[0664] In one embodiment, the anionic amphiphile is an organic sulfate or sulfonate, preferably selected from sodium lauryl sulfate, sodium hexadecanesulfonate and sodium dodecylbenzenesulfonate or any mixture thereof.

[0665] In one embodiment, the anionic amphiphile is an organic phosphonate, preferably selected from octadecylphosphonic acid and dodecylphosphonic acid or any mixture thereof.

[0666] In one embodiment, the anionic amphiphile is an anionic phospholipid, preferably selected from phosphatidylserine, phosphatidylglycerol and phosphatidic acid or any mixture thereof.

[0667] In one embodiment, the anionic amphiphile is selected from:

[0668] carboxylic acid;

[0669] Half esters of dicarboxylic acids and cholesterol;

[0670] Half esters of dicarboxylic acids and diacylglycerols;

[0671] Phosphate esters with diacylglycerol;

[0672] or any mixture thereof.

[0673] In one embodiment, the anionic amphiphile is selected from:

[0674] Cholesteryl hemisuccinate (CHEMS);

[0675] dimyristoyl hemisuccinate (DMGS);

[0676] dioleoylglyceryl hemisuccinate (DOGS);

[0677] or any mixture thereof.

[0678] In one embodiment, the anionic amphiphile is CHEMS. In one embodiment, the anionic amphiphile is DMGS. In one embodiment, the anionic amphiphile is DOGS.

[0679] In one embodiment, the anionic amphiphile is present in an amount of 0 to 50 mol % of the total lipids present in the lipid mixture. In one embodiment, the anionic amphiphile is present in an amount of 5 to 45 mol % of the total lipids present in the lipid mixture. The term "lipid mixture" in this context applies to the lipid mixture component of the aqueous dispersion and the nucleic acid-lipid particle.

[0680] Grafted lipid

[0681] The compositions described herein may also contain grafted lipids. In this specification, the term "grafted lipid" in its broadest sense refers to a lipid or lipid-like material (in the broadest aspect or preferred aspect) as defined above conjugated to a polymer (in the broadest aspect or preferred aspect) as defined below.

[0682] As used herein, " polymer " has its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may be all identical, or in some cases, more than one type of repeating unit may be present in a polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as an albumen. In some cases, another part, such as a targeting moiety, may also be present in the polymer. If there are more than one type of repeating units in the polymer, the polymer is referred to as a "copolymer". The repeating units forming the copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeating unit (e.g., a first block), and one or more regions each comprising a second repeating unit (e.g., a second block), etc. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0683] In one embodiment, the grafted lipid can serve as a stealth lipid. In this specification, the term "stealth lipid" refers to a stealth polymer (defined below) conjugated to a lipid (as defined herein). In this specification, the term "stealth polymer" refers to a polymer (defined above) having the following characteristics: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge. In certain embodiments, stealth polymers are designed to spatially stabilize lipid particles by forming a protective hydrophilic layer that protects the hydrophobic lipid layer. In certain embodiments, when such lipid particles are administered in vivo, stealth polymers can reduce their binding to serum proteins and / or the resulting absorption of the reticuloendothelial system.

[0684] In one embodiment, the grafted lipid is a lipid that is conjugated to polyethylene glycol (also referred to as a PEG-lipid or a PEGylated lipid). The term "PEGylated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety. The lipid of PEGylation is known in the art. The PEG-lipid can comprise 5-1000, 5-500, 5-100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100 or 10-50 ethylene glycol repeating units, which can be continuous.

[0685] In certain embodiments, the PEG-conjugated lipid (PEGylated lipid) is a lipid having the following general structure:

[0686]

[0687] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 12 and R 13 Each of w is independently a straight or branched alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester linkages; and w has an average value ranging from 30 to 60.

[0688] In certain embodiments of this formula, R 12 and R 13 Each of is independently a linear alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[0689] In certain embodiments of this formula, R 12 and R 13 In certain embodiments, R 12 and R 13 Each of is a straight chain alkyl chain containing 12 carbon atoms. In certain embodiments, R 12 and R 13 Each of is a straight chain alkyl chain containing 14 carbon atoms. In certain embodiments, R 12 and R 13 Each of is a straight alkyl chain containing 16 carbon atoms.

[0690] In certain embodiments of this formula, R 12 and R 13 In certain embodiments, R 12 and R 13 One of them is a straight alkyl chain containing 12 carbon atoms, and R 12 and R 13 The other is a straight alkyl chain containing 14 carbon atoms.

[0691] In certain embodiments of this formula, w has an average value in the range of 40 to 50, such as an average value of 45.

[0692] In certain embodiments of the formula, w is in a range such that the PEG portion of the PEGylated lipid has an average molecular weight of about 400 to about 6000 g / mol, such as about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. In certain embodiments, R 12 and R 13 Each of is a linear alkyl chain containing 14 carbon atoms and w has an average value of 45.

[0693] Various PEG-conjugated lipids are known in the art and include, but are not limited to, PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butane dioate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl In some embodiments of the present invention, the PEG portion of the PEGylated lipid has an average molecular weight of about 400 to about 6000 g / mol, such as about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 1700 to about 3000 g / mol, or about 1800 to about 2200 g / mol. In one embodiment, the PEG portion of the PEGylated lipid has an average molecular weight of about 2000 g / mol.

[0694] In certain embodiments, the PEG-conjugated lipid (PEGylated lipid) is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide. In certain embodiments, the PEGylated lipid has the following structure:

[0695]

[0696] Other examples of grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids, poly(vinylpyrrolidone) (PVP)-conjugated lipids; poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA)-conjugated lipids; poly(dehydroalanine) (pDha)-conjugated lipids; poly(aminoethoxyethoxyacetic acid) (pAEEA)-conjugated lipids and poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA)-conjugated lipids.

[0697] In one embodiment, the grafted lipid is a polysarcosine conjugated lipid, also referred to herein as a sarcosinated lipid or pSar-lipid. The term "sarcosinated lipid" refers to a molecule comprising a lipid portion and a polysarcosine (poly(N-methylglycine)) portion having the repeating unit shown below:

[0698]

[0699] wherein x represents the number of sarcosine units. The polysarcosine may comprise 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, preferably 10 to 70 sarcosine units, preferably 15 to 50 sarcosine units, more preferably 20 to 30 sarcosine units, even more preferably 21 to 25 sarcosine units.

[0700] In one embodiment, the grafted lipid comprises a polysarcosine moiety (as defined and exemplified above) bonded at its carbonyl end to (C 6-30 In one embodiment, the grafted lipid comprises a polysarcosine moiety (as defined and exemplified above) whose carbonyl end is bonded to a (C 12-20 In one embodiment, the grafted lipid comprises a polysarcosine moiety (as defined and exemplified above) whose carbonyl end is bonded to a (C 14 alkyl)amine (as defined and exemplified above), and having its amino terminus optionally bonded to an acetyl group.

[0701] In a particularly preferred embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) having the following structure:

[0702] where n is 23.

[0703] In a particularly preferred embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 acetate (C14-pSar 23Ac) having the following structure:

[0704] where n is 23.

