Preparation and storage of a liposomal RNA formulation suitable for treatment

Abstract

The present disclosure relates to a method for preparing RNA lipoplex particles for delivering RNA to target tissues after parenteral administration, particularly after intravenous administration, and a composition comprising such RNA lipoplex particles. The present disclosure also relates to a method that enables the preparation of RNA lipoplex particles in a manner that complies with industrial GMP. Furthermore, the present disclosure relates to a method and composition for preserving RNA lipoplex particles without substantial loss of product quality, particularly without substantial loss of RNA activity.

Description

Technical Field

[0001] The present disclosure relates to methods for preparing RNA lipoplex particles for delivering RNA to target tissues after parenteral administration, particularly after intravenous administration, and to compositions comprising such RNA lipoplex particles. The present disclosure also relates to methods that enable the preparation of RNA lipoplex particles in a manner compliant with industrial GMP. Further, the present disclosure relates to methods and compositions for storing RNA lipoplex particles without substantially losing the quality of the product, particularly without substantially losing RNA activity. The RNA lipoplex particle formulations described herein can be frozen or dehydrated by lyophilization, spray drying, or related methods that enable a long shelf life of the product compared to liquid storage. In one embodiment, the RNA lipoplex particles comprise single-stranded RNA such as mRNA encoding a peptide or protein of interest, such as a pharmaceutically active peptide or protein. The RNA is taken up by the cells of the target tissue and the RNA is translated into the encoded peptide or protein, which can exhibit its physiological activity. The peptide or protein of interest can be a peptide or protein comprising one or more epitopes for inducing or enhancing an immune response against one or more epitopes. The methods and compositions described herein are suitable for use in a manner compliant with pharmaceutical requirements, more specifically the manufacturing requirements in accordance with GMP, and the quality requirements of pharmaceuticals for parenteral application.

Background Art

[0002] The use of RNA for delivering foreign genetic information to target cells provides an attractive alternative to DNA. The advantages of using RNA include transient expression and non-transforming properties. Since RNA does not need to enter the nucleus to be expressed and furthermore cannot be integrated into the host genome, the risk of carcinogenesis is eliminated.

[0003] RNA can be delivered by so-called lipoplex formulations in which the RNA binds to liposomes composed of a mixture of cationic lipids and helper lipids to form injectable nanoparticle formulations. However, developing a formulation for delivering biologically active RNA to target tissues even after storage of the formulation remains an unmet need. Furthermore, developing a method for manufacturing injectable RNA lipoplex particle formulations in compliance with GMP that results in a long shelf life remains an unmet need.

[0004] Therefore, there is a need to provide a formulation for delivering biologically active RNA to target tissues such that the delivered RNA is efficiently translated into the peptide or protein it encodes. Furthermore, there is a need to provide such a formulation with good storage stability without substantially losing the quality of the product, particularly without substantially losing the biological activity of the RNA.

[0005] The inventors have surprisingly discovered that the RNA lipoplex particle formulations described herein meet the above requirements.

Summary of the Invention

Means for Solving the Problems

[0006] I. Method for Preparing RNA Lipoplex Particles, RNA Lipoplex Particles, and Compositions Containing RNA Lipoplex Particles In a first aspect, the present disclosure relates to a method for preparing RNA lipoplex particles with improved biological activity, RNA lipoplex particles prepared according to the present disclosure, and compositions containing such RNA lipoplex particles. The RNA lipoplex particles, and compositions containing the RNA lipoplex particles are useful for delivering RNA to target tissues after parenteral administration, particularly after intravenous administration. The RNA lipoplex particles are prepared using liposomes obtained by injecting a high-concentration solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the RNA lipoplex product is about 1 nm -1A single Bragg peak is observed at, and the peak width is less than 0.2 nm, which is characteristic of a specific pattern in X-ray scattering. -1 than, and is characterized by a specific pattern in X-ray scattering.

[0007] In one embodiment, the liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). The concentration of DOPE in the lipid mixture is higher than the equilibrium solubility of DOPE in ethanol alone. At room temperature, DOPE has a solubility of about 50 mM alone and a solubility of 100 mM or more in combination with DOTMA. The lipid solution for forming liposomes that result in highly active lipoplexes can have a total lipid concentration of 270 mM or more (e.g., 90 mM or more of DOPE). By raising the temperature, even higher concentration ethanol solutions can be obtained. Liposomes obtained from lipid solutions where the concentration of DOPE exceeds the equilibrium solubility are significantly larger than liposomes obtained from lipid solutions where the concentration of DOPE is below the equilibrium solubility. The liposome size increases monotonically with the concentration in ethanol.

[0008] The liposomes prepared according to the present disclosure can be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the RNA is incubated with NaCl prior to mixing to adjust the specific ionic strength required for increased activity of the lipoplex. In these lipoplexes formed from these large liposomes, the biological activity is significantly enhanced, as demonstrated by in vitro and in vivo experiments. These lipoplexes with enhanced activity can be clearly distinguished from low-activity lipoplexes by specific physicochemical parameters, such as (i) a decrease in the peak width of the Bragg peak, and (ii) various separation profiles in dispersion analysis methods for size measurements such as field-flow fractionation. Low-activity lipoplexes are on average smaller. Furthermore, they also have different elution profiles that may be related to parameters such as molecular structure, shape, and interaction with the bulk phase.

[0009] Thus, in this aspect, the present disclosure relates to a method of generating a liposomal colloid, comprising injecting an ethanol solution of a lipid into an aqueous phase to generate a liposomal colloid, wherein the concentration of at least one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of at least one lipid in ethanol.

[0010] In one embodiment, the method comprises heating the lipid solution to increase the concentration of the lipid in the lipid solution. In one embodiment, the lipid solution is heated to a temperature of at least about 40 °C or at least about 60 °C.

[0011] In one embodiment, the lipid solution is a solution of a mixture of two or more different lipids.

[0012] In one embodiment, the concentration of one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of the lipid in ethanol.

[0013] In one embodiment, the total lipid concentration in the lipid solution is about 180 mM to about 600 mM, about 300 mM to about 600 mM, or about 330 mM.

[0014] In one embodiment, the lipid solution comprises at least one cationic lipid and at least one additional lipid.

[0015] In one embodiment, the concentration of the additional lipid in the lipid solution corresponds to or is higher than the equilibrium solubility of the additional lipid in ethanol.

[0016] In one embodiment, the at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0017] In one embodiment, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0018] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0019] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0020] In one embodiment, the lipid solution comprises DOTMA and DOPE in a molar ratio of from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0021] In one embodiment, the concentration of DOPE in the lipid solution is at least about 60 mM or at least about 90 mM.

[0022] In one embodiment, the lipid solution is injected into the aqueous phase at a stirring rate of the aqueous phase of about 50 rpm to about 150 rpm.

[0023] In one embodiment, the aqueous phase is water.

[0024] In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase contains, for example, about 5 mM of acetic acid.

[0025] In one embodiment, the method further comprises stirring the liposome colloid.

[0026] In one embodiment, the liposome colloid is stirred for about 15 minutes to about 60 minutes, or about 30 minutes.

[0027] The present disclosure further relates to a method for producing a liposome colloid, which includes injecting a lipid solution containing DOTMA and DOPE in ethanol at a molar ratio of about 2:1 into water being stirred at a stirring speed of about 150 rpm to produce a liposome colloid, wherein the concentrations of DOTMA and DOPE in the lipid solution are about 330 mM.

[0028] In one embodiment, the method for producing liposomes does not include a step of extruding the liposomes.

[0029] The present disclosure further relates to a liposome colloid obtainable by a method for producing liposomes.

[0030] In one embodiment, the liposomes have an average diameter of at least about 250 nm.

[0031] In one embodiment, the liposomes have an average diameter in the range of about 250 nm to about 800 nm.

[0032] In one embodiment, the liposomes have a polydispersity index of less than about 0.5, less than about 0.4 or less than about 0.3.

[0033] In one embodiment, the liposomes are cationic liposomes.

[0034] In one embodiment, the liposomes contain at least one cationic lipid and at least one additional lipid.

[0035] In one embodiment, at least one cationic lipid includes 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0036] In one embodiment, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0037] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0038] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0039] In one embodiment, the liposome comprises DOTMA and DOPE in a molar ratio of from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0040] The present disclosure further relates to a method of preparing RNA lipoplex particles, the method comprising adding the above liposome colloid to a solution containing RNA.

[0041] In one embodiment, in the X-ray scattering pattern, the RNA lipoplex is characterized by a single Bragg peak at about 1 nm -1 where the peak width is less than 0.2 nm -1 and smaller than.

[0042] In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm or about 350 nm to about 400 nm.

[0043] The present disclosure further relates to a composition comprising RNA lipoplex particles that can be obtained as described above.

[0044] In one embodiment, the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0045] In one embodiment, the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0046] The present disclosure further RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, and a composition comprising RNA lipoplex particles, wherein the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0, and the RNA lipoplex particles are characterized by a single Bragg peak at about 1 nm -1 with a peak width less than 0.2 nm -1 relates to a composition.

[0047] In one embodiment, the composition further comprises sodium chloride at a concentration of about 10 mM to about 300 mM, about 45 mM to about 300 mM, about 10 mM to about 50 mM, or about 80 mM to about 150 mM.

[0048] In one embodiment, the composition further comprises a buffer.

[0049] In one embodiment, the composition further comprises a chelating agent.

[0050] In one embodiment, the RNA lipoplex particles described in this aspect under I. have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm.

[0051] In one embodiment, the RNA lipoplex particles have a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3.

[0052] In one embodiment, at least one cationic lipid includes 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0053] In one embodiment, at least one additional lipid includes 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol), and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC).

[0054] In one embodiment, at least one cationic lipid includes 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), and at least one additional lipid includes 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0055] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, about 3:1 to about 1:1, or about 2:1.

[0056] In one embodiment, the RNA lipoplex particles include DOTMA and DOPE in a molar ratio of about 10:0 to 1:9, about 4:1 to 1:2, about 3:1 to about 1:1, or about 2:1, and the charge ratio of the positive charge in DOTMA to the negative charge in RNA is about 1:2 to 1.9:2.

[0057] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0058] In one embodiment, the EDTA is at a concentration of about 0.25 mM to about 5 mM, or about 2.5 mM.

[0059] In one embodiment, the composition further comprises an adjuvant.

[0060] In one embodiment, the composition is formulated for systemic administration.

[0061] In one embodiment, the systemic administration is by intravenous administration.

[0062] The present disclosure further relates to the described composition for therapeutic use.

[0063] II. Method for Preparing RNA Lipoplex Particles in a Manner Compliant with Industrial GMP In a second aspect, the present disclosure relates to a method that enables the preparation of RNA lipoplex particles in a manner compliant with industrial GMP.

[0064] In one embodiment of the present disclosure, a fluid path system is used for the GMP-compliant production of pharmaceutical RNA lipoplex particle products, thereby enabling precise control of the mixing ratio of RNA and liposomes, which is important for the quality of the products. In one embodiment, the fluid path includes mixing a liposome solution and an RNA solution in a 1:1 (volume / volume) manner, where the concentrations of the components are selected to accurately maintain the intended charge ratio. In one embodiment, the RNA is incubated with NaCl prior to mixing to adjust the specific ionic strength required for the activity of the lipoplex. In one embodiment, a Y-type mixing setup based entirely on disposable materials is realized. The hydrodynamics are optimized to maintain particle characteristics and avoid clogging. In contrast, when using commercially available microfluidic devices, clogging occurs after a while, making it impossible to apply GMP.

[0065] In one embodiment, lipoplexes are produced by incubating RNA with cationic liposomes, where the mixing ratio and conditions are precisely controlled by using a syringe pump (perfusion pump) in which two syringes, one containing liposomes and one containing RNA, are preferentially and concurrently inserted into the same pump within the syringe pump. The pistons of both pumps are moved forward by the same drive device, thereby precisely controlling the relative volumes to be mixed. At the selected process conditions, the same syringe is used for both solutions, thereby enabling precise 1:1 (v / v) mixing conditions. By adjusting the concentrations of the two solutions prior to mixing, the ratio of RNA to liposomes (cationic lipids) is precisely controlled.

[0066] Accordingly, in this aspect, the present disclosure relates to a method for the continuous flow production of RNA lipoplex particles, the method comprising mixing a solution containing RNA and a solution containing cationic liposomes under controlled mixing conditions of the RNA and the cationic liposomes.

[0067] In one embodiment, the solution containing cationic liposomes is the liposome colloid described above.

[0068] In one embodiment, the solution containing RNA and the solution containing cationic liposomes are aqueous solutions.

[0069] In one embodiment, a flow rate is used that enables the mixing of the solution containing RNA and the solution containing cationic liposomes.

[0070] In one embodiment, the flow is characterized by a Reynolds number greater than 300, or from about 500 to about 2100.

[0071] In one embodiment, the controlled mixing conditions include controlling the mixing ratio of the solution containing RNA and the solution containing cationic liposomes.

[0072] In one embodiment, the controlled mixing conditions include controlling the relative volumes of a solution containing the RNA to be mixed and a solution containing the cationic liposomes.

[0073] In one embodiment, the mixing ratio of the RNA to the cationic liposomes is controlled by using the same mixing volume (v / v) of the solution containing the RNA and the solution containing the cationic liposomes and adjusting the concentrations of the RNA and the cationic liposomes in each solution.

[0074] In one embodiment, the controlled mixing conditions are selected to maintain the properties of the RNA-lipoplex particles while avoiding clogging.

[0075] In one embodiment, the method includes using a Y-shaped or T-shaped mixing element.

[0076] In one embodiment, the Y-shaped or T-shaped mixing element has a diameter of about 1.2 mm to about 50 mm.

[0077] In one embodiment, the method includes using a mixing element in which fluids from two tubes or hoses are combined, for example, without internal static mixing elements such as split and recombine, alternating herringbone channels, zigzag channels or twist channels, or three-dimensional serpentine paths, such as a Y-shaped or T-shaped mixing element. The mixing element can have a diameter of 1.2 mm to 50.0 mm.

[0078] In one embodiment, the method includes using an apparatus in which two syringes, one containing a solution of cationic liposomes and one containing a solution of RNA, are inserted in parallel into the same or two holders, and the pistons of the apparatus are pushed out by one or two precision actuators. In one embodiment, the method includes using a syringe pump in which two syringes, one containing a solution of cationic liposomes and one containing a solution of RNA, are inserted in parallel into the same pump.

[0079] In one embodiment, the method includes using a pressure vessel, a membrane pump, a gear pump, a magnetic levitation pump, a peristaltic pump, an HPLC / FPLC pump, or any other piston pump, optionally in combination with a flow sensor that may have a feedback loop for online control and real-time adjustment of flow rate.

[0080] In one embodiment, a mixture of a solution containing RNA and a solution containing liposomes contains sodium chloride at a concentration of about 45 mM to about 300 mM or has an ionic strength corresponding to a concentration of sodium chloride of about 45 mM to about 300 mM.

[0081] In one embodiment, the RNA solution contains sodium chloride at a concentration of about 90 mM to about 600 mM or has an ionic strength corresponding to a concentration of sodium chloride of about 90 mM to about 600 mM.

[0082] In one embodiment, a mixture of a solution containing RNA and a solution containing liposomes has an ionic strength of at least about 50 mM.

[0083] In one embodiment, in the X-ray scattering pattern, the RNA lipoplex is characterized by a single Bragg peak at about 1 nm -1 where the peak width is less than 0.2 nm -1 and smaller.

[0084] In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm.

[0085] The present disclosure further relates to a composition comprising RNA lipoplex particles obtainable as described above.

[0086] In one embodiment, the RNA lipoplex particles contain at least one cationic lipid and at least one additional lipid.

[0087] In one embodiment, the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive charge to negative charge in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0088] The present disclosure further provides an RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, and a composition comprising RNA lipoplex particles, wherein the ratio of positive charge to negative charge in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0, and the RNA lipoplex particles are characterized by a single Bragg peak at about 1 nm -1 with a peak width less than 0.2 nm. -1 The present disclosure relates to a composition.

[0089] In one embodiment, the composition further comprises sodium chloride at a concentration of from about 10 to about 300 mM, from about 45 mM to about 300 mM, from about 10 mM to about 50 mM, or from about 80 mM to about 150 mM.

[0090] In one embodiment, the composition further comprises a buffer.

[0091] In one embodiment, the composition further comprises a chelating agent.

[0092] In one embodiment, the RNA lipoplex particles described in this aspect under II. have an average diameter in the range of from about 200 to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.

[0093] In one embodiment, the RNA lipoplex particles have a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3.

[0094] In one embodiment, at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0095] In one embodiment, at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC).

[0096] In one embodiment, at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0097] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0098] In one embodiment, the RNA lipoplex particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1, and the charge ratio of the positive charge in DOTMA to the negative charge in RNA is from about 1:2 to 1.9:2.

[0099] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0100] In one embodiment, the concentration of EDTA is from about 0.25 mM to about 5 mM, or about 2.5 mM.

[0101] In one embodiment, the composition further comprises an adjuvant.

[0102] In one embodiment, the composition is formulated for systemic administration.

[0103] In one embodiment, the systemic administration is by intravenous administration.

[0104] The present disclosure further relates to the described composition for therapeutic use.

[0105] III. Methods and Compositions for Preserving RNA Lipoplex Particles In a third aspect, the present disclosure relates to methods and compositions for preserving RNA lipoplex particles without substantially losing the quality of the product, particularly without substantially losing RNA activity. In particular, the present disclosure relates to formulations that enable freezing, lyophilization, or spray drying of RNA lipoplex particles without substantially losing the quality of the RNA lipoplex particles, particularly without substantially losing RNA activity.

[0106] The RNA lipoplex particle formulations described herein can be frozen or dehydrated by lyophilization, spray drying, or related methods that enable a long shelf life of the product compared to liquid storage.

[0107] To enable freezing, a stabilizer (cryoprotectant) is added. In one embodiment, the lipoplex is diluted with a stabilizer (cryoprotectant) after manufacture, which allows adjustment of the ionic strength, preferably reduction of the ionic strength, and adjustment of the appropriate concentration of the stabilizer. For freezing of the product, the stabilizer concentration may be higher than the value for obtaining physiological osmolality. In that case, for administration, the product is diluted with a suitable aqueous phase (e.g., water for injection, physiological saline) to adjust the desired osmolality and ionic strength. As stabilizers, not only sugars such as glucose, sucrose, or trehalose, but also other compounds such as dextran can be used.

[0108] Surprisingly, according to the present disclosure, it has been found that the RNA lipoplex formulation containing the stabilizer described herein can also be lyophilized. In the case of lyophilization, the required stabilizer (lyoprotectant) concentration can be lower than that in the case of freezing, and the acceptable NaCl concentration (ionic strength) can be higher than that in the case of freezing. When large-scale economical dehydration is required, the product can also be spray-dried.

[0109] The pH of some RNA lipoplex formulations is adjusted to a value lower than the normal physiological range and the normal pH optimal for RNA storage in the bulk phase. The optimal pH is about 6.2, and the preferred range is about 5.7 to about 6.7. In other formulations, the ideal pH can be even lower. The local pH inside the RNA lipoplex is assumed to be higher than the bulk phase pH due to the positive charge of the cationic lipid.

[0110] In an embodiment of the present disclosure where the RNA lipoplex composition is frozen for storage, the composition may be thawed, and optionally, the osmolality, ionic strength and / or pH of the composition may be adjusted by adding an aqueous liquid. The resulting composition can be administered to a subject.

[0111] In an embodiment of the present disclosure where the RNA lipoplex composition is lyophilized or freeze-dried for storage, the composition may be reconstituted by adding an aqueous liquid, and optionally, the osmolality, ionic strength and / or pH of the composition may be adjusted by adding an aqueous liquid. The resulting composition can be administered to a subject.

[0112] Thus, in this aspect, the present disclosure relates to a method for preparing a frozen composition comprising RNA lipoplex particles, the method comprising: (i) providing an aqueous composition comprising RNA lipoplex particles and a stabilizer; and (ii) freezing the composition.

[0113] In one embodiment, the freezing is at a temperature of about -15°C to about -40°C, or about -30°C.

[0114] In one embodiment, the composition is stored at a storage temperature of, for example, about -15°C to about -40°C, or about -20°C.

[0115] In one embodiment, the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0116] In one embodiment, providing an aqueous composition comprising RNA lipoplex particles and a stabilizer includes providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles. Accordingly, a method of preparing a composition for freezing includes providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles.

[0117] In one embodiment, adding a stabilizer to an aqueous composition comprising RNA lipoplex particles reduces the ionic strength of the aqueous composition comprising RNA lipoplex particles

[0118] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex particles and the stabilizer is higher than the value required for physiological osmolarity.

[0119] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, particularly to avoid substantially losing RNA activity after storing the composition at a temperature of about -15°C to about -40°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0120] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising the RNA lipoplex and the stabilizer is from about 5% to about 35.0% (w / v), from about 10% to about 30.0% (w / v), from about 12.5% to about 25.0% (w / v), or about 22.0% (w / v).

[0121] In one embodiment, the pH of the aqueous composition comprising the RNA lipoplex and the stabilizer is lower than the normal pH optimal for RNA preservation.

[0122] In one embodiment, the aqueous composition comprising the RNA lipoplex and the stabilizer contains sodium chloride at a concentration of about 10 mM to about 50 mM or has an ionic strength corresponding to a concentration of sodium chloride of about 10 mM to about 50 mM.

[0123] In one embodiment, the aqueous composition comprising the RNA lipoplex and the stabilizer has an ionic strength corresponding to a concentration of sodium chloride of about 20 mM.

[0124] In one embodiment, the RNA lipoplex particles can be obtained by the method described above under I. and II.

[0125] In one embodiment, the method of preparing the frozen composition further comprises storing the frozen composition comprising the RNA lipoplex particles. The composition can be stored at a temperature corresponding to or essentially corresponding to the freezing temperature, or at a temperature higher or lower than the freezing temperature. Generally, the composition is stored at a temperature of about -15°C to about -40°C, for example about -20°C.

[0126] The present disclosure further relates to a composition comprising RNA lipoplex particles obtainable by the above method of preparing a frozen composition. The present disclosure also relates to a composition comprising RNA lipoplex particles obtainable by the above method of preparing a composition for freezing.

[0127] In one embodiment, the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0128] In one embodiment, the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0129] In one embodiment, the composition further comprises sodium chloride at a concentration of about 10 mM to about 50 mM.

[0130] The present disclosure further provides an RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, an RNA lipoplex particle comprising: an RNA lipoplex particle in which the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0; sodium chloride at a concentration of 0 mM to about 40 mM; a stabilizer; and a composition comprising the same.

[0131] In one embodiment, the composition further comprises a buffer.

[0132] In one embodiment, the amount of RNA in the composition is from about 0.01 mg / mL to about 1 mg / mL, from about 0.05 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL.

[0133] In one embodiment, the sodium chloride is at a concentration of about 20 mM to about 30 mM.

[0134] In one embodiment, the sodium chloride is at a concentration of about 20 mM.

[0135] In one embodiment, the sodium chloride is at a concentration of about 30 mM.

[0136] In one embodiment, the concentration of the stabilizer in the composition is higher than the value required for physiological osmolality.

[0137] In one embodiment, the concentration of the stabilizer in the composition is from about 5 to about 35 weight / volume percent (% w / v) or from about 12.5 to about 25 weight / volume percent (% w / v).

[0138] In one embodiment, the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0139] In one embodiment, the stabilizer is sucrose at a concentration of from about 5 to about 25 weight / volume percent (% w / v).

[0140] In one embodiment, the sucrose is at a concentration of from about 15% (w / v) to about 25% (w / v).

[0141] In one embodiment, the sucrose is at a concentration of from about 20% (w / v) to about 25% (w / v).

[0142] In one embodiment, the sucrose is at a concentration of about 22% (w / v).

[0143] In one embodiment, the sucrose is at a concentration of about 20% (w / v).

[0144] In one embodiment, the composition has a pH lower than the normal pH optimal for RNA preservation.

[0145] In one embodiment, the composition has a pH of from about 5.7 to about 6.7, or about 6.2.

[0146] In one embodiment, the buffer is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

[0147] In one embodiment, the HEPES is at a concentration of from about 2.5 mM to about 10 mM, or about 7.5 mM.

[0148] In one embodiment, the composition further comprises a chelating agent.

[0149] The present disclosure further provides RNA encoding a peptide or protein comprising at least one epitope at a concentration of about 0.05 mg / mL, and DOTMA and DOPE at a molar ratio of about 2:1, an RNA lipoplex particle comprising an RNA lipoplex particle in which the ratio of positive charge to negative charge in the RNA lipoplex particle is about 1.3:2.0, and sodium chloride at a concentration of about 20 mM, and sucrose at a concentration of about 22% (w / v), and HEPES at a concentration of about 7.5 mM with a pH of about 6.2, and EDTA at a concentration of about 2.5 mM, relates to a composition comprising.

[0150] In one embodiment, the composition is in a liquid state or a frozen state.

[0151] In one embodiment, the frozen composition is stable at a temperature of about -15°C to about -40°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0152] In one embodiment, the frozen composition is stable at a temperature of about -15°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0153] In one embodiment, the frozen composition is stable at a temperature of about -15°C for at least 2 months.

[0154] In one embodiment, the frozen composition is stable at a temperature of about -20°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0155] In one embodiment, the frozen composition is stable at a temperature of about -20°C for at least 2 months.

[0156] In one embodiment, the frozen composition is stable at a temperature of about -30°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0157] In one embodiment, the frozen composition is stable at a temperature of about -30°C for at least 2 months.

[0158] The present disclosure further relates to an aqueous composition comprising RNA lipoplex particles, which can be obtained by thawing the above frozen composition and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

[0159] In one embodiment, the osmolality of the composition is from about 200 mOsmol / kg to about 450 mOsmol / kg.

[0160] In one embodiment, the composition comprises sodium chloride at a concentration of about 80 mM to about 150 mM.

[0161] In one embodiment, the RNA lipoplex particles can be obtained by the methods described above under I. and II.

[0162] The present disclosure further relates to a method for preparing a dehydrated, e.g., lyophilized or spray-dried, composition comprising RNA lipoplex particles, the method comprising: (i) providing an aqueous composition comprising RNA lipoplex particles and a stabilizer; and (ii) dehydrating the composition, e.g., by lyophilization or spray drying.

[0163] In one embodiment, the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0164] In one embodiment, providing an aqueous composition comprising RNA lipoplex particles and a stabilizer includes providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles. Thus, a method of preparing a composition for dehydration, for example, lyophilization or spray drying, includes providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles.

[0165] In one embodiment, when a stabilizer is added to an aqueous composition comprising RNA lipoplex particles, the ionic strength of the aqueous composition comprising RNA lipoplex particles decreases.

[0166] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex particles and the stabilizer is higher than the value required for physiological osmolarity.

[0167] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, in particular, to avoid substantially losing RNA activity after storing the composition for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[0168] In one embodiment, the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex and the stabilizer is about 5% to about 35.0% (w / v), about 10% to about 30.0% (w / v), about 12.5% to about 25.0% (w / v), or about 22.0% (w / v).

[0169] In one embodiment, the pH of the aqueous composition comprising RNA lipoplex and the stabilizer is lower than the normal pH optimal for RNA storage.

[0170] In one embodiment, an aqueous composition comprising an RNA lipoplex and a stabilizer comprises sodium chloride at a concentration of about 10 mM to about 80 mM or about 10 mM to about 50 mM, or has an ionic strength corresponding to a concentration of sodium chloride of about 10 mM to about 80 mM or about 10 mM to about 50 mM.

[0171] In one embodiment, an aqueous composition comprising an RNA lipoplex and a stabilizer has an ionic strength corresponding to a concentration of sodium chloride of about 20 mM, about 40 mM, about 60 mM or about 80 mM.

[0172] In one embodiment, RNA lipoplex particles can be obtained by the methods described above under I. and II.

[0173] In one embodiment, a method of preparing a dehydrated, e.g., lyophilized or spray-dried, composition further comprises storing the lyophilized or spray-dried composition comprising RNA lipoplex particles. Generally, the composition is stored at a temperature of about -15°C to about -40°C, such as about -20°C. In certain embodiments, the composition is stored at a temperature higher than 0°C, such as about 25°C or about 4°C, or at room temperature, for example.

[0174] The present disclosure further relates to a composition comprising RNA lipoplex particles obtainable by the above-described method of preparing a dehydrated, e.g., lyophilized or spray-dried, composition. The present disclosure also relates to a composition comprising RNA lipoplex particles obtainable by the above-described method of preparing a composition for dehydration, e.g., lyophilization or spray-drying.

[0175] In one embodiment, RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0176] In one embodiment, the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particles is about 1:2 to about 1.9:2, or about 1.3:2.0.

[0177] In one embodiment, the composition further comprises sodium chloride at a concentration of about 10 mM to about 80 mM or about 10 mM to about 50 mM.

[0178] The present disclosure further provides RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, an RNA-lipoplex particle comprising an RNA-lipoplex particle having a ratio of positive to negative charges in the RNA-lipoplex particle of about 1:2 to about 1.9:2, or about 1.3:2.0, sodium chloride at a concentration of 10 mM to about 80 mM, a stabilizer, and a composition comprising the same.

[0179] In one embodiment, the composition further comprises a buffer.

[0180] In one embodiment, the amount of RNA in the composition is about 0.01 mg / mL to about 1 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL.

[0181] In one embodiment, the sodium chloride is at a concentration of about 20 mM to about 30 mM.

[0182] In one embodiment, the sodium chloride is at a concentration of about 20 mM.

[0183] In one embodiment, the sodium chloride is at a concentration of about 30 mM.

[0184] In one embodiment, the concentration of the stabilizer in the composition is higher than the value required for physiological osmolarity.

[0185] In one embodiment, the concentration of the stabilizer in the composition is about 5 to about 35 weight / volume percent (% w / v) or about 10 to about 25 weight / volume percent (% w / v).

[0186] In one embodiment, the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0187] In one embodiment, the stabilizer is trehalose at a concentration of about 5 to about 35 weight / volume percent (% w / v).

[0188] In one embodiment, the trehalose is at a concentration of about 5% (w / v) to about 25% (w / v).

[0189] In one embodiment, the trehalose is at a concentration of about 10% (w / v) to about 25% (w / v).

[0190] In one embodiment, the trehalose is at a concentration of about 10% (w / v).

[0191] In one embodiment, the trehalose is at a concentration of about 15% (w / v).

[0192] In one embodiment, the composition has a pH lower than the normal pH optimal for RNA preservation.

[0193] In one embodiment, the composition has a pH of about 5.7 to about 6.7, or about 6.2.

[0194] In one embodiment, the buffer is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

[0195] In one embodiment, the HEPES is at a concentration of about 2.5 mM to about 10 mM, or about 7.5 mM.

[0196] In one embodiment, the composition further comprises a chelating agent.

[0197] The present disclosure further relates to RNA encoding a peptide or protein comprising at least one epitope at a concentration of about 0.05 mg / mL, and DOTMA and DOPE at a molar ratio of about 2:1, an RNA lipoplex particle comprising an RNA lipoplex particle having a ratio of positive to negative charges in the RNA lipoplex particle of about 1.3:2.0, and sodium chloride at a concentration of about 20 mM, and trehalose at a concentration of about 10% (w / v), and HEPES at a concentration of about 7.5 mM with a pH of about 6.2, and EDTA at a concentration of about 2.5 mM, and relates to a composition comprising the same.

[0198] In one embodiment, the composition is in a liquid state, or in a dehydrated state, for example, a lyophilized or freeze-dried state.

[0199] In one embodiment, the dehydrated composition, for example, a lyophilized or freeze-dried composition, is stable for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months. In one embodiment, the composition is stored at a temperature higher than 0 °C, for example, about 25 °C or about 4 °C, or for example, at room temperature.

[0200] In one embodiment, the dehydrated composition, for example, a lyophilized or freeze-dried composition, is stable for at least 1 month.

[0201] In one embodiment, the dehydrated composition, for example, a lyophilized or freeze-dried composition, is stable for at least 2 months.

[0202] The present disclosure further relates to an aqueous composition comprising RNA lipoplex particles that can be obtained by reconstituting the dehydrated, e.g., lyophilized or freeze-dried, composition described above and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

[0203] In one embodiment, the osmolality of the composition is from about 150 mOsmol / kg to about 450 mOsmol / kg.

[0204] In one embodiment, the composition comprises sodium chloride at a concentration of from about 80 mM to about 150 mM.

[0205] In one embodiment, the RNA lipoplex particles can be obtained by the methods described above under I. and II.

[0206] In one embodiment, the RNA lipoplex particles described in this aspect under III. are characterized by a single Bragg peak at about 1 nm -1 where the peak width is less than 0.2 nm -1 and smaller.

[0207] In one embodiment, the RNA lipoplex particles described in this aspect under III. have an average diameter in the range of from about 200 to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm or from about 350 nm to about 400 nm.

[0208] In one embodiment, the RNA lipoplex particles have a polydispersity index of less than about 0.5, less than about 0.4 or less than about 0.3.

[0209] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0210] In one embodiment, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0211] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0212] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0213] In one embodiment, the RNA lipoplex particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1, and the charge ratio of the positive charge in DOTMA to the negative charge in RNA is from about 1:2 to 1.9:2.

[0214] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0215] In one embodiment, EDTA is at a concentration of from about 0.25 mM to about 5 mM, or about 2.5 mM.

[0216] In one embodiment, the composition further comprises an adjuvant.

[0217] In one embodiment, the composition is formulated for systemic administration.

[0218] In one embodiment, systemic administration is by intravenous administration.

[0219] The present disclosure further relates to the described compositions for therapeutic use.

[0220] The present disclosure further relates to a method of preparing an aqueous composition comprising RNA lipoplex particles, comprising thawing the above-described frozen composition, or reconstituting the above-described lyophilized or spray-dried composition, and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

[0221] In one embodiment, an aqueous liquid is added to obtain an osmolality of the composition of about 200 mOsmol / kg to about 450 mOsmol / kg.

[0222] In one embodiment, an aqueous liquid is added to obtain a concentration of sodium chloride of about 80 mM to about 150 mM.

[0223] Some of the RNA lipoplex formulations described herein, which are suitable for storing RNA lipoplex particles without substantially losing the quality of the product, particularly without substantially losing RNA activity, do not require modification of the product, particularly dilution of the product with an aqueous phase (e.g., water for injection, physiological saline), to adjust the desired osmolality and ionic strength prior to administration. Such RNA lipoplex formulations can be administered directly after storage of the product, optionally after thawing or reconstitution. In embodiments of the present disclosure where the RNA lipoplex composition is frozen for storage, the composition can be thawed and administered without the need to adjust the osmolality, ionic strength, and / or pH of the composition.

[0224] Accordingly, the present disclosure provides RNA, as well as at least one cationic lipid and at least one additional lipid, comprising RNA lipoplex particles, sodium chloride at a concentration of about 10 mM or less, a stabilizer at a concentration of about 10% weight / volume percent (% w / v) or less, a buffer, Relates to a composition comprising.

[0225] In one embodiment, sodium chloride is at a concentration of about 5 mM to about 10 mM. In one embodiment, sodium chloride is at a concentration of about 7.5 mM or less, for example, about 5 mM to about 7.5 mM. In one embodiment, sodium chloride is at a concentration of about 6.5 mM or about 7.5 mM.

[0226] In one embodiment, the concentration of salts and / or stabilizers in the composition is on the order of the value required for physiological osmolality. In one embodiment, the osmolality resulting from the dissolved components including ionic and non-ionic components is on the order of the value required for physiological osmolality.

[0227] In one embodiment, the concentration of the stabilizer in the composition is about 5 to about 10% (w / v). In one embodiment, the concentration of the stabilizer in the composition is about 5 to about 7.5% (w / v). In one embodiment, the concentration of the stabilizer in the composition is about 7.5 to about 10% (w / v).

[0228] In one embodiment, the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0229] In one embodiment, the stabilizer is sucrose or trehalose. In one embodiment, the stabilizer is sucrose at a concentration of about 5 to about 10% (w / v). In one embodiment, sucrose is at a concentration of about 10% (w / v). In one embodiment, the stabilizer is trehalose at a concentration of about 5 to about 10% (w / v). In one embodiment, trehalose is at a concentration of about 10% (w / v).

[0230] In one embodiment, the buffer is selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), histidine, acetic acid / sodium acetate, and MES (2-(N-morpholino)ethanesulfonic acid). In one embodiment, the buffer is HEPES, histidine or MES. In one embodiment, the buffer is HEPES or MES. In one embodiment, the buffer is HEPES.

[0231] In one embodiment, the composition has a pH of 6.0 - 7.2, 6.0 - 7.0, 6.2 - 7.0, 6.5 - 7.0 or 6.5 - 6.7. In one embodiment, the composition has a pH of about 6.5 or 6.7.

[0232] In one embodiment, the buffer is present at a concentration of 2.5 mM to 10 mM. In one embodiment, the buffer is present at a concentration of 2.5 mM to 5 mM. In one embodiment, the buffer is present at a concentration of 5 mM to 10 mM. In one embodiment, the buffer is present at a concentration of 5 mM to 7.5 mM. In one embodiment, the buffer is present at a concentration of 7.5 mM to 10 mM. In one embodiment, the buffer is present at a concentration of about 7.5 mM.

[0233] In one embodiment, the buffer is HEPES at a concentration of about 7.5 mM or less, for example 2.5 mM to 7.5 mM or 5 mM to 7.5 mM, where the pH is about 6.5 or about 6.7.

[0234] In one embodiment, at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP). In one embodiment, at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC). In one embodiment, at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0235] In one embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0236] In one embodiment, the RNA lipoplex particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1.

[0237] In one embodiment, the composition further comprises a chelating agent. In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In one embodiment, the concentration of EDTA is about 3.5 mM or less, or from about 0.25 mM to about 3.5 mM or from about 0.25 mM to about 2.5 mM.

[0238] In one embodiment of the compositions described herein, the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive charge to negative charge in the composition is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0239] The present disclosure further provides RNA encoding a peptide or protein comprising at least one epitope, DOTMA and DOPE in a molar ratio of about 2:1, an RNA lipoplex particle comprising, an RNA lipoplex particle having a ratio of positive to negative charges in the composition of about 1.3:2.0, sodium chloride at a concentration of about 7.5 mM, sucrose at a concentration of about 10% (w / v), HEPES at a concentration of about 7.5 mM with a pH of about 6.5 or about 6.7, EDTA at a concentration of about 2.5 mM, relates to a composition comprising.

[0240] In one embodiment of the composition described herein, the RNA lipoplex particles have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm or about 350 nm to about 400 nm.

[0241] In one embodiment of the composition described herein, the amount of RNA in the composition is about 0.01 mg / mL to about 1 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, about 0.05 mg / mL, or about 0.02 mg / mL.

[0242] In one embodiment of the composition described herein, the composition further comprises an adjuvant.

[0243] In one embodiment of the composition described herein, the composition is in a liquid state, a frozen state or a dehydrated state.

[0244] In one embodiment, the composition is a frozen composition and is stable at a temperature of about -15°C for at least 1 month. In one embodiment, the composition is a frozen composition and is stable at a temperature of about -15°C for at least 2 months. In one embodiment, the composition is a frozen composition and is stable at a temperature of about -15°C for at least 4 months. In one embodiment, the composition is a frozen composition and is stable at a temperature of about -15°C for at least 6 months.

[0245] The present disclosure further relates to a liquid composition comprising RNA lipoplex particles, which can be obtained by thawing the frozen composition described herein. In one embodiment, the liquid composition has the above composition.

[0246] The present disclosure further relates to a liquid composition comprising RNA lipoplex particles, which can be obtained by dissolving the dehydrated composition described herein. In one embodiment, the liquid composition has the above composition.

[0247] In one embodiment, the liquid composition described herein is an aqueous composition.

[0248] In one embodiment, the composition, particularly the liquid composition described herein, can be administered directly to a subject.

[0249] In one embodiment, the composition described herein is a pharmaceutical composition.

[0250] In one embodiment, the composition described herein is formulated for systemic administration.

[0251] In one embodiment, systemic administration is by intravenous administration.

[0252] The present disclosure further relates to the composition described herein for therapeutic use.

[0253] The present disclosure further relates to a method of preparing a liquid composition for direct administration to a subject, the method comprising thawing a frozen composition described herein. The present disclosure further relates to a method of preparing a liquid composition for direct administration to a subject, the method comprising dissolving a dehydrated composition described herein. In one embodiment of the method described herein, the liquid composition is an aqueous composition. In one embodiment, the liquid composition has the above-described composition. In the therapeutic applications described herein, the liquid composition described herein is administered to a subject.

[0254] Further embodiments are as follows: 1. A method of generating a liposomal colloid, comprising injecting an ethanol solution of lipids into an aqueous phase to generate a liposomal colloid, wherein the concentration of at least one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of at least one lipid in ethanol.

[0255] 2. The method of embodiment 1, wherein the lipid solution is a solution of a mixture of two or more different lipids.

[0256] 3. The method of embodiment 1 or 2, wherein the concentration of one lipid in the lipid solution corresponds to or is higher than the equilibrium solubility of the lipid in ethanol at room temperature.

[0257] 4. The method of any one of embodiments 1 to 3, wherein the total lipid concentration in the lipid solution is about 180 mM to about 600 mM, about 300 mM to about 600 mM, or about 330 mM.

[0258] 5. The method of any one of embodiments 1 to 4, wherein the lipid solution comprises at least one cationic lipid and at least one additional lipid.

[0259] 6. The method of embodiment 5, wherein the concentration of the additional lipid in the lipid solution corresponds to or is higher than the equilibrium solubility of the additional lipid in ethanol.

[0260] 7. The method of embodiment 5 or 6, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0261] 8. The method of any one of embodiments 5 to 7, wherein at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC).

[0262] 9. The method of any one of embodiments 5 to 8, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0263] 10. The method of any one of embodiments 5 to 9, wherein the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0264] 11. The method of any one of embodiments 1 to 10, wherein the lipid solution comprises DOTMA and DOPE in a molar ratio of from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0265] 12. The method of any one of embodiments 8 to 11, wherein the concentration of DOPE in the lipid solution is at least about 60 mM or at least about 90 mM.

[0266] 13. The method of any one of embodiments 1 to 12, wherein the lipid solution is injected into the aqueous phase at a stirring rate of about 50 rpm to about 150 rpm of the aqueous phase.

[0267] 14. The method according to any one of embodiments 1 to 13, wherein the aqueous phase is water.

[0268] 15. The method according to any one of embodiments 1 to 14, further comprising stirring the liposome colloid.

[0269] 16. The method according to any one of embodiments 1 to 15, wherein the liposome colloid is stirred for about 15 minutes to about 60 minutes, or about 30 minutes.

[0270] 17. A method for producing a liposome colloid, comprising injecting a lipid solution containing DOTMA and DOPE in ethanol at a molar ratio of about 2:1 into water stirred at a stirring speed of about 150 rpm to produce a liposome colloid, wherein the concentrations of DOTMA and DOPE in the lipid solution are about 330 mM.

[0271] 18. A liposome colloid obtainable by the method according to any one of embodiments 1 to 17.

[0272] 19. The liposome colloid according to embodiment 18, wherein the liposome has an average diameter of at least about 250 nm.

[0273] 20. The liposome colloid according to embodiment 18 or 19, wherein the liposome has an average diameter in the range of about 250 nm to about 800 nm.

[0274] 21. The liposome colloid according to any one of embodiments 18 to 20, wherein the liposome is a cationic liposome.

[0275] 22. The liposome colloid according to any one of embodiments 18 to 21, wherein the liposome contains at least one cationic lipid and at least one additional lipid.

[0276] 23. The liposomal colloid of embodiment 22, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0277] 24. The liposomal colloid of embodiment 22 or 23, wherein at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC).

[0278] 25. The liposomal colloid of any one of embodiments 22 to 24, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0279] 26. The liposomal colloid of any one of embodiments 22 to 25, wherein the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0280] 27. The liposomal colloid of any one of embodiments 18 to 26, wherein the liposome comprises DOTMA and DOPE in a molar ratio of from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0281] 28. A method for preparing RNA - lipoplex particles, the method comprising adding a liposomal colloid of any one of embodiments 18 to 27 to a solution containing RNA.

[0282] 29. A method for the continuous flow production of RNA lipoplex particles, the method comprising mixing a solution containing RNA and a solution containing cationic liposomes under controlled mixing conditions of the RNA and the cationic liposomes.

[0283] 30. The method of embodiment 29, wherein the solution containing cationic liposomes is one of the liposome colloids of any one of embodiments 18 to 27.

[0284] 31. The method of embodiment 29 or 30, wherein the solution containing RNA and the solution containing cationic liposomes are aqueous solutions.

[0285] 32. The method of any one of embodiments 29 to 31, wherein a flow rate that enables mixing of the solution containing RNA and the solution containing cationic liposomes is used.

[0286] 33. The method of any one of embodiments 29 to 32, wherein the flow has a Reynolds number greater than 300 or from about 500 to about 2100.

[0287] 34. The method of any one of embodiments 29 to 33, wherein the controlled mixing conditions include controlling the mixing ratio of the solution containing RNA and the solution containing cationic liposomes.

[0288] 35. The method of any one of embodiments 29 to 34, wherein the controlled mixing conditions include controlling the relative volumes of the solution containing RNA and the solution containing cationic liposomes to be mixed.

[0289] 36. The method of any one of embodiments 29 to 35, wherein the mixing ratio of RNA and cationic liposomes is controlled by using the same mixing volume (v / v) of the solution containing RNA and the solution containing cationic liposomes and adjusting the concentrations of RNA and cationic liposomes in each solution.

[0290] 37. A method according to any one of embodiments 29 to 36, wherein the controlled mixing conditions are selected to maintain the properties of the RNA lipoplex particles while avoiding clogging.

[0291] 38. A method according to any one of embodiments 29 to 37, comprising using a Y-shaped or T-shaped mixing element.

[0292] 39. A method according to any one of embodiments 29 to 38, wherein the Y-shaped or T-shaped mixing element has a diameter of about 1.2 mm to about 50 mm.

[0293] 40. A method according to any one of embodiments 29 to 39, comprising using a syringe pump in which two syringes, one containing a solution comprising cationic liposomes and one containing a solution comprising RNA, are inserted in parallel into the same pump.

[0294] 41. A method according to any one of embodiments 29 to 40, comprising using a pressure vessel, a membrane pump, a gear pump, a magnetic levitation pump or a peristaltic pump in combination with a flow sensor optionally having a feedback loop for online control and real-time adjustment of the flow rate.

[0295] 42. A method according to any one of embodiments 29 to 41, wherein the mixture of the solution containing RNA and the solution containing liposomes contains sodium chloride at a concentration of about 45 mM to about 300 mM or has an ionic strength corresponding to a concentration of sodium chloride of about 45 mM to about 300 mM.

[0296] 43. A method according to any one of embodiments 29 to 42, wherein the mixture of the solution containing RNA and the solution containing liposomes has an ionic strength of at least about 50 mM.

[0297] 44. In the X-ray scattering pattern, the RNA lipoplex is characterized by a single Bragg peak at about 1 nm -1 and the peak width is less than 0.2 nm -1 A method according to any one of embodiments 28 to 43.

[0298] 45. A method according to any one of embodiments 28 to 44, wherein the RNA lipoplex particles have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm or about 350 nm to about 400 nm.

[0299] 46. A method for preparing a frozen composition comprising RNA lipoplex particles, the method comprising: (i) providing an aqueous composition comprising RNA lipoplex particles and a stabilizer; and (ii) freezing the composition.

[0300] 47. The method of embodiment 46, wherein the freezing is at a temperature of about -15 °C to about -40 °C, or about -30 °C.

[0301] 48. The method of embodiment 47, wherein the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0302] 49. The method according to any one of embodiments 46 to 48, wherein providing an aqueous composition comprising RNA lipoplex particles and a stabilizer comprises providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles.

[0303] 50. The method according to any one of embodiments 46 to 49, wherein adding a stabilizer to an aqueous composition comprising RNA lipoplex particles reduces the ionic strength of the aqueous composition comprising RNA lipoplex particles.

[0304] 51. The method according to any one of embodiments 46 to 50, wherein the concentration of the stabilizer in the aqueous composition comprising RNA lipoplex particles and the stabilizer is higher than the value required for physiological osmolality.

[0305] 52. The concentration of the stabilizer in the aqueous composition containing the RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, and in particular, substantially loses RNA activity after storing the composition at a temperature of about -15°C to about -40°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months or at least 36 months. The method according to any one of embodiments 46 to 51 is sufficient to avoid this.

[0306] 53. The pH of the aqueous composition containing the RNA lipoplex and the stabilizer is lower than the normal pH optimal for RNA storage. The method according to any one of embodiments 46 to 52.

[0307] 54. The aqueous composition containing the RNA lipoplex and the stabilizer contains sodium chloride at a concentration of about 10 mM to about 50 mM or has an ionic strength corresponding to a concentration of sodium chloride of about 10 mM to about 50 mM. The method according to any one of embodiments 46 to 53.

[0308] 55. The aqueous composition containing the RNA lipoplex and the stabilizer has an ionic strength corresponding to a concentration of sodium chloride of about 20 mM. The method according to any one of embodiments 46 to 54.

[0309] 56. The RNA lipoplex particles can be obtained by the method according to any one of embodiments 28 to 44. The method according to any one of embodiments 46 to 55.

[0310] 57. A composition comprising RNA lipoplex particles obtainable by the method according to any one of embodiments 28 to 45.

[0311] 58. The composition of embodiment 57, wherein the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0312] 59. A composition according to embodiment 57 or 58, wherein the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0313] 60. An RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, and an RNA lipoplex particle comprising a composition, wherein the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0, and the RNA lipoplex particles are characterized by a single Bragg peak at about 1 nm -1 with a peak width smaller than 0.2 nm -1 composition.

[0314] 61. A composition according to any one of embodiments 57 to 60, further comprising sodium chloride at a concentration of about 10 to about 300 mM, about 45 mM to about 300 mM, about 10 mM to about 50 mM or about 80 mM to about 150 mM.

[0315] 62. A composition according to any one of embodiments 57 to 61, further comprising a buffer.

[0316] 63. A composition according to any one of embodiments 57 to 62, further comprising a chelating agent.

[0317] 64. A composition comprising RNA lipoplex particles obtainable by a method according to any one of embodiments 46 to 56.

[0318] 65. A composition according to embodiment 64, wherein the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0319] 66. A composition according to embodiment 64 or 65, wherein the RNA encodes a peptide or protein comprising at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0.

[0320] 67. A composition according to any one of embodiments 64 to 66, further comprising sodium chloride at a concentration of about 10 mM to about 50 mM.

[0321] 68. An RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, and an RNA lipoplex particle comprising: an RNA lipoplex particle wherein the ratio of positive to negative charges in the RNA lipoplex particles is from about 1:2 to about 1.9:2, or about 1.3:2.0; sodium chloride at a concentration of 0 mM to about 40 mM; a stabilizer; and a composition comprising the same. 69. A composition according to any one of embodiments 64 to 68, further comprising a buffer.

[0322] 70. A composition according to any one of embodiments 64 to 69, wherein the amount of RNA in the composition is from about 0.01 mg / mL to about 1 mg / mL, from about 0.05 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL.

[0323] 71. A composition according to any one of embodiments 67 to 70, wherein the sodium chloride is at a concentration of about 20 mM to about 30 mM.

[0324] 72. A composition according to any one of embodiments 67 to 71, wherein the sodium chloride is at a concentration of about 20 mM.

[0325] 73. A composition according to any one of embodiments 67 to 71, wherein the sodium chloride is at a concentration of about 30 mM.

[0326] 74. A composition according to any one of embodiments 64 to 73, wherein the concentration of the stabilizer in the composition is higher than the value required for physiological osmolality.

[0327] 75. A composition according to any one of embodiments 64 to 74, wherein the concentration of the stabilizer in the composition is from about 5 to about 35 weight / volume percent (% w / v) or from about 12.5 to about 25 weight / volume percent (% w / v).

[0328] 76. A composition according to any one of embodiments 64 to 75, wherein the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

[0329] 77. A composition according to any one of embodiments 64 to 76, wherein the stabilizer is sucrose at a concentration of from about 5 to about 25 weight / volume percent (% w / v).

[0330] 78. The composition of embodiment 77, wherein the sucrose is at a concentration of from about 15% (w / v) to about 25% (w / v).

[0331] 79. The composition of embodiment 77, wherein the sucrose is at a concentration of from about 20% (w / v) to about 25% (w / v).

[0332] 80. The composition of embodiment 77, wherein the sucrose is at a concentration of about 22% (w / v).

[0333] 81. The composition of embodiment 77, wherein the sucrose is at a concentration of about 20% (w / v).

[0334] 82. A composition according to any one of embodiments 64 to 81, having a pH lower than the normal pH optimal for RNA preservation.

[0335] 83. A composition according to any one of embodiments 64 to 82, having a pH of from about 5.7 to about 6.7, or about 6.2.

[0336] 84. A composition according to any one of embodiments 68 to 83, wherein the buffer is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

[0337] 85. A composition according to embodiment 84, wherein HEPES is at a concentration of about 2.5 mM to about 10 mM, or about 7.5 mM.

[0338] 86. A composition according to any one of embodiments 64 to 85, further comprising a chelating agent.

[0339] 87. RNA encoding a peptide or protein comprising at least one epitope at a concentration of about 0.05 mg / mL, and DOTMA and DOPE in a molar ratio of about 2:1, an RNA lipoplex particle comprising, an RNA lipoplex particle having a ratio of positive charge to negative charge in the RNA lipoplex particle of about 1.3:2.0, and sodium chloride at a concentration of about 20 mM, and sucrose at a concentration of about 22% (w / v), and HEPES at a concentration of about 7.5 mM with a pH of about 6.2, and EDTA at a concentration of about 2.5 mM, and a composition comprising.

[0340] 88. A composition according to any one of embodiments 64 to 87, which is in a liquid state or a frozen state.

[0341] 89. A frozen composition according to embodiment 88, which is stable at a temperature of about -15 °C to about -40 °C for at least one month.

[0342] 90. A frozen composition according to embodiment 88, which is stable at a temperature of about -15 °C for at least one month.

[0343] 91. A frozen composition according to embodiment 88, which is stable at a temperature of about -15 °C for at least two months.

[0344] 92. The frozen composition of embodiment 88 that is stable at a temperature of about -20 °C for at least 1 month.

[0345] 93. The frozen composition of embodiment 88 that is stable at a temperature of about -20 °C for at least 2 months.

[0346] 94. The frozen composition of embodiment 88 that is stable at a temperature of about -30 °C for at least 1 month.

[0347] 95. The frozen composition of embodiment 88 that is stable at a temperature of about -30 °C for at least 2 months.

[0348] 96. An aqueous composition containing RNA lipoplex particles that can be obtained by thawing the frozen composition of any one of embodiments 88 to 95 and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

[0349] 97. The composition of embodiment 96, wherein the osmolality of the composition is about 200 mOsmol / kg to about 450 mOsmol / kg.

[0350] 98. The composition of embodiment 96 or 97, containing sodium chloride at a concentration of about 80 mM to about 150 mM.

[0351] 99. The composition of any one of embodiments 64 to 98, wherein the RNA lipoplex particles can be obtained by the method of any one of embodiments 28 to 45. 100. The RNA lipoplex particles are characterized by a single Bragg peak at about 1 nm -1 and the peak width is less than 0.2 nm -1 The composition of any one of embodiments 64 to 99

[0352] 101. The composition of any one of embodiments 57 to 100, wherein the RNA lipoplex particles have an average diameter in the range of about 200 to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm or about 350 nm to about 400 nm.

[0353] 102. A composition according to any one of embodiments 58 to 63, 65 to 86, and 88 to 101, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA) and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP).

[0354] 103. A composition according to any one of embodiments 58 to 63, 65 to 86, and 88 to 102, wherein at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC).

[0355] 104. A composition according to any one of embodiments 58 to 63, 65 to 86, and 88 to 103, wherein at least one cationic lipid comprises 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), and at least one additional lipid comprises 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE).

[0356] 105. A composition according to any one of embodiments 58 to 63, 65 to 86, and 88 to 104, wherein the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 3:1 to about 1:1, or about 2:1.

[0357] 106. A composition according to any one of embodiments 58 to 63, 65 to 86, and 88 to 105, wherein the RNA lipoplex particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1, and the charge ratio of the positive charge in DOTMA to the negative charge in RNA is from about 1:2 to 1.9:2.

[0358] 107. A composition according to any one of embodiments 63, 86, and 88 to 106, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0359] 108. The composition of embodiment 107, wherein the EDTA is at a concentration of about 0.25 mM to about 5 mM, or about 2.5 mM.

[0360] 109. A composition according to any one of embodiments 57 to 108, further comprising an adjuvant.

[0361] 110. A composition according to any one of embodiments 57 to 109, formulated for systemic administration.

[0362] 111. The composition of embodiment 110, wherein the systemic administration is by intravenous administration.

[0363] 112. A composition according to any one of embodiments 57 to 111, for therapeutic use.

[0364] 113. A method of preparing an aqueous composition comprising RNA lipoplex particles, the method comprising thawing a frozen composition according to any one of embodiments 88 to 112 and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

[0365] 114. The method of embodiment 113, wherein the aqueous liquid is added to obtain an osmolality of the composition of about 200 mOsmol / kg to about 450 mOsmol / kg.

[0366] 115. The method of embodiment 113 or 114, wherein the aqueous liquid is added to obtain a concentration of sodium chloride of about 80 mM to about 150 mM. BRIEF DESCRIPTION OF THE DRAWINGS

[0367]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

Figure 56

Figure 57

Figure 58

Figure 59

Figure 60

Figure 61

Figure 62

Figure 63

Figure 64

Figure 65

Figure 66

Figure 67

Figure 68

Figure 69

Figure 70

Figure 71

Mode for Carrying Out the Invention

[0368] The present disclosure will be described in detail below, but it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, and these may vary. It should also be understood that the terms used herein are for the purpose of describing only particular embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0369] Preferably, the terms used herein are defined as described in ''A multilingual glossary of biotechnological terms:(IUPAC Recommendations)'', H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0370] The practice of the present disclosure uses conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989), unless otherwise indicated.

[0371] Elements of the present disclosure are described below. While these elements are recited with specific embodiments, it should be understood that they may be combined in any way and in any number to create additional embodiments. The examples and embodiments described variously should not be construed as limiting the present disclosure to only the explicitly described embodiments. This description is to be understood as disclosing and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Further, any rearrangement and combination of all the elements described should be considered to be disclosed by this description unless otherwise specifically indicated in the context.

[0372] The term "about" means approximately or nearly and, in the context of a numerical value or range described herein, in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0373] As used in the context of the present disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references should be construed to include both the singular and the plural unless otherwise specifically indicated herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise specifically indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise specifically indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the present disclosure and is not limiting of the claims. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0374] Unless otherwise specified, the term "comprising" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprising", further members may optionally exist. However, it is contemplated as a particular embodiment of the present disclosure that the term "comprising" encompasses the possibility that no further members exist, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of".

[0375] Several references are cited throughout the text of this specification. Each reference cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety, whether above or below. Nothing in this specification should be construed as an admission that the present disclosure had the right to precedence over such disclosure.

[0376] Definitions The following provides definitions that apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Terms that are not defined have the meanings generally recognized in their technical fields.

[0377] Terms such as "reduce" or "inhibit" as used herein mean the ability to cause an overall decrease at a level of, for example, about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, i.e., reduction to zero or substantially zero.

[0378] Terms such as "increase" or "enhance" in one embodiment relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0379] As used herein, "physiological pH" refers to a pH of about 7.5.

[0380] As used in the present disclosure, "% w / v" refers to weight / volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (mL).

[0381] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Thus, the ionic strength I is given by the following equation

Number

[0382] According to the present disclosure, in one embodiment, the term "ionic strength" relates to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, due to the presence of chelating agents, their concentration or effective concentration (presence of free ions) is, in one embodiment, low enough to prevent RNA degradation. In one embodiment, the concentration or effective concentration of divalent ions is lower than the catalytic level for hydrolysis of the phosphodiester bond between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or essentially absent.

[0383] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0384] The Reynolds number is a dimensionless number and can be calculated using the following formula

Number

[0385] The term "freezing" typically relates to the solidification of a liquid accompanied by the removal of heat.

[0386] The term "lyophilize" or "lyophilization" refers to the lyophilization of a substance by freezing the substance and then reducing the ambient pressure to directly sublimate the frozen medium in the substance from the solid phase to the gas phase.

[0387] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with a fluid atomized (sprayed) in a vessel (spray dryer), where the solvent from the droplets formed therein evaporates, resulting in a dry powder.

[0388] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing stage.

[0389] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying stage.

[0390] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0391] The term "recombinant" in the context of the present disclosure means "produced via genetic engineering". In one embodiment, a "recombinant object" in the context of the present disclosure does not occur naturally.

[0392] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in a living organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in the laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but that may be discovered and / or isolated from natural sources in the future.

[0393] The term "equilibrium solubility" refers to the concentration of a solute at which the rate of solute dissolution is the same as the rate of solute precipitation from the solution. In one embodiment, this term relates to the respective concentrations at room temperature.

[0394] As used herein, the term "room temperature" refers to a temperature above 4°C, preferably about 15°C to about 40°C, about 15°C to about 30°C, about 15°C to about 24°C, or about 16°C to about 21°C. Such temperatures include 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0395] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecular complexes. In one embodiment, the term "particle" relates to micro-sized or nano-sized structures, for example, micro-sized or nano-sized dense structures.

[0396] In the context of the present disclosure, the term "RNA lipoplex particle" relates to a particle containing a lipid, particularly a cationic lipid, and RNA. The electrostatic interaction between the positively charged liposome and the negatively charged RNA results in the complexation and spontaneous formation of RNA lipoplex particles. The positively charged liposome can generally be synthesized using a cationic lipid such as DOTMA and an additional lipid such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0397] As used in the present disclosure, "nanoparticle" refers to a particle that contains RNA and at least one cationic lipid and has an average diameter suitable for intravenous administration.

[0398] The term "average diameter" refers to the so-called Z having a dimension of length 平均And the hydrodynamic diameter of the particles, measured by dynamic light scattering (DLS) with data analysis using a so-called cumulant algorithm that provides as a result a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of the particles is used synonymously with this Z 平均 value.

[0399] The term "polydispersity index" is used herein as a measure of the size distribution of a population of particles, for example nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0400] As used herein, "subvisible particles" refers to particles having an average diameter of less than 100 micrometers (μm). The number of subvisible particles can be measured in the present disclosure using light obscuration to indicate the degree of aggregation of RNA lipoplex particles. In some embodiments, the number of subvisible particles having an average diameter of 10 μm or greater is measured. In other embodiments, the number of subvisible particles having an average diameter of 25 μm or greater is measured.

[0401] The term "ethanol injection technique" refers to the process by which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, such as the formation of lipid vesicles such as liposomes. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Such a colloidal liposome dispersion is formed in one embodiment using the ethanol injection technique as follows: an ethanol solution containing lipids such as a cationic lipid like DOTMA and additional lipids is injected into the aqueous solution while stirring. In one embodiment, the RNA lipoplex particles described herein can be obtained without using an extrusion step.

[0402] The term "extrude" or "extrusion" refers to the production of particles having a fixed cross-sectional profile. In particular, this term refers to the miniaturization of particles by forcing them through a filter having defined pores.

[0403] The term "trehalose" always refers to both trehalose anhydrate and trehalose dihydrate. All concentrations are given with respect to trehalose dihydrate.

[0404] The term "EDTA" refers to disodium ethylenediaminetetraacetate. All concentrations are given with respect to the disodium salt of EDTA.

[0405] The diameter of the RNA lipoplex particles The RNA lipoplex particles described herein, in one embodiment, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0406] For example, the RNA lipoplex particles described herein, generated by the process described herein, exhibit a polydispersity index of less than about 0.5, less than about 0.4 or less than about 0.3. By way of example, the RNA lipoplex particles can exhibit a polydispersity index in the range of about 0.1 to about 0.3.

[0407] Lipid In one embodiment, the lipid solutions, liposomes, and RNA lipoplex particles described herein contain a cationic lipid. As used herein, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix by electrostatic interactions. Generally, cationic lipids have a lipophilic moiety such as a sterol, acyl, or diacyl chain, and the head group of the lipid typically bears a positive charge. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacetyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE) and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). DOTMA, DOTAP, DODAC and DOSPA are preferred. In certain embodiments, at least one cationic lipid is DOTMA and / or DOTAP. In one embodiment, at least one cationic lipid is DOTMA, particularly (R)-DOTMA.

[0408] To adjust the overall ratio of positive to negative charges and the physical stability of the RNA lipoplex particles, additional lipids may be incorporated. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid having a net charge of zero. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In certain embodiments, the second lipid is DOPE, cholesterol, and / or DOPC.

[0409] In certain embodiments, the RNA lipoplex particles contain both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE. Without wishing to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid can affect important RNA lipoplex particle properties such as charge, particle size, stability, tissue selectivity, and the biological activity of the RNA. Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, or from about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0410] RNA In the present disclosure, the term "RNA" relates to nucleic acid molecules containing ribonucleotide residues. In preferred embodiments, the RNA comprises all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can refer to the addition of non-nucleotide substances to internal RNA nucleotides or to the ends (one or both) of the RNA. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0411] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is generated by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is generated by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

[0412] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template 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. The cDNA can be obtained by reverse transcription of RNA.

[0413] In certain embodiments of the present disclosure, the RNA in the RNA-lipoplex composition described herein is at a concentration of about 0.01 mg / mL to about 1 mg / mL or about 0.05 mg / mL to about 0.5 mg / mL. In certain embodiments, the RNA is at a concentration of about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL or about 0.50 mg / mL. In an exemplary embodiment, the RNA is at a concentration of about 0.02 mg / mL or about 0.05 mg / mL.

[0414] In one embodiment, the RNA can have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine and pseudouridine.

[0415] In some embodiments, the RNA according to the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA can be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7 position. Providing a 5' cap or 5' cap analog to the RNA can be achieved by in vitro transcription where the 5' cap is co-transcriptionally expressed on the RNA strand or ligated to the RNA post-transcriptionally using a capping enzyme.

[0416] In some embodiments, the RNA according to the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA molecule such as an mRNA molecule. The untranslated region (UTR) can be present 5' (upstream) (5'-UTR) and / or 3' (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, when present, is located at the 5' end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (when present), for example, directly adjacent to the 5' cap. The 3'-UTR, when present, is located at the 3' end downstream of the stop codon of the protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (when present), for example, directly adjacent to the poly(A) sequence.

[0417] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" typically relates to a sequence of adenyl (A) residues located at the 3'-end of an RNA molecule. According to the present disclosure, in one embodiment, the poly(A) sequence comprises at least about 20, at least about 40, at least about 80 or at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200 or up to about 150 A nucleotides, particularly about 120 A nucleotides.

[0418] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into a peptide or protein.

[0419] With respect to RNA, the term "expression" or "translation" relates to a process within the ribosomes of a cell by which a strand of mRNA induces the construction of an amino acid sequence so as to produce a peptide or protein.

[0420] In one embodiment, after administration of the RNA lipoplex particles described herein, at least a portion of the RNA is delivered to the target cells. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cells to produce a peptide or protein. In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells such as professional antigen-presenting cells within the spleen. In one embodiment, the target cells are dendritic cells of the spleen. Thus, the RNA lipoplex particles described herein can be used to deliver RNA to such target cells. Accordingly, the present disclosure also relates to a method for delivering RNA to target cells of a subject, the method comprising administering to the subject the RNA lipoplex particles described herein. In one embodiment, the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cells to produce a peptide or protein.

[0421] In one embodiment, the RNA encodes a pharmaceutically active peptide or protein.

[0422] According to the present disclosure, the term "encoded by the RNA" means that when the RNA is present in a suitable environment such as within the cells of the target tissue, it can direct the construction of amino acids to produce the peptide or protein that it encodes during the translation process. In one embodiment, the RNA can interact with the cellular translation machinery that enables the translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., within the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or produce them on the surface.

[0423] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance containing about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" refers to a large peptide, particularly a peptide having at least about 151 amino acids, but the terms "peptide" and "protein" are generally used synonymously herein.

[0424] A "pharmaceutically active peptide or protein" has a positive or beneficial effect on the condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, a pharmaceutically active peptide or protein has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide and can also refer to its pharmaceutically active fragments. This term can also include pharmaceutically active analogs of the peptide or protein.

[0425] Examples of pharmaceutically active proteins include, but are not limited to, cytokines and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony stimulating factors (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active antigens, such as bacterial antigens, parasitic antigens or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative enzymes, steroid producing enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochrome, adenylate cyclase or guanylate cyclase, neuraminidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (such as growth hormone or growth factor binding proteins), transcription and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin and myosin, etc.), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified factor VIII, anticoagulant factors, etc.).

[0426] The term "immunologically active compound" relates to any compound that modifies the immune response, for example, by inducing and / or suppressing the maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and also downregulate other aspects of the immune response. For example, the immune response can be shifted from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants.

[0427] In one embodiment, a pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or protein to a subject elicits an immune response against one or more antigens or one or more epitopes in the subject, which can be therapeutic or partially or fully protective.

[0428] The term "antigen" relates to an agent that contains an epitope capable of generating an immune response. The term "antigen" particularly includes proteins and peptides. In one embodiment, an antigen is presented by cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. An antigen or its processing product, such as a T cell epitope, is bound in one embodiment by a T cell receptor or a B cell receptor, or by an immunoglobulin molecule such as an antibody. Thus, an antigen or its processing product can react specifically with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-related antigen such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0429] The term "disease-related antigen" is used in its broadest sense to refer to any antigen related to a disease. A disease-related antigen is a molecule that contains epitopes that stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-related antigen or its epitope can be used for therapeutic purposes. A disease-related antigen can be related to an infection by a microorganism, typically a microbial antigen, or to cancer, typically a tumor.

[0430] The term "tumor antigen" refers to components of cancer cells that can be derived from the cytoplasm, cell surface, and cell nucleus. In particular, this term refers to antigens that are produced intracellularly or as surface antigens on tumor cells.

[0431] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., that can induce an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0432] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., that can induce an immune response in an individual. A bacterial antigen can be derived from the cell wall or cytoplasmic membrane of a bacterium.

[0433] The term "epitope" refers to a part or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. The epitope of an antigen can include contiguous or non-contiguous portions of the antigen and can be about 5 to about 100 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0434] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by T cells when presented in association with MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" relate to a complex of genes that includes MHC class I molecules and MHC class II molecules and is present in all vertebrates. MHC proteins or MHC molecules are important for signal transduction between lymphocytes and antigen-presenting cells or diseased cells in the immune response, and MHC proteins or MHC molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. In the case of class II MHC / peptide complexes, the bound peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides can also be effective.

[0435] In certain embodiments of the present disclosure, the RNA encodes at least one epitope. In certain embodiments, the epitope is derived from a tumor antigen. The tumor antigen can be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen can also be a "neoantigen" that is specific to an individual's tumor and has not been previously recognized by the immune system. A neoantigen or neoepitope can result from one or more cancer-specific mutations in the genome of a cancer cell that cause an amino acid change. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, claudin-6, claudin-18.2 and claudin-12, cell surface proteins of the claudin family such as c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / melanA, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT and WT-1.

[0436] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets in the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes capable of inducing a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to the patient. Dendritic cells (DCs) present in the spleen are antigen-presenting cells particularly involved in the RNA expression of immunogenic epitopes or antigens such as tumor epitopes. The use of multiple epitopes has been shown to enhance the therapeutic effect in tumor vaccine compositions. Rapid sequencing of the tumor mutanome can provide multiple epitopes for personalized vaccines that can be encoded by the RNAs described herein, for example, as a single polypeptide in which the epitopes are optionally separated by linkers. In certain embodiments of the present disclosure, the RNA encodes at least 1 epitope, at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes or at least 10 epitopes. Exemplary embodiments include RNAs encoding at least 5 epitopes (referred to as "pentaepitopes") and RNAs encoding at least 10 epitopes (referred to as "decaepitopes").

[0437] Charge ratio The charge of the RNA lipoplex particles of the present disclosure is the sum of the charges present in at least one cationic lipid and the charges present in the RNA. The charge ratio is the ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA is calculated by the following formula: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in the RNA)]. The concentration of the RNA and the amount of at least one cationic lipid can be determined by methods commonly used by those skilled in the art.

[0438] In the first embodiment, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is from about 1.9:2 to about 1:2. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0 or about 1:2.0. In one embodiment, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is 1.3:2.0. In another embodiment, the RNA lipoplex particles described herein can have an equal number of positive and negative charges at physiological pH, resulting in RNA lipoplex particles having a net neutral charge ratio.

[0439] In the second embodiment, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is from about 6:1 to about 1.5:1. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 6.0:1.0, about 5.9:1.0, about 5.8:1.0, about 5.7:1.0, about 5.6:1.0, about 5.5:1.0, about 5.4:1.0, about 5.3:1.0, about 5.2:1.0, about 5.1:1.0, about 5.0:1.0, about 4.9:1.0, about 4.8:1.0, about 4.7:1.0, about 4.6:1.0, about 4.5:1.0, about 4.4:1.0, about 4.3:1.0, about 4.2:1.0, about 4.1:1.0, about 4.0:1.0, about 3.9:1.0, about 3.8:1.0, about 3.7:1.0, about 3.6:1.0, about 3.5:1.0, about 3.4:1.0, about 3.3:1.0, about 3.2:1.0, about 3.1:1.0, about 3.0:1.0, about 2.9:1.0, about 2.8:1.0, about 2.7:1.0, about 2.6:1.0, about 2.5:1.0, about 2.4:1.0, about 2.3:1.0, about 2.2:1.0, about 2.1:1.0, about 2.0:1.0, about 1.9:1.0, about 1.8:1.0, about 1.7:1.0, about 1.6:1.0 or about 1.5:1.0.

[0440] In RNA-based immunotherapy, targeting a distinct organ such as the pancreas is necessary to avoid autoimmune responses in other organs and potential toxicity. According to the present disclosure, RNA can target various cells, tissues or organs.

[0441] It has been found that RNA lipoplex particles having a charge ratio according to a first embodiment can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, in one embodiment, RNA accumulation and / or RNA expression occurs in the spleen after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA within the spleen. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression does not occur or essentially does not occur in the lung and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs within antigen-presenting cells, such as professional antigen-presenting cells, in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA within such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0442] It has been found that RNA lipoplex particles with a charge ratio according to the second embodiment can be used to preferentially target lung tissue or lung cells. Thus, in one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occur in the lung. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the lung. Thus, when RNA expression in a tissue other than the spleen is desired, in embodiments described herein in connection with the charge ratio according to the first embodiment, for example, a charge ratio of about 1:2 to about 1.9:2, the charge ratio according to the second embodiment, for example, a charge ratio of about 6:1 to about 1.5:1, can be used in place of the charge ratio according to the first embodiment. In these and other embodiments described herein, an RNA other than an RNA encoding a peptide or protein comprising at least one epitope, for example, an RNA encoding a pharmaceutically active peptide or protein described herein, can be used. In one embodiment, the pharmaceutically active peptide or protein is a cytokine and / or treatment of lung cancer is intended.

[0443] Composition comprising RNA lipoplex particles A. Salts and ionic strength According to the present disclosure, the compositions described herein can include salts such as sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmolality agent for preconditioning RNA before mixing with at least one cationic lipid. Certain embodiments contemplate alternative organic or inorganic salts to sodium chloride in the present disclosure. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and the sodium salt of ethylenediaminetetraacetic acid (EDTA).

[0444] Generally, a composition comprising the RNA lipoplex particles described herein preferably contains sodium chloride at a concentration in the range of 0 mM to about 500 mM, about 5 mM to about 400 mM, or about 10 mM to about 300 mM. In one embodiment, the composition comprising the RNA lipoplex particles has an ionic strength corresponding to such a sodium chloride concentration.

[0445] Generally, compositions for forming RNA lipoplex particles from RNA and liposomes such as those described herein, and compositions obtained from such formation, have a high sodium chloride concentration or a high ionic strength. In one embodiment, the sodium chloride is at a concentration of at least 45 mM. In one embodiment, the sodium chloride is at a concentration of about 45 mM to about 300 mM or about 50 mM to about 150 mM. In one embodiment, the composition has an ionic strength corresponding to such a sodium chloride concentration.

[0446] Generally, compositions for storing RNA lipoplex particles, for example, compositions for freezing RNA lipoplex particles such as those described herein, have a low sodium chloride concentration or a low ionic strength. In one embodiment, sodium chloride is at a concentration of 0 mM to about 50 mM, 0 mM to about 40 mM, or about 10 mM to about 50 mM. In certain embodiments, sodium chloride is at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM. In preferred embodiments, sodium chloride is at a concentration of about 20 mM, about 30 mM, or about 40 mM. In an exemplary embodiment, sodium chloride is at a concentration of 20 mM. In another exemplary embodiment, sodium chloride is at a concentration of 30 mM. In one embodiment, the composition has an ionic strength corresponding to such a sodium chloride concentration.

[0447] Generally, a composition obtained by thawing a frozen RNA lipoplex particle composition and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid has a high sodium chloride concentration or a high ionic strength. In one embodiment, sodium chloride is at a concentration of about 50 mM to about 300 mM or about 80 mM to about 150 mM. In one embodiment, the composition has an ionic strength corresponding to such a sodium chloride concentration.

[0448] B. Stabilizer The compositions described herein may include a stabilizer to avoid substantially losing the quality of the product, particularly losing substantially the RNA activity, during freezing, lyophilization or spray drying, and during storage of the frozen composition, lyophilized composition or spray dried composition. Such compositions are also referred to herein as being stable. Typically, the stabilizer is present prior to the freezing, lyophilization or spray drying process and remains in the resulting frozen preparation, lyophilized preparation or freeze-dried preparation. This can be used, for example, to protect RNA lipoplex particles during freezing, lyophilization or spray drying, and during storage of the frozen preparation, lyophilized preparation or freeze-dried preparation, to reduce or prevent aggregation, particle disintegration, RNA degradation and / or other types of damage.

[0449] In one embodiment, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

[0450] In one embodiment, the stabilizer is a monosaccharide. As used herein, the term "monosaccharide" refers to a single carbohydrate unit (e.g., simple sugar) that cannot be hydrolyzed into simpler carbohydrate units. Exemplary monosaccharide stabilizers include glucose, fructose, galactose, xylose, ribose, and the like.

[0451] In one embodiment, the stabilizer is a disaccharide. As used herein, the term "disaccharide" refers to a compound or chemical moiety formed by two monosaccharide units linked to each other via a glycosidic bond, e.g., via a 1-4 bond or a 1-6 bond. Disaccharides can be hydrolyzed into two monosaccharides. Exemplary disaccharide stabilizers include sucrose, trehalose, lactose, maltose, and the like.

[0452] The term "trisaccharide" means three sugars that are linked together to form one molecule. Examples of trisaccharides include raffinose and melezitose.

[0453] In one embodiment, the stabilizer is an oligosaccharide. As used herein, the term "oligosaccharide" refers to a compound or chemical moiety formed by 3 to about 15, preferably 3 to about 10, monosaccharide units that are linked to each other via glycosidic bonds, for example, via 1-4 bonds or 1-6 bonds, to form a linear, branched or cyclic structure. Exemplary oligosaccharide stabilizers include cyclodextrin, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. The oligosaccharide can be oxidized or reduced.

[0454] In one embodiment, the stabilizer is a cyclic oligosaccharide. As used herein, the term "cyclic oligosaccharide" refers to a compound or chemical moiety formed by 3 to about 15, preferably 6, 7, 8, 9 or 10, monosaccharide units that are linked to each other via glycosidic bonds, for example, via 1-4 bonds or 1-6 bonds, to form a cyclic structure. Exemplary cyclic oligosaccharide stabilizers include cyclic oligosaccharides that are distinct compounds such as α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin.

[0455] Other exemplary cyclic oligosaccharide stabilizers include compounds that contain a cyclodextrin moiety within a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. The cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to a dicarbonyl form. As used herein, the term "cyclodextrin moiety" refers to a cyclodextrin (e.g., α, β or γ-cyclodextrin) radical that is incorporated into or is part of a larger molecular structure, such as a polymer. The cyclodextrin moiety can be linked directly or via any linker to one or more other moieties. The cyclodextrin moiety can be oxidized or reduced, for example, oxidized to a dicarbonyl form.

[0456] Carbohydrate stabilizers, such as cyclic oligosaccharide stabilizers, can be derivatized carbohydrates. For example, in one embodiment, the stabilizer is a derivatized cyclic oligosaccharide, such as a derivatized cyclodextrin, such as 2-hydroxypropyl-β-cyclodextrin, such as a partially etherified cyclodextrin (e.g., a partially etherified β-cyclodextrin).

[0457] Exemplary stabilizers include polysaccharides. As used herein, the term "polysaccharide" refers to a compound or chemical moiety formed by at least 16 monosaccharide units that are linked to each other via glycosidic bonds, for example, via 1 to 4 bonds or 1 to 6 bonds, to form a linear, branched, or cyclic structure, and includes polymers that contain a polysaccharide as part of their backbone structure. In the backbone, the polysaccharide can be linear or cyclic. Exemplary polysaccharide stabilizers include glycogen, amylose, cellulose, dextran, maltodextrin, and the like.

[0458] In one embodiment, the stabilizer is a sugar alcohol. As used herein, the term "sugar alcohol" refers to a reduction product of a "sugar" and indicates that every oxygen atom within the simple sugar alcohol molecule is present in the form of a hydroxyl group. A sugar alcohol is a "polyol". This term refers to a chemical compound containing three or more hydroxyl groups and is synonymous with another common term, polyhydric alcohol. Examples of sugar alcohols include, but are not limited to, sorbitol, mannitol, maltitol, lactitol, erythritol, glycerin, xylitol, or inositol.

[0459] According to the present disclosure, a pharmaceutical composition containing sucrose as a stabilizer is provided. Without wishing to be bound by theory, sucrose promotes cryoprotection of the composition, thereby preventing aggregation of RNA lipoplex particles and functioning to maintain the chemical and physical stability of the composition. Certain embodiments contemplate alternative stabilizers to sucrose in the present disclosure. Alternative stabilizers include, but are not limited to, trehalose, glucose, fructose, arginine, glycerin, mannitol, proline, sorbitol, glycine betaine, and dextran. In certain embodiments, the alternative stabilizer to sucrose is trehalose.

[0460] In one embodiment, the stabilizer is at a concentration of about 5% (w / v) to about 35% (w / v) or about 10% (w / v) to about 25% (w / v). In certain embodiments, the stabilizer is at a concentration of about 10% (w / v), about 11% (w / v), about 12% (w / v), about 13% (w / v), about 14% (w / v), about 15% (w / v), about 16% (w / v), about 17% (w / v), about 18% (w / v), about 19% (w / v), about 20% (w / v), about 21% (w / v), about 22% (w / v), about 23% (w / v), about 24% (w / v), or about 25% (w / v). In a preferred embodiment, the stabilizer is at a concentration of about 15% (w / v) to about 25% (w / v). In another preferred embodiment, the stabilizer is at a concentration of about 20% (w / v) to about 25% (w / v). In an exemplary embodiment, the stabilizer is at a concentration of about 25% (w / v). In another exemplary embodiment, the stabilizer is at a concentration of about 22% (w / v). In embodiments of the present disclosure, the stabilizer is sucrose or trehalose. In one embodiment of the present disclosure, the stabilizer is sucrose. In one embodiment of the present disclosure, the stabilizer is trehalose.

[0461] According to the present disclosure, the RNA lipoplex particle composition described herein has a stabilizer concentration suitable for the stability of the composition, particularly the stability of RNA lipoplex particles and the stability of RNA.

[0462] C. pH and buffer According to the present disclosure, the RNA lipoplex particle composition described herein has a pH suitable for the stability of the RNA lipoplex particles, particularly the stability of the RNA. In one embodiment, the RNA lipoplex particle composition described herein has a pH of from about 5.7 to about 6.7. In certain embodiments, the composition has a pH of about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6 or about 6.7.

[0463] According to the present disclosure, a composition comprising a buffer is provided. Without wishing to be bound by theory, the use of the buffer maintains the pH of the composition during the manufacture, storage and use of the composition. In certain embodiments of the present disclosure, the buffer may be sodium bicarbonate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) or phosphate buffered saline (PBS). Other suitable buffers may be acetic acid in salt, citric acid in salt, boric acid in salt and phosphoric acid in salt.

[0464] In some embodiments, the buffer has a pH of from about 5.7 to about 6.7. In certain embodiments, the buffer has a pH of about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6 or about 6.7. In one embodiment, the buffer is HEPES. In a preferred embodiment, HEPES has a pH of from about 5.7 to about 6.7. In certain embodiments, HEPES has a pH of about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6 or about 6.7. In an exemplary embodiment, HEPES has a pH of about 6.2.

[0465] In yet another embodiment, the buffer has a concentration of from about 2.5 mM to about 10 mM. In certain embodiments where HEPES is the buffer, the concentration of HEPES is about 2.5 mM, about 2.75 mM, 3.0 mM, about 3.25 mM, about 3.5 mM, about 3.75 mM, about 4.0 mM, about 4.25 mM, about 4.5 mM, about 4.75 mM, about 5.0 mM, about 5.25 mM, about 5.5 mM, about 5.75 mM, about 6.0 mM, about 6.25 mM, about 6.5 mM, about 6.75 mM, about 7.0 mM, about 7.25 mM, about 7.5 mM, about 7.75 mM, about 8.0 mM, about 8.25 mM, about 8.5 mM, about 8.75 mM, about 9.0 mM, about 9.25 mM, about 9.5 mM, about 9.75 mM or about 10.0 mM. In a preferred embodiment, HEPES is at a concentration of about 7.5 mM.

[0466] D. Chelating Agent Certain embodiments of the present disclosure contemplate the use of chelating agents. A chelating agent refers to a chemical compound that can form at least two coordination covalent bonds with metal ions, thereby generating a stable water-soluble complex. Without wishing to be bound by theory, in the present disclosure, chelating agents reduce the concentration of free divalent ions that would otherwise induce accelerated RNA degradation. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, sodium dithiocarbamate, penicillamine, calcium pentetate, sodium salts of pentetate, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexane tetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), bis(aminoethyl) glycol ether-N,N,N',N'-tetraacetic acid, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof. In certain embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

[0467] In some embodiments, the EDTA is at a concentration of about 0.25 mM to about 5 mM. In certain embodiments, the EDTA is at a concentration of about 0.25 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM or about 5.0 mM. In a preferred embodiment, the EDTA is at a concentration of about 2.5 mM.

[0468] E. Exemplary Compositions of the Present Disclosure In an exemplary embodiment, the composition of the RNA lipoplex particles comprises DOTMA and DOPE in a molar ratio of about 2:1 to about 1:1, RNA at a concentration of about 0.05 mg / mL encoding at least one epitope, wherein at physiological pH, the charge ratio of positive to negative charges in the RNA lipoplex particles is about 1.3:2.0, sodium chloride at a concentration of about 20 mM, sucrose at a concentration of about 22% (w / v), HEPES at a concentration of about 7.5 mM with a pH of about 6.2, and EDTA at a concentration of about 2.5 mM. In a further specific embodiment, the RNA encodes 5 epitopes or 10 epitopes.

[0469] In another exemplary embodiment, the composition of the RNA lipoplex particles comprises DOTMA and DOPE in a molar ratio of about 2:1 to about 1:1, RNA at a concentration of about 0.05 mg / mL encoding at least one epitope, wherein at physiological pH, the charge ratio of positive to negative charges in the RNA lipoplex particles is about 1.3:2.0, sodium chloride at a concentration of about 30 mM, sucrose at a concentration of about 20% (w / v), HEPES at a concentration of about 7.5 mM with a pH of about 6.2, and EDTA at a concentration of about 2.5 mM. In a further specific embodiment, the RNA encodes 5 epitopes or 10 epitopes.

[0470] F. Stability of the Compositions of the Present Disclosure As used herein, "stable" refers to a composition in which measured values of various physicochemical parameters are within defined ranges. In one embodiment, the composition is analyzed to evaluate stability according to various parameters. According to the present disclosure, stability parameters include, but are not limited to, the average diameter of RNA lipoplex particles, polydispersity index, RNA integrity, RNA content, pH, osmolality, and the number of subvisible particles. One of ordinary skill in the art will be able to measure such parameters using conventional experimental techniques and measurement means. For example, stability parameters may be evaluated using dynamic light scattering (DLS), light obscuration, spectroscopic measurement, agarose gel electrophoresis, a bioanalyzer, or any other suitable technique. In one embodiment, the bioanalyzer is an Agilent 2100 Bioanalyzer (Agilent Technologies) that can measure both RNA integrity and RNA content. In one embodiment, the bioanalyzer is a Fragment Analyzer manufactured by Advanced Analytical.

[0471] While not wishing to be bound by theory, DLS measurements are useful for analyzing parameters associated with the RNA lipoplex particles of the present disclosure. In one embodiment, DLS can be used to determine the average diameter of RNA lipoplex particles, expressed in terms of Z-avg (a measure of average particle size). In another embodiment, DLS can be used to determine the polydispersity index of RNA lipoplex particles, which indicates the size and weight distribution of the RNA lipoplex particles.

[0472] In certain embodiments, the composition is stable when the measured value of the stability parameter is within a defined range. In one embodiment of a stable composition, the RNA lipoplex particles have an average diameter that is no more than ±20%, ±10%, ±5% or ±3% different from the original average diameter (i.e., the average diameter before freezing, freeze-drying, or spray-drying and thawing or reconstitution) after storage, for example, after storage at a temperature of about -15°C to about -40°C. In one embodiment of a stable composition, the RNA lipoplex particles have an average diameter that is 20%, 10%, 5% or 3% or less compared to the original average diameter (i.e., the average diameter before freezing, freeze-drying, or spray-drying and thawing or reconstitution) after storage, for example, after storage at a temperature of about -15°C to about -40°C. In another embodiment of a stable composition, the RNA lipoplex particles have a polydispersity index that is no more than ±20%, ±10%, ±5% or ±3% different from the original polydispersity index (i.e., the polydispersity index before freezing, freeze-drying, or spray-drying and thawing or reconstitution) after storage, for example, after storage at a temperature of about -15°C to about -40°C. In one embodiment, the stable composition has no more than 6000 subvisible particles with a diameter of 10 μm or greater after storage, for example, after storage at a temperature of about -15°C to about -40°C. In one embodiment, the stable composition has no more than 600 subvisible particles with a diameter of 25 μm or greater after storage, for example, after storage at a temperature of about -15°C to about -40°C.

[0473] In one embodiment of a stable composition, the integrity of the RNA is at least 80 percent after storage, for example, after storage at a temperature of about -15°C to about -40°C.

[0474] In one embodiment, the composition is stable at a storage temperature of about -15°C to about -40°C. In certain embodiments, the composition is stable at a temperature of about -15°C, about -16°C, about -17°C, about -18°C, about -19°C, about -20°C, about -21°C, about -22°C, about -23°C, about -24°C, about -25°C, about -26°C, about -27°C, about -28°C, about -29°C, about -30°C, about -31°C, about -32°C, about -33°C, about -34°C, about -35°C, about -36°C, about -37°C, about -38°C, about -39°C or about -40°C. In preferred embodiments, the composition is stable at a temperature of about -15°C, about -20°C, about -30°C or about -40°C.

[0475] In one embodiment, the composition is stable at a temperature of about -15°C to about -40°C when the pharmaceutical composition is protected from light. In preferred embodiments, the composition is stable at a temperature of about -15°C to about -25°C when the composition is protected from light.

[0476] In one embodiment, the composition is stable at a temperature of about -15°C to about -40°C for at least 1 month to about 24 months. In certain embodiments, the composition is stable at a temperature of about -15°C to about -40°C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, at least 25 months, at least 26 months, at least 27 months, at least 28 months, at least 29 months, at least 30 months, at least 31 months, at least 32 months, at least 33 months, at least 34 months, at least 35 months or at least 36 months.

[0477] In a preferred embodiment, the composition is stable at a temperature of about -15 °C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months or at least 6 months.

[0478] In another preferred embodiment, the composition is stable at a temperature of about -20 °C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months or at least 6 months.

[0479] In yet another preferred embodiment, the composition is stable at a temperature of about -30 °C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months or at least 6 months.

[0480] In one embodiment, the composition is frozen at a temperature of about -15 °C to about -40 °C and is stable after thawing to a temperature of about 4 °C to about 25 °C (ambient temperature). In another embodiment, the composition is frozen at a temperature of about -15 °C to about -40 °C in multiple freeze-thaw cycles and is stable after thawing to a temperature of about 4 °C to about 25 °C (ambient temperature).

[0481] G. Physical state of the compositions of the present disclosure In embodiments, the compositions of the present disclosure are liquid or solid. Non-limiting examples of solids include frozen or lyophilized forms. In preferred embodiments, the composition is liquid.

[0482] Pharmaceutical compositions of the present disclosure Compositions comprising the RNA lipoplex particles described herein are useful as pharmaceutical compositions or agents for therapeutic or prophylactic treatment, or for the preparation thereof.

[0483] The particles of the present disclosure can be administered in any suitable pharmaceutical composition form.

[0484] The term "pharmaceutical composition" preferably relates to a formulation containing a therapeutically active agent together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known as pharmaceutical formulations in the art. In the context of the present disclosure, the pharmaceutical composition comprises the RNA lipoplex particles described herein.

[0485] The pharmaceutical compositions of the present disclosure preferably contain one or more adjuvants or can be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include groups of heterologous compounds such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (such as alum), bacterial products (such as Bordetella pertussis toxin) or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, chemokines. Chemokines can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-γ, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide (registered trademark) ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0486] The pharmaceutical compositions according to the present disclosure are generally applied in "pharmaceutically effective amounts" and "pharmaceutically acceptable preparations".

[0487] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredient of the pharmaceutical composition.

[0488] The term "pharmaceutically effective amount" refers to an amount that, alone or together with additional dosages, achieves a desired reaction or desired effect. In the case of the treatment of a particular disease, the desired reaction preferably relates to the inhibition of the course of the disease. This includes slowing down the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired reaction in the treatment of a disease can also be the delay or prevention of the onset of the above-mentioned disease or the above-mentioned condition. The effective amount of the particles or compositions described herein depends on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of the treatment, the type of concomitant treatment (if any), the particular route of administration, and similar factors. Accordingly, the dosage of the particles or compositions described herein can depend on such various parameters. If the patient's response is insufficient with the initial dosage, higher dosages (or substantially higher dosages achieved by different, more local routes of administration) can be used.

[0489] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0490] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0491] 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.

[0492] The term "diluent" relates to an agent that dilutes and / or thins. Further, the term "diluent" includes any one or more of fluids, liquids or solid suspensions and / or mixed media. Examples of suitable diluents include ethanol, glycerol and water.

[0493] The term "carrier" refers to a component, which can be natural, synthetic, organic or inorganic, with which the active ingredient is combined to facilitate, enhance or enable the administration of a pharmaceutical composition. The carrier used herein can be one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and in particular biocompatible polylactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure contains isotonic saline.

[0494] 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 edit. 1985).

[0495] Pharmaceutical carriers, excipients or diluents can be selected with respect to the intended route of administration and standard pharmaceutical practice.

[0496] Route of administration of the pharmaceutical composition of the present disclosure In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration can include enteral administration, including absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any method other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration.

[0497] Product In one aspect, the RNA lipoplex particles described herein are present in a pharmaceutical composition. In another aspect, the composition described herein is a pharmaceutical composition.

[0498] In one aspect, the present disclosure relates to a vial containing the pharmaceutical composition described herein. In another aspect, the present disclosure relates to a syringe containing the pharmaceutical composition described herein.

[0499] Use of the pharmaceutical composition of the present disclosure The RNA lipoplex particles described herein can be used for the therapeutic treatment or prophylactic treatment of various diseases, particularly diseases in which providing a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, providing an antigen or epitope derived from a virus can be useful for the treatment of viral diseases caused by the above virus. Providing a tumor antigen or epitope can be useful for the treatment of cancer diseases in which cancer cells express the above tumor antigen.

[0500] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease can be caused by factors derived from the outside, such as an infectious disease, or by internal dysfunctions, such as an autoimmune disease. In humans, the term "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual or similar problems in those in contact with the individual. In this broader sense, a disease sometimes includes injuries, disabilities, disorders, syndromes, infections, single symptoms, deviant behaviors, and atypical changes in structure and function, although in other situations and for other purposes, these may be considered distinguishable categories. Since many people suffer from diseases and live with them, diseases usually affect an individual not only physically but also emotionally, as they can change a person's outlook on life and personality.

[0501] In this context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject aimed at combating a condition such as a disease or disorder. This term is intended to include any range of treatments for a given condition that a subject is suffering from, for example, to relieve symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or reduce symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, and to prevent a condition, and includes the administration of a therapeutically effective compound for preventing a condition, where prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0502] The term "therapeutic treatment" relates to any treatment that improves the health state of an individual and / or extends (increases) lifespan. The above treatments can eliminate a disease in an individual, stop or delay the onset of a disease in an individual, inhibit or delay the onset of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrences in an individual who currently has or has previously had a disease.

[0503] The terms "preventive treatment" or "prophylactic treatment" relate to any treatment aimed at preventing a disease from occurring in an individual. The terms "preventive treatment" or "prophylactic treatment" are used interchangeably herein.

[0504] The terms "individual" and "subject" are used interchangeably herein. These refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse or primate) that can or is susceptible to suffering from a disease or disorder (e.g., cancer), and may or may not have a disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus include adults, the elderly, children and neonates. In embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0505] The term "patient" means an individual or subject for treatment, particularly an affected individual or subject.

[0506] In one embodiment of the present disclosure, the objective is to provide an immune response against disease cells expressing an antigen, such as cancer cells expressing a tumor antigen, and to treat diseases such as cancer diseases in which cells expressing an antigen such as a tumor antigen are involved.

[0507] A pharmaceutical composition comprising the RNA lipoplex particles described herein, which comprises RNA encoding a peptide or protein comprising one or more antigens or one or more epitopes, may be administered to a subject to induce an immune response in the subject that may be therapeutic or may be partially or fully protective against one or more antigens or one or more epitopes. One skilled in the art will understand that one of the principles of immunotherapy and vaccination is based on the fact that an immune defense response against a disease occurs by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease being treated. Thus, the pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. As such, the pharmaceutical compositions described herein are useful for prophylactic and / or therapeutic treatment of diseases in which an antigen or epitope is involved.

[0508] As used herein, "immune response" refers to an integrated bodily response to an antigen, or to a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses directed to cells expressing an antigen and characterized by presentation of the antigen by class I or class II MHC molecules. The cellular response is associated with T lymphocytes, which can be classified as helper T cells (also called CD4+ T cells) that play a central role by controlling the immune response, or killer cells (also called cytotoxic T cells, CD8+ T cells or CTLs) that induce apoptosis in infected or cancerous cells. In one embodiment, administration of the pharmaceutical compositions of the present disclosure includes stimulating an anti-tumor CD8+ T cell response against cancer cells expressing one or more tumor antigens. In certain embodiments, the tumor antigen is presented by class I MHC molecules.

[0509] The present disclosure contemplates immune responses that can be protective, defensive, prophylactic and / or therapeutic. As used herein, "inducing (or induction of) an immune response" can indicate that an immune response to a particular antigen did not exist prior to induction, or that there was a basal level of immune response to a particular antigen prior to induction and this was enhanced after induction. Thus, "inducing (or induction of) an immune response" includes "enhancing (or enhancement of) an immune response".

[0510] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0511] The term "immunization" or "vaccination" represents the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0512] In one embodiment, the present disclosure contemplates an embodiment in which the RNA lipoplex particles described herein that target spleen tissue are administered. The RNA encodes, for example, a peptide or protein that includes an antigen or epitope, as described herein. The RNA is taken up by antigen-presenting cells in the spleen, such as dendritic cells, and expresses the peptide or protein. Following any processing and presentation by the antigen-presenting cells, an immune response to the antigen or epitope occurs, and prophylactic and / or therapeutic treatment of a disease in which the antigen or epitope is involved can be provided. In one embodiment, the immune response induced by the RNA lipoplex particles described herein includes presentation of a fragment thereof, such as an antigen or epitope, by antigen-presenting cells such as dendritic cells and / or macrophages, and activation of cytotoxic T cells by this presentation. For example, the peptide or protein encoded by the RNA or a processing product thereof can be presented by major histocompatibility complex (MHC) proteins expressed on the antigen-presenting cells. The MHC-peptide complex can then be recognized by immune cells such as T cells or B cells, leading to their activation.

[0513] Accordingly, in one embodiment, the RNA in the RNA lipoplex particles described herein is delivered to the spleen and / or expressed within the spleen after administration. In one embodiment, the RNA lipoplex particles are delivered to the spleen to activate spleen antigen-presenting cells. Accordingly, in one embodiment, delivery of the RNA and / or expression of the RNA within the antigen-presenting cells occurs after administration of the RNA lipoplex particles. The antigen-presenting cells can be professional antigen-presenting cells or non-professional antigen-presenting cells. Professional antigen-presenting cells can be dendritic cells and / or macrophages, and even more preferably spleen dendritic cells and / or spleen macrophages.

[0514] Accordingly, the present disclosure relates to the RNA lipoplex particles described herein, or a pharmaceutical composition comprising the RNA lipoplex particles, for inducing or enhancing an immune response, preferably an immune response against cancer.

[0515] In a further embodiment, the present disclosure relates to the RNA lipoplex particles described herein, or a pharmaceutical composition comprising the RNA lipoplex particles, for use in the prophylactic treatment and / or therapeutic treatment of diseases involving an antigen, preferably cancer diseases.

[0516] In a further embodiment, the present disclosure is a method for delivering an antigen, or an epitope of an antigen, to an antigen-presenting cell such as a professional antigen-presenting cell in the spleen, or for expressing an antigen, or an epitope of an antigen, in an antigen-presenting cell such as a professional antigen-presenting cell in the spleen, the method comprising administering to a subject the RNA lipoplex particles described herein, or a pharmaceutical composition comprising the RNA lipoplex particles. In one embodiment, the antigen is a tumor antigen. In this aspect, the antigen, or the epitope of the antigen, is preferably encoded by the RNA in the RNA lipoplex particles.

[0517] In one embodiment, systemic administration of the RNA lipoplex particles described herein, or a pharmaceutical composition comprising the RNA lipoplex particles, results in targeting and / or accumulation of the RNA lipoplex particles or RNA in the spleen and not in the lungs and / or liver. In one embodiment, the RNA lipoplex particles release RNA and / or enter cells in the spleen. In one embodiment, systemic administration of the RNA lipoplex particles described herein, or a pharmaceutical composition comprising the RNA lipoplex particles, delivers RNA to antigen-presenting cells in the spleen. In certain embodiments, the antigen-presenting cells in the spleen are dendritic cells or macrophages.

[0518] In a further embodiment, the present disclosure relates to a method for inducing or enhancing an immune response in a subject, the method comprising administering to the subject an RNA lipoplex particle as described herein, or a pharmaceutical composition comprising the RNA lipoplex particle. In an exemplary embodiment, the immune response is against cancer.

[0519] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf foreign pathogens within the macrophage and kill the foreign pathogens by hydrolytic and oxidative attacks that result in the degradation of the pathogens. Peptides derived from the degraded proteins are displayed on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the B cell surface, leading to the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophage is a splenic macrophage.

[0520] The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells first change into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation ability. Immature dendritic cells constantly sample the surrounding environment of pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated to become mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small fragments, and when mature, present these fragments on the cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as co-receptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to the lymph nodes. Here, they function as antigen-presenting cells and also activate B cells by presenting antigens together with helper T cells and killer T cells, as well as non-antigen-specific co-stimulatory signals. Thus, dendritic cells can actively induce immune responses related to T cells or B cells. In one embodiment, the dendritic cells are spleen dendritic cells.

[0521] The term "antigen-presenting cell" (APC) is one of various cells that can display, acquire, and / or present at least one antigen or antigenic fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0522] The term "professional antigen-presenting cell" relates to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When a T cell interacts with an MHC class II molecule complex on the membrane of an antigen-presenting cell, the antigen-presenting cell produces co-stimulatory molecules that induce activation of the T cell. Professional antigen-presenting cells include dendritic cells and macrophages.

[0523] The term "non-professional antigen-presenting cell" relates to antigen-presenting cells that do not constitutively express MHC class II molecules but express them upon stimulation by certain cytokines such as interferon γ. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0524] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells such as antigen-presenting cells.

[0525] The term "antigen-involved disease" or "epitope-involved disease" refers to any disease in which an antigen or epitope is involved, e.g., a disease characterized by the presence of an antigen or epitope. The disease in which an antigen or epitope is involved can be an infectious disease, or a cancer disease or simply cancer. As described above, the antigen can be a disease-related antigen such as a tumor-associated antigen, a viral antigen, or a bacterial antigen, and the epitope can be derived from such an antigen.

[0526] The term "infectious disease" refers to any disease that can be transmitted from individual to individual or from organism to organism and is caused by a microbial agent (e.g., the common cold). 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, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza and influenza.

[0527] The term "cancer disease" or "cancer" typically refers to or represents a physiological state of an individual characterized by disordered cell growth. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas and leukemias. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma or uveal melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancers of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancers of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic cancer, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal cord tumors, gliomas, meningiomas, and pituitary adenomas. The term "cancer" according to the present disclosure also includes cancer metastases.

[0528] Combination strategies in cancer treatment may be desirable due to the resulting synergistic effects, which can be considerably more potent than the effects of single-agent therapy approaches. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. As used herein, an "immunotherapeutic agent" relates to any agent that may be involved in the activation of a specific immune response and / or one or more immune effector functions. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve a therapeutic effect on cancer cells through various mechanisms including inducing apoptosis, blocking components of signaling pathways, or inhibiting the growth of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or bind to complement proteins to bring about direct cytotoxicity known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afucosylated rituximab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72),apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), canzumab mertansine (mucin CanAg), canzumab ravtansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitaxizumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), daratumumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), drozitumab (DR5), eculizumab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH900105), figitumumab (IGF-1 receptor), flanvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gebotuzumab (IL-Ιβ), girenuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovomab (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labeluximab (CEA), lexatumumab (TRAIL-R2), revirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL-5), miratuzumab (CD74), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), nacolomab tafenatox (C242 antigen), naputumab estafenatox (5T4), namatumumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), orlaratumumab (PDGF-Ra) Omalizumab (human scatter factor receptor kinase), Oportuzumab Monatox (EpCAM), Oregovomab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pintumomab (adenocarcinoma antigen), Puriteumab (vimentin), Racotumomab (N-glycolylneuraminic acid), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrotuzumab (FAP), Siltuximab (IL-6), Tabalumab (BAFF), Tocilizumab Tetraxetan (alpha-fetoprotein), Tositumomab (CD20), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Tucotuzumab Celmoleukin (EpCAM), Ublituximab (MS4A1), Urelumab (4-1BB), Volociximab (integrin α5β1), Bortumomab (tumor antigen CTAA 16.88), Zalutumumab (EGFR) and Zanomlimab (CD4).

[0529] In one embodiment, the immunotherapeutic agent is a PD-1 axis-binding antagonist. PD-1 axis-binding antagonists include, but are not limited to, PD-1-binding antagonists, PD-L1-binding antagonists, and PD-L2-binding antagonists. Aliases of "PD-1" include CD279 and SLEB2. Aliases of "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Aliases of "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, the PD-1-binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In certain embodiments, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1-binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In a particular embodiment, the PD-L1-binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2-binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In a particular embodiment, the PD-L2-binding partner is PD-1. The PD-1-binding antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1-binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). Examples of anti-PD-1 antibodies include, but are not limited to, MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.

[0530] In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising the extracellular portion or the PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg and PD-L2-Fc), a fusion soluble receptor described in International Publication No. WO 2010 / 027827 and International Publication No. WO 2011 / 066342.

[0531] In one embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody including, but not limited to, YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab).

[0532] In one embodiment, the immunotherapeutic agent is a PD-1 binding antagonist. In another embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody. In an exemplary embodiment, the anti-PD-L1 antibody is atezolizumab.

[0533] The citation of documents and tests referred to herein is not intended as an admission that any of the above is pertinent prior art. All statements regarding the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the content of these documents.

[0534] The following description is presented to enable a person skilled in the art to make and use various embodiments. The description of specific devices, techniques, and applications is provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded the scope consistent with the claims.

Examples

[0535] (Example 1) Materials The important materials used in the experiments described below were as follows: TIFF0007692837000003.tif202153

[0536] (Example 2) Preparation of lipid mixtures DOTMA / DOPE lipid mixtures were prepared at different lipid concentrations in ethanol with a lipid molar ratio of 2:1. The solutions were prepared as follows: · Weigh the DOPE lipid. · Calculate the amount of DOTMA to maintain a 2:1 DOTMA / DOPE % molar ratio. · Weigh the DOTMA lipid. · Calculate the amount of absolute ethanol required to dissolve the lipid. · Weigh the absolute ethanol. · Dissolve the lipid in ethanol using a 37 °C water bath.

[0537] The solubility of DOPE and DOPE / DOTMA in ethanol was investigated by preparing 300 mM DOPE or 330 mM DOPE / DOTMA (66:33) ethanol solutions. The lipid was dissolved by incubating the lipid solution at 37 °C for 20 minutes. The DOPE solution was centrifuged at 17000 G for 1 hour and the DOPE concentration in the supernatant was measured by HPLC. The DOTMA / DOPE solution was filtered through a 0.22 μm PES syringe filter and the lipid concentration in the filtrate was measured by HPLC.

[0538] Additional lipid mixtures can be prepared in a similar manner by following the basic steps described in this example, provided that other lipids are used as starting materials and / or other molar ratios are calculated.

[0539] (Example 3) Preparation of liposomes Liposomes were prepared by ethanol injection as follows: Using a 1 mL syringe equipped with a 0.9×40 mm needle, while stirring at 120 rpm, a 0.2 mL ethanol solution of DOTM / DOPE or (other lipid solution) was injected into 9.8 mL of water. The liposome colloids were stirred for 30 minutes. The liposomes were either filtered through a 0.45 or 1.2 or 5 μm CA syringe filter or not filtered. The liposome colloids were stored at 4 - 8°C. Using a 50 mM intermediate step, DOTMA / DOPE lipid solutions were prepared at different total lipid concentrations from 100 to 400 mM and at a 66:33 mol% ratio.

[0540] (Example 4) Preparation of RNA Lipoplexes RNA lipoplex formulations were prepared as follows by first mixing an RNA solution (e.g., Luc - RNA solution) with an NaCl solution to pre - condense the Luc - RNA. Thereafter, the liposome colloid and the Luc - RNA - NaCl solution were mixed to form the RNA lipoplex. The RNA lipoplex formulations were incubated at room temperature for 10 minutes and stored at 4 - 8°C. For example, various RNA lipoplex formulations were prepared using an N / P ratio of 0.65. The RNA concentration in these various RNA lipoplex formulations was 0.1 mg / mL and the NaCl concentration was 50 mM. Various liposome precursors (different sizes) were used for the preparation of the RNA lipoplexes.

[0541] (Example 5) Dynamic Light Scattering Using a Nicomp instrument (PSS, Santa Barbara, USA), the liposome size and RNA lipoplex size were measured by a known method of dynamic light scattering (DLS). The liposome samples were diluted with water up to a total lipid concentration of 1 mM. The RNA lipoplex samples were diluted 1:5 using 0.9% NaCl solution. The samples were measured in 5×50 mm culture tubes (Kimble, USA).

[0542] (Example 6) Optical Mask - Dynamic Light Scattering Particle counting / measurement was performed on various liposome and RNA lipoplex formulations in the size range of 0.5 - 5 μm using an Accusizer A7000 machine (PSS, Santa Barbara, USA). Three measurements were taken with a volume of 5 mL (2.5 μL of sample / 20 mL of free particle water). The results obtained represented the average value of the particle amounts from the three measurements.

[0543] (Example 7) Small-angle X-ray scattering The internal structure parameters of various RNA lipoplex formulations prepared using various liposome precursors were measured by small-angle X-ray scattering (SAXS). SAXS is a technique that can quantify the nanoscale density differences in a sample by analyzing the elastic scattering behavior of X-rays as they pass through the material and recording those scatterings at small angles. For every RNA formulation tested, the correlation length and d-spacing parameters were calculated. RNA lipoplex formulations were prepared using an N / P ratio of 0.65, 0.1 mg / mL of RNA, and 112 mM NaCl.

[0544] (Example 8) Agarose gel electrophoresis The amount of free RNA in various RNA lipoplex samples was measured by gel electrophoresis. The measurement was performed using a 1% agarose gel containing sodium hypochlorite. The RNA lipoplex samples were diluted 1:6 using a DNA loading dye. 12 μL of the diluted RNA lipoplex sample was carefully loaded onto the agarose gel. Electrophoresis was carried out at 80 V for a running time of 40 minutes.

[0545] (Example 9) HPLC Using a Sunfire C18 2.5μm 4.6×75mm column (Waters, Massachusetts, USA) and a wavelength of 205 nm, the lipid concentration in various liposome formulations was measured by HPLC (Agilent technologies, Santa Clara, USA). Mobile phase A was a mixture of 70% methanol / 30% isopropanol / 0.1% TFA, and mobile phase B was a mixture of 55% methanol / 15% isopropanol / 30% water / 0.1% TFA. The liposome samples were either diluted with water or not diluted up to a total lipid concentration of 3 mM.

[0546] (Example 10) Cell culture: RNA transfection in dendritic cells in vitro RNA lipoplex formulations were prepared using various liposome precursors and Luc-RNA. For cell culture experiments, the RNA lipoplexes were diluted with 0.9% NaCl solution up to 0.01 mg / mL of RNA. The RNA transfection efficiency of various RNA lipoplex formulations was investigated on human dendritic cells seeded in medium or whole blood.

[0547] (Example 11) Animal model: Splenic targeting and RNA transfection in dendritic cells The transfection efficiency of various RNA lipoplex formulations was investigated in BALB / c mice. 20 μg of formulated RNA lipoplexes were injected retroorbitally, and luciferase expression in dendritic cells (spleen target) was measured 6 hours later. RNA lipoplexes were prepared using Luc-RNA and various liposome precursors, small or large liposomes obtained from crude colloids, and small and large liposomes after 0.45μm filtration, with an N / P ratio of 0.65 and 112 mM NaCl.

[0548] (Example 12) Solubility test A high-concentration DOPE-containing solution was prepared (in a supersaturated state) to test whether it could be used for ethanol injection in liposome production (next section). The results obtained from this series of solubility tests are shown in Tables 1 and 2: [Table 1] [Table 2]

[0549] According to these results, DOPE purchased from various suppliers has an equilibrium solubility of approximately 50 - 60 mM (room temperature) in ethanol. However, when preparing a co-solution with the cationic lipid DOTMA, using a DOTMA / DOPE co-solution with a molar ratio of 66:33, the equilibrium solubility increases significantly, for example, up to approximately 90 - 100 mM.

[0550] (Example 13) Production of liposomes of various sizes Using the protocol described in Example 3, liposomes were produced by ethanol injection. After ethanol injection, the filtration process was not performed. The lipid concentration in ethanol was varied while all other parameters were kept fixed. Liposome size was measured by dynamic light scattering (DLS) as described in Examples 5 and 6.

[0551] As an example, the results for liposomes obtained from a DOTMA / DOPE mixture with a molar ratio of 66:33 are shown in Table 3 (below) as well as in Figures 1 and 2. [Table 3]

[0552] As described above, the obtained size (Z-average) of the liposomes increased with the lipid concentration in the ethanol solution used for ethanol injection. Therefore, the lipid concentration in ethanol can be efficiently used to control the size of the liposomes. Interestingly, the size change was most prominent when the DOPE concentration was below and above the equilibrium solubility of DOPE in ethanol alone. When the DOPE concentration exceeded 50 mM (total lipid concentration of 150 mM), the obtained liposome size dramatically increased from less than 50 nm to over 500 nm (Figure 1). However, beyond the dissolution limit, the liposome size further increased monotonically as the lipid concentration increased.

[0553] A 0.5-μm liposome fraction was present in all liposome preparations, and this liposome fraction was high in liposomes prepared using a 300-mM lipid solution and decreased in liposomes prepared at lipid concentrations lower or higher than that. Furthermore, 0.6-μm and 0.7-μm liposome fractions were measured in liposome preparations prepared at high lipid concentrations (Figure 2). After ethanol injection of the high-concentration lipid solution, large liposomes were formed. The total amount of liposomes formed was less compared to the liposome preparations prepared at low lipid concentrations in the lipid solution. The results obtained represent the average value of the particle amounts of three measurements.

[0554] (Example 14) Production of RNA Lipoplexes from Liposomes of Various Sizes Using various liposome precursors, RNA lipoplexes were produced as described in Example 4, in which case the size of the liposomes for their formation was varied while all other parameters were kept fixed. During the dynamic light scattering (DLS) experiments described in Examples 5 and 6, the liposome size (Z-average) and polydispersity index (PDI) were determined.

[0555] Results regarding the effect of lipid concentration for liposome preparation on the RNA lipoplex size (Z-average) (and thus on the liposome precursor size) are shown in Table 4 (below) as well as the corresponding Figures 3 and 4. [Table 4]

[0556] According to this series of tests, RNA lipoplexes obtained from small liposomes were smaller than those obtained from large liposomes. RNA lipoplexes obtained from liposomes produced using a solution of 300 mM or more were approximately twice the size of those obtained from liposomes from a 150 mM stock solution. No correlation was seen between liposome size and RNA lipoplex size in any of the RNA lipoplexes prepared using large liposomes (Figure 3).

[0557] The amount of RNA-lipoplexes obtained increased with the size of the liposomes used for their formation. In formulations prepared using large liposomes, a large amount of large RNA-lipoplex particles were measured, corroborating the data obtained from dynamic light scattering measurements (Figure 4). The results obtained represent the average value of the particle amounts of three measurements.

[0558] (Example 15) Small-angle X-ray scattering (SAXS) from various lipoplexes Small-angle X-ray scattering experiments were performed as described using RNA lipoplexes obtained from liposomes from stock solutions of various concentrations. For example, RNA lipoplexes were formed from DOTMA / DOPE 2 / 1 (mol / mol) liposomes and RNA with a charge ratio of 1.3:2. Liposomes were produced by ethanol injection as described from lipid concentrations in ethanol of 100 mM, 300 mM, and 400 mM.

[0559] Diffraction curves obtained from small-angle X-ray scattering measurements of RNA lipoplexes formed using liposomes prepared at charge ratios of 4 / 1 (upper) and 1.3 / 2. The diffraction curves of liposomes for lipoplex formation obtained using mM lipid stock solutions in 400 mM, 300 mM, and 100 mM ethanol are shown in Fig. 5.

[0560] The scattering pattern contains a single Bragg peak at approximately 1 nm -1 This is a typical diffraction pattern of spleen-targeted lipoplexes using excess (negatively charged) RNA for lipoplex formation. If the lipoplexes are not negatively charged, the scattering profile is completely different. As an example, when measuring RNA lipoplexes with a charge ratio of + / - 4 / 1, much less prominent peaks were observed. Similarly, secondary peaks (although of low intensity) are also distinguishable. In fact, a general feature of the X-ray scattering profile of lipoplexes is that there can be several peaks that can be equidistant or have other spacings depending on the phase state. Here. Conversely, only a single peak is determined. Also, the peak width varies depending on the concentration of the stock solution originally used for liposome production. The higher the concentration in ethanol, the lower the peak width. The Bragg peak indicates that the lipoplexes are regularly arranged, where the repeat distance (d-spacing) is given from the peak position as follows:

Number

[0561] where q is the momentum transfer,

Number

[0562] The peak width Δq decreases as the number of repeating units in the stack increases. In the case of a liquid crystal array, the correlation length can be given as follows:

Number

[0563] For the lipoplex product here, a clear correlation can be derived between the scattering pattern, the aforementioned lipid concentration in ethanol for liposome production, and the biological activity. The peak position is invariant, but the peak width varies monotonically with the applied lipid concentration in ethanol. An increase in the lipid concentration in ethanol (resulting in an increase in liposome size) corresponds to a decrease in the peak width and thus an increase in the correlation length. At the same time, the biological activity increases with the increase in the correlation length. Therefore, the above-described lipoplex with improved activity, produced from liposomes using a high-concentration lipid stock solution in ethanol, can be identified by distinct structural features. Furthermore, the above-described lipoplex with improved activity can also be distinguished from the less active lipoplex by other methods such as asymmetric flow field-flow fractionation (AF4).

[0564] (Example 16) Transfection efficiency of RNA lipoplexes in human dendritic cells Using various liposome precursors, Luc-RNA lipoplexes were produced as described, in which case the size of the liposomes for their formation was varied (by changing the starting lipid concentration for their formation), and all other parameters were kept fixed. Subsequently, the RNA transfection efficiency of various RNA lipoplex formulations was investigated in human dendritic cells seeded in medium or whole blood.

[0565] Results showing the in vitro transfection efficiency (in human dendritic cells), determined by measuring the luciferase expression and the corresponding biological activity of various RNA lipoplexes prepared using various liposome precursors, are shown in FIG. 6.

[0566] Biological activity (in vitro RNA transfection) increases monotonically with the correlation length of the RNA lipoplex. As the correlation length increases, the population of lipid bilayers in the RNA lipoplex becomes more uniform.

[0567] (Example 17) Asymmetric flow field-flow fractionation of various lipoplexes Today, another difference in RNA lipoplexes was determined using asymmetric flow field-flow fractionation (AF4), a common and state-of-the-art method for the fractionation and separation of particles in suspension. According to AF4 theory, particles with similar properties and equal sizes should elute simultaneously.

[0568] Some results regarding AF4 measurements of lipoplexes obtained from two different types of liposomes produced from either a 150 mM stock solution in ethanol or a 400 mM stock solution in ethanol are shown in Figure 7.

[0569] In summary, the field-flow fractionation measurements demonstrate that lipoplexes derived from liposomes obtained from a 150 mM lipid concentration in ethanol are qualitatively and quantitatively different from those obtained from a 400 mM lipid concentration. The 150 mM-derived lipoplexes are smaller, but counterintuitively, they elute more slowly, indicating that there must be a qualitative difference (shape, media interaction, charge) between these two fractions. The RNA lipoplexes obtained from the 150 mM ethanol solution are smaller in size but elute more slowly. This indicates that RNA lipoplexes derived from liposomes produced using a 150 mM lipid solution in ethanol are on average smaller than those obtained from 400 mM lipids in ethanol. Even with the same size, the 150 mM-derived lipoplexes have physicochemical properties different from those of the 400 mM-derived lipoplexes. These differences in size and physicochemical properties correlate with the relatively high biological activity of the 400 mM-derived RNA lipoplexes.

[0570] (Example 18) Biological activity of RNA lipoplexes in vitro The luciferase signal, and thus the biological activity, increases monotonically with the lipid starting concentration and thus the liposome size used for RNA lipoplex formation, as determined by cell culture transfection experiments (dendritic cells) using RNA lipoplexes of various sizes encoding luciferase, prepared from liposomes as described, various lipid stocks and / or various lipid concentrations. The corresponding results are shown in FIGS. 6, 8 and 9.

[0571] In summary, RNA lipoplexes generated from high lipid concentrations for liposome production show significantly higher activity in vitro.

[0572] (Example 19) Biological activity of RNA lipoplexes in vivo RNA lipoplexes prepared using large liposomes result in significantly higher luciferase expression and corresponding biological activity, as determined by cell culture transfection experiments (dendritic cells) using RNA lipoplexes of various sizes encoding luciferase, prepared from liposomes of various sizes.

[0573] Non-limiting examples of this series of tests are shown in FIGS. 10 and 11. RNA lipoplexes prepared using large liposomes obtained from 360 mM of native colloid result in significantly more in vivo expression signal than small RNA lipoplexes obtained from 200 mM of native colloid.

[0574] (Example 20) Automated production of RNA lipoplexes For the automated batch production of RNA lipoplexes, a generally applicable procedure was developed, which is shown in Figure 12. All steps are carried out using a pre-sterilized disposable fluid path that enables safe and aseptic handling of the materials. First, the concentration of RNA is adjusted to match the liposome concentration, and NaCl is added to concentrate the RNA. Thereby, the RNA solution is adjusted to an RNA concentration that allows mixing of the same volume of RNA and liposomes. Both solutions of RNA and liposomes are transferred to large-volume syringes, both syringes are attached to a single syringe pump, and the two pistons of the syringes are driven simultaneously. After RNA lipoplex formation, a cryoprotectant solution is added to adjust the final concentration of the formulation. After filling the formulation into glass bottles, the formulation is frozen as a concentrate for long-term storage.

[0575] An important quality attribute of RNA lipoplexes is the charge ratio, which is adjusted by the mixing ratio of RNA and liposomes. An automated and scalable industrial manufacturing process for RNA lipoplexes was developed that enables efficient control of the mixing ratio. In small-scale (≤10 liters) manufacturing processes, control of the mixing of two aqueous solutions of the same volume containing liposomes and RNA is achieved by using a single perfusion pump that drives two large-dose syringes filled with RNA or liposomes simultaneously. To pump even larger volumes (≥10 liters) equally, a pumping system such as a pressure vessel, diaphragm pump, gear pump, magnetic levitation pump, or peristaltic pump is used in combination with a flow sensor having a feedback loop for online control of the flow rate and real-time adjustment.

[0576] For the automated production of RNA lipoplexes, a static mixing element is required to ensure efficient mixing of the aqueous solutions containing RNA and liposomes. Commercially available microfluidic mixing elements, including serpentine paths and embedded structures to promote mixing, as well as prototype mixing elements with equivalent structures, were found to clog during production. Therefore, these mixing elements are not suitable for the automated production of RNA lipoplexes. Y-shaped and T-shaped mixing elements with diameters ranging from 1.2 mm to 50.0 mm were found to be suitable for the automated production of RNA lipoplexes.

[0577] Methods: RNA lipoplexes were prepared by using various mixing elements (Table 1). During the production of lipoplexes, the mixing elements were observed by the operator, and deposition of materials or clogging was recorded.

[0578] Results: Clogging was observed after the preparation of 3 mL of RNA lipoplexes using a commercially available microfluidic chip (NanoAssemblr™, Precision Nanosystems, Vancouver, Canada). Similar observations were made during the testing of additional prototype microfluidic mixing elements (Table 5). Deposition, particularly clogging of the elements, was observed in any (micro)fluidic mixing element with a structure equivalent to the commercially available mixing elements, whereas it was not observed when Y-shaped or T-shaped mixing elements were used. In addition to the above-described Y-shaped mixing element with a diameter of 2.4 mm, mixing elements with larger diameters were tested. Since no limitation on the diameter of the Y-shaped mixing element was found, the above-described method is considered suitable for the preparation of RNA lipoplexes with Y-shaped mixing elements having a diameter of up to 50 mm.

Table 5

[0579] When using a Y-shaped or T-shaped mixing element, a minimum flow rate is required to achieve sufficient mixing. The preparation of RNA lipoplexes can be carried out by mixing two aqueous solutions containing RNA and liposomes by using a Y-shaped or T-shaped mixing element with an inner diameter of 1.6 - 50 mm. To ensure efficient mixing, the Reynolds number resulting from the flow rate for mixing should not be less than about 300. To ensure efficient mixing, the ratio of the flow rate to the diameter of the mixing element should not be less than about 150. When experimentally testing a Reynolds number of up to about 2100, it was found to be suitable for automated manufacturing. The data also supports that even higher flow rates are achievable. This is due to the fact that at a Reynolds number of 2100, the conditions are already in the turbulent flow mode, so similar mixing conditions are predicted even at higher flow rates.

[0580] Method: RNA lipoplexes were prepared by pumping an RNA solution and a liposome solution using a single perfusion pump. RNA lipoplex formation was carried out using representative Y-shaped mixing elements with inner diameters of 2.4 and 3.2 mm. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS) measurements. The Reynolds number resulting from the investigated combinations of the flow rate and the diameter of the mixing element was calculated theoretically using an equation (Figure 13). The ratio of the flow rate to the diameter of the mixing element was calculated by dividing the flow rate (cm 3 / min) by the diameter of the mixing element (cm). The coefficient is given as a dimensionless number.

[0581] Results: When manufacturing RNA lipoplexes using combinations of flow rates and mixing elements where the theoretically calculated Reynolds number is less than about 300, RNA lipoplexes with increased particle size and polydispersity are formed (Figure 13 and Table 6). To ensure reproducible formation of RNA lipoplexes with desired particle characteristics, the Reynolds number should be at least about 300 theoretically (Figure 13 and Figure 14 and Table 6), and the ratio of the flow rate to the diameter of the mixing element should be at least about 150. A 2.4 mm mixing element was found to enable efficient and reproducible mixing of RNA and liposomes at a wide range of flow rates (60 - 240 mL / min). Since no upper limit was found during these tests, the upper limit of the Reynolds number or the ratio of the flow rate to the diameter of the mixing element is not predicted.

Table 6

[0582] (Example 21) Effect of RNA concentration RNA lipoplexes were prepared using a Y-shaped mixing element with a representative dimension (2.4 mm). To identify the concentration range in which RNA lipoplexes can be prepared in this setup, the RNA concentration was systematically varied from 0.05 mg / mL to 0.5 mg / mL. To investigate the stability of the formed lipoplexes, the final formulation was adjusted to 0.05 mg / mL RNA, 22% sucrose, and 20 mM NaCl, and the formulation was frozen three times.

[0583] RNA lipoplex formation at RNA concentrations of 0.1 - 0.5 mg / mL during RNA lipoplex formation results in equivalent particle characteristics (Figure 15). The particle characteristics of such particles are also preserved even after being frozen three times, indicating a manufacturing process that is very robust with respect to variations in RNA concentration (Figure 16). Since there is no indication of a limit to the RNA concentration during RNA lipoplex preparation, the described setup is considered suitable for RNA lipoplex preparation at RNA concentrations up to 5 mg / mL.

[0584] The described process for the automated production of RNA lipoplexes enables the reproducible preparation of stable RNA lipoplexes with various charge ratios.

[0585] Method: To demonstrate the robustness of the semi - automated preparation of RNA lipoplexes with respect to the charge ratio, this parameter was systematically varied from 1.0:2.0 to 2.1:2.0, and from 2.0:1.0 to 5.0:1.0. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS) measurements.

[0586] Results: At charge ratios from 1.0:2.0 to 2.1:2.0, no influence of charge ratio variation on RNA lipoplex size and polydispersity was observed (Figure 17). Thus, the range from 1.0:2.0 to 2.1:2.0 is considered to result in RNA lipoplex preparations of equivalent quality. Furthermore, at charge ratios from 3.0:1.0 to 5.0:1.0, stable particles with defined size and polydispersity were formed (Figure 18).

[0587] (Example 22) Salt concentration during RNA lipoplex formation For the automated production of RNA lipoplexes with high biological activity, controlled ionic conditions during RNA lipoplex formation are required. To ensure biological activity, RNA lipoplex formation must be carried out in the presence of 45 - 300 mM NaCl. For example, other ionic compounds such as EDTA, HEPES, etc. can contribute to the ionic strength and potentially reduce the required minimum concentration of NaCl.

[0588] Method: RNA lipoplexes were automatically prepared with various concentrations of NaCl during RNA lipoplex formation. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS) measurements. Furthermore, the biological activity of the lipoplexes was investigated by measuring the luciferase signal in vitro.

[0589] Results: By adjusting the ionic strength, particle properties could be controlled. An increase in salt concentration during production induced a slight increase in particle size (Figure 19). The salt concentration was found to affect biological activity. An increase in salt concentration during production induced an increase in biological activity (Figure 20). Therefore, the NaCl concentration during RNA lipoplex formation should not be less than 45 mM NaCl.

[0590] (Example 23) Stabilization of RNA Lipoplexes To stabilize the RNA in RNA lipoplexes both in the presence and absence of cryoprotectants, buffer systems such as HEPES, acetic acid / sodium acetate, and sodium phosphate can be used in the pH range of 5.5 - 6.7 for the stabilization of RNA in the lipoplexes. The sodium carbonate system was found not to provide an equivalent stabilization effect.

[0591] Method: For investigation of the optimal pH range and testing the compatibility of various buffers including various pH ranges (HEPES pH 6.8 - 8.2, acetic acid / sodium acetate pH 3.7 - 5.6, sodium phosphate pH 5.8 - 8.0 and sodium carbonate pH 6.2 - 8.6). In the absence of cryoprotectant, RNA lipoplexes were first incubated under stress conditions (40 °C). RNA integrity was analyzed by capillary electrophoresis over 21 days. To investigate the optimal pH range in the presence of an exemplary cryoprotectant, RNA lipoplexes were incubated at 40 °C, in the presence of HEPES and sucrose, and RNA integrity was analyzed for 21 days.

[0592] Results: Equivalent results were obtained with the buffer systems HEPES, acetic acid / sodium acetate, and sodium phosphate, while the carbonate system did not provide equivalent stabilization of RNA (Figure 21). RNA integrity is dependent on the pH value of the formulation. The optimal pH range was identified to be in the range of 5.5 - 7.4. In the presence of the exemplary cryoprotectant sucrose, a pH range of 5.5 - 8.0 that provides the best stabilization of RNA was identified (Figure 22).

[0593] Divalent metal ions can originate from the RNA synthesis process, formulation excipients, or glass containers and may affect RNA stability. Disodium EDTA forms stable water-soluble complexes with alkaline earth ions and heavy metal ions. Disodium EDTA contributes to the concentration of ions present during RNA lipoplex formation, thereby reducing the concentration of NaCl required for the preparation of bioactive RNA lipoplexes. By the described process, it is possible to form RNA lipoplexes in the presence of EDTA (0 - 20 mM).

[0594] Method: RNA lipoplexes were formed in the presence of increasing concentrations of EDTA (up to 18 mM) and, after dilution, incubated at 40 °C with decreasing EDTA content (0.1 mM - 5.4 mM). RNA integrity was analyzed as an important physicochemical parameter over 21 days.

[0595] Results: It was found that forming RNA lipoplexes in the presence of high concentrations of EDTA (up to 18 mM) resulted in particle characteristics equivalent to those prepared in the presence of lower concentrations. No significant differences were seen between different groups containing 0.01% (w / v) (0.26 mM) - 0.2% (w / v) (5.2 mM) EDTA during storage (Figure 23). Since disodium EDTA contributes to the concentration of ions present during RNA lipoplex formation and can act as a scavenger for divalent metal ions that may reduce RNA integrity, the presence of an EDTA concentration of up to 20 mM during RNA lipoplex formation is considered advantageous.

[0596] (Example 24) Optimization of NaCl and cryoprotectant content During the manufacture, long-term storage, and application to patients of RNA lipoplexes, it is necessary to adjust the ionic conditions (Figure 24). The concentration of NaCl can be 45 - 300 mM during RNA lipoplex formation, 10 - 50 mM during long-term storage of RNA lipoplexes in the frozen state, and 80 - 150 mM after thawing and dilution with physiological saline.

[0597] For each NaCl concentration of ≤ 70 mM, the respective content of cryoprotectant that should not be reduced to ensure the stabilization of particle properties during multiple freezings was found. As cryoprotectants, monosaccharides and disaccharides such as glucose, sucrose, mannitol, trehalose, sorbitol, triol as glycerin, and mixtures thereof can be used at a concentration of 12.5 - 35.0% (w / v). The stabilizing effect of sorbitol is low compared to the latter compound, and arginine does not efficiently stabilize RNA lipoplexes during freezing. [Table 7] [Table 8]

[0598] Method: To identify suitable cryoprotectants, various representative compounds were investigated for cryoprotectant systems containing monosaccharides (glucose and sorbitol), disaccharides (sucrose and trehalose), amino acids (arginine, proline), triol (glycerin), and mixtures of different sugars (mannitol and sucrose) (Figs. 25 and 26). Thus, RNA lipoplexes were frozen in the presence of these compounds at increasing concentrations. To identify the minimum content of cryoprotectant at a specific concentration of NaCl, RNA lipoplexes were frozen in the presence of increasing amounts of sucrose or trehalose as representative cryoprotectants (Table 7). To determine the concentration range of cryoprotectants to be investigated in detail, the samples were first frozen once. In a third experiment, the effect of particle size stabilization was verified by freezing RNA lipoplexes up to 10 times in the presence of the cryoprotectant trehalose (Table 8).

[0599] Results: Arginine clearly destabilizes RNA lipoplexes, while other cryoprotectants are generally applicable for the stabilization of RNA lipoplexes during freezing (Figures 25 and 26). Glycerol, mannitol, and sucrose (1:1, w:w), proline, and sorbitol can be used as cryoprotectants for RNA lipoplexes. In addition to arginine, none of the additional stabilizers tested seemed suitable, indicating that a wide range of amino acids, sugars, and mixtures of such compounds are suitable for the stabilization of RNA lipoplexes during freezing. The stabilizing effect of sorbitol is low compared to the latter compound.

[0600] A detailed investigation of the required amounts of cryoprotectants at each concentration of NaCl (Table 8) revealed a direct correlation between the NaCl concentration and the required concentration of cryoprotectant after a single freeze-thaw cycle (Figures 27 and 28). With 20% (w / v) sucrose or trehalose dihydrate, acceptable preservation of particle size was seen after a single freeze-thaw cycle with 0 - 60 mM NaCl, but insufficient stabilization was seen with lower percentages of cryoprotectant (e.g., ≤15% for 60 mM NaCl; ≤10% for 40 mM NaCl; ≤5% for 20 mM NaCl). No difference was seen between sucrose and trehalose within the investigated range.

[0601] Analysis of the particle size of RNA lipoplexes after 1, 2, 3, 5, and 10 freeze-thaw cycles in the presence of the combinations of NaCl and trehalose shown in Table 8 gave the following results. At a concentration of 50 mM NaCl, ≥12.5% trehalose was sufficient for the stabilization of RNA lipoplex particle properties even after 10 freeze-thaw cycles (Figure 29), while at 70 mM NaCl, ≥12.5% was required for minimal stabilization and ≥22.5% was required for accurate preservation of particle size (Figure 30). At 90 mM NaCl, ≥15.0% trehalose was required for minimal stabilization, and accurate preservation of particle size could not be achieved even at the highest investigated concentration of 27.5% (Figure 31).

[0602] (Example 25) Combination of salt and cryoprotectant for long-term storage For long-term storage at a given temperature, the combination of NaCl and cryoprotectant listed in Table 9 should be used.

[0603] Method: To investigate the minimum content of cryoprotectant for long-term storage at -15 °C to -30 °C at a specific concentration of NaCl, RNA lipoplexes were frozen in the presence of a combination of NaCl and either sucrose or trehalose. The samples were frozen at -30 °C and then transferred to each temperature (-15 or -30 °C) for long-term storage. After the specified storage time, the samples were analyzed, and the preservation of colloidal stability was analyzed by measuring the particle size using PCS. These experiments were conducted at a total RNA concentration of 0.05 mg / mL.

[0604] Results: In the presence of up to 70 mM NaCl, the particle properties of RNA lipoplexes could be preserved during freezing, but in these experiments, additional effects contributing to the destabilization of colloidal stability could be observed. Also, for example, in the combination of 60 mM NaCl and 20% sucrose, acceptable stabilization of particle properties was confirmed after freezing, and at a storage temperature of -15 °C, the particle size of these formulations increased significantly over time (Figures 32 and 33). This effect was delayed during storage at -30 °C (Figures 34 and 35).

[0605] The stability of RNA lipoplexes at a given NaCl content depends on the amount of cryoprotectant. The amount of cryoprotectant required for stabilization increases as the salt content in the storage solution increases. This effect is independent of the type of sugar used as the cryoprotectant. The composition of RNA lipoplexes for long-term storage in the frozen state at -15 °C or -30 °C should contain the cryoprotectant content listed in Table 9. [Table 9]

[0606] (Example 26) Long-term preservation using various cryoprotectants When using 10 - 40 mM NaCl, stabilization over 9 months in the presence of 22% (w / v) monosaccharide or disaccharide is possible. These formulations can be frozen at -15 to -40 °C and stored at their respective temperatures for long-term preservation. In the presence of 12.6 - 16.8% (w / v) dextran, 10 - 30 mM NaCl is achievable. These experiments were conducted at a total RNA concentration of 0.05 mg / mL.

[0607] Method: To investigate the minimum content of typical cryoprotectants such as mixtures containing sucrose, trehalose, glucose, and dextran required for long-term preservation at -20 °C, RNA lipoplexes were frozen with a fixed cryoprotectant content in combination with various NaCl concentrations. For monomeric or dimeric molecular cryoprotectants as glucose, sucrose, and trehalose, a concentration of 22% (w / v) was adjusted. These formulations were frozen, stored at -15 to -40 °C, and long-term stability was investigated by measuring the particle size using PCS after a defined storage time.

[0608] For formulations containing a mixture of polymeric dextran, the compositions listed in Table 10 were adjusted. Samples were frozen and stored at -20 °C. After the defined storage time, the samples were analyzed, and the preservation of colloidal stability was analyzed by measuring the particle size.

[0609] Results: For all monomeric or dimeric molecular cryoprotectants investigated, a maximum NaCl concentration was found that should not be exceeded to ensure long-term stabilization of RNA lipoplexes in the frozen state. 60 and 80 mM NaCl led to rapid destabilization of the lipoplexes, but when the NaCl concentration was ≤ 40 mM when stored at -20 °C, the colloidal properties could be stored for at least 9 months (Table 10 and Figures 36 - 38). For sucrose, trehalose, and glucose, no difference in the stabilization effect was observed. For formulations containing 20 mM NaCl, when the samples were frozen and stored at -15 to -40 °C, no difference in long-term stability was observed (Figure 39).

[0610] When preparations containing dextran were investigated using even lower cryoprotectant contents (12.6 - 16.8% (w / v)), the stabilization effect was equivalent or even better when compared to monosaccharides or disaccharides (Figure 40).

Table 10

[0611] (Example 27) Lyophilization a) Freeze - thaw To determine the most effective cryoprotectant / lyoprotectant concentration, a freeze - thaw test was performed. RNA lipoplex preparations were freeze - thawed in 5 mM HEPES, 80 mM NaCl, 2.6 mM EDTA with 10%, 15%, 20%, 25% and 30% trehalose added. Particle sizes were determined before and after storage at - 20°C. Particle aggregation was observed in preparations frozen in the absence of trehalose, and their particle sizes were not determined. According to Figure 41, preparations frozen with cryoprotectant / lyoprotectant showed concentration - dependent cryoprotection, and the particle size increased as the cryoprotectant / lyoprotectant concentration decreased. At 22% w / v trehalose, only a minimal increase in particle size was observed, and Sf / Si (Sf = final size, Si = initial size) was 1.04, which is still considered acceptable as it is less than 1.3. At even lower trehalose concentrations, the Sf / Si ratio was even higher. The Sf / Si ratio obtained from the freeze - thaw test correlated with the Sf / Si ratio obtained from the same preparation after lyophilization and reconstitution.

[0612] b) Reconstitution and particle stability RNA lipoplex formulations prepared with 5 mM HEPES, 2.6 mM EDTA, NaCl concentrations from 0 mM to 80 mM, and trehalose concentrations of 10% or 22% were lyophilized. All lyophilized samples showed a good cake appearance. The samples were reconstituted to their original volume using 0.9% NaCl solution. All lyophilized RNA lipoplex formulations dissolved immediately upon reconstitution with 0.9% NaCl solution or WFI. The particle size changes of lyophilized RNA lipoplex formulations prepared in 22% trehalose were determined after reconstitution with 0.9% NaCl solution or water.

[0613] According to Figure 42, in lyophilized RNA lipoplex formulations containing trehalose, the particle size remained almost stable after lyophilization and reconstitution. When the lyophilized samples were reconstituted with 0.9% NaCl, only a slight decrease in the size of the RNA lipoplexes was observed compared to the lyophilized samples reconstituted with water. A correlation was observed between the ratio of NaCl to trehalose and particle stability. In formulations prepared with low concentrations of trehalose and high NaCl concentrations, the size of the RNA lipoplex particles increased.

[0614] c) Cell culture experiments using lyophilized RNA lipoplex formulations In vitro transfection experiments were performed with lyophilized RNA lipoplex formulations encoding luciferase prepared in 22% trehalose at various NaCl concentrations. The lyophilized samples were reconstituted with 0.9% NaCl solution.

[0615] According to Figure 43, lyophilized RNA lipoplex formulations prepared with 22% trehalose and various NaCl concentrations showed similar Luc-RNA transfection levels in dendritic cells. No correlation was seen between the NaCl concentration present in the RNA lipoplex formulation and in vitro RNA transfection. The lyophilized samples showed similar or better Luc-RNA transfection compared to the fresh RNA lipoplex control.

[0616] d) Stability test For stability tests at 4 °C, 25 °C and 40 °C (1 month and 6 months), lyophilized RNA lipoplex formulations containing 10% trehalose, 22% trehalose and 0 mM, 20 mM, 40 mM, 60 mM and 80 mM NaCl were investigated. After reconstitution to the original volume using 0.9% NaCl solution, the samples were characterized for particle size and RNA integrity (full-length RNA %).

[0617] According to Figures 44 and 45, the sizes of the various RNA lipoplexes did not change significantly over time, independent of formulation or storage temperature. Interestingly, a small amount of cryoprotectant (e.g., 10%) is sufficient to maintain particle stability in the lyophilized formulation, while a larger amount (e.g., 22%) is required for the frozen formulation. The RNA integrity (full-length RNA %) in the lyophilized samples after storage at 4 °C for 6 months varied between 94% and 100%, and for the RNA(lip) formulation stored at 25 °C it varied between 85% and 94%. However, no correlation was identified between the ratio of trehalose to NaCl concentration or storage time.

[0618] (Example 28) Preparation and testing of RNA lipoplex particles Preparation of RNA lipoplexes For the automated batch manufacture of RNA lipoplexes, all steps are performed using pre-sterilized disposable fluid paths that allow for the safe and aseptic handling of materials. First, the concentration of RNA is adjusted to match the liposome concentration, and NaCl is added to concentrate the RNA. Thereby, the RNA solution is adjusted to an RNA concentration that allows for the mixing of the same volume of RNA and liposomes. Both solutions of RNA and liposomes are transferred to large-volume syringes, both syringes are attached to a single syringe pump, and the two pistons of the syringes are driven simultaneously. After RNA lipoplex formation, the final concentration of the formulation is adjusted by adding a cryoprotectant solution. After filling the formulation into glass vials, the formulation is frozen as a concentrate for long-term storage.

[0619] An important quality attribute of RNA lipoplexes is the charge ratio, which is adjusted by the mixing ratio of RNA and liposomes. An automated and scalable industrial manufacturing process for RNA lipoplexes was developed that enables efficient control of the mixing ratio. In small-scale (≤10 liters) manufacturing processes, control of the mixing of two aqueous solutions of the same volume containing liposomes and RNA is achieved by using a single perfusion pump that simultaneously drives two large-dose syringes filled with RNA or liposomes. To pump even larger volumes (≥10 liters) equally, a pumping system such as a pressurized vessel, diaphragm pump, gear pump, magnetic levitation pump, or peristaltic pump is used in combination with a flow sensor having a feedback loop for online control of flow rate and real-time adjustment.

[0620] Automated RNA lipoplex manufacturing requires a mixing element that ensures efficient mixing of the aqueous solutions containing RNA and liposomes. Commercially available microfluidic mixing elements containing serpentine paths and embedded structures to facilitate mixing, as well as prototype mixing elements having equivalent structures, were found to clog during manufacturing. Therefore, these mixing elements are not suitable for the automated manufacture of RNA lipoplexes. Y-type and T-type mixing elements having diameters of 1.2 mm to 50.0 mm were found to be suitable for the automated manufacture of RNA lipoplexes.

[0621] 1. Composition Examples 1.1 Composition of the Formulation Formulation 1 contains the following: · Approximately 10% (or less) trehalose / sucrose · ≤10 mM NaCl · ≤7.5 mM HEPES (or histidine as a second option) · pH 6.5 or pH 6.7 · ≤3.5 mM EDTA.

[0622] Formulation 1 enables the freezing of RNA lipoplexes under buffer conditions, which allows for parenteral administration directly to patients without further dilution or the use of additional processes. Low salt content does not reduce activity. The osmolality of the formulation is suitable for direct intravenous injection.

[0623] The formulation is obtained by mixing the same volume of an RNA solution and a liposome solution in a fluid path setting where the concentration and buffer conditions are optimized. The concentration of the cationic lipid in the liposome and RNA is an accurately defined molar ratio (charge ratio) of 1.3:2. The solutions of liposome and RNA are provided under the following conditions 1.2 Composition of RNA · RNA concentration of approximately 0.3 mg / mL (however, the exact molar ratio to the cationic lipid in the liposome) · Approximately 18 mM HEPES · Approximately 18 mM EDTA*2Na*2H2O · pH 6.2 - 7.0

[0624] To compensate for the pH shift caused by acetic acid present in the liposome, the pH of the RNA drug buffer is adjusted to pH 7.0.

[0625] The concentration of RNA is adjusted to match the concentration of DOTMA in the liposome by dilution with (normal) saline. 1.3 Composition of Liposome · Approximately 0.5 mM - 0.7 mM DOTMA · Approximately 0.2 - 0.4 mM DOPE · ≤ 5 mM acetic acid (preferably 2 mM)

[0626] The addition of acetic acid extends the shelf life of the liposome.

[0627] 2. Examples of Further Suitable Buffers and Optimal pH Ranges To stabilize RNA in RNA lipoplexes both in the presence and absence of cryoprotectants, buffer systems such as HEPES, histidine, and acetic acid / sodium acetate can be used in the pH range of 5.5 - 7.0 for the stabilization of RNA in lipoplexes.

[0628] Method: To investigate the optimal pH range and test the suitability of various buffers, buffers encompassing various pH ranges (HEPES pH 6.0 - 7.2, histidine pH 5.8 - 7.0, acetate pH 5.5 - 5.8, MES pH 6.0 - 7.0) are tested. In the presence of an exemplary cryoprotectant, RNA lipoplexes are incubated under accelerated conditions (25 °C). RNA integrity is analyzed by capillary electrophoresis over 60 days. Further, in the presence of an exemplary cryoprotectant, RNA lipoplexes are incubated for up to 2 years under predicted storage conditions (-15 °C). RNA integrity is analyzed by capillary electrophoresis.

[0629] Results: For the buffer systems HEPES, histidine, acetate, and MES, equivalent results were obtained for the preservation of the particle size and polydispersity of RNA lipoplexes combined with reduced amounts of sucrose and NaCl both in the liquid (Figures 46, 47, 54, 55) and frozen states (Figures 50, 51, 58 - 60). RNA integrity is dependent on the pH value of the formulation. The optimal pH range was identified to be in the range of pH 6.5 - 7.0 (Figures 48, 49, 53, 57, and 62). All of the buffer types tested were found to efficiently preserve the adjusted pH (Figures 52, 56, and 61).

[0630] Example 2.1: HEPES, histidine, and acetate are suitable for the stabilization of RNA lipoplexes In the following tests, three buffer systems (HEPES, histidine, and acetate) were investigated for their ability to stabilize RNA lipoplexes formed using liposomes containing 5 mM acetic acid. The respective RNA lipoplexes were investigated under accelerated conditions (25 °C) and in the frozen state (-15 °C). A pH range of 5.5 - 7.0 was tested, with different pH ranges being encompassed by the respective buffer systems (HEPES pH 6.2 - 7.0, histidine pH 5.8 - 7.0, and acetate pH 5.5 - 5.8).

Table 11

[0631] Liquid storage at 25 °C (accelerated) Figures 46 and 47 demonstrate that in the presence of any buffer system, both particle size and polydispersity are well maintained during storage under accelerated conditions for all systems.

[0632] Figure 48 demonstrates that in the presence of any buffer system, the adjusted pH is well maintained during storage under accelerated conditions for all systems.

[0633] Figure 49 shows that under accelerated conditions (25 °), the best stability was found in samples containing the HEPES buffer, while a decrease in the stability of the RNA in the RNA lipoplexes was revealed for the histidine and acetate buffers.

[0634] For the HEPES buffer system, pH values above 6.2 show significantly improved stability compared to lower values. For the best stability, a pH around 6.5 - 7.0 was identified.

[0635] For histidine, a similar pH-dependence is observed. However, the overall stability is lower when compared to formulations buffered with HEPES. Acetate shows lower integrity than both other systems, and 5.8 shows better stability than pH 5.5.

[0636] Frozen storage (-15 °C) Figures 50 and 51 show the colloidal stability of samples buffered in the same system as previously investigated in the liquid state at 25°C. Figures 50 and 51 demonstrate that HEPES, histidine, and acetate are equally suitable for the preservation of the particle size and polydispersity of RNA lipoplexes in the frozen state (-15°C). Furthermore, in the presence of low concentrations of NaCl, it can be seen that low concentrations of sucrose are sufficient for the efficient preservation of the polydispersity of RNA lipoplexes in the frozen state.

[0637] Figure 52 shows the pH values of samples buffered in the same system as previously investigated in the liquid state at 25°C. It has been demonstrated that all of the buffer systems (HEPES, histidine, and acetate) can maintain the pH within the adjusted range in the frozen state (-15°C).

[0638] Figure 53 shows the stability of samples buffered in the same system as previously investigated in the liquid state at 25°C. Overall, there is no clear trend regarding the preservation of RNA integrity in the frozen state (-15°C). Specific variations may be caused by errors given by the experimental conditions. The somewhat lower integrity in the HEPES buffer system at pH 7.0 may be due to such experimental artifacts, since no degradation over time is observed in this system.

[0639] Example 2.2: MES is similarly suitable for the stabilization of RNA lipoplexes In the following tests, two of the most preferred buffer systems (HEPES and histidine) identified in the previous tests were compared with an additional buffer system (MES). RNA lipoplexes were formed using liposomes containing 2 mM acetic acid. Each RNA lipoplex was investigated under accelerated conditions (25°C) and in the frozen state (-15°C). Here, the pH range of 6.0 - 7.0, which was found to be the optimal range in the previous tests, was focused on.

Table 12

[0640] Liquid storage (accelerated) at 25°C As shown in Figure 55, in this new test as well, no differences between buffer systems were observed with respect to polydispersity and particle size during storage under accelerated conditions. MES showed behavior equivalent to histidine and HEPES.

[0641] As shown in Figure 56, all buffer systems were able to maintain the pH within the adjusted range during storage under accelerated conditions. Thus, like HEPES and histidine, MES is suitable for fixing the respective pH of the RNA lipoplex formulation.

[0642] Figure 57 shows stability data obtained from various buffer systems during storage under accelerated conditions (25 °C). HEPES and MES showed equivalent stabilization of RNA integrity, while histidine showed slightly decreased stability. The optimal pH with HEPES, MES, or histidine buffer was found to be at a pH of approximately 6.5 - 7.0.

[0643] Frozen storage (-15 °C) Figure 58 demonstrates that in the presence of any buffer system, particle size and polydispersity are well maintained upon freezing of the formulation, and in the presence of low concentrations of NaCl, low concentrations of sucrose are sufficient to efficiently stabilize the particle size of the RNA lipoplex upon freezing.

[0644] Figures 59 and 60 demonstrate that in the presence of any buffer system, particle size and polydispersity are well maintained during storage in the frozen state. Furthermore, it can be seen that in the presence of low concentrations of NaCl, low concentrations of sucrose are sufficient to efficiently stabilize the colloidal properties of the RNA lipoplex in the frozen state.

[0645] Figure 61 demonstrates that all of the investigated buffer systems (HEPES, histidine, MES) were able to maintain the pH within the adjusted range in the frozen state.

[0646] Figure 62 shows stability data obtained from various buffer systems in the frozen state. None of the buffer systems investigated (HEPES, MES, or histidine) showed equivalent stabilization of RNA integrity, and there was no clear trend indicating an optimal pH within the previously optimized pH ranges investigated.

[0647] 3. Further examples of suitable buffer concentrations For the long-term stabilization of RNA lipoplexes in both the liquid and frozen states, the buffer concentrations required for both adjusted pH stabilization and preservation of RNA integrity were optimized. To ensure pH stabilization and RNA integrity, RNA lipoplexes were formulated with 10% (w / v) sucrose and 6.5 mM NaCl. Samples were stored under accelerated conditions in the presence of an exemplary buffer system HEPES having the following concentrations: · 2.5 mM HEPES · 5.0 mM HEPES · 7.5 mM HEPES · 10.0 mM HEPES

[0648] Using the following exemplary pH values, the effect of stabilization was investigated: · pH 6.2 · pH 6.7 · pH 7.2

[0649] Method: All RNA lipoplexes were automatically prepared in a single batch, and different concentrations of HEPES were adjusted by adding cryoprotectant solutions containing different amounts of HEPES. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS) measurements. Furthermore, RNA integrity was analyzed by capillary electrophoresis and the pH was measured.

[0650] Results: All buffer concentrations tested were found to efficiently preserve the particle size and polydispersity of RNA lipoplexes in the liquid state (Figures 63, 64). Furthermore, the pH value was efficiently maintained (Figure 65), and the maintenance of RNA integrity as a function of pH was independent of the concentration of each buffer (Figure 66). As a conclusion, buffer concentrations of 2.5 mM to 10.0 mM were found to equally efficiently stabilize RNA integrity in lipoplex formulations. The optimal pH was found to be in the range of pH 6.7 - 7.2.

[0651] In the following tests, the concentration of HEPES required for pH stabilization of RNA lipoplexes was investigated. RNA lipoplexes were formed using liposomes containing 5 mM acetic acid, and various concentrations of HEPES were adjusted by using various cryoprotectant solutions. Each RNA lipoplex was investigated under accelerated conditions (25 °C). The pH range of 6.2 - 7.2 was tested.

Table 13

[0652] Liquid storage (accelerated) at 25 °C In Figures 63 and 64, it can be seen that the particle size and polydispersity of RNA lipoplexes are preserved at each pH value with any buffer concentration investigated during storage under accelerated conditions (25 °C).

[0653] Figure 65 shows the measured pH values of RNA lipoplexes. Samples were stored in the presence of 2.5 mM to 10.0 mM HEPES. It was found that a low HEPES concentration of 2.5 mM was sufficient for the stabilization of the adjusted pH volume.

[0654] Figure 66 shows the stability data obtained from various buffer concentrations. Under accelerated conditions, all buffer concentrations investigated were found to equally efficiently stabilize RNA integrity. The best stabilization of RNA integrity was seen at a pH of approximately 6.7 - 7.2. RNA lipoplexes stored at pH 6.2 showed slightly decreased RNA integrity.

[0655] 4. Examples of more suitable concentrations of NaCl and cryoprotectant The following shows an example where the adjusted ionic conditions during long-term storage of RNA lipoplexes and during application to patients can be the same. To obtain an injectable product, no dilution or other modification of the product after thawing is required. In addition to the already investigated concentration range of NaCl during storage, even lower NaCl concentrations (5 - 10 mM) are also suitable for long-term storage. Such RNA lipoplexes can be administered directly after thawing and do not require an additional dilution step.

[0656] For NaCl concentrations ≤ 10 mM, a respective content of cryoprotectant of 6.0 - 16.0% (w / v) that should not be reduced to ensure stabilization of particle properties upon multiple freezings was found.

[0657] Method: As a representative cryoprotectant, sucrose was investigated at a concentration of 10% (w / v). A detailed description of the experiment is shown in Section 2.

[0658] Results: Analyzing the particle size of RNA lipoplexes during storage in the frozen state, an exemplary concentration of 10% (w / v) sucrose in combination with an NaCl concentration ≤ 10.0 mM was found to be sufficient for efficient long-term stabilization of RNA lipoplexes. Colloidal stability in the frozen state (Figs. 50, 51, 58 - 60 and 71), and the RNA degradation rate in RNA lipoplexes stabilized by the reported formulations were confirmed by accelerated (Figs. 49, 57, 66 and 70) and freeze (Figs. 53 and 62) stability tests. Using histidine as a buffer in combination with a decrease in sugar content and a decrease in NaCl content showed results equivalent to those of this formulation (22% sucrose and 20 mM NaCl).

[0659] Example 4.1: Stabilization of RNA lipoplexes with reduced sucrose and NaCl concentrations In the following tests, the long-term stability of RNA lipoplexes in the frozen state was investigated using a combination of low concentrations of NaCl and sucrose in the presence of either HEPES or histidine as a buffer at pH 6.5.

Table 14

[0660] Liquid storage (accelerated) at 25 °C Figures 67 and 68 demonstrate that in systems with reduced NaCl and sucrose content, the particle size and polydispersity are well maintained during storage under accelerated conditions. The particle size and polydispersity are equivalent to those of the present formulation containing 22% sucrose and 20 mM NaCl.

[0661] Figure 69 demonstrates that the summarized system can maintain the pH within the adjusted range during storage under accelerated conditions. As an explanation for the slight measured pH increase, a malfunction of the pH meter used was identified.

[0662] Figure 70 shows the stability data obtained from formulations containing low concentrations of sucrose and NaCl. Compared with the present formulation containing 20 mM NaCl and 22% sucrose, the new formulation shows improved RNA integrity. Consistent with previous tests, this improved stabilization may be due to the improved pH value of 6.5. Consistent with previous tests, RNA integrity is better preserved when using HEPES compared to histidine.

[0663] Frozen storage (-15 °C) Figure 71 demonstrates that the described system can stabilize the colloidal properties as the particle size of the formulation. Any of the formulations can be frozen with minimal change in particle size.

Claims

1. RNA, and at least one cationic lipid and at least one additional lipid, an RNA-lipoplex particle comprising the same, sodium chloride at a concentration of 5 mM or more and 7.5 mM or less, a stabilizer at a concentration of 10% weight / volume percent (% w / v) or less, a buffer, comprising, wherein the at least one cationic lipid comprises 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), wherein the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and wherein the stabilizer is sucrose at a concentration of 5-10% (w / v).

2. The composition according to claim 1, wherein the sodium chloride is at a concentration of 6.5 mM or 7.5 mM.

3. The buffer is selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), histidine, acetic acid / sodium acetate, and MES (2-(N-morpholino)ethanesulfonic acid), and / or the composition has a pH of 6.0 to 7.2, and / or the buffer is present at a concentration of 2.5 mM to 10 mM, the composition according to claim 1 or 2.

4. The at least one cationic lipid comprises 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), The composition according to any one of claims 1 to 3, wherein the RNA lipoplex particles contain DOTMA and DOPE in a molar ratio of 4:1 to 1:

2.

5. The composition according to any one of claims 1 to 4, further comprising a chelating agent.

6. The composition according to claim 5, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

7. The composition according to claim 6, wherein the EDTA is at a concentration of 3.5 mM or less.

8. The composition according to any one of claims 1 to 7, wherein the RNA encodes a peptide or protein containing at least one epitope, and the ratio of positive charge to negative charge in the RNA lipoplex particles is 1:2 to 1.9:

2.

9. RNA encoding a peptide or protein containing at least one epitope, and DOTMA and DOPE in a molar ratio of 2:1, An RNA lipoplex particle comprising, The ratio of positive charge to negative charge in the RNA lipoplex particle is 1.3:2.0, and Sodium chloride at a concentration of 7.5 mM, and Sucrose at a concentration of 10% (w / v), and The pH is 6.5 or 6.7, and HEPES at a concentration of 7.5 mM, and EDTA at a concentration of 2.5 mM, and The composition according to any one of claims 1 to 8.

10. The composition according to any one of claims 1 to 9, wherein the amount of RNA in the composition is 0.01 mg / mL to 1 mg / mL.

11. The composition according to any one of claims 1 to 10, further comprising an adjuvant.

12. The composition according to any one of claims 1 to 10, which is for a liquid state, a frozen state or a lyophilized RNA lipoplex formulation.

13. The composition in a frozen state according to claim 12, which is stable at a temperature of -15°C for at least one month.

14. The composition in a liquid state according to claim 12, which is an aqueous composition.

15. The composition in a liquid state according to claim 14, wherein the aqueous composition can be directly administered to a subject.

16. The composition is a pharmaceutical composition, and / or The composition according to any one of claims 1 to 15, wherein the composition is formulated for systemic administration.

17. The systemic administration is by intravenous administration, and / or The composition according to claim 16, wherein the composition is for therapeutic use.

18. A method for preparing a liquid composition for direct administration to a subject, comprising RNA lipoplex particles, comprising: (a) thawing the composition in a frozen state according to claim 12, or (b) dissolving the lyophilized RNA lipoplex formulation according to claim 12.

19. The method according to claim 18, wherein the liquid composition is an aqueous composition.

Images