Preparation and storage of liposomal RNA formulations suitable for treatment.

Abstract

The present invention provides formulations for delivering biologically active RNA to target tissues, such that the delivered RNA is efficiently translated into the peptide or protein it encodes. [Solution] A composition is provided comprising RNA lipoplex particles containing RNA, at least one cationic lipid, and at least one additional lipid; sodium chloride at a concentration of about 10 mM or less; a stabilizer at a concentration greater than about 10% by weight / volume (%w / v) and less than about 15% by weight / volume (%w / v); and a buffer.

Description

[Technical Field]

[0001] This disclosure relates to methods for preparing RNA lipoplex particles for delivering RNA to target tissue after parenteral administration, particularly after intravenous administration, and to compositions comprising such RNA lipoplex particles. This disclosure also relates to methods enabling the preparation of RNA lipoplex particles in a manner compliant with industrial GMP. Furthermore, this 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. The RNA lipoplex particle formulations described herein can be frozen or dehydrated by lyophilization, spray drying, or related methods, which allow for a longer 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 cells of the target tissue, and the RNA can be translated into the encoded peptide or protein to exhibit its physiological activity. The peptide or protein of interest may be a peptide or protein comprising one or more epitopes to induce or enhance an immune response to one or more epitopes. The methods and compositions described herein are suitable for use in a manner that conforms to the requirements of pharmaceuticals, more specifically, to the requirements of GMP-compliant manufacturing and the quality requirements of pharmaceuticals for parenteral administration. [Background technology]

[0002] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. Advantages of using RNA include transient expression and non-transforming properties. RNA does not need to enter the nucleus to be expressed and cannot be integrated into the host genome, thus eliminating the risk of cancer.

[0003] RNA can be delivered by so-called lipoplex formulations, in which RNA binds to liposomes composed of a mixture of cationic lipids and helper lipids to form injectable nanoparticle formulations. However, there is still an unmet need to develop formulations that deliver biologically active RNA to target tissues even after the formulation has been stored. Furthermore, there is still an unmet need to develop methods for manufacturing injectable RNA lipoplex particle formulations in compliance with GMP, which will result in a long shelf life. [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, there is a need to provide formulations 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 formulations with good storage stability without substantially losing product quality, and in particular without substantially losing the biological activity of the RNA. [Means for solving the problem]

[0005] To our surprise, the inventors have discovered that the RNA lipoplex particle formulation described herein satisfies the above requirements.

[0006] I. Method for preparing RNA lipoplex particles, RNA lipoplex particles, and compositions containing RNA lipoplex particles In a first aspect, the disclosure relates to a method for preparing RNA lipoplex particles with improved biological activity, RNA lipoplex particles prepared according to the disclosure, and compositions comprising such RNA lipoplex particles. RNA lipoplex particles and compositions comprising RNA lipoplex particles are useful for delivering RNA to target tissues after parenteral administration, particularly after intravenous administration. 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 approximately 1 nm in size. -1 A single Bragg peak was observed with a peak width of 0.2 nm. -1 It is characterized by a specific pattern in X-ray scattering that is smaller than [a certain value].

[0007] In one embodiment, liposomes and RNA lipoplex particles contain 1,2-di-O-octadecenyl-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 alone has a solubility of about 50 mM, and together with DOTMA, it has a solubility of 100 mM or more. The lipid solution for forming liposomes that yield highly active lipoplexes may have a total lipid concentration of 270 mM or more (e.g., 90 mM or more of DOPE). By increasing the temperature, even higher concentrations of ethanol solutions can be obtained. Liposomes obtained from a lipid solution where the concentration of DOPE is above the equilibrium solubility are significantly larger than liposomes obtained from a lipid solution where the concentration of DOPE is below the equilibrium solubility. Liposome size increases monotonically with increasing concentration in ethanol.

[0008] Liposomes prepared according to this disclosure can be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the RNA is incubated with NaCl before mixing to adjust the specific ionic strength required for increased lipoplex activity. Lipoplexes formed from these large liposomes exhibit significantly increased biological activity, as demonstrated by in vitro and in vivo experiments. These highly active lipoplexes can be clearly distinguished from less active lipoplexes by certain physicochemical parameters, such as (i) a decrease in the peak width of the Bragg peak, and (ii) different separation profiles in dispersed analytical methods for sizing, such as field flow fractionation. Less active lipoplexes are, on average, smaller. Furthermore, they also have different elution profiles, which may be related to parameters such as molecular three-dimensional structure, shape, and interaction with the bulk phase.

[0009] Accordingly, in this embodiment, the present disclosure relates to a method for producing liposome colloids, comprising injecting an ethanol solution of lipids into an aqueous phase to produce liposome colloids, 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 includes heating the lipid solution to increase the concentration of lipids 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 a single lipid 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 from about 180 mM to about 600 mM, from 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-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0017] In one embodiment, the 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, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0019] In one embodiment, the molar ratio of the at least one cationic lipid to the 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 speed of the aqueous phase of from 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 comprises acetic acid, for example, in an amount of about 5 mM.

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

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

[0027] The present disclosure further relates to 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 the liposome colloid, wherein the concentration of DOTMA and DOPE in the lipid solution is about 330 mM.

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

[0029] The present disclosure further relates to a liposome colloid obtainable by the 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 ranging from 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 liposome comprises at least one cationic lipid and at least one additional lipid.

[0035] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (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-trimethylammoniumpropane (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 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.

[0039] In one embodiment, the liposome contains DOTMA and DOPE in molar ratios of approximately 10:0 to 1:9, 4:1 to 1:2, 3:1 to 1:1, or 2:1.

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

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

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

[0043] This disclosure further relates to compositions comprising RNA lipoplex particles that can be obtained as described above.

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

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

[0046] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, At least one cationic lipid and at least one additional lipid, A composition comprising RNA lipoplex particles containing, The ratio of positive to negative charges in RNA lipoplex particles is approximately 1:2 to 1.9:2, or approximately 1.3:2.0. RNA lipoplex particles are approximately 1 nm in size. -1 It features a single Bragg peak with a peak width of 0.2 nm. -1Regarding compositions smaller than this.

[0047] In one embodiment, the composition further comprises sodium chloride at concentrations 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 buffering agent.

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

[0050] In one embodiment, the RNA lipoplex particles described in this embodiment 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 comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0053] 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).

[0054] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one additional lipid comprises 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 contain DOTMA and DOPE in molar ratios of approximately 10:0 to 1:9, approximately 4:1 to 1:2, approximately 3:1 to approximately 1:1, or approximately 2:1, and the charge ratio of positive charge in DOTMA to negative charge in RNA is approximately 1:2 to 1.9:2.

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

[0058] In one embodiment, the EDTA concentration is approximately 0.25 mM to approximately 5 mM, or approximately 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, systemic administration is performed by intravenous administration.

[0062] This disclosure further relates to compositions described for therapeutic use.

[0063] II. Method for preparing RNA lipoplex particles in accordance with industrial GMP. In a second aspect, the disclosure relates to a method for preparing RNA lipoplex particles in a manner compliant with industrial GMP.

[0064] In one embodiment of this disclosure, a fluid pathway system is used for GMP-compliant manufacturing of pharmaceutical RNA lipoplex particle products, thereby enabling precise control of the RNA-liposome mixing ratio, which is critical to product quality. In one embodiment, the fluid pathway includes mixing a liposome solution and an RNA solution in a 1:1 (volume / volume) manner, where the component concentrations are selected to precisely maintain the intended charge ratio. In one embodiment, the RNA is incubated with NaCl before mixing to adjust the specific ionic strength required for lipoplex activity. In one embodiment, a Y-type mixing setup based entirely on disposable materials is realized. Fluid dynamics are optimized to maintain particle properties and avoid clogging. In contrast, commercially available microfluidic devices tend to clog after a while, making GMP compliance impossible.

[0065] In one embodiment, lipoplex is 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 the other containing RNA, are preferentially inserted in parallel into the same pump within the syringe pump. The pistons of both pumps are moved forward by the same drive mechanism, thereby precisely controlling the relative volume to be mixed. Under selected process conditions, identical syringes are used for both solutions, thereby enabling precise 1:1 (v / v) mixing conditions. The ratio of RNA to liposomes (cationic lipids) is precisely controlled by adjusting the concentrations of the two solutions before mixing.

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

[0070] In one embodiment, the flow is characterized by a Reynolds number greater than 300, or between approximately 500 and approximately 2100.

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

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

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

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

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

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

[0077] In one embodiment, the method involves combining fluids from two tubes or hoses and using a mixing element in which there is no internal static mixing element, such as a Y-shaped or T-shaped mixing element, which is, for example, a splitting and recombining element, an alternating herringbone channel, a zigzag channel or a twisted channel, or a three-dimensional meandering path. The mixing element may have a diameter of 1.2 mm to 50.0 mm.

[0078] In one embodiment, the method involves using a device in which two syringes, one containing a solution containing cationic liposomes and the other containing a solution containing RNA, are inserted in parallel into the same or two holders, and the piston of the device is drawn out by one or two precision actuators. In one embodiment, the method involves using a syringe pump in which two syringes, one containing a solution containing cationic liposomes and the other containing a solution containing RNA, are inserted in parallel into the same pump.

[0079] In one embodiment, the method includes using a pressurized vessel, membrane pump, gear pump, magnetic levitation pump, peristaltic pump, HPLC / FPLC pump, or any other piston pump, optionally in combination with a flow sensor that optionally has a feedback loop for online control and real-time adjustment of the 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 at 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 at 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 has about 1 nm -1 characterized by a single Bragg peak at , wherein the peak width is 0.2 nm -1 less than .

[0084] In one embodiment, the RNA lipoplex particles have an average diameter ranging from 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 comprise 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 relates to an RNA encoding a peptide or protein comprising at least one epitope, at least one cationic lipid and at least one additional lipid, a composition comprising RNA lipoplex particles comprising the above, 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, the RNA lipoplex particles are characterized by a single Bragg peak at about 1 nm -1 and the peak width is 0.2 nm -1 less than .

[0089] In one embodiment, the composition further comprises sodium chloride at concentrations 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.

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

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

[0092] In one embodiment, the RNA lipoplex particles described in this embodiment under Section II 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.

[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-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (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-di-O-octadecenyl-3-trimethylammoniumpropane (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 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.

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

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

[0100] In one embodiment, the EDTA concentration is approximately 0.25 mM to approximately 5 mM, or approximately 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, systemic administration is performed by intravenous administration.

[0104] This disclosure further relates to compositions described for therapeutic use.

[0105] III. Methods and compositions for preserving RNA lipoplex particles In a third aspect, the disclosure relates to methods and compositions for preserving RNA lipoplex particles without substantially losing the quality of the product, and in particular without substantially losing RNA activity. In particular, the 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, and in particular 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, which allows for a longer shelf life of the product compared to liquid storage.

[0107] To enable freezing, a stabilizer (freeze-protective agent) is added. In one embodiment, the lipoplex is diluted with a stabilizer (freeze-protective agent) after preparation, thereby adjusting the ionic strength, preferably by reducing it, and allowing for adjustment of the appropriate concentration of the stabilizer. For freezing of the product, the stabilizer concentration may be higher than the value required to obtain the physiological gravimetric 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 gravimetric osmolality and ionic strength. As stabilizers, sugars such as glucose, sucrose, or trehalose, as well as other compounds such as dextran, can be used.

[0108] Surprisingly, this disclosure reveals that RNA lipoplex formulations containing the stabilizers described herein can also be lyophilized. In the case of lyophilization, the required stabilizer (lyophilization protective agent) concentration can be lower than in the case of freezing, and the acceptable NaCl concentration (ionic strength) can be higher than in the case of freezing. If large-scale economic 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 preservation in the bulk phase. The optimal pH is approximately 6.2, with a preferred range of approximately 5.7 to 6.7. In other formulations, the ideal pH may be even lower. The local pH within the RNA lipoplex is assumed to be higher than the bulk phase pH due to the positive charge of cationic lipids.

[0110] In embodiments of this disclosure in which the RNA lipoplex composition is frozen for storage, the composition may be thawed, and optionally the osmolality by weight, ionic strength, and / or pH of the composition may be adjusted by adding an aqueous solution. The resulting composition may be administered to a subject.