[0705] In one embodiment, the grafted lipid is a polyoxazoline (POX)-conjugated and / or polyoxazine (POZ)-conjugated lipid and / or a POX / POZ-conjugated lipid, also referred to herein as a conjugate of a POX and / or POZ polymer and one or more hydrophobic chains, or an oxazolinylated and / or oxazinylated lipid or POX and / or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising a lipid portion and a polyoxazoline portion, wherein the polyoxazoline portion (pOx) has the repeating units shown below. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising a lipid portion and a polyoxazine portion, wherein the polyoxazine (pOz) portion has the repeating units shown below. The term "oxazolinylated / oxazinylated lipid" or "POX / POZ-lipid" or "POXZ-lipid" refers to a molecule comprising a lipid portion and a portion of a copolymer of polyoxazoline and polyoxazine, i.e., a polymer having repeating units pOx and pOz as shown below:

[0706]

[0707] wherein x represents the number of pOx and / or pOz units. The total number of pOx and / or pOz repeating units in the polymer may comprise 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, preferably 10 to 70 pOx and / or pOz units.

[0708] In one embodiment, the grafted lipid is a poly(vinyl pyrrolidone) (PVP)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, in its broadest aspect or preferred aspect) of poly(vinyl pyrrolidone) (PVP) conjugated to a lipid. The term "poly(vinyl pyrrolidone)" or "PVP" refers to a polymer having vinyl pyrrolidine repeating units (i.e., the repeating units shown below).

[0709]

[0710] In one embodiment, the grafted lipid is a poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, in its broadest aspect or preferred aspects) of poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid. The term "poly(N-(2-hydroxypropyl)methacrylamide" or "pHPMA" refers to a polymer having the repeating units shown below.

[0711]

[0712] In one embodiment, the grafted lipid is a poly(dehydroalanine) (pDha)-conjugated lipid. The term "pDha" refers to a polymer having the repeating unit shown below.

[0713]

[0714] In one embodiment, the grafted lipid is an amphiphilic oligoethylene glycol (OEG)-conjugated lipid. Examples of amphiphilic oligoethylene glycol (OEG)-conjugated lipids include poly (aminoethyl-ethylene glycol acetyl) (pAEEA) and / or poly (methylaminoethyl-ethylene glycol acetyl) (pmAEEA). The terms "pAEEA" and "pmAEAA" refer to polymers having repeating units as shown below:

[0715]

[0716] Wherein x represents the total number of pAEEA and / or pmAEEA units in the polymer. The total number of pAEEA and / or pmAEEA repeating units in the polymer may comprise 1 to 100, 5 to 50, 5 to 25, 7 to 14, preferably 10 to 20, more preferably 12 to 16.

[0717] In the broadest or preferred aspects, the lipid moiety of the (pAEEA)-conjugated lipid can be any of those defined above for lipids. In one embodiment, the lipid moiety is a tocopherol or tocotrienol residue. In one embodiment, the lipid moiety is α-tocopherol. In one embodiment, the lipid moiety is β-tocopherol. In one embodiment, the lipid moiety is γ-tocopherol. In one embodiment, the lipid moiety is δ-tocopherol. In one embodiment, the lipid moiety is α-tocotrienol. In one embodiment, the lipid moiety is β-tocotrienol. In one embodiment, the lipid moiety is γ-tocotrienol. In one embodiment, the lipid moiety is δ-tocotrienol.

[0718] In one embodiment, the grafted lipid is α-tocopherol pAEEA14.

[0719] In one embodiment, the grafted lipid is a peptide-conjugated lipid. The compositions described herein may also contain a lipid conjugated to a binding moiety. In certain embodiments, the lipid with a binding moiety covalently attached thereto comprises compound L-X1-P-X2-B as further described herein. Preferably, the binding moiety is a peptide, and the compositions described herein may also contain a peptide-conjugated lipid. In this specification, the term "peptide-conjugated lipid" refers in its broadest sense to a lipid or lipid-like material as defined above that is conjugated to a peptide (in the broadest or preferred aspects). In this respect, "peptide" is synonymous with "polypeptide" and "protein". In one embodiment, the peptide comprises an ALFA-tag (i.e., the peptide-conjugated lipid may be an ALFA-conjugated lipid). Such peptide-conjugated lipids are described in more detail in US63 / 305,905 (not disclosed at the time of submission).

[0720] In certain embodiments, the peptide-conjugated lipid comprises a compound of formula (A):

[0721] L-X1-P-X2-B(A)

[0722] in

[0723] P contains polymers;

[0724] L comprises a hydrophobic moiety attached to the first end of the polymer;

[0725] B comprises a binding moiety attached to the second end of the polymer;

[0726] X1 is absent or is the first linking moiety; and

[0727] X2 does not exist or is the second connecting part.

[0728] In certain embodiments, X1 comprises a carbonyl group.

[0729] In certain embodiments, X2 comprises the reaction product of a maleimide group and a thiol or cysteine ​​group of a compound comprising a binding moiety.

[0730] In certain embodiments, the hydrophobic moiety is a lipid or is contained in a lipid. In certain embodiments, the lipid comprises a phospholipid, for example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0731] In certain embodiments, the polymer provides stealth properties, increases circulation half-life, and / or reduces nonspecific protein binding or cell adhesion.

[0732] In certain embodiments, the polymer comprises polyethylene glycol (PEG). The average molecular weight of the PEG may range from 200 to 10,000, preferably from 500 to 5000, more preferably from 1000 to 4000, and most preferably from 2000.

[0733] In certain embodiments, the hydrophobic moiety having a binding moiety covalently linked thereto comprises distearoyl-glycero-phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0734] In certain embodiments, the binding moiety covalently linked to the hydrophobic moiety comprises a peptide, preferably, the binding moiety comprises an ALFA-tag.

[0735] In certain embodiments, the ALFA-tag comprises the amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 are:

[0736] AA0 is Pro or deleted;

[0737] AA1 is Ser, Gly, Thr, or Pro;

[0738] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0739] AA3 is Leu, Ile, or Val;

[0740] AA4 is Glu or Gln;

[0741] AA5 is Glu or Gln;

[0742] AA6 is Glu or Gln;

[0743] AA7 is Leu, Ile, or Val;

[0744] AA8 is Arg, Ala, Gln, or Glu;

[0745] AA9 is Arg, Ala, Gln, or Glu;

[0746] AA10 is Arg;

[0747] AA11 is Leu;

[0748] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0749] AA13 is Glu, Lys, Pro, Ser, Ala, Asp or is deleted; and

[0750] The AA14 is Pro or it's deleted.

[0751] In certain embodiments, the ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0752] In certain embodiments, the ALFA-tag comprises a cyclized amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side chains of any two amino acids (X1, X2) of AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 are covalently linked; and

[0753] The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 that are not X1 and X2 are:

[0754] AA0 is Pro or deleted;

[0755] AA1 is Ser, Gly, Thr, or Pro;

[0756] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0757] AA3 is Leu, Ile, or Val;

[0758] AA4 is Glu or Gln;

[0759] AA5 is Glu or Gln;

[0760] AA6 is Glu or Gln;

[0761] AA7 is Leu, Ile, or Val;

[0762] AA8 is Arg, Ala, Gln, or Glu;

[0763] AA9 is Arg, Ala, Gln, or Glu;

[0764] AA10 is Arg;

[0765] AA11 is Leu;

[0766] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0767] AA13 is Glu, Lys, Pro, Ser, Ala, Asp or is deleted; and

[0768] The AA14 is Pro or it's deleted.

[0769] In certain embodiments, X1 and X2 are separated by 2 or 3 amino acids.

[0770] In certain embodiments, AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AA10 is X1 and AA13 is X2, AA6 is X1 and AA10 is X2, or AA4 is X1 and AA8 is X2.

[0771] In certain embodiments, the ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of:

[0772] a.-AA0-AA1-AA2-AA3-AA4-ring(X1-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-,

[0773] b.-AA0-AA1-AA2-AA3-AA4-ring(X1-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-,

[0774] c.-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-ring(X1-Arg-Leu-AA12-X2)-AA14-,

[0775] d.-AA0-AA1-AA2-AA3-AA4-AA5-ring(X1-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-,

[0776] e.-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-ring(X1-Arg-Leu-X2)-AA13-AA14-,

[0777] f.-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-ring(X1-Leu-AA12-X2)-AA14-,

[0778] g.-AA0-AA1-AA2-AA3-AA4-AA5-ring(X1-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and

[0779] h.-AA0-AA1-AA2-AA3-ring(X1-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-,

[0780] wherein the side chains of the amino acid residues X1 and X2 are covalently linked;

[0781] AA0 is Pro or deleted;

[0782] AA1 is Ser, Gly, Thr, or Pro;

[0783] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0784] AA3 is Leu, Ile, or Val;

[0785] AA4 is Glu or Gln;

[0786] AA5 is Glu or Gln;

[0787] AA6 is Glu or Gln;

[0788] AA7 is Leu, Ile, or Val;

[0789] AA8 is Arg, Ala, Gln, or Glu;

[0790] AA9 is Arg, Ala, Gln, or Glu;

[0791] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0792] AA13 is Glu, Lys, Pro, Ser, Ala, Asp or is deleted; and

[0793] The AA14 is Pro or it's deleted.

[0794] In certain embodiments, X1 and X2 in the peptides disclosed herein are covalently linked through an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0795] In certain embodiments, the cyclized amino acid sequences described herein are generated by linking the amino group of the side chain of one of X1 and X2 via an amide bond to the carboxyl group of the side chain of the other of X1 and X2. The amino group of the side chain of an amino acid having a pendant amine group (e.g., lysine or a lysine derivative) and the carboxyl group of the side chain of an acidic amino acid (e.g., aspartic acid, glutamic acid, or a derivative thereof) can be used to generate the cyclized amino acid sequence via an amide bond.

[0796] In certain embodiments, the cyclized amino acid sequences described herein are generated by linking the sulfhydryl group of the side chain of one of X1 and X2 via a disulfide bond to the sulfhydryl group of the side chain of the other of X1 and X2. Sulfhydryl-containing amino acids include cysteine ​​and other sulfhydryl-containing amino acids, such as Pen.

[0797] In certain embodiments, X1 and X2 are independently selected from Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, provided that when X1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen. when X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap or DDap, X2 is Glu, DGlu, Asp or DAsp; and when X1 is Cys, DCys, hCys, DhCys, Pen or DPen, X2 is Cys, DCys, hCys, DhCys, Pen or DPen.

[0798] In certain embodiments, X1 is Glu and X2 is Lys. In certain embodiments, -ring(Glu--------Lys)-, -c(Glu-------Lys)-, -ring(E-------K)-, -c(E--------K)-, -E------K-ring or -ringE----ringK comprises the following structure:

[0799]

[0800] In certain embodiments, X1 is Lys and X2 is Glu. In certain embodiments, -ring (Lys------Glu)-, -c(Lys------Glu)-, -ring (K------E)-, -c(K------E)-, -K-----E-ring or ring K------ring E comprises the following structure:

[0801]

[0802] In certain embodiments, X1 is Cys and X2 is Cys. In certain embodiments, -Cyc(Cys------Cys)-, C(Cys------Cys)-, -Cyc(C------C)-, -C(C--------C)-, -C----C-Cyc or -Cyc-----Cyc-C comprises the following structure:

[0803] In certain embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Cycle(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In certain other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Cycle(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In certain other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Cycle(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In certain other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Cycle(Lys-Arg-Leu-Thr-Glu)-.

[0804] The cyclic peptides can have different cyclic bridging moieties that form the ring structure. Preferably, a chemically stable bridging moiety is included in the ring structure, such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole. The following are examples illustrating the variability of the bridging moiety in the peptide:

[0805]

[0806] The peptide-conjugated lipid can be contained in a lipid mixture as described herein and incorporated into the aqueous dispersion. The peptide-conjugated lipid may not be contained in a lipid mixture, but may be subsequently added to the lipid particles contained in the dispersed phase of the aqueous dispersion. The peptide-conjugated lipid may not be contained in a lipid mixture, but may be subsequently added to the nucleic acid-lipid particles. When the peptide-conjugated lipid is added to the lipid particles contained in the dispersed phase of the aqueous dispersion or is added to the nucleic acid-lipid particles, the amount of the added peptide-conjugated lipid can replace the steroid (e.g., cholesterol) of the corresponding amount in the particle. The peptide-conjugated lipid is typically added to the particle in a final molar ratio of 0.1-0.3 mol%, optionally about 0.2 mol%, of the total lipids.

[0807] When the nucleic acid-lipid particle comprises a peptide-conjugated lipid, this allows for functionalization of the nucleic acid-lipid particle. For example, a binding portion of a peptide that specifically binds to the peptide-conjugated lipid can bind to the nucleic acid-lipid particle, wherein the binding portion can also bind to a target cell (e.g., by specifically binding to a target cell surface antigen). This can provide targeted delivery of nucleic acids contained within the functionalized nucleic acid-lipid particle. The binding portion of the peptide that specifically binds to the peptide-conjugated lipid can be an ALFA-tag binding portion.

[0808] In certain embodiments, the ALFA-tag binding portion comprises an antibody or antibody fragment, for example, a camelid VHH domain. In certain embodiments, the ALFA-tag binding portion comprises a single domain antibody (sdAb), NbALFA-nanobody. In certain embodiments, the ALFA-tag binding portion comprises a single domain antibody, for example, a camelid VHH domain comprising a CDR1 sequence VTX1SALNAMAMG, wherein X1 is I or V, a CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D or S, and a CDR3 sequence LEDRVDSFHDY.

[0809] In certain embodiments, the ALFA-tag binding moiety comprises a single domain antibody, e.g., a Camelidae VHH domain, comprising the CDR1 sequence GVTX1SALNAMAMG, wherein X1 is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0810] In certain embodiments, the ALFA-tag binding portion comprises a single domain antibody, e.g., a Camelidae VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYR QAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVS LQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence or a fragment thereof, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence. In certain embodiments, the amino acid sequence comprises CDR1, CDR2, and CDR3 sequences as described above.

[0811] In certain embodiments, the ALFA-tag binding moiety comprises a bispecific antibody that targets an ALFA-tag and a cell surface antigen. In certain embodiments, the ALFA-tag binding moiety comprises a portion that binds a peptide containing an ALFA-tag and a portion that targets a cell surface antigen.

[0812] In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.5 to 10 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.2 to 5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1 to 2.5 mol % of the total lipids present in the lipid mixture. The grafted lipid may comprise a mixture of: (i) a grafted lipid selected from pSar-conjugated lipids; POX-conjugated lipids; POZ-conjugated lipids, PVP-conjugated lipids; pHPMA-conjugated lipids; pDha-conjugated lipids; pAEEA-conjugated lipids and pmAEEA-conjugated lipids, and (ii) a peptide-conjugated lipid. The term "lipid mixture" in this context applies to the lipid mixture component of the aqueous dispersion and the nucleic acid-lipid particles.

[0813] In one embodiment, the grafted lipid is a PEG-conjugated lipid and is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is a PEG-conjugated lipid and is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is a PEG-conjugated lipid and is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is a PEG-conjugated lipid and is present in the lipid mixture in an amount of about 1.8 mol% of the total lipid present in the lipid mixture. The term "lipid mixture" is applicable to the lipid mixture components of the aqueous dispersion (typically containing pre-LNP) and the nucleic acid-lipid particle in this context.

[0814] In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of about 1.8 mol% of the total lipids present in the lipid mixture. The term "lipid mixture" in this context applies to the lipid mixture components of the aqueous dispersion (typically containing pre-LNP) and the nucleic acid-lipid particles.

[0815] In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of about 1.8 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of 3 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly (sarcosine) (pSar) conjugated lipid and is present in the lipid mixture in an amount of about 4 mol % of the total lipid present in the lipid mixture. The term "lipid mixture" is applicable to the lipid mixture components of the aqueous dispersion (typically containing pre-LNP) and the nucleic acid-lipid particle in this context.

[0816] In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of about 1.8 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of 3 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) or n-tetradecyl poly(sarcosine) 23 (C14-pSar 23) acetate, and is present in the lipid mixture in an amount of about 4 mol% of the total lipids present in the lipid mixture. The term "lipid mixture" in this context applies to both the aqueous dispersion (typically containing the pre-LNP) and the lipid mixture component of the nucleic acid-lipid particle.

[0817] In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA) conjugated lipid and is present in the lipid mixture in an amount of 0.5 to 10 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA) conjugated lipid and is present in the lipid mixture in an amount of 0.2 to 5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA) conjugated lipid and is present in the lipid mixture in an amount of 1 to 2.5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA) conjugated lipid and is present in the lipid mixture in an amount of 1.8 to 2 mol % of the total lipids present in the lipid mixture.

[0818] In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 1.8 to 2 mol% of the total lipids present in the lipid mixture. The term "lipid mixture" in this context is applicable to the lipid mixture components of the aqueous dispersion (typically containing pre-LNP) and the nucleic acid-lipid particles.

[0819] Pharmaceutical composition

[0820] The nucleic acid-lipid particle compositions described herein can be used as or in the preparation of a pharmaceutical composition or medicament for therapeutic or prophylactic treatment.

[0821] The nucleic acid-lipid particle compositions described herein can be administered in the form of any suitable pharmaceutical composition.

[0822] The term "pharmaceutical composition" refers to a composition comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition can be used to treat, prevent a disease or disorder or alleviate its severity by administering the pharmaceutical composition to a subject. In certain embodiments, the therapeutically effective agent is or comprises an active ingredient as described herein. In the context of the present disclosure, the pharmaceutical composition comprises a nucleic acid as described herein. In certain embodiments, the therapeutically effective agent is or comprises a nucleic acid described in the present disclosure, which comprises a nucleic acid sequence (e.g., ORF) encoding one or more polypeptides (e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins).

[0823] In certain embodiments, when the nucleic acid is mRNA, the mRNA integrity of the initial pharmaceutical composition (i.e., after its preparation, but before freezing, lyophilization or storage) is at least 50%, preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, such as at least 90%.

[0824] In certain embodiments, the size (Z) of the particles of the initial pharmaceutical composition (ie, after its preparation but before freezing, lyophilization or storage) is 平均 ) is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, and more preferably between about 40 nm and about 120 nm.

[0825] In certain embodiments, the polydispersity index (PDI) of the particles of the initial pharmaceutical composition (ie, after preparation, but before freezing, lyophilization, or storage) is less than 0.3, preferably less than 0.2, more preferably less than 0.1.

[0826] The pharmaceutical compositions of the present disclosure can be in frozen form or "ready-to-use form" (i.e., a form that can be immediately administered to a subject, particularly in liquid form, e.g., without any processing, such as thawing, reconstitution, or dilution). Therefore, before administering a pharmaceutical composition in a storable form, the storable form must be processed or transferred into a ready-to-use form or an administrable form. For example, a frozen pharmaceutical composition must be thawed. Ready-to-use injections can be placed in containers such as vials, ampoules, or syringes, wherein the container can contain one or more doses.

[0827] In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

[0828] In certain embodiments, the pharmaceutical compositions are in frozen form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature within the range of about -90°C to about -10°C, such as about -90°C to about -40°C, or about -40°C to about -25°C, or about -25°C to about -10°C, or about -20°C.

[0829] In certain embodiments of the pharmaceutical composition in frozen form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored at -20°C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, and more preferably at least 6 months.

[0830] In certain embodiments of the pharmaceutical composition in frozen form, when the nucleic acid is mRNA, the mRNA integrity after thawing of the frozen pharmaceutical composition is at least 90%, at least 95%, at least 97%, at least 98% or essentially 100% of the initial mRNA integrity, for example, after thawing of a frozen composition that has been stored at -20°C for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months or at least 36 months, preferably at least 4 weeks.

[0831] In certain embodiments of the pharmaceutical composition in frozen form, the size of the particles (Z 平均 ) and / or size distribution and / or PDI are substantially equal to the size (Z) of the particles of the initial pharmaceutical composition before freezing. 平均 ) and / or size distribution and / or PDI. For example, if a ready-to-use pharmaceutical composition is prepared from a frozen pharmaceutical composition as described herein, it is preferred that the size (Z 平均 ) and / or size distribution and / or PDI are substantially equal to the initial size (Z) of the particles contained in the frozen pharmaceutical composition before freezing. 平均 ) and / or size distribution and / or PDI.

[0832] In certain embodiments, when the nucleic acid is mRNA, the mRNA particle size and mRNA integrity of the pharmaceutical composition are substantially equivalent to the mRNA particle size and mRNA integrity of the initial pharmaceutical composition (i.e., before the pharmaceutical composition is frozen for the first time) after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, and more preferably after five or more freeze / thaw cycles.

[0833] In certain embodiments, the pharmaceutical compositions are in liquid form and can be stored at a temperature in the range of about 0° C. to about 20° C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature in the range of about 1° C. to about 15° C., such as about 2° C. to about 10° C., or about 2° C. to about 8° C., or at a temperature of about 5° C.

[0834] In certain embodiments, when the nucleic acid is mRNA, the mRNA integrity of the pharmaceutical composition upon storage is at least 70%, preferably at least 80%, more preferably at least 90% of the initial mRNA integrity (i.e., the mRNA integrity of the initial pharmaceutical composition).

[0835] In certain embodiments of the pharmaceutical composition in liquid form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months or at least 24 months, preferably at least 4 weeks. For example, the liquid pharmaceutical composition can be 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.

[0836] In certain embodiments of the pharmaceutical composition in liquid form, when the nucleic acid is mRNA, when, for example, stored at 0°C or higher for at least 1 week, the mRNA integrity of the liquid composition makes it possible to achieve a desired effect, for example, inducing an immune response. For example, when, for example, stored at 0°C or higher for at least 1 week (such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months or at least 6 months), the mRNA integrity of the liquid composition can be at least 90% of the mRNA integrity of the initial composition (i.e., the mRNA integrity before the composition is stored). In certain embodiments, after storing for at least 4 weeks (e.g., at least 3 months), preferably at 0°C or higher temperatures, such as about 2°C to about 8°C, the mRNA integrity of the composition is at least 90% of the mRNA integrity before storage.

[0837] In certain embodiments, when the nucleic acid is mRNA, the initial mRNA integrity of the pharmaceutical composition (i.e., after its preparation, but before storage) is at least 50%, and after storage for at least 1 week (such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, or at least 3 months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, the mRNA integrity of the pharmaceutical composition is at least 90% of the initial mRNA integrity.

[0838] In certain embodiments of the pharmaceutical composition in liquid form, the size of the particles of the pharmaceutical composition (Z 平均 ) (and / or size distribution and / or polydispersity index (PDI)) such that the desired effect can be achieved, for example, inducing an immune response. For example, when stored, for example, at 0° C. or higher for at least 1 week, the size (Z 平均 ) (and / or size distribution and / or polydispersity index (PDI)) are substantially equal to the size (Z average) (and / or size distribution and / or PDI) of the particles of the initial pharmaceutical composition (ie, before storage).

[0839] In certain embodiments, for example, after storage of the pharmaceutical composition at 0°C or higher for at least 1 week, the size (Z 平均 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm. In certain embodiments, after storage of the pharmaceutical composition, for example at 0° C. or higher for at least 1 week, the PDI of the particles is less than 0.3, preferably less than 0.2, more preferably less than 0.1.

[0840] In certain embodiments, after storage of the pharmaceutical composition, for example at 0°C or higher for at least 1 week, the size (Z 平均 ) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and after storage of the pharmaceutical composition, for example, at 0° C. or higher for at least 1 week, the size of the particles (Z 平均 ) ) and / or size distribution and / or PDI) are substantially equal to the size of the particles before storage (Z 平均 ) ) and / or size distribution and / or PDI. In certain embodiments, after storage of the pharmaceutical composition, for example at 0°C or higher for at least 1 week, the size of the particles (Z 平均) is from about 50 nm to about 500 nm, preferably from about 40 nm to about 200 nm, more preferably from about 40 nm to about 120 nm, and the PDI of the particles is less than 0.3 (preferably less than 0.2, more preferably less than 0.1) after storage of the pharmaceutical composition, for example at 0° C. or higher for at least 1 week.

[0841] The pharmaceutical compositions according to the present disclosure are typically administered in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0842] The term "pharmaceutically acceptable" refers to non-toxic materials that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0843] The term "pharmaceutically effective amount" refers to an amount that, alone or in combination with another dose, achieves a desired response or desired effect. In the case of treating a specific disease, the desired response preferably involves inhibiting the progression of the disease. This includes slowing the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be to delay the onset of the disease or condition or to prevent the onset of the disease or condition. The effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severity of the disease, the patient's individual parameters (including age, physiological condition, size and weight), the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Therefore, the dosage of the particles or pharmaceutical compositions described herein may depend on various such parameters. If the patient's response to the initial dose is insufficient, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) may be used.

[0844] In certain embodiments, the pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, i.e., a pharmaceutical composition that can be used to induce an immune response) is formulated as a single dose in a container (e.g., a vial). In certain embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In certain embodiments, the multi-dose formulation includes at least 2 doses per bottle. In certain embodiments, the multi-dose formulation includes a total of 2-20 doses per bottle, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per bottle. In certain embodiments, the volume of each dose in the vial is equal. In certain embodiments, the volume of the first dose is different from that of the subsequent dose.

[0845] "Stable" multi-dose formulations preferably do not exhibit unacceptable levels of microbial growth and exhibit substantially no or no breakdown or degradation of the active biomolecule component(s). As used herein, "stable" immunogenic compositions include formulations that are still capable of eliciting a desired immunological response when administered to a subject.

[0846] The pharmaceutical compositions of the present disclosure may contain a buffer (particularly, a nucleic acid (such as RNA) composition from which the pharmaceutical composition is prepared), a preservative, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure, particularly ready-to-use pharmaceutical compositions, comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0847] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0848] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0849] The term "diluent" refers to a diluent and / or thinning agent. In addition, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and / or a mixed medium. Examples of suitable diluents include ethanol and water.

[0850] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which the active ingredients are combined to promote, enhance, or enable the administration of the pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0851] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro, ed. 1985).

[0852] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0853] In one embodiment, the compositions described herein, such as pharmaceutical compositions or ready-to-use pharmaceutical compositions described herein, can be administered intravenously, intra-arterially, subcutaneously, intradermally, intradermally, intradermally, intranodules, intramuscularly, or intratumorally. In certain embodiments, the (pharmaceutical) composition is formulated for topical or systemic administration. Systemic administration can include enteral administration (which involves absorption by the gastrointestinal tract) or parenteral administration. "Parenteral administration" as used herein means administration in any manner other than by the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for intramuscular administration.

[0854] Medical uses and treatments

[0855] The nucleic acid-lipid particles described herein and pharmaceutical compositions comprising them can be used for therapeutic or prophylactic treatment of various diseases, particularly diseases in which providing a peptide or protein to a subject can produce a therapeutic or prophylactic effect. For example, providing an antigen or epitope derived from a virus can be used to treat or prevent a viral disease caused by the virus. Providing a tumor antigen or epitope can be used to treat a cancerous disease in which cancer cells express the tumor antigen. Providing a functional protein or enzyme can be used to treat a genetic disorder characterized by a dysfunctional protein, such as in a lysosomal storage disease (e.g., mucopolysaccharidosis) or factor deficiency. Providing a cytokine or cytokine fusion protein may help regulate the tumor microenvironment.

[0856] Thus, in one aspect, disclosed is a nucleic acid-lipid particle or pharmaceutical composition as defined herein for use in medicine.

[0857] In one embodiment, there is provided a nucleic acid-lipid granule or pharmaceutical composition as defined herein, for delivering nucleic acid (such as mRNA) to a cell. In one embodiment, there is provided a nucleic acid-lipid granule or pharmaceutical composition as defined herein, for transfecting a cell with nucleic acid (such as mRNA). In one embodiment, there is provided a nucleic acid-lipid granule or pharmaceutical composition as defined herein for use in preparing a medicine, for delivering nucleic acid (such as mRNA) to a cell. In one embodiment, there is provided a nucleic acid-lipid granule or pharmaceutical composition as defined herein for use in preparing a medicine, for transfecting a cell with nucleic acid (such as mRNA). In one embodiment, there is provided a method for delivering nucleic acid (such as mRNA) to a cell, the method comprising administering a nucleic acid-lipid granule or pharmaceutical composition as defined herein to the cell. In one embodiment, there is provided a method for transfecting a cell with nucleic acid (such as mRNA), the method comprising adding a nucleic acid-lipid granule or pharmaceutical composition as defined herein to the cell;With the mixture of the composition and the cell incubated for a sufficient amount of time. In certain embodiments, particularly those of pharmaceutically active proteins encoded by said nucleic acid (such as mRNA), the mixture of said compositions and cells is incubated enough to allow the time for pharmaceutically active protein expression. In certain embodiments, the time of said sufficient amount is at least 1 hour (such as at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or at most about 48 hours (such as at most about 36 or at most about 24 hours). In certain embodiments, the mixture of said compositions and cells is incubated in the presence of serum (such as human serum).

[0858] The cell can be any cell capable of receiving a nucleic acid (such as mRNA) to produce a therapeutic effect. In one embodiment, the cell is a hepatocyte. In one embodiment, the cell is a spleen cell. In one embodiment, the cell is a lung cell.

[0859] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in treating a disease that can be treated with nucleic acid (such as mRNA). In one embodiment, there is provided a composition as defined herein for use in the preparation of a medicine for use in treating a disease that can be treated with nucleic acid (such as mRNA). In one embodiment, there is provided a method for treating a disease that can be treated with nucleic acid (such as mRNA) in a subject in need thereof, the method comprising administering a nucleic acid-lipid particle or a pharmaceutical composition as defined herein to the subject.

[0860] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided the use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein in the preparation of a medicament for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided a method for the prophylactic and / or therapeutic treatment of a disease involving an antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

[0861] In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in inducing an immune response. In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in preparing a medicine for use in inducing an immune response.

[0862] In one embodiment, there is provided a nucleic acid-lipid particle or pharmaceutical composition as defined herein for use in treating cancer. In one embodiment, there is provided use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the preparation of a medicament for treating cancer. In one embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or pharmaceutical composition as defined herein.

[0863] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition that affects an individual's body. A disease is generally interpreted as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originating from an external source (such as an infectious disease), or it may be caused by an internal dysfunction (such as an autoimmune disease). In humans, "disease" is generally used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems or death in the individual who is ill, or a condition that causes similar problems in people who come into contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behaviors, and atypical changes in structure and function, while in other cases and for other purposes, these may be considered as distinguishable categories.

[0864] The term "infectious disease" refers to any disease caused by a microbial agent that can be transmitted between individuals or between organisms. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, the infectious disease can be, for example, a sexually transmitted disease (e.g., chlamydia, gonorrhea, or syphilis), SARS, a coronavirus disease (e.g., COVID-19), acquired immunodeficiency syndrome (AIDS), measles, chickenpox, cytomegalovirus infection, herpes simplex virus (e.g., HSV-1, HSV-2), hepatitis (such as hepatitis B or hepatitis C), influenza (flu, such as human influenza, swine influenza, canine influenza, equine influenza, and avian influenza), HPV infection, herpes zoster, rabies, , common cold, gastroenteritis, rubella, mumps, anthrax, cholera, diphtheria, foodborne illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infections, Lyme disease, Rocky Mountain spotted fever, chlamydia, whooping cough, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, diamoebiasis, giardiasis, amoebic dysentery, coccidiosis, toxoplasmosis, sarcocystis, rhinosporidiosis, and balantidiasis.

[0865] In certain embodiments, the nucleic acid-lipid particles or pharmaceutical compositions described herein can be used to treat or prevent infectious diseases.

[0866] In the context of the present invention, the term "treatment," "treating," or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full range of treatments for the specific condition from which the subject is suffering, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, slow the progression of the disease, disorder, or condition, relieve or alleviate symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, as well as prevention of the condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, condition, or disorder, and includes the administration of an active compound to prevent the occurrence of symptoms or complications.

[0867] The term "therapeutic treatment" refers to any treatment that improves health and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in the individual, prevent or slow the progression of the disease in the individual, inhibit or slow the progression of the disease in the individual, reduce the frequency or severity of symptoms in the individual, and / or reduce the recurrence of the disease in an individual who currently has or has had the disease.

[0868] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0869] The terms "individual" and "subject" are used interchangeably herein. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates), or any other non-mammalian animals, including poultry (chickens), fish, or any other animal species that may suffer from or be susceptible to a disease or disorder (e.g., cancer, infectious diseases), but may or may not suffer from the disease or disorder, or may require preventive intervention (such as vaccination), or may require intervention (such as by protein replacement). In many embodiments, the individual is a human being. Unless otherwise indicated, the terms "individual" and "subject" do not refer to a specific age, and therefore encompass adults, the elderly, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0870] The term "patient" refers to an individual or subject in need of treatment, particularly one who is ill.

[0871] In certain embodiments of the present disclosure, the goal is to provide protection against infectious diseases through vaccination.

[0872] In certain embodiments of the present disclosure, it is an object to provide a secreted therapeutic protein, such as an antibody, bispecific antibody, cytokine, cytokine fusion protein, enzyme, to a subject, particularly a subject in need thereof.

[0873] In certain embodiments of the present disclosure, it is an object to provide protein replacement therapy, such as the production of erythropoietin, Factor VII, Von Willebrand Factor, β-galactosidase, α-N-acetylglucosaminidase, to a subject, particularly a subject in need thereof.

[0874] In certain embodiments of the present disclosure, the goal is to modulate / reprogram immune cells in the blood.

[0875] In certain embodiments, the compositions described herein contain mRNA encoding SARS-CoV-2 S protein, its immunogenic variant, or an immunogenic fragment of SARS-CoV-2 S protein or its immunogenic variant (hereinafter referred to as "SARS-CoV-2S nucleic acid composition", which explicitly includes SARS-CoV-2S RNA composition), which induces an antibody response, in particular a neutralizing antibody response, to a group of different S protein variants (such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants) in the subject after administration to the subject. In certain embodiments, the group of different S protein variants includes at least 5, at least 10, at least 15 or even more S protein variants. In certain embodiments, such S protein variants include variants with amino acid modifications in the RBD domain and / or variants with amino acid modifications outside the RBD domain. In certain embodiments, the SARS-CoV-2S nucleic acid composition described herein induces an immune response (cellular and / or antibody response, in particular a neutralizing antibody response) targeting VOC-202012 / 01 in the subject after administration to the subject.

[0876] In certain embodiments, the SARS-CoV-2S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) targeting 501.V2 in a subject following administration to the subject.

[0877] In certain embodiments, the SARS-CoV-2S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) targeting “Cluster 5” in a subject following administration to the subject.

[0878] In certain embodiments, the SARS-CoV-2S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) targeting “B.1.1.28” in a subject after administration to the subject.

[0879] In certain embodiments, the SARS-CoV-2S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) targeting “B.1.1.248” in a subject after administration to the subject.

[0880] In certain embodiments, the SARS-CoV-2S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in a subject that targets the Omicron (B.1.1.529) variant following administration to the subject.

[0881] Those skilled in the art will appreciate that one of the principles of immunotherapy and vaccination is based on the fact that by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease to be treated, an immunoprotective response against the disease is generated. Thus, the pharmaceutical compositions described herein are suitable for inducing or enhancing an immune response. Thus, the pharmaceutical compositions described herein can be used for the prophylactic and / or therapeutic treatment of diseases involving antigens or epitopes.

[0882] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual for the purpose of inducing an immune response, eg, for therapeutic or prophylactic reasons. Example

[0883] The examples provide detailed information about the manufacturing process according to the present invention, highlighting the problems overcome by the present invention using a model formulation comprising an ionizable lipid (HY501), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol as helper lipids and C14 amine-terminated polysarcosine (NH-Psar) 23 as a stealth moiety. The final drug product formulated in the present invention uses luciferase as the RNA payload.

[0884] Example 1: Formulations containing grafted lipids

[0885] The preparation is carried out in two steps: (i) preparing preformed lipid nanoparticles (ie, aqueous dispersion) and (ii) mixing the preformed lipid nanoparticles with mRNA. The details of each step are further described below.

[0886] (i) Preparation of HY501 / C14PSar(23)-NH preformed lipid nanoparticles by solvent injection

[0887] Preformed lipid nanoparticles were prepared by mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM acetic acid (AcOH), pH approximately 3.5) in a fluidic path. Mixing was achieved using a syringe pump and a T-piece as a mixing element at a total flow rate of 200 mL / min and a volume ratio of 1:4 (organic phase: aqueous phase). The lipid mixture (50.0 mM total concentration) consisted of the cationic ionizable lipid HY-501, cholesterol, DSPC, and C14-Psar(23)-NH in a molar ratio of 47.5:38.5:10:4, respectively, dissolved in isopropanol. The organic solvent in the resulting crude colloidal nanoparticles was removed by diafiltration in 5 mM AcOH using a hollow fiber (mPES / 100 kD, REPLIGEN) using tangential flow filtration (TFF) and concentrated if necessary. After TFF, the nanoparticles were diluted with 40% sucrose in 5 mM AcOH to a sucrose concentration of 10%, filtered through a 0.22 μm polyethersulfone (PES) filter, and stored at -20°C until further use for RNA-lipid particle preparation. A schematic diagram of the preparation of preformed lipid nanoparticles is reproduced in Figure 1.

[0888] (ii) Mixing of preformed lipid nanoparticles with mRNA

[0889] RNA-lipid particles were prepared by compounding an aqueous dispersion of preformed lipid nanoparticles with an N1-methylpseudouridine-modified mRNA (modRNA) encoding a model antigen. This was accomplished by mixing equal volumes of RNA and preformed lipid nanoparticles in a T-shaped mixing channel (2.4 mm internal diameter) at a total flow rate of 360 mL / min using a semi-automated process.

[0890] The RNA phase was prepared by diluting the modRNA to an RNA concentration of 0.25 mg / mL in its native buffer (i.e., 10 mM HEPES / 0.1 mM EDTA, pH 7). The second phase contained an aqueous dispersion of preformed lipid nanoparticles containing HY501, cholesterol, DSPC, and NH-Psar at a molar ratio of 47.5:38.5:10:4, respectively. The preformed lipid nanoparticles were provided in 5 mM acetic acid and 10% (w / v) sucrose (pH 4.5). If necessary, the preformed lipid nanoparticles were further diluted to the target concentration with their native buffer matrix. The two phases were mixed at an N / P ratio of 6 and a total flow rate of 360 mL / min.

[0891] After mixing the two streams of RNA and preformed lipid particles, the crude RNA-lipid nanoparticles with an RNA concentration of 0.125 mg / mL were conditioned by diluting with a storage matrix containing 60 mM HEPES, 30% (w / v) sucrose to a final RNA concentration of 0.1 mg / mL and a target pH of approximately 5.5. In the next step, the RNA-lipid particles were sterile filtered using a 0.22 μm polyethersulfone (PES) filter and filled into vials. All preparation processes were performed at room temperature. The composition of the formulation is shown in Table 1. Figure 2 A schematic diagram of the RNA-lipid particle preparation process is also depicted in FIG.

[0892] Table 1: Composition of exemplary RNA-lipid particles used in the present invention

[0893] Components concentration RNA 0.10mg / mL HEPES 3.81 mg / mL EDTA 0.01mg / mL HY501 1.38 mg / mL NH-Psar 0.28 mg / mL DSPC 0.30mg / mL cholesterol 0.57 mg / mL Acetic acid 0.12 mg / mL sucrose 100.00mg / mL

[0894] Stability of preformed lipid nanoparticles

[0895] The stability of preformed lipid nanoparticles was studied. Figure 3 As shown, the colloidal stability of the nanoparticles was observed to be maintained under liquid (2-8°C and 25°C) and frozen (-20°C) conditions over the 3 months evaluated so far.

[0896] Long-term stability of RNA-lipid particles

[0897] The long-term stability of RNA-lipid particles was investigated in stability studies. Figure 4 As shown, no changes in particle size, polydispersity, or RNA integrity were observed over time when the drug product was stored frozen at -20°C or at -80°C.

[0898] The biological function of the RNA-lipid granule (LNP 2) prepared by the method of the present invention is compared with the biological function of the RNA-lipid granule (LNP 1) prepared by conventional LNP preparation technology. Here, model antigen is used to compare two kinds of pharmaceutical products in vivo by intramuscular administration route. The administration scheme is to carry out initial administration for the 1st day, and then to strengthen administration after 21 days.

[0899] like Figure 5 As shown, all experimental groups showed a higher CD8+ peptide pool than CD4+ peptide pool. Compared with the particles (LNP 1) prepared by the traditional LNP preparation process (one-step method), the RNA-lipid particles (LNP 2) prepared by the two-step method of the present invention showed a higher T-cell response.

[0900] Example 2: Preparation of DODMA / C14-Psar(23)-Ac preformed lipid nanoparticles by ethanol injection

[0901] The stability of preformed lipid nanoparticles was studied. Preformed lipid nanoparticles were prepared by mixing an organic phase containing dissolved lipids with an aqueous phase (5mM AcOH, pH 3.5) in a fluid path. A syringe pump and a T-piece were used as mixing elements to achieve mixing at a total flow rate of 200mL / min and a volume ratio of 1:4 (organic phase: aqueous phase). The lipid mixture consisted of a cationic ionizable lipid (DODMA), cholesterol, DSPC, and C14-PSar(23)-Ac in a molar ratio of 47.5:38.5:10:4, dissolved in ethanol. The crude colloidal nanoparticles were filtered in 5mM AcOH using a hollow fiber (mPES / 100kD, REPLIGEN) using tangential flow filtration (TFF) to remove the organic solvent and concentrate to 2 times (2X). After TFF, the nanoparticles were diluted to a sucrose concentration of 10% if necessary, filtered through a 0.22 μm polyethersulfone (PES) filter and stored at 4° C. until further use in RNA-lipid particle preparation.

[0902] studied the stability of preformed lipid nanoparticles and observed that the colloidal stability of the nanoparticles was maintained at 2–8 °C and 25 °C for the 3 months evaluated so far, e.g. Figure 6 shown.

[0903] Example 3: Preparation of DODMA / DMG-PEG2k preformed lipid nanoparticles by ethanol injection

[0904] The effect of different buffer conditions on the stability of preformed lipid nanoparticles was studied. To prepare preformed lipid nanoparticles, the lipid mixture consisted of a cationic ionizable lipid (DODMA), cholesterol, DSPC, and DMG-PEG2k dissolved in ethanol at a molar ratio of 47.5:40.7:10:1.8, and was mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 200 mL / min and a volume ratio of 1:4 (organic phase: aqueous phase) using a standard syringe pump-based device and a T-piece as a mixing element. The results were shown in Figure 2. Figure 7 The crude colloidal nanoparticles were dialyzed against (i) 5 mM AcOH, (ii) 40 mM acetate buffer, or (iii) 10 mM HEPES in a 10K molecular weight cutoff (MWCO) Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA) to remove the organic solvent. Following dialysis, the nanoparticles were diluted to a sucrose concentration of 10%, filtered through a 0.22 μm polyethersulfone (PES) filter, and subjected to freeze-thaw and stability studies under various conditions.

[0905] Freeze-thaw studies were performed by cycling the nanoparticles from -80°C (overnight) to room temperature (25°C) (2h) at least 3 times. Before the next freezing cycle, the nanoparticles were mixed between the thawing and freezing cycles by gentle inversion. The particle size and polydis...

Claims

1. Aqueous dispersions having an aqueous mobile phase and a dispersed phase; in: The dispersed phase comprises a lipid mixture including cationic ionizable lipids; and The aqueous mobile phase comprises an anion of an aqueous acid; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA, and The aqueous mobile phase contains a cryoprotectant.

2. The aqueous dispersion of claim 1, having a pH of 2.5 to 5.

5.

3. The aqueous dispersion of claim 2, having a pH of 2.5 to 4.

5.

4. The aqueous dispersion of any one of claims 1 to 3, wherein the molar ratio between the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid is between 20:1 and 1:

20.

5. The aqueous dispersion of any one of claims 1 to 4, wherein the molar ratio between the cationic lipid or cationic ionizable lipid and the anion of the aqueous acid is between 5:1 and 1:

5.

6. The aqueous dispersion of any one of claims 1 to 5, wherein the aqueous acid is an inorganic acid or a water-soluble organic acid.

7. The aqueous dispersion of any one of claims 1 to 6, wherein the aqueous acid is acetic acid, malic acid, or succinic acid.

8. The aqueous dispersion of any one of claims 1 to 7, wherein the concentration of the aqueous acid is in the range of 1-20 mM.

9. The aqueous dispersion of any one of claims 1 to 8, wherein the concentration of the aqueous acid is in the range of 2.5 to 10 mM.

10. The aqueous dispersion of any one of claims 1 to 9, wherein the cationic lipid or cationic ionizable lipid is selected from: [(4-Hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315); 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); Heptatriacontriacontac-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl) 9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319); Di-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonaneamido)-nonadecanedioate (A9); (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); Heptadec-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanoylcarbonyloxyoctyl}-amino] 4-(dimethylamino)butanoate (HY501); 2-(Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2); 4-((Di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutane-4-amine (HYAM-2); ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405); (2-(4-(dimethylamino)butyryl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405); Diheptadecan-9-yl 3,3′-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); 7,7'-((4-Hydroxybutyl)azanediyl)-bis(N-hexyl-N-octyheptane-1-sulfonamide) (BNT-51); 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octyhleptane-1-sulfonamide) (BNT-52); Compounds with the following structure or any mixture thereof.

11. The aqueous dispersion of any one of claims 1 to 10, wherein the lipid mixture further comprises one or more additional lipids.

12. The aqueous dispersion of claim 11, wherein the one or more additional lipids comprise a neutral or zwitterionic lipid.

13. The aqueous dispersion of claim 12, wherein the one or more additional lipids comprise a neutral or zwitterionic phospholipid.

14. The aqueous dispersion of claim 13, wherein the neutral or zwitterionic phospholipid is selected from: Distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); Dimyristoylphosphatidylcholine (DMPC); Dipalmitoylphosphatidylcholine (DPPC); Palmitoyloleoyl-phosphatidylcholine (POPC); dioleoylphosphatidylethanolamine (DOPE); 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG); N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); or any mixture thereof.

15. The aqueous dispersion of claim 14, wherein the neutral or zwitterionic phospholipid is distearoylphosphatidylcholine (DSPC).

16. The aqueous dispersion of any one of claims 11 to 15, wherein the one or more additional lipids comprises a steroid.

17. The aqueous dispersion of claim 16, wherein the steroid is cholesterol.

18. The aqueous dispersion of any one of claims 11 to 15, wherein the one or more additional lipids comprises a grafted lipid.

19. The aqueous dispersion of claim 18, wherein the grafted lipid is selected from the group consisting of a poly(alkylene glycol)-conjugated lipid, a poly(sarcosine)-conjugated lipid, a poly(oxazoline) (POX)-conjugated lipid; a poly(oxazine) (POZ)-conjugated lipid; a poly(vinylpyrrolidone) (PVP)-conjugated lipid; a poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA)-conjugated lipid; a poly(dehydroalanine) (pDha)-conjugated lipid; a poly(aminoethoxyethoxyacetic acid) (pAEEA)-conjugated lipid; and a poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA)-conjugated lipid; or any mixture thereof.

20. The aqueous dispersion of any one of claims 11 to 19, wherein the one or more additional lipids comprises a peptide-conjugated lipid.

21. The aqueous dispersion of claim 20, wherein the peptide-conjugated lipid is an ALFA-tag-conjugated lipid.

22. A freeze-dried composition comprising the aqueous dispersion of any one of claims 1 to 21.

23. A frozen composition comprising the aqueous dispersion of any one of claims 1 to 21, wherein the frozen composition is at a temperature between -15°C and -90°C.

24. A method of forming the aqueous dispersion of any one of claims 1 to 21, the method comprising mixing: (i) a lipid mixture comprising cationically ionizable lipids; (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant; to produce an aqueous dispersion comprising the anion of the aqueous acid.

25. A method of forming the aqueous dispersion of any one of claims 1 to 23, the method comprising: (a) Mixing: (i) a lipid mixture comprising cationically ionizable lipids; and (ii) an aqueous phase comprising an aqueous acid; to produce a first intermediate aqueous dispersion comprising an anion of the aqueous acid; and (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce the aqueous dispersion.

26. form the method for the lipid granule that contains RNA, described method comprises mixing the aqueous dispersion described in any one of claim 1 to 21 and the aqueous solution that comprises RNA, to produce the described lipid granule that contains RNA.

27. The method of claim 26, wherein the cryoprotectant in the aqueous dispersion is selected from sucrose, trehalose or glucose or a mixture thereof.

28. The method of claim 27, wherein the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 15% to about 25%.

29. The method of any one of claims 26 to 28, wherein the method does not comprise further steps of dialysis, filtration, dilution or addition of cryoprotectants.

30. The method of any one of claims 26 to 29, wherein the RNA is mRNA.

31. The method of claim 30, wherein the mRNA encodes one or more patient-specific antigens suitable for personalized cancer therapy.

32. A lipid particle comprising RNA obtained or obtainable by the method of any one of claims 26 to 31.

33. lipid particle according to claim 32, described lipid particle is lipid nanoparticle.

34. The lipid particle of claim 32 or 33, for medical use.

35. The lipid particle of claim 32 or 33, for use in treating cancer.

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