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

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

[0113] In one embodiment, freezing occurs at a temperature of approximately -15°C to approximately -40°C, or approximately -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 containing RNA lipoplex particles and a stabilizer includes providing an aqueous composition containing RNA lipoplex particles and adding a stabilizer to the aqueous composition containing RNA lipoplex particles. Therefore, a method for preparing a composition for freezing includes providing an aqueous composition containing RNA lipoplex particles and adding a stabilizer to the aqueous composition containing RNA lipoplex particles.

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

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

[0119] In one embodiment, the concentration of the stabilizer in an aqueous composition containing RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, and in particular, to avoid substantial loss of RNA activity after the composition has been stored 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 containing 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).

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

[0122] In one embodiment, the aqueous composition containing RNA lipoplex and a 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 at about 10 mM to about 50 mM.

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

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

[0125] In one embodiment, a method for preparing a frozen composition further includes storing the frozen composition containing RNA lipoplex particles. The composition may 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 disclosure further relates to a composition comprising RNA lipoplex particles that can be obtained by the above-described method for preparing a frozen composition. The disclosure also relates to a composition comprising RNA lipoplex particles that can be obtained by the above-described method for preparing a composition for freezing.

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

[0128] In one embodiment, the RNA encodes a peptide or protein containing at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particle is 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] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1:2 to approximately 1.9:2, or approximately 1.3:2.0, Sodium chloride at concentrations of 0 mM to approximately 40 mM, This relates to a composition containing a stabilizer.

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

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

[0133] In one embodiment, the sodium chloride concentration is approximately 20 mM to approximately 30 mM.

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

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

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

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

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

[0141] In one embodiment, the sucrose concentration is approximately 20% (w / v) to approximately 25% (w / v).

[0142] In one embodiment, the sucrose concentration is approximately 22% (w / v).

[0143] In one embodiment, the sucrose concentration is approximately 20% (w / v).

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

[0145] In one embodiment, the composition has a pH of 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 concentration of HEPES is approximately 2.5 mM to approximately 10 mM, or approximately 7.5 mM.

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

[0149] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, at a concentration of approximately 0.05 mg / mL, and DOTMA and DOPE are used in a molar ratio of approximately 2:1. RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1.3:2.0, Sodium chloride at a concentration of approximately 20 mM, Sucrose at a concentration of approximately 22% (w / v), HEPES at a concentration of approximately 7.5 mM with a pH of approximately 6.2, This relates to a composition containing EDTA at a concentration of approximately 2.5 mM.

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

[0151] In one embodiment, the frozen composition is stable at a temperature of approximately -15°C to approximately -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 approximately -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 for at least two months at a temperature of about -15°C.

[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 for at least two months at a temperature of about -20°C.

[0156] In one embodiment, the frozen composition is stable at a temperature of approximately -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 for at least two months at a temperature of approximately -30°C.

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

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

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

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

[0162] The disclosure further relates to a method for preparing a dehydrated, for example, lyophilized or spray-dried composition containing RNA lipoplex particles, comprising: (i) providing an aqueous composition comprising RNA lipoplex particles and a stabilizer; and (ii) dehydrating the composition, for example, 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 comprises providing an aqueous composition comprising RNA lipoplex particles and adding a stabilizer to the aqueous composition comprising RNA lipoplex particles. Therefore, a method for preparing a composition for dehydration, such as freeze-drying or spray-drying, comprises 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 containing RNA lipoplex particles, the ionic strength of the aqueous composition containing RNA lipoplex particles decreases.

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

[0167] In one embodiment, the concentration of the stabilizer in an aqueous composition containing RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, and in particular, to avoid substantial loss of RNA activity after the composition has been stored for at least one month, at least six months, at least twelve months, at least twenty-four months, or at least thirty-six months.

[0168] In one embodiment, the concentration of the stabilizer in the aqueous composition containing 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 containing RNA lipoplex and a stabilizer is lower than the normal pH that is optimal for RNA preservation.

[0170] In one embodiment, the aqueous composition comprising RNA lipoplex and a stabilizer contains 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 RNA lipoplex and a stabilizer has an ionic strength corresponding to sodium chloride concentrations 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 method described above under I. and II.

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

[0174] The disclosure further relates to compositions comprising RNA lipoplex particles that can be obtained by the above-described methods for preparing a dehydrated, for example, lyophilized or spray-dried composition. The disclosure also relates to compositions comprising RNA lipoplex particles that can be obtained by the above-described methods for preparing a composition for dehydration, for example, lyophilization or spray-drying.

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

[0176] In one embodiment, the RNA encodes a peptide or protein containing at least one epitope, and the ratio of positive to negative charges in the RNA lipoplex particle 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] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1:2 to approximately 1.9:2, or approximately 1.3:2.0, Sodium chloride at concentrations of 10 mM to approximately 80 mM, This relates to a composition containing a stabilizer.

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

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

[0181] In one embodiment, the sodium chloride concentration is approximately 20 mM to approximately 30 mM.

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

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

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

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

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

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

[0190] In one embodiment, the trehalose concentration is approximately 10% (w / v).

[0191] In one embodiment, the trehalose concentration is approximately 15% (w / v).

[0192] In one embodiment, the composition has a pH lower than the normal pH that is 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 concentration of HEPES is approximately 2.5 mM to approximately 10 mM, or approximately 7.5 mM.

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

[0197] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, at a concentration of approximately 0.05 mg / mL, and DOTMA and DOPE are used in a molar ratio of approximately 2:1. RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1.3:2.0, Sodium chloride at a concentration of approximately 20 mM, Trehalose at a concentration of approximately 10% (w / v), HEPES at a concentration of approximately 7.5 mM with a pH of approximately 6.2, This relates to a composition containing EDTA at a concentration of approximately 2.5 mM.

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

[0199] In one embodiment, a dehydrated, for example, lyophilized or freeze-dried composition is stable for at least one month, at least six months, at least twelve months, at least twenty-four months, or at least thirty-six months. In one embodiment, the composition is stored at a temperature above 0°C, for example, about 25°C or about 4°C, or for example, at room temperature.

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

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

[0202] This disclosure further relates to aqueous compositions comprising RNA lipoplex particles, which can be obtained by reconstituting the above-described dehydrated, for example, lyophilized or freeze-dried compositions and optionally adjusting the gravimetric osmolality and ionic strength by adding an aqueous liquid.

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

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

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

[0206] In one embodiment, the RNA lipoplex particles described in this embodiment under III. are approximately 1 nm in size. -1 It is characterized by a single Bragg peak, where the peak width is 0.2 nm. -1 It is smaller than that.

[0207] In one embodiment, the RNA lipoplex particles described in this embodiment under III. 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.

[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-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (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-trimethylammoniumpropane (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 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.

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

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

[0215] In one embodiment, the EDTA concentration is approximately 0.25 mM to approximately 5 mM, or approximately 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 performed by intravenous administration.

[0219] This disclosure further relates to compositions described for therapeutic use.

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

[0221] In one embodiment, an aqueous liquid is added to obtain a composition with a weight osmolality of approximately 200 mOsmol / kg to approximately 450 mOsmol / kg.

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

[0223] Several RNA lipoplex formulations described herein, suitable for preserving RNA lipoplex particles without substantially losing product quality, 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 gravimetric osmolality and ionic strength before administration. Such RNA lipoplex formulations can be administered directly after storage of the product, and optionally after thawing or reconstitution. In embodiments of this disclosure in which the RNA lipoplex composition is frozen for storage, the composition can be thawed and administered without the need to adjust the gravimetric osmolality, ionic strength, and / or pH of the composition.

[0224] Therefore, this disclosure is, RNA, and At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, Sodium chloride at a concentration of approximately 10 mM or less, A stabilizer at a concentration of approximately 10% by weight / volume (%w / v) or less, This relates to a composition containing a buffering agent.

[0225] In one embodiment, the sodium chloride concentration is about 5 mM to about 10 mM. In another embodiment, the sodium chloride concentration is about 7.5 mM or less, for example, about 5 mM to about 7.5 mM. In yet another embodiment, the sodium chloride concentration is about 6.5 mM or about 7.5 mM.

[0226] In one embodiment, the concentration of salts and / or stabilizers in the composition is approximately the value required for physiological osmolality. In one embodiment, the osmolality resulting from the dissolved components, including ionic and nonionic components, is approximately 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 another embodiment, the concentration of the stabilizer in the composition is about 5 to about 7.5% (w / v). In yet another 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, the 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, the 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, acetate / 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 approximately 6.5 or 6.7.

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

[0233] In one embodiment, the buffer is HEPES with a pH of about 6.5 or about 6.7 and 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.

[0234] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (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-di-O-octadecenyl-3-trimethylammoniumpropane (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 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.

[0236] In one embodiment, the RNA lipoplex particles contain DOTMA and DOPE in molar ratios of approximately 10:0 to 1:9, approximately 4:1 to 1:2, approximately 3:1 to approximately 1:1, or approximately 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 about 0.25 mM to about 3.5 mM or 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 containing at least one epitope, and the ratio of positive charge to negative charge in the composition is about 1:2 to about 1.9:2, or about 1.3:2.0.

[0239] This disclosure will be furthered, RNA encoding a peptide or protein containing at least one epitope, DOTMA and DOPE are used in a molar ratio of approximately 2:1. RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge in the composition is approximately 1.3:2.0, Sodium chloride at a concentration of approximately 7.5 mM, Sucrose at a concentration of approximately 10% (w / v), HEPES at a concentration of approximately 7.5 mM with a pH of approximately 6.5 or 6.7, This relates to a composition containing EDTA at a concentration of approximately 2.5 mM.

[0240] In one embodiment of the compositions 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 compositions 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 compositions described herein, the composition further comprises an adjuvant.

[0243] In one embodiment of the compositions 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 that is stable for at least one month at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least two months at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least four months at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least six months at a temperature of about -15°C.

[0245] This 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] This 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, a composition, in particular a 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 performed by intravenous administration.

[0252] This disclosure further relates to compositions described herein for therapeutic use.

[0253] This disclosure further relates to a method for preparing a liquid composition for direct administration to a subject, comprising RNA lipoplex particles, comprising thawing a frozen composition as described herein. This disclosure further relates to a method for preparing a liquid composition for direct administration to a subject, comprising RNA lipoplex particles, comprising dissolving a dehydrated composition as 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 composition. In the therapeutic application described herein, the liquid composition described herein is administered to a subject.

[0254] Therefore, this disclosure is, RNA, and At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, Sodium chloride at a concentration of approximately 10 mM or less, A stabilizer with a concentration exceeding approximately 10% by weight / volume (%w / v) and less than approximately 15% by weight / volume (%w / v), This relates to a composition containing a buffering agent.

[0255] In one embodiment, the sodium chloride concentration is approximately 5 mM to approximately 10 mM. In another embodiment, the sodium chloride concentration is approximately 8.5 mM or less, for example, approximately 5 mM to approximately 8.5 mM. In yet another embodiment, the sodium chloride concentration is approximately 8.2 mM.

[0256] In one embodiment, the concentration of salts and / or stabilizers in the composition is approximately the value required for physiological osmolality. In one embodiment, the osmolality resulting from the dissolved components, including ionic and nonionic components, is approximately the value required for physiological osmolality.

[0257] In one embodiment, the concentration of the stabilizer in the composition is approximately 11 to 14% (w / v). In another embodiment, the concentration of the stabilizer in the composition is approximately 12 to 14% (w / v). In yet another embodiment, the concentration of the stabilizer in the composition is approximately 13% (w / v).

[0258] 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.

[0259] In one embodiment, the stabilizer is sucrose or trehalose. In one embodiment, the stabilizer is sucrose at a concentration of about 12-14% (w / v). In one embodiment, the sucrose is at a concentration of about 13% (w / v). In one embodiment, the stabilizer is trehalose at a concentration of about 12-14% (w / v). In one embodiment, the trehalose is at a concentration of about 13% (w / v).

[0260] In one embodiment, the buffer is selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), histidine, acetate / 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.

[0261] In one embodiment, the composition has a pH of 6.0-7.5, 6.5-7.5, 6.5-7.3, 6.5-7.2, 6.7-7.2, or 6.5-7.0. In one embodiment, the composition has a pH of approximately 6.7.

[0262] 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 7.5 mM. In another embodiment, the buffer is present at a concentration of 4 mM to 6 mM. In another embodiment, the buffer is present at a concentration of approximately 5 mM.

[0263] In one embodiment, the buffer is HEPES with a pH of approximately 6.7 and a concentration of approximately 7.5 mM or less, for example, 2.5 mM to 7.5 mM or 4 mM to 6 mM, for example, approximately 5 mM.

[0264] In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (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-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0265] 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.

[0266] In one embodiment, the RNA lipoplex particles contain DOTMA and DOPE in molar ratios of approximately 10:0 to 1:9, approximately 4:1 to 1:2, approximately 3:1 to approximately 1:1, or approximately 2:1.

[0267] In one embodiment, the composition further comprises a chelating agent. In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), particularly disodium EDTA. In one embodiment, the concentration of EDTA is about 3.5 mM or less, or about 0.25 mM to about 3.5 mM or about 0.25 mM to about 2.5 mM.

[0268] In one embodiment of the composition 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.

[0269] The present disclosure further relates to an RNA encoding a peptide or protein comprising at least one epitope, DOTMA and DOPE in a molar ratio of about 2:1, which is an RNA lipoplex particle comprising an RNA lipoplex particle wherein the ratio of positive charge to negative charge in the composition is about 1.3:2.0, and sodium chloride at a concentration of about 8.2 mM, and sucrose at a concentration of about 13% (w / v), and HEPES at a concentration of about 5 mM, having a pH of about 6.7, and EDTA at a concentration of about 2.5 mM.

[0270] In one embodiment of the composition described herein, the RNA lipoplex particles 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.

[0271] In one embodiment of the composition described herein, 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 0.025 mg / mL.

[0272] In one embodiment of the composition described herein, the composition further comprises an acid. In one embodiment, the acid is present in a liposome-stabilizing amount. In one embodiment, the acid is present in an amount that reduces DOPE hydrolysis. In one embodiment, the acid is acetic acid or HCl. In one embodiment, the acid is acetic acid. In one embodiment, the acid is acetic acid at a concentration of about 0.09 mM.

[0273] In one embodiment of the compositions described herein, the composition further comprises an adjuvant.

[0274] In one embodiment of the compositions described herein, the composition is in a liquid state, a frozen state, or a dehydrated state.

[0275] In one embodiment, the composition is a frozen composition that is stable for at least one month at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least two months at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least four months at a temperature of about -15°C. In one embodiment, the composition is a frozen composition that is stable for at least six months at a temperature of about -15°C.

[0276] This 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.

[0277] This 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.

[0278] In one embodiment, the liquid composition described herein is an aqueous composition.

[0279] In one embodiment, a composition, in particular a liquid composition described herein, can be administered directly to a subject.

[0280] In one embodiment, the composition described herein is a pharmaceutical composition.

[0281] In one embodiment, the composition described herein is formulated for systemic administration.

[0282] In one embodiment, systemic administration is performed by intravenous administration.

[0283] The present disclosure further relates to the compositions described herein for therapeutic use.

[0284] The present disclosure further relates to a method of preparing a liquid composition comprising RNA lipoplex particles 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 comprising RNA lipoplex particles 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 comprises the composition described above. In the therapeutic applications described herein, the liquid composition described herein is administered to a subject.

[0285] Further embodiments are as follows: 1. A method of producing a liposome colloid, comprising injecting an ethanol solution of a lipid into an aqueous phase to produce the liposome colloid, wherein the concentration of at least one lipid among the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of the at least one lipid in ethanol.

[0286] 2. The method of embodiment 1, wherein the lipid solution is a solution of a mixture of two or more different lipids.

[0287] 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.

[0288] 4. The method of any one of embodiments 1 to 3, wherein the total lipid concentration in the lipid solution is from about 180 mM to about 600 mM, from about 300 mM to about 600 mM, or about 330 mM.

[0289] 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.

[0290] 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.

[0291] 7. The method of Embodiment 5 or 6, wherein at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0292] 8. 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).

[0293] 9. Any one of Embodiments 5 to 8, wherein at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0294] 10. Any one of Embodiments 5 to 9, wherein 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.

[0295] 11. Any one of Embodiments 1 to 10, wherein the lipid solution contains DOTMA and DOPE in molar ratios of 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.

[0296] 12. 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.

[0297] 13. Any one of Embodiments 1 to 12, wherein the lipid solution is injected into the aqueous phase at an aqueous phase stirring speed of approximately 50 rpm to approximately 150 rpm.

[0298] 14. Any one of Embodiments 1 to 13, wherein the aqueous phase is water.

[0299] 15. Any one of embodiments 1 to 14, further comprising stirring the liposome colloid.

[0300] 16. Any one of Embodiments 1 to 15, wherein the liposome colloid is stirred for about 15 to about 60 minutes, or about 30 minutes.

[0301] 17. A method for producing liposome colloids, comprising injecting a lipid solution containing DOTMA and DOPE in ethanol in a molar ratio of about 2:1 into water being stirred at a stirring speed of about 150 rpm to produce liposome colloids, wherein the concentration of DOTMA and DOPE in the lipid solution is about 330 mM.

[0302] 18. A liposome colloid that can be obtained by any one of the methods of Embodiments 1 to 17.

[0303] 19. A liposome colloid of Embodiment 18, wherein the liposomes have an average diameter of at least about 250 nm.

[0304] 20. A liposome colloid of Embodiment 18 or 19, wherein the liposomes have an average diameter in the range of approximately 250 nm to approximately 800 nm.

[0305] 21. A liposome colloid according to any one of embodiments 18 to 20, wherein the liposomes are cationic liposomes.

[0306] 22. A liposome colloid according to any one of embodiments 18 to 21, wherein the liposome comprises at least one cationic lipid and at least one additional lipid.

[0307] 23. A liposome colloid of Embodiment 22, wherein at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0308] 24. A liposome 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).

[0309] 25. Any one of the liposome colloids of Embodiments 22 to 24, wherein 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).

[0310] 26. Any one of embodiments 22 to 25, wherein 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.

[0311] 27. A liposome colloid according to any one of Embodiments 18 to 26, wherein the liposomes contain DOTMA and DOPE in molar ratios of approximately 10:0 to approximately 1:9, approximately 4:1 to approximately 1:2, approximately 3:1 to approximately 1:1, or approximately 2:1.

[0312] 28. A method for preparing RNA lipoplex particles, comprising adding one liposome colloid of any one of embodiments 18 to 27 to an RNA-containing solution.

[0313] 29. A method for continuous flow production of RNA lipoplex particles, comprising mixing an RNA-containing solution with a cationic liposome-containing solution under controlled mixing conditions of RNA and cationic liposomes.

[0314] 30. The method of Embodiment 29, wherein the solution containing cationic liposomes is a liposome colloid of any one of Embodiments 18 to 27.

[0315] 31. The method of Embodiment 29 or 30, wherein the solution containing RNA and the solution containing cationic liposomes are aqueous solutions.

[0316] 32. Any one of embodiments 29 to 31, wherein a flow rate is used that allows mixing of an RNA-containing solution and a cationic liposome-containing solution.

[0317] 33. Any one of the embodiments 29 to 32, wherein the flow is characterized by a Reynolds number greater than 300, or between approximately 500 and approximately 2100.

[0318] 34. Any one of Embodiments 29 to 33, wherein controlled mixing conditions control the mixing ratio of the RNA-containing solution to the cationic liposome-containing solution.

[0319] 35. Any one of Embodiments 29 to 34, wherein controlled mixing conditions control the relative volume of the solution containing the RNA to be mixed and the solution containing the cationic liposomes.

[0320] 36. Any one of Embodiments 29 to 35, wherein the mixing ratio of RNA to cationic liposomes is controlled by using the same mixing volume (v / v) of the RNA-containing solution and the cationic liposome-containing solution, and adjusting the concentrations of RNA and cationic liposomes in each solution.

[0321] 37. Any one of embodiments 29 to 36, wherein controlled mixing conditions are selected to maintain the properties of RNA lipoplex particles while avoiding clogging.

[0322] 38. Any one of embodiments 29 to 37, comprising using a Y-type or T-type mixed element.

[0323] 39. Any one of embodiments 29 to 38, wherein the Y-type or T-type mixed element has a diameter of about 1.2 mm to about 50 mm.

[0324] 40. Any one of the embodiments 29 to 39, comprising using a syringe pump in which two syringes, one containing a solution containing cationic liposomes and the other containing a solution containing RNA, are inserted in parallel into the same pump.

[0325] 41. Any one of embodiments 29 to 40, including using a pressurized vessel, membrane pump, gear pump, magnetic levitation pump, or peristaltic pump in combination with a flow sensor which optionally has a feedback loop for online control and real-time adjustment of the flow rate.

[0326] 42. Any one of Embodiments 29 to 41, wherein the mixture of the RNA-containing solution and the liposome-containing solution 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 at about 45 mM to about 300 mM.

[0327] 43. Any one of Embodiments 29 to 42, wherein the mixture of the RNA-containing solution and the liposome-containing solution has an ionic strength of at least about 50 mM.

[0328] 44. In the X-ray scattering pattern, RNA lipoplexes are approximately 1 nm -1 It features a single Bragg peak with a peak width of 0.2 nm. -1 A method smaller than any one of embodiments 28 to 43.

[0329] 45. Any one of Embodiments 28 to 44, wherein the RNA lipoplex particles have an average diameter in the range of approximately 200 to approximately 800 nm, approximately 250 to approximately 700 nm, approximately 400 to approximately 600 nm, approximately 300 nm to approximately 500 nm, or approximately 350 nm to approximately 400 nm.

[0330] 46. ​​A method for preparing a frozen composition containing RNA lipoplex particles, comprising (i) providing an aqueous composition containing RNA lipoplex particles and a stabilizer, and (ii) freezing the composition.

[0331] 47. The method of Embodiment 46, wherein freezing occurs at a temperature of approximately -15°C to approximately -40°C, or approximately -30°C.

[0332] 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.

[0333] 49. A method of 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 an aqueous composition comprising RNA lipoplex particles.

[0334] 50. A method in which a stabilizer is added to an aqueous composition containing RNA lipoplex particles, thereby reducing the ionic strength of the aqueous composition containing RNA lipoplex particles, as described in any one of Embodiments 46 to 49.

[0335] 51. Any one of Embodiments 46 to 50, wherein the concentration of the stabilizer in an aqueous composition comprising RNA lipoplex particles and the stabilizer is higher than the value required for the physiological gravimetric osmolality.

[0336] 52. Any one of Embodiments 46 to 51, wherein the concentration of the stabilizer in an aqueous composition comprising RNA lipoplex and the stabilizer is sufficient to maintain the quality of the RNA lipoplex particles, and in particular to avoid substantial loss of RNA activity after the composition has been stored 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.

[0337] 53. Any one of Embodiments 46 to 52, wherein the pH of the aqueous composition comprising RNA lipoplex and a stabilizer is lower than the normal pH optimal for RNA preservation.

[0338] 54. Any one of Embodiments 46 to 53, wherein the aqueous composition comprising RNA lipoplex and a 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 at a concentration of about 10 mM to about 50 mM.

[0339] 55. Any one of Embodiments 46 to 54, wherein an aqueous composition comprising RNA lipoplex and a stabilizer has an ionic strength corresponding to a concentration of sodium chloride of about 20 mM.

[0340] 56. Any one method of Embodiments 46 to 55, wherein RNA lipoplex particles can be obtained by any one method of Embodiments 28 to 44.

[0341] 57. A composition comprising RNA lipoplex particles that can be obtained by any one of the methods of Embodiments 28 to 45.

[0342] 58. The composition of Embodiment 57, wherein the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0343] 59. The composition of Embodiment 57 or 58, 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 particle is about 1:2 to about 1.9:2, or about 1.3:2.0.

[0344] 60. RNA encoding a peptide or protein containing at least one epitope, At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing A composition containing, The ratio of positive to negative charges in RNA lipoplex particles is approximately 1:2 to 1.9:2, or approximately 1.3:2.0. RNA lipoplex particles are approximately 1 nm in size. -1 It features a single Bragg peak with a peak width of 0.2 nm. -1 A composition smaller than that.

[0345] 61. Any one of the compositions of Embodiments 57 to 60, further comprising sodium chloride in concentrations of approximately 10 to 300 mM, approximately 45 mM to 300 mM, approximately 10 mM to 50 mM, or approximately 80 mM to 150 mM.

[0346] 62. Any one of embodiments 57 to 61, further comprising a buffering agent.

[0347] 63. Any one of embodiments 57 to 62, further comprising a chelating agent.

[0348] 64. A composition comprising RNA lipoplex particles, which can be obtained by any one of the methods of Embodiments 46 to 56.

[0349] 65. The composition of Embodiment 64, wherein the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.

[0350] 66. The composition of Embodiment 64 or 65, 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 particle is about 1:2 to about 1.9:2, or about 1.3:2.0.

[0351] 67. Any one of the compositions from Embodiments 64 to 66, further comprising sodium chloride at a concentration of approximately 10 mM to approximately 50 mM.

[0352] 68. RNA encoding a peptide or protein containing at least one epitope, At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1:2 to approximately 1.9:2, or approximately 1.3:2.0, Sodium chloride at concentrations of 0 mM to approximately 40 mM, Stabilizers and, A composition containing the following: 69. A composition comprising any one of embodiments 64 to 68, further comprising a buffering agent.

[0353] 70. Any one of the compositions from Embodiments 64 to 69, wherein the amount of RNA in the composition is approximately 0.01 mg / mL to approximately 1 mg / mL, approximately 0.05 mg / mL to approximately 0.5 mg / mL, or approximately 0.05 mg / mL.

[0354] 71. Any one of the compositions from Embodiments 67 to 70, wherein the sodium chloride is concentrated at a concentration of approximately 20 mM to approximately 30 mM.

[0355] 72. Any one of the compositions from Embodiments 67 to 71, wherein the sodium chloride concentration is approximately 20 mM.

[0356] 73. Any one of the compositions from Embodiments 67 to 71, wherein the sodium chloride concentration is approximately 30 mM.

[0357] 74. Any one of the compositions from Embodiments 64 to 73, wherein the concentration of the stabilizer in the composition is higher than the value required for the physiological gravimetric osmolality.

[0358] 75. Any one of the compositions from Embodiments 64 to 74, wherein the concentration of the stabilizer in the composition is about 5 to about 35 wt / volume percent (%w / v) or about 12.5 to about 25 wt / volume percent (%w / v).

[0359] 76. Any one of the compositions 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.

[0360] 77. Any one of the compositions from Embodiments 64 to 76, wherein the stabilizer is sucrose in a concentration of about 5 to about 25% by weight / volume (%w / v).

[0361] 78. The composition of Embodiment 77, wherein sucrose is present at a concentration of approximately 15% (w / v) to approximately 25% (w / v).

[0362] 79. The composition of Embodiment 77, wherein sucrose is present at a concentration of approximately 20% (w / v) to approximately 25% (w / v).

[0363] 80. The composition of Embodiment 77, wherein sucrose is present at a concentration of approximately 22% (w / v).

[0364] 81. The composition of Embodiment 77, wherein sucrose is present at a concentration of approximately 20% (w / v).

[0365] 82. Any one of embodiments 64 to 81 having a pH lower than the normal pH that is optimal for RNA preservation.

[0366] 83. Any one of the compositions from Embodiments 64 to 82 having a pH of approximately 5.7 to approximately 6.7, or approximately 6.2.

[0367] 84. Any one of the compositions from Embodiments 68 to 83, wherein the buffer is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

[0368] 85. The composition of Embodiment 84, wherein HEPES is concentrated at a concentration of approximately 2.5 mM to approximately 10 mM, or approximately 7.5 mM.

[0369] 86. Any one of embodiments 64 to 85, further comprising a chelating agent.

[0370] 87. RNA encoding a peptide or protein containing at least one epitope at a concentration of approximately 0.05 mg / mL, and DOTMA and DOPE are used in a molar ratio of approximately 2:1. RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is approximately 1.3:2.0, Sodium chloride at a concentration of approximately 20 mM, Sucrose at a concentration of approximately 22% (w / v), HEPES at a concentration of approximately 7.5 mM with a pH of approximately 6.2, EDTA at a concentration of approximately 2.5 mM, A composition containing the following:

[0371] 88. A composition from any one of embodiments 64 to 87, which is in a liquid state or a frozen state.

[0372] 89. The freezing composition of Embodiment 88, which is stable for at least one month at a temperature of approximately -15°C to approximately -40°C.

[0373] 90. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -15°C for at least one month.

[0374] 91. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -15°C for at least two months.

[0375] 92. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -20°C for at least one month.

[0376] 93. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -20°C for at least two months.

[0377] 94. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -30°C for at least one month.

[0378] 95. The freeze-forming composition of Embodiment 88, which is stable at a temperature of approximately -30°C for at least two months.

[0379] 96. An aqueous composition comprising RNA lipoplex particles, which can be obtained by thawing any one of the frozen compositions of Embodiments 88 to 95 and optionally adjusting the gravimetric osmolality and ionic strength by adding an aqueous liquid.

[0380] 97. The composition of Embodiment 96, wherein the weight osmolality of the composition is approximately 200 mOsmol / kg to approximately 450 mOsmol / kg.

[0381] 98. The composition of Embodiment 96 or 97, comprising sodium chloride at a concentration of approximately 80 mM to approximately 150 mM.

[0382] 99. Any one composition from any one of embodiments 64 to 98, wherein RNA lipoplex particles can be obtained by any one method from any one of embodiments 28 to 45. 100. RNA lipoplex particles are approximately 1 nm in size. -1 It features a single Bragg peak with a peak width of 0.2 nm. -1 A composition smaller than any one of embodiments 64 to 99

[0383] 101. Any one of Embodiments 57 to 100, wherein the RNA lipoplex particles have an average diameter in the range of approximately 200 to approximately 800 nm, approximately 250 to approximately 700 nm, approximately 400 to approximately 600 nm, approximately 300 nm to approximately 500 nm, or approximately 350 nm to approximately 400 nm.

[0384] 102. Any one composition of Embodiments 58 to 63, 65 to 86, and 88 to 101, wherein at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0385] 103. Any one composition 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).

[0386] 104. Any one of the compositions of Embodiments 58 to 63, 65 to 86, and 88 to 103, wherein at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0387] 105. Any one composition 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 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.

[0388] 106. Any one of the compositions of Embodiments 58 to 63, 65 to 86, and 88 to 105, wherein the RNA lipoplex particles contain 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 positive charge in DOTMA to negative charge in RNA is about 1:2 to 1.9:2.

[0389] 107. Any one of embodiments 63, 86, and 88 to 106, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0390] 108. The composition of Embodiment 107, wherein the EDTA is concentrated at a concentration of approximately 0.25 mM to approximately 5 mM, or approximately 2.5 mM.

[0391] 109. Any one of embodiments 57 to 108, further comprising an adjuvant.

[0392] 110. Any one of the compositions from Embodiments 57 to 109, formulated for systemic administration.

[0393] 111. The composition of Embodiment 110, wherein systemic administration is by intravenous administration.

[0394] 112. Any one of the compositions from embodiments 57 to 111 for therapeutic use.

[0395] 113. A method for preparing an aqueous composition containing RNA lipoplex particles, comprising thawing one of the frozen compositions of any one of embodiments 88 to 112, and optionally adjusting the gravimetric osmolality and ionic strength by adding an aqueous liquid.

[0396] 114. The method of Embodiment 113, wherein an aqueous liquid is added to obtain a composition with a weight osmolality of approximately 200 mOsmol / kg to approximately 450 mOsmol / kg.

[0397] 115. The method of Embodiment 113 or 114, wherein an aqueous liquid is added to obtain sodium chloride at a concentration of approximately 80 mM to approximately 150 mM. [Brief explanation of the drawing]

[0398] [Figure 1]This study shows the correlation between lipid concentration in the lipid stock solution and liposome size. Liposomes were prepared by ethanol injection into water (no filtration process was performed after ethanol injection). Liposome size increased with lipid concentration in ethanol. This example uses a lipid mixture DOTMA / DOPE with a 66:33% molar ratio. [Figure 2] This shows the amount of particles present in DOTMA / DOPE liposome formulations prepared using various lipid solutions of different concentrations. [Figure 3] This study shows the effect of liposome precursor size (the unfiltered liposome colloid used) on RNA-lipoplex size. When small liposomes were used for their formation, small RNA-lipoplexes were obtained. No clear correlation was observed between liposome size and RNA-lipoplex size in RNA-lipoplexes prepared using large liposomes. [Figure 4] This shows the amount of particles present in RNA-lipoplex formulations prepared using various liposome precursors (unfiltered liposomes). RNA-lipoplex formulations prepared using large liposomes show an increase in the amount of 0.5 μm particles. Liposomes were prepared by ethanol injection using various lipid stock solutions of different concentrations. [Figure 5] The diffraction curves obtained from SAXS measurements of RNA-lipoplexes formed using liposomes prepared with a 4 / 1 (top) charge ratio and a 1.3 / 2 charge ratio are shown. Here, liposomes for lipoplex formation were obtained using mM lipid stock solutions in 400 mM, 300 mM, and 100 mM ethanol. [Figure 6] This paper shows the correlation length and transfection efficiency in vitro (in human dendritic cells) of various RNA-lipoplexes prepared using various liposome precursors. Biological activity (in vitro RNA transfection) increases monotonically with the correlation length of the RNA-lipoplex. Liposomes were prepared by ethanol injection and with various lipid stocks prepared at different lipid concentrations. [Figure 7]The AF4 measurements of lipoplexes obtained from two different types of liposomes prepared from either a 150 mM stock solution in ethanol or a 400 mM stock solution in ethanol are shown. [Figure 8] This shows the in vitro transfection efficiency of various RNA-lipoplexes in dendritic cells. Luciferase signaling increases monotonically with the liposome size used for RNA-lipoplex formation. [Figure 9] This shows the in vitro transfection efficiency of various RNA-lipoplexes in dendritic cells. Luciferase signaling increases monotonically with liposome size. [Figure 10] This shows the in vivo transfection efficiency of RNA-lipoplex formulations 6 hours after application. RNA-lipoplexes were prepared using small liposomes and large liposomes (non-filterable liposomes). RNA-lipoplexes prepared using large liposomes resulted in higher luciferase expression. [Figure 11] In vivo imaging of RNA-lipoplexes 6 hours after application is shown. RNA-lipoplexes were prepared using small liposomes and large liposomes. A) RNA-lipoplex prepared using small liposomes obtained from biocolloids. B) RNA-lipoplex prepared using large liposomes obtained from biocolloids. C) RNA-lipoplex prepared using small filterable liposomes. D) RNA-lipoplex prepared using large filterable liposomes. Relatively high bioluminescence signal obtained from RNA-lipoplexes prepared using large liposomes. [Figure 12] This document outlines a general procedure for automated batch production of RNA lipoplexes. [Figure 13] This shows the Z-mean and polydispersity of RNA lipoplexes prepared using Y-type mixed elements with an inner diameter of 3.2 mm at various flow rates. [Figure 14] This shows the Z-mean and polydispersity of RNA lipoplexes prepared using Y-type mixed elements with an inner diameter of 2.4 mm at various flow rates. [Figure 15] This shows the correlation between particle size and RNA concentration during lipoplex formation. PCS measurements were performed before freezing. [Figure 16] This shows the correlation between particle characteristics and RNA concentration during lipoplex formation after three freeze-thaw cycles. [Figure 17] This shows the correlation between particle properties and the charge ratio (positive:negative) in the range of 1.0:2.0 to 2.1:2.0. [Figure 18] This shows the correlation between particle properties and the charge ratio (positive:negative) in the range of 2.0:1.0 to 5.0:1.0. [Figure 19] This shows the correlation between particle characteristics during lipoplex formation and NaCl concentration. [Figure 20] This shows the correlation between biological activity during lipoplex formation and NaCl concentration. [Figure 21] This chart shows RNA integrity measurements in cryoprotectant-free RNA (LIP) compositions after accelerated storage at +40°C, using various buffers (HEPES; sodium acetate; sodium phosphate; sodium carbonate) at several pH values. The bars indicate increasing storage time from left (3 days) to right (21 days). [Figure 22] This graph shows the RNA integrity measurements in RNA lipoplex formulations using sucrose as a cryoprotectant and HEPES as a buffer at several pH values ​​after accelerated storage at +40°C. The various bars indicate increasing storage time from left (1 day) to right (21 days). [Figure 23] This demonstrates the integrity of RNA in lipoplexes stored at 40°C using various EDTA content levels. [Figure 24] This is a schematic diagram showing that the concentrations of NaCl and cryoprotectant were optimized in each step of the process. [Figure 25] This shows the Z-mean and polydispersity of RNA lipoplexes frozen in the presence of gradually increasing alternative cryoprotective agents. [Figure 26] This indicates the number of invisible particles ≥10 μm in RNA lipoplex formulations frozen in the presence of gradually increasing amounts of alternative cryoprotective agents. [Figure 27]This shows the measured particle size in RNA lipoplex formulations containing 5-20% w / v sucrose (X-axis) and various low NaCl concentrations, before and after freezing at -30°C. [Figure 28] The graph shows the measured particle size in RNA lipoplex formulations containing 5-20% w / v trehalose dihydrate (x axis) and various low NaCl concentrations, before and after freezing at -30°C. [Figure 29] The measured particle sizes in RNA lipoplex formulations containing 50 mM NaCl and various amounts (%w / v) of trehalose dihydrate after multiple freeze-thaw cycles are shown. [Figure 30] The measured particle sizes in RNA lipoplex formulations containing 70 mM NaCl and various amounts (%w / v) of trehalose dihydrate after multiple freeze-thaw cycles are shown. [Figure 31] The measured particle sizes in RNA lipoplex formulations containing 90 mM NaCl and various amounts (%w / v) of trehalose dihydrate after multiple freeze-thaw cycles are shown. [Figure 32] This graph shows particle size measurements in RNA lipoplex formulations containing 5-20% w / v sucrose and varying low NaCl concentrations after 8 months of storage at -15°C. The different bars indicate increasing storage time from left (0 months) to right (8 months). For sucrose / NaCl combinations with fewer than 8 bars, the particle size exceeded the specification at the following two time points, and the analysis was stopped. [Figure 33] This graph shows particle size measurements in RNA lipoplex formulations containing 5-20% w / v sucrose and varying low NaCl concentrations after 8 months of storage at -30°C. The different bars indicate increasing storage time from left (0 months) to right (8 months). For sucrose / NaCl combinations with fewer than 8 bars, the particle size exceeded specifications at the following two time points, and the analysis was stopped. [Figure 34]This graph shows particle size measurements in RNA lipoplex formulations containing 5-20% w / v trehalose dihydrate and varying low NaCl concentrations, after storage at -15°C for 8 months. The varying bars indicate increasing storage time from left (0 months) to right (8 months). For trehalose / NaCl combinations with fewer than 8 bars, the particle size exceeded specifications at the following two time points, and the analysis was stopped. [Figure 35] This graph shows particle size measurements in RNA lipoplex formulations containing 5-20% w / v trehalose dihydrate and varying low NaCl concentrations, after storage at -30°C for 8 months. The varying bars indicate increasing storage time from left (0 months) to right (8 months). For trehalose / NaCl combinations with fewer than 8 bars, the particle size exceeded specifications at the following two time points, and the analysis was stopped. [Figure 36] The measured particle sizes in RNA lipoplex formulations containing 22% w / v sucrose and having various low NaCl concentrations, after storage at -20°C, are shown. [Figure 37] The measured particle sizes in RNA lipoplex formulations containing 22% w / v trehalose dihydrate and having various low NaCl concentrations, after storage at -20°C, are shown. [Figure 38] The measured particle sizes in RNA lipoplex formulations containing 22% w / v glucose and having various low NaCl concentrations, after storage at -20°C, are shown. [Figure 39] The measured particle size in RNA lipoplex formulations containing 22% w / v sucrose and 20 mM NaCl, frozen and stored at -15 to -40°C, is shown. [Figure 40] The measured particle sizes of frozen RNA lipoplexes in compositions containing the cryoprotective agent combinations listed in Table 10 after storage at -20°C are shown. [Figure 41] This shows the effect of trehalose concentration in RNA lipoplex preparations [RNA(lip)] preparations after freeze-thawing, or after lyophilization and reconstitution. [Figure 42]This shows the particle size changes of lyophilized RNA (lip) preparations prepared in 22% trehalose after reconstitution using 0.9% NaCl solution or WFI (water for injection). [Figure 43] This study demonstrates Luc-RNA in vitro transfection of lyophilized RNA lipoplex preparations [RNA(lip)] prepared in 22% trehalose at various NaCl concentrations. Lyophilized samples were reconstituted using a 0.9% NaCl solution. (Texturized column: liquid control) [Figure 44] This shows the Z-mean diameter of lyophilized RNA lipoplex formulations [RNA(lip)] prepared at various trehalose / NaCl ratios, stored at 2–8°C or 25°C, after reconstitution using a 0.9% NaCl solution. The formulations were reconstituted to their original volume after lyophilization. [Figure 45] This study shows the RNA integrity (full-length RNA%) of lyophilized RNA (lip) formulated with various trehalose / NaCl ratios after reconstitution using 0.9% NaCl solution and storage at 2–8°C or 25°C. The formulations were reconstituted to their original volume after lyophilization and diluted 1:1 with 0.9% NaCl solution (0.01 mg / mL RNA) for cell culture experiments. [Figure 46] This shows the measured particle sizes of RNA lipoplex compositions containing various buffers and having different pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 47] This shows the measured polydispersity of RNA lipoplex compositions containing various buffers and having varying pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limit of 0.5 for the polydispersity index. [Figure 48] This shows the pH measurements of RNA lipoplex compositions containing various buffering substances and having various pH values ​​after storage under accelerated conditions (+25°C). [Figure 49]This shows the measured RNA integrity of RNA lipoplex compositions containing various buffers and at various pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limit of ≥80% complete full-length RNA. [Figure 50] This shows the measured particle size of RNA lipoplex compositions containing various buffering materials and having various pH levels after storage at -15°C. The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 51] This shows the measured polydispersity of RNA lipoplex compositions containing various buffers and having various pH values ​​after storage at -15°C. The dotted line indicates the predicted specification limit of the polydispersity index, which is 0.5. 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 frozen RNA lipoplex. [Figure 52] The pH measurements of RNA lipoplex compositions containing various buffering substances and having various pH values ​​after storage at -15°C are shown. [Figure 53] This shows the measured RNA integrity of RNA lipoplex compositions containing various buffers and at various pH levels after storage at -15°C. The dotted line indicates the predicted specification limit of ≥80% complete full-length RNA. [Figure 54] This shows the measured particle sizes of RNA lipoplex compositions containing various buffers and having different pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 55] This graph shows the measured polydispersity index of RNA lipoplex compositions containing various buffers and with varying pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limit of 0.5 for the polydispersity index. [Figure 56] This shows the pH measurements of RNA lipoplex compositions containing various buffering substances and having various pH values ​​after storage under accelerated conditions (+25°C). [Figure 57]This shows the measured RNA integrity of RNA lipoplex compositions containing various buffers and having different pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the specification limit of ≥80% complete full-length RNA. [Figure 58] This shows the particle size changes of RNA lipoplex compositions containing various buffering substances and having various pH levels before and after freezing. The dotted line indicates the predicted specification limits of particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 59] This shows the measured particle size of RNA lipoplex compositions containing various buffering materials and having various pH levels after storage at -15°C. The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 60] This shows the measured polydispersity of RNA lipoplex compositions containing various buffers and having varying pH levels after storage at -15°C. The dotted line indicates the predicted specification limit of 0.5 for the polydispersity index. [Figure 61] The pH measurements of RNA lipoplex compositions containing various buffering substances and having various pH values ​​after storage at -15°C are shown. [Figure 62] This shows the measured RNA integrity of RNA lipoplex compositions containing various buffers and at various pH levels after storage at -15°C. The dotted line indicates the predicted specification limit of ≥80% complete full-length RNA. [Figure 63] This shows the measured particle sizes of RNA lipoplex compositions with various HEPES buffer concentrations and pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). The particle size measured after 50 days is considered an outliner. [Figure 64] This shows the measured polydispersity index of RNA lipoplex compositions with various HEPES buffer concentrations and pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the specification limit of 0.5 for the polydispersity index. [Figure 65]The pH measurements of RNA lipoplex compositions with various HEPES buffer concentrations and pH values ​​after storage under accelerated conditions (+25°C) are shown. [Figure 66] This shows the measured RNA integrity of RNA lipoplex compositions with various HEPES buffer concentrations and pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the specification limit of ≥80% complete full-length RNA. [Figure 67] This shows the measured particle sizes of RNA lipoplex compositions containing various buffers and having different pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limits for particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 68] This graph shows the measured polydispersity index of RNA lipoplex compositions containing various buffers and with varying pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limit of 0.5 for the polydispersity index. [Figure 69] This shows the pH measurements of RNA lipoplex compositions containing various buffering substances and having various pH values ​​after storage under accelerated conditions (+25°C). [Figure 70] This shows the measured RNA integrity of RNA lipoplex compositions containing various buffers and at various pH levels after storage under accelerated conditions (+25°C). The dotted line indicates the predicted specification limit of ≥80% complete full-length RNA. [Figure 71] This shows the particle size changes of RNA lipoplex compositions containing low concentrations of sucrose and NaCl before and after freezing. The dotted line indicates the predicted specification limits of particle size (upper limit: 700 nm, lower limit: 250 nm). [Figure 72]Summary of changes to the formulation. A: Table A shows the differences between the current formulation and the proposed formulation. In order to simplify the manufacturing process and reduce production costs, the following aspects were investigated and the following changes should be implemented: 1. Acidification with 1.1 mM acetic acid improved liposome stability. Changes to the RNA drug substance buffer for process simplification (addition of 100 mM NaCl to compensate for the pH shift induced by acetic acid and a change in pH to 7.0, as well as improved RNA stability). Reducing the RNA concentration in the formulation from 0.05 mg / mL to 0.025 mg / mL will result in a ready-to-use product that can be administered directly without dilution. There will be no need for bedside dilution, and handling of low doses will be easier. Reduce the ionic strength from 7.5 mM HEPES to 5.0 mM HEPES without affecting pH stability. Reduction to HEPES. The EDTA disodium salt content remained unchanged and was found to contribute as a buffer. EDTA disodium salt stabilizes the pH during formulation production. The NaCl content for RNA conditioning remains unchanged. The reduction in NaCl in the formulation allows for a reduction in sucrose content due to a decrease in solution ionic strength. The reduction to 13% (w / v) sucrose leads to cost savings without affecting stability and is essential for an almost physiological gravimetric osmolality. Bedside dilution is not required. The pH value is raised to pH 6.7 to improve RNA stabilization. B: Table B shows the ranges investigated for the development of the proposed formulation. The table includes the investigated concentration ranges and pH ranges for RNA, DOTMA, DOPE, HEPES, EDTA disodium salt, sucrose, NaCl, and acetic acid. [Figure 73]Investigation of the stability of DOPE in liposomes at different acetic acid concentrations as a function of time, measured at three different temperatures. A: Liposomes stored at 4°C with 0mM–3.0mM acetic acid. B: Liposomes stored at 25°C with 0mM–1.6mM acetic acid. C: Liposomes stored at 40°C with 0mM–3.0mM acetic acid. Acidification was found to result in a decrease in the hydrolysis rate of DOPE in liposomes. Stability increased with increasing acetic acid concentration at all measured temperatures. An acetic acid concentration of 1.1mM is preferred as it can be achieved by slightly modifying existing liposome manufacturing processes. The stabilizing effect can already be observed at low concentrations of acetic acid. There is a linear correlation of the stabilizing effect upon addition of acetic acid, which plateaus above approximately 1mM acetic acid concentration. [Figure 74] RNA API Buffer and RNA Concentration: Adjustments for Process Simplification The developed RNA API formulations are shown in this table. This allows for the processing of RNA APIs into RNA lipoplexes without the need for concentration and tonic adjustments. Fixed, slightly increased buffer and EDTA disodium salt concentrations, as well as pH adjustments, enhance process reproducibility and allow for compensation of pH shifts caused by acetate in liposomes (predictable pH shifts during RNA lipoplex formation). Process parameters such as RNA concentration and ionic conditions remain similar during RNA lipoplex formation, with a low risk of change. The stability of the RNA API is improved at pH 7.0. [Figure 75] RNA concentration adjustment to 0.025 mg / mL. An RNA concentration of 25 μg / mL in the formulation results in a dose volume corresponding to a large mark on a 1 mL syringe for all scenarios under consideration. These dose volumes are easier for HCPs to accurately measure and administer. [Figure 76]HEPES content: reduced to 5.0 mM. A: Graph A shows the RNA stability in formulations over time at 25°C for various buffering systems (HEPES, histidine, and sodium acetate). Within the relevant pH range (pH 6.4–7.0), HEPES provides some unique stabilizing effect on RNA in the formulation (better than histidine, MES (not shown), and sodium acetate (pH 5.5–5.8)) and significantly enhances RNA stability in the formulation. HEPES helps control pH during mixing of RNA with liposomes containing acetate. The buffering capacity of HEPES at pH 6–7 was found to be sufficient for long-term pH stabilization. B: Graph B shows the RNA integrity in formulations over time at a selected pH of 6.7 when incubated at 25°C for various HEPES concentrations. The concentration range of 2.5 mM–10.0 mM was found to yield comparable pH and RNA stabilization. To reduce the ion load, the HEPES concentration in the formulation was reduced from 7.5 mM to 5.0 mM. [Figure 77]EDTA disodium salt content: Unchanged at 2.5 mM. A: Figure A shows the particle size of RNA lipoplexes stored at -15°C using various amounts of EDTA disodium salt and various pH levels. RNA lipoplexes prepared without EDTA disodium salt yielded larger particle sizes compared to RNA lipoplexes prepared using a drug substance buffer containing EDTA disodium salt. The particle size of RNA lipoplexes from EDTA disodium salt-containing formulations was equivalent for 0.4 mM to 2.5 mM EDTA disodium salt and pH 6.5 and 7.0. During RNA lipoplex formation, the EDTA disodium salt concentrations were identical in the two groups containing EDTA disodium salt. The final EDTA disodium salt concentration was adjusted by subsequent dilution of the RNA lipoplex. Disodium EDTA contributes to pH stabilization during RNA lipoplex formation (0 mM disodium EDTA results in a pH shift during RNA lipoplex formation, increased RNA lipoplex size, and decreased RNA stability in the formulation (Figure 77B)). B: Graph B shows the time course of RNA integrity in the formulation when incubated at 25°C for various disodium EDTA concentrations. RNA stabilization was comparable among 0.4 mM to 2.5 mM disodium EDTA in the formulation, with slightly higher RNA stabilization observed at pH 7.0 compared to pH 6.5. In the absence of disodium EDTA in the active pharmaceutical ingredient buffer (0 mM disodium EDTA in the formulation), a pH shift occurs during RNA lipoplex formation, resulting in decreased RNA stability in the formulation and increased RNA lipoplex size (Figure 77A)). [Figure 78] The NaCl concentration was reduced to 8.2 mM. The particle sizes of RNA lipoplexes stored at -15°C with varying amounts of NaCl and sucrose are shown in the figure. NaCl content is important for the long-term colloidal stability of RNA lipoplexes in the frozen state (lower is better). The NaCl concentration during RNA lipoplex formation should remain the same, but the NaCl concentration in the DP should be reduced to the lowest possible value. [Figure 79]Sucrose as a cryoprotectant ensures the colloidal stability of the formulation. A: Particle size of RNA lipoplexes with worst-case compositions (higher ion load and increased RNA concentration in the formulation) compared to the current nominal formulation (BM1). Various groups had different sucrose concentrations (8%(w / v)~14%(w / v); BM1=22%(w / v)) and were stored at -15°C for 12 months. Due to the increased concentration of ionic components, the RNA lipoplex size increased when the formulation was frozen (8%(w / v)~14%(w / v) sucrose). Higher sucrose concentrations (12%(w / v)~14%(w / v)) resulted in better stabilization of RNA lipoplex size than lower sucrose concentrations (8%(w / v)~12%(w / v)). Over time, there was only a slight increase in RNA lipoplex particle size. B: The AF4 size distribution profiles of RNA lipoplexes with worst-case compositions (higher ion loading and increased RNA concentration in the formulation) and nominal compositions containing 10% (w / v) sucrose were compared to the current nominal formulation. Various groups had different sucrose concentrations (8% (w / v) to 14% (w / v); BM1 = 22% (w / v)) and were stored at -15°C for 12 months. When the samples were analyzed using the AF4 system, the components eluted as a function of hydrodynamic radius (Rh). In general, all particles showed light scattering peaks at elution times of 25 to 70 minutes, with slightly different peak shapes at higher elution times. Furthermore, samples with worst-case compositions and reduced sucrose concentrations showed a shift in peak maximum value toward higher elution times, indicating changes in particle size over time. The elution profile of the nominal composition containing 10% (w / v) sucrose shows a slight shift compared to worst-case samples containing 12% (w / v) and 14% (w / v) sucrose. Equivalent elution profiles of the worst-case samples containing 12% (w / v) and 14% (w / v) sucrose indicate that increasing sucrose concentrations beyond 12% (w / v) does not result in further improvements in stability. Based on AF4 data, a sucrose concentration of at least 12% (w / v) should be considered sufficient to ensure the long-term stability of the formulation, even under worst-case conditions. [Figure 80]pH value: Increase to pH 6.7. A: RNA integrity in lipoplex formulations containing HEPES buffer at various pH values ​​(pH 6.2, 6.7, and 7.2) when incubated at 25°C. The optimal pH range for HEPES was found to be pH 6.7 to pH 7.2, which resulted in significantly better RNA stabilization than pH 6.2. In the frozen state, no difference was observed between pH 5.5 and 7.2 when combined with various buffer systems (data not shown). To optimize RNA stability in the liquid state, the pH of the RNA lipoplex formulation should be changed to pH 6.7. B: RNA integrity in lipoplex formulations containing HEPES buffer and 2.5 mM EDTA disodium salt at various pH values ​​(pH 6.0, 6.5, and 7.0) when incubated at 25°C. Consistent with the results shown in Figure 80A, the optimal pH range was found to be between pH 6.5 and pH 7.0, but pH 6.0 resulted in significantly lower RNA stabilization. Furthermore, the chelating effect of EDTA disodium salt was pH-dependent, and no difference in RNA stabilization in the formulation was observed between pH 6.5 and pH 7.0. To optimize RNA stability in the liquid state, the pH of the RNA lipoplex formulation should be changed to pH 6.7. [Figure 81]Comparison of formulations with various sucrose concentrations and formulations containing acetic acid in liposomes, as well as formulations without acetic acid. A: Table A shows a detailed description of the formulations tested. The described formulations containing 10% (w / v) sucrose, both with and without acetic acid in liposomes, were compared to a benchmark formulation containing 22% (w / v) sucrose. B: The particle size of RNA lipoplex formulations containing 22% (w / v) sucrose (INEST 2.1), 10% (w / v) sucrose (INEST 2.X w / o), and 10% (w / v) sucrose and 2 mM acetic acid (INEST 2.X with AcOH), prepared in triplicates (#1-#3), was measured by PCS. No differences in size and polydispersity were observed for the various products. Batch-to-batch reproducibility was given for all individual formulations. C: In vitro luciferase signal intensity of RNA lipoplex formulations containing 22% (w / v) sucrose (INEST 2.1), 10% (w / v) sucrose (INEST 2.X w / o), and 10% (w / v) sucrose and 2mM acetate (INEST 2.X with AcOH) in triple-layered liposomes (#1-#3). In vitro translation was equivalent for all formulations. D: In vitro luciferase signal intensity of RNA lipoplex formulations containing 22% (w / v) sucrose (INEST 2.1), 10% (w / v) sucrose (INEST 2.X w / o), and 10% (w / v) sucrose and 2mM acetate (INEST 2.X with AcOH) in triple-layered liposomes (#1-#3). The in vivo volusiferase signal intensity is equivalent for all formulations. [Figure 82]Stability of various formulations with varying sucrose concentrations, containing acetic acid in liposomes, and not containing acetic acid. A: Table A provides a detailed description of the formulations tested. The described formulations containing 10% (w / v) sucrose, both containing and not containing acetic acid in liposomes, were compared to a benchmark formulation containing 22% (w / v) sucrose. B: The particle sizes of RNA lipoplex formulations containing 22% (w / v) sucrose (INEST 2.1), 10% (w / v) sucrose (INEST 2.X w / o), and 10% (w / v) sucrose and 2 mM acetic acid (INEST 2.X with AcOH), prepared in triplicates (#1-#3), were measured by PCS after storage at -20°C for 13 months. No size differences were observed for the various products. All formulations measured showed comparable stability. In vitro luciferase signal intensity of RNA lipoplex formulations containing 22% (w / v) sucrose (INEST 2.1), 10% (w / v) sucrose (INEST 2.X w / o), and 10% (w / v) sucrose and 2 mM acetate (INEST 2.X with AcOH) in triple-linked liposomes (#1-#3) prepared after storage at -20°C for 13 months. In vitro translation was equivalent for all formulations. [Modes for carrying out the invention]

[0399] This disclosure is 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 that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of this disclosure, and that the scope of this disclosure is limited only by the accompanying claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0400] Preferably, the terms used herein are defined as those found in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0401] Unless otherwise indicated, the implementation of this disclosure will utilize conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in 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).

[0402] The elements of this disclosure are described below. These elements are listed along with specific embodiments, but it should be understood that they may be combined in any way and in any number to create additional embodiments. The various examples and embodiments described should not be construed as limiting this disclosure to only the embodiments expressly described. This description should be understood as disclosing and encompassing embodiments that combine the expressly described embodiments with any number of disclosed elements. Furthermore, any rearrangement and combination of all described elements should be considered disclosed by this description unless specifically indicated in the context.

[0403] The term “about” means approximately or nearly, and in the context of numerical values ​​or ranges described herein, in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the numerical values ​​or ranges enumerated or claimed.

[0404] In the context describing this disclosure (particularly in the context of the claims), the terms “one” and “it,” and similar references, should be interpreted as encompassing both singular and plural, unless otherwise specifically indicated herein or unless the context clearly contradicts it. Enumerations of value ranges herein are intended simply as a way of concisely referring to each distinct value belonging to that range individually. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein may be carried out in any preferred order, unless otherwise specifically indicated herein or unless the context clearly contradicts it. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended solely to better illustrate this disclosure and does not impose limitations on the claims. No language herein should be interpreted as referring to any unclaimed element essential to the practice of this disclosure.

[0405] Unless otherwise specified, the term “contains” is used in the context of this Document to indicate that there may be additional members in addition to the members of the list introduced by “contains.” However, in certain embodiments of this Disclosure, the term “contains” is intended to include the possibility that there may be no additional members; that is, for the purposes of these embodiments, “contains” should be understood to mean “consisting of.”

[0406] Throughout this specification, several sources are referenced. Each source referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that this disclosure had no prior rights to such disclosure.

[0407] definition The following definitions apply to all aspects of this disclosure. Unless otherwise indicated, the following terms have the meanings set forth below. Terms that are not defined have the meanings that are widely recognized in their respective art.

[0408] As used herein, terms such as “reduce” or “inhibit” mean the ability to produce an overall reduction of, for example, a level of 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 include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0409] In one embodiment, terms such as “increase” or “enhance” 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%.

[0410] As used herein, "physiological pH" refers to a pH of approximately 7.5.

[0411] As used in this disclosure, "%w / v" refers to weight / volume percentage, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0412] The term "ionic strength" refers to the mathematical relationship between the number of different types of ions in a given solution and their respective charges. Therefore, the ionic strength I is given by the following equation:

number

[0413] According to this disclosure, the term “ionic strength” in one embodiment refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, the presence of a chelating agent causes their concentration or effective concentration (presence of free ions) to be sufficiently low in one embodiment to prevent RNA degradation. In one embodiment, the concentration or effective concentration of divalent ions is lower than the catalytic level for hydrolysis of phosphodiester bonds 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.

[0414] "Osmolality by weight" refers to the concentration of a particular solute, expressed as the number of osmoles of solute per kilogram of solvent.

[0415] The Reynolds number is a dimensionless number and can be calculated using the following form:

number

[0416] The term "freezing" usually refers to the solidification of a liquid, typically accompanied by the removal of heat.

[0417] The term "freeze-drying" refers to the process of freeze-drying a substance by freezing it and then reducing the surrounding pressure, which causes the freezing medium within the substance to sublimate directly from the solid phase to the gas phase.

[0418] The term "spray drying" refers to the process of drying a substance by mixing a heated gas with a liquid atomized (sprayed) in a container (spray dryer). The solvent then evaporates from the droplets formed, resulting in a dry powder.

[0419] The term "freezing agent" refers to a substance added to a formulation to protect the active ingredient during the freezing process.

[0420] The term "freeze-drying protective agent" refers to a substance added to a formulation to protect the active ingredient during the drying process.

[0421] The term "reconstruct" refers to the process of adding a solvent, such as water, to a dried product to return it to its original liquid state or other liquid state.

[0422] In the context of this disclosure, the term “recombinant” means “produced by genetic engineering.” In one embodiment, the “recombinant object” in the context of this disclosure does not exist in nature.

[0423] As used herein, the term “naturally occurring” refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that are present in living organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring. The term “found in nature” means “present in nature” and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0424] The term "equilibrium solubility" refers to the concentration of a solute at which the rate at which it dissolves is equal to the rate at which it precipitates from the solution. In one embodiment, this term refers to the respective concentrations at room temperature.

[0425] As used herein, the term “room temperature” refers to temperatures greater than 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.

[0426] In the context of this disclosure, the term “particle” refers to a structured entity formed by a molecule or molecular complex. In one embodiment, the term “particle” refers to a micro-sized or nano-sized structure, for example, a micro-sized or nano-sized dense structure.

[0427] In the context of this disclosure, the term “RNA lipoplex particles” refers to particles containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and additional lipids such as DOPE. In one embodiment, the RNA lipoplex particles are nanoparticles.

[0428] As used in this disclosure, “nanoparticles” refers to particles comprising RNA and at least one cationic lipid, having an average diameter suitable for intravenous administration.

[0429] The term "average diameter" refers to the so-called Z, which has the dimension of length. 平均 This refers to the mean hydrodynamic diameter of a particle, measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "mean diameter", "diameter", or "size" of a particle is this Z 平均 It is used synonymously with the value.

[0430] The term "polydispersion index" is used herein as a measure of the size distribution of a particle aggregate, such as a collection of nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0431] As used herein, “invisible particles” refers to particles having an average diameter of less than 100 micrometers (μm). The number of invisible particles can be measured in this disclosure using light occlusion to indicate the degree of aggregation of RNA lipoplex particles. In some embodiments, the number of invisible particles having an average diameter of 10 μm or more is measured. In other embodiments, the number of invisible particles having an average diameter of 25 μm or more is measured.

[0432] The term "ethanol injection technique" refers to the process in which a lipid-containing ethanol solution is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicles, including liposomes. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. In one embodiment, such a colloidal liposome dispersion is formed using the ethanol injection technique as follows: an ethanol solution containing lipids, such as cationic lipids like DOTMA and additional lipids, is injected into an aqueous solution while stirring. In one embodiment, the RNA lipoplex particles described herein can be obtained without using an extrusion process.

[0433] The term "extrude" refers to the creation of particles with a fixed cross-sectional profile. In particular, the term refers to the miniaturization of particles, where the particles are forced to pass through a filter with defined pores.

[0434] The term trehalose always refers to both trehalose anhydrous and trehalose dihydrate. Any concentration of trehalose is given in relation to trehalose dihydrate.

[0435] The term EDTA refers to ethylenediaminetetraacetate disodium salt. Any concentration is given in relation to EDTA disodium salt.

[0436] Diameter of RNA lipoplex particles In one embodiment, the RNA lipoplex particles described herein 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, RNA lipoplex particles have an average diameter of approximately 200 nm, approximately 225 nm, approximately 250 nm, approximately 275 nm, approximately 300 nm, approximately 325 nm, approximately 350 nm, approximately 375 nm, approximately 400 nm, approximately 425 nm, approximately 450 nm, approximately 475 nm, approximately 500 nm, approximately 525 nm, approximately 550 nm, approximately 575 nm, approximately 600 nm, approximately 625 nm, approximately 650 nm, approximately 700 nm, approximately 725 nm, approximately 750 nm, approximately 775 nm, approximately 800 nm, approximately 825 nm, approximately 850 nm, approximately 875 nm, approximately 900 nm, approximately 925 nm, approximately 950 nm, approximately 975 nm, or approximately 1000 nm. In one embodiment, RNA lipoplex particles have an average diameter in the range of approximately 250 nm to approximately 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.

[0437] For example, RNA lipoplex particles produced by the processes described herein exhibit a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3. As an example, RNA lipoplex particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3.

[0438] Lipids In one embodiment, the lipid solutions, liposomes, and RNA lipoplex particles described herein contain cationic lipids. As used herein, “cationic lipids” refers to lipids that have a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix through electrostatic interactions. Generally, cationic lipids have lipophilic moieties such as sterol, acyl, or diacyl chains, and the lipid head group typically carries a positive charge. Examples of cationic lipids, but not limited to, include 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; dioctadecyldimethylammonium chloride (DODAC), 2,3- Examples include di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium 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.

[0439] Additional lipids may be incorporated to adjust the overall positive-to-negative charge ratio and physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipids are neutral lipids. As used herein, “neutral lipids” refers to lipids 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, ceramides, sphingomyelin, cephalin, cholesterol, and cerebrosides. In certain embodiments, the second lipid is DOPE, cholesterol, and / or DOPC.

[0440] In certain embodiments, RNA lipoplex particles contain both cationic lipids and additional lipids. In exemplary embodiments, the cationic lipid is DOTMA and the additional lipid is DOPE. While we do not wish to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid may affect important RNA lipoplex particle properties such as charge, particle size, stability, tissue selectivity, and the bioactivity of RNA. Therefore, in some embodiments, 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, or about 3:1 to about 1:1. In certain embodiments, the molar ratio may 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 exemplary embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is approximately 2:1.

[0441] RNA In this disclosure, the term “RNA” refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, 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, double-stranded RNA, single-stranded RNA, isolated RNA such as 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 alteration of one or more nucleotides. Such alterations may 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 intended herein that nucleotides in RNA may be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogues of naturally occurring RNA.

[0442] In certain embodiments of this disclosure, RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is 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, RNA is produced by in vitro transcription or chemosynthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to nucleic acids comprising deoxyribonucleotides.

[0443] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for regulating transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0444] In certain embodiments of this 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, RNA is present in concentrations of approximately 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.10 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.20 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, and 0.25 mg The concentrations are approximately 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.30 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.50 mg / mL. In exemplary embodiments, the RNA concentration is approximately 0.02 mg / mL or approximately 0.05 mg / mL.

[0445] In one embodiment, RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine and pseudouridine.

[0446] In some embodiments, the RNA according to this disclosure includes a 5' cap. In one embodiment, the RNA according to this disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified by a 5' cap analogue. The term “5' cap” refers to a 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 position 7. Providing a 5' cap or a 5' cap analogue to RNA can be achieved by in vitro transcription, either by co-transcriptional expression of the 5' cap on the RNA strand or by capping enzymes that can be attached to the RNA post-transcriptionally.

[0447] In some embodiments, the RNA according to this disclosure includes a 5'-UTR and / or a 3'-UTR. The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) may be present on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if 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 (if present), for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end downstream of the termination codon of the protein-coding region, but the term “3'-UTR” preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0448] In some embodiments, the RNA according to this disclosure comprises a 3'-poly(A) sequence. The term “poly(A) sequence” refers to a sequence of adenyl (A) residues typically located at the 3' end of an RNA molecule. According to this 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.

[0449] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. RNA can then be translated into peptides or proteins.

[0450] In relation to RNA, the terms "expression" or "translation" refer to the process within a cell's ribosome in which a chain of mRNA is guided to construct an amino acid sequence to make up a peptide or protein.

[0451] In one embodiment, after administration of the RNA lipoplex particles described herein, at least a portion of the RNA is delivered to 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 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 in the spleen. In one embodiment, the target cells are dendritic cells in the spleen. Thus, the RNA lipoplex particles described herein can be used to deliver RNA to such target cells. Therefore, this disclosure also relates to a method for delivering RNA to target cells, comprising administering the RNA lipoplex particles described herein to the target. In one embodiment, the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is RNA encoding a peptide or protein, and the RNA is translated by the target cells to produce a peptide or protein.

[0452] In one embodiment, the RNA encodes a pharmaceutically active peptide or protein.

[0453] According to this disclosure, the term “RNA-encoded” means that RNA, when present in a suitable environment such as within the cells of a target tissue, can induce the construction of amino acids to produce the peptide or protein it encodes during the translation process. In one embodiment, RNA can interact with cellular translation mechanisms that enable the translation of peptides or proteins. Cells may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or produce them on their surface.

[0454] According to this disclosure, the term “peptide” includes oligopeptides and polypeptides and refers to substances containing about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100 or about 150 consecutive amino acids linked to one another by peptide bonds. The term “protein” refers to a large peptide, in particular a peptide having at least about 151 amino acids, but the terms “peptide” and “protein” are generally used as synonyms herein.

[0455] A "pharmaceutically active peptide or protein," when provided to a subject in a therapeutically effective amount, exerts a positive or beneficial effect on the subject's condition or disease state. In one embodiment, a pharmaceutically active peptide or protein may have therapeutic or palliative properties and may 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 may have prophylactic properties and may 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" may include the entire protein or polypeptide and may also refer to its pharmaceutically active fragments. The term may also include pharmaceutically active analogues of the peptide or protein.

[0456] 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 factors (G-CSF), granulocyte-macrophage colony-stimulating factors (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, adresins, seretins, homing receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, and immune Scientifically active antigens (e.g., bacterial antigens, parasitic antigens or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormones, catecholamines, gonadotrophins, 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). Enzymes involved in hormone biosynthesis or degradation, steroid-producing enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylyl cyclase or guanylate cyclase, neuramidases, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription factors and translation factors, tumor growth inhibitory proteins (e.g., those that inhibit angiogenesis). Examples include proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (such as 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, and anticoagulant factors).

[0457] The term “immunologically active compound” refers to any compound that alters the immune response by altering humoral immunity, for example, by inducing and / or suppressing the maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or stimulating antibody production by B cells. Immunologically active compounds may have potent immunostimulatory activity, including, but are not limited to, antiviral and antitumor activity, and may also downregulate other aspects of the immune response, for example, by shifting the immune response away from the TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants.

[0458] In one embodiment, the 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 induces an immune response in the subject against one or more antigens or one or more epitopes, which may be therapeutic, partially, or completely protective.

[0459] The term "antigen" refers to an active substance containing an epitope that can elicit an immune response. The term "antigen" includes proteins and peptides in particular. 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 conjugated, 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-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, and the epitope is derived from such an antigen.

[0460] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or humoral antibody response to the disease. Therefore, disease-associated antigens or their epitopes may be used for therapeutic purposes. Disease-associated antigens may be associated with microorganisms, typically infections caused by microbial antigens, or with cancer, typically tumors.

[0461] The term "tumor antigen" refers to components of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus. In particular, this term refers to antigens produced intracellularly or as surface antigens on tumor cells.

[0462] The term "viral antigen" refers to any viral component that possesses antigenic properties, i.e., can induce an immune response in an individual. Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0463] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties, that is, can induce an immune response in an individual. Bacterial antigens can originate from the bacterial cell wall or cytoplasmic membrane.

[0464] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by T cells, B cells, or antibodies. An antigen epitope may include a continuous or discontinuous portion of the antigen and may be about 5 to about 100 amino acids long. In one embodiment, an epitope is about 10 to about 25 amino acids long. The term "epitope" includes T cell epitopes.

[0465] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in association with an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or disease cells in immune responses, and MHC proteins or 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 cell surface and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is typically about 8 to 10 amino acids long, but longer or shorter peptides may also be effective. In the case of class II MHC / peptide complexes, the bound peptide is typically about 10 to 25 amino acids long, and especially about 13 to 18 amino acids long, although longer and shorter peptides may also be effective.

[0466] In certain embodiments of this disclosure, the RNA encodes at least one epitope. In certain embodiments, the epitope is derived from a tumor antigen. The tumor antigen may be a “standard” antigen that is commonly known to be expressed in various cancers. The tumor antigen may also be a “neoantigen” that is specific to the tumor of an individual and has not been previously recognized by the immune system. A neoantigen or neoepitope may arise from one or more cancer-specific mutations in the genome of a cancer cell that result in amino acid changes. 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, and other cell surface proteins of the claudin family, 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 / Melan A, MC1R, Myosin / m, MUC1 Examples include 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.

[0467] 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 that can induce a robust immune response within the host. RNA can be used to deliver patient-specific tumor epitopes to patients. 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 therapeutic effects in tumor vaccine compositions. Rapid sequencing of tumor mutanomes may provide multiple epitopes for personalized vaccines encoded by the RNAs described herein, for example, as single polypeptides, with the epitopes optionally isolated by linkers. In certain embodiments of this disclosure, the RNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. An exemplary embodiment includes RNA encoding at least five epitopes (referred to as "pentatopes") and RNA encoding at least ten epitopes (referred to as "decatopes").

[0468] charge ratio The charge of the RNA lipoplex particles of this 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 = [(concentration of cationic lipid (mol)) * (total number of positive charges in the cationic lipid)] / [(concentration of RNA (mol)) * (total number of negative charges in the RNA)]. The concentration of RNA and the amount of at least one cationic lipid can be determined by a person skilled in the art using a method commonly used by those skilled in the art.

[0469] In the first embodiment, the charge ratio of positive to negative charges in RNA lipoplex particles at physiological pH is about 1.9:2 to about 1:2. In certain embodiments, the charge ratio of positive to negative charges in 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 RNA lipoplex particles at physiological pH is 1.3:2.0. In another embodiment, the RNA lipoplex particles described herein may result in RNA lipoplex particles having an equal number of positive and negative charges at physiological pH and having a net neutral charge ratio.

[0470] In the second embodiment, the charge ratio of positive to negative charges in RNA lipoplex particles at physiological pH is approximately 6:1 to approximately 1.5:1. In certain embodiments, the charge ratio of positive to negative charges in RNA lipoplex particles at physiological pH is approximately 6.0:1.0, 5.9:1.0, 5.8:1.0, 5.7:1.0, 5.6:1.0, 5.5:1.0, 5.4:1.0, 5.3:1.0, 5.2:1.0, 5.1:1.0, 5.0:1.0, 4.9:1.0, 4.8:1.0, 4.7:1.0, 4.6:1.0, 4.5:1.0, 4.4:1.0, 4.3:1.0, 4.2:1.0, 4.1:1.0, and 4.0:1.0. The ratios are approximately 0, 3.9:1.0, 3.8:1.0, 3.7:1.0, 3.6:1.0, 3.5:1.0, 3.4:1.0, 3.3:1.0, 3.2:1.0, 3.1:1.0, 3.0:1.0, 2.9:1.0, 2.8:1.0, 2.7:1.0, 2.6:1.0, 2.5:1.0, 2.4:1.0, 2.3:1.0, 2.2:1.0, 2.1:1.0, 2.0:1.0, 1.9:1.0, 1.8:1.0, 1.7:1.0, 1.6:1.0, or 1.5:1.0.

[0471] In RNA-based immunotherapy, targeting a specific organ, such as the spleen, is necessary to avoid autoimmune responses in other organs and potential toxicity. According to this disclosure, RNA can target a variety of cells, tissues, or organs.

[0472] It has been found that RNA lipoplex particles having a charge ratio according to the 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 RNA lipoplex particles. In this way, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression does not occur at all or is essentially absent in the lungs and / or liver after administration of RNA lipoplex particles. In one embodiment, RNA accumulation and / or RNA expression occurs in the spleen within antigen-presenting cells, such as professional antigen-presenting cells, after administration of RNA lipoplex particles. 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.

[0473] It has been found that RNA lipoplex particles having the charge ratio according to the second embodiment can be used to preferentially target lung tissue or lung cells. Thus, in one embodiment, RNA accumulation and / or RNA expression occur in the lung after administration of RNA lipoplex particles. In this way, the RNA lipoplex particles of this disclosure can be used to express RNA in the lung. Therefore, if RNA expression is desired in tissue other than the spleen, in the embodiments described herein, 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 instead of the charge ratio according to the first embodiment, for example, a charge ratio of about 1:2 to about 1.9:2, in relation to the charge ratio according to the first embodiment. In these and other embodiments described herein, RNA other than RNA encoding a peptide or protein containing at least one epitope, for example, RNA encoding a pharmaceutically active peptide or protein as described herein, may be used. In one embodiment, the pharmaceutically active peptide or protein is a cytokine and / or is intended for the treatment of lung cancer.

[0474] Composition containing RNA lipoplex particles A. Salt and Ionic Strength According to this disclosure, the compositions described herein may include salts such as sodium chloride. While not wishing to be bound by theory, sodium chloride functions as an ionic osmolality agent for pre-preparing RNA before mixing with at least one cationic lipid. Certain embodiments of this disclosure intend alternative organic or inorganic salts to sodium chloride. 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 sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0475] Generally, compositions containing RNA lipoplex particles as described herein preferably contain sodium chloride at concentrations ranging from 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 containing RNA lipoplex particles has an ionic strength corresponding to such a sodium chloride concentration.

[0476] 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 high ionic strength. In one embodiment, the sodium chloride concentration is at least 45 mM. In one embodiment, the sodium chloride concentration is about 45 mM to about 300 mM or about 50 mM to about 150 mM. In one embodiment, the composition has ionic strength corresponding to such sodium chloride concentrations.

[0477] Generally, compositions for preserving RNA lipoplex particles, such as those described herein, have low sodium chloride concentrations or low ionic strengths. In one embodiment, the sodium chloride concentration is 0 mM to about 50 mM, 0 mM to about 40 mM, or about 10 mM to about 50 mM. In a particular embodiment, the sodium chloride concentration is 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, or about 25 mM. The concentrations are approximately 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, or 50 mM. In a preferred embodiment, the sodium chloride is concentrated at approximately 20 mM, 30 mM, or 40 mM. In an exemplary embodiment, the sodium chloride is concentrated at 20 mM. In another exemplary embodiment, the sodium chloride is concentrated at 30 mM. In one embodiment, the composition has an ionic strength corresponding to such a sodium chloride concentration.

[0478] Generally, compositions obtained by thawing a frozen RNA lipoplex particle composition and, optionally, adjusting the osmolality by weight and ionic strength by adding an aqueous solution, have a high sodium chloride concentration or high ionic strength. In one embodiment, the sodium chloride concentration is 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.

[0479] B. Stabilizers The compositions described herein may include stabilizers to prevent substantial loss of product quality, particularly substantial loss of RNA activity, during freezing, lyophilization, or spray drying, and during storage of the frozen, lyophilized, or spray-dried compositions. Such compositions are also referred to herein as stable. Typically, the stabilizer is present before the freezing, lyophilization, or spray-driing process and remains in the resulting frozen, lyophilized, or freeze-dried preparation. It can be used, for example, to protect RNA lipoplex particles during freezing, lyophilization, or spray drying, and during storage of the frozen, lyophilized, or freeze-dried preparation, in order to reduce or prevent aggregation, particle disintegration, RNA degradation, and / or other types of damage.

[0480] In one embodiment, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0481] In one embodiment, the stabilizer is a monosaccharide. As used herein, the term “monosaccharide” refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to further simpler carbohydrate units. Exemplary monosaccharide stabilizers include glucose, fructose, galactose, xylose, and ribose.

[0482] 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 glycosidic bonds, for example, through 1 to 4 bonds or 1 to 6 bonds. Disaccharides can be hydrolyzed to two monosaccharides. Exemplary disaccharide stabilizers include sucrose, trehalose, lactose, and maltose.

[0483] The term "trisaccharide" refers to three sugars that combine to form a single molecule. Examples of trisaccharides include raffinose and meletitose.

[0484] 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 linked to one another via glycosidic bonds, for example, 1 to 4 bonds or 1 to 6 bonds, to form a linear, branched, or cyclic structure. Exemplary oligosaccharide stabilizers include cyclodextrin, raffinose, meletitose, maltotriose, stachyose, acarbose, and the like. Oligosaccharides can be oxidized or reduced.

[0485] 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 linked to each other via glycosidic bonds, for example, 1 to 4 bonds or 1 to 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.

[0486] Other exemplary cyclic oligosaccharide stabilizers include compounds containing a cyclodextrin moiety within a relatively large molecular structure, such as a polymer containing a cyclic oligosaccharide moiety. Cyclic oligosaccharides can be oxidized or reduced, for example, 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 part of a relatively large molecular structure, such as a polymer. A cyclodextrin moiety can be linked directly to one or more other moieties or via any linker. A cyclodextrin moiety can be oxidized or reduced, for example, to a dicarbonyl form.

[0487] 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., partially etherified β-cyclodextrin).

[0488] Examples of stabilizers include polysaccharides. As used herein, the term “polysaccharide” refers to a compound or chemical moiety formed by at least 16 monosaccharide units linked to one another 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 polysaccharides as part of their skeletal structure. In the skeleton, the polysaccharides may be linear or cyclic. Examples of polysaccharide stabilizers include glycogen, amylase, cellulose, dextran, and maltodextrin.

[0489] In one embodiment, the stabilizer is a sugar alcohol. As used herein, the term “sugar alcohol” refers to the reduction product of a “sugar,” indicating that any oxygen atoms in a simple sugar alcohol molecule are present in the form of a hydroxyl group. Sugar alcohols are also “polyols.” This term refers to a chemical compound containing three or more hydroxyl groups and is synonymous with another conventional term, polyhydric alcohol. Examples of sugar alcohols include, but are not limited to, sorbitol, mannitol, maltitol, lactitol, erythritol, glycerin, xylitol, or inositol.

[0490] This disclosure provides pharmaceutical compositions comprising sucrose as a stabilizer. While not wishing to be bound by theory, sucrose functions to promote cryoprotection of the composition, thereby preventing aggregation of RNA lipoplex particles and maintaining the chemical and physical stability of the composition. Certain embodiments of this disclosure envision alternative stabilizers to sucrose. 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.

[0491] In one embodiment, the stabilizer is concentrated at a concentration of approximately 5% (w / v) to approximately 35% (w / v) or approximately 10% (w / v) to approximately 25% (w / v). In a specific embodiment, the stabilizer is concentrated at a concentration of approximately 10% (w / v), approximately 11% (w / v), approximately 12% (w / v), approximately 13% (w / v), approximately 14% (w / v), approximately 15% (w / v), approximately 16% (w / v), approximately 17% (w / v), approximately 18% (w / v), approximately 19% (w / v), approximately 20% (w / v), approximately 21% (w / v), approximately 22% (w / v), approximately 23% (w / v), approximately 24% (w / v), or approximately 25% (w / v). In one preferred embodiment, the stabilizer is concentrated at a concentration of about 15% (w / v) to about 25% (w / v). In another preferred embodiment, the stabilizer is concentrated at a concentration of about 20% (w / v) to about 25% (w / v). In one exemplary embodiment, the stabilizer is concentrated at a concentration of about 25% (w / v). In another exemplary embodiment, the stabilizer is concentrated at a concentration of about 22% (w / v). In embodiments of this disclosure, the stabilizer is sucrose or trehalose. In one embodiment of this disclosure, the stabilizer is sucrose. In one embodiment of this disclosure, the stabilizer is trehalose.

[0492] According to this disclosure, the RNA lipoplex particle compositions described herein have stabilizer concentrations suitable for the stability of the composition, particularly the stability of the RNA lipoplex particles and the stability of the RNA.

[0493] C. pH and buffering agent According to this disclosure, the RNA lipoplex particle compositions described herein have 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 about 5.7 to about 6.7. In a particular embodiment, 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.

[0494] This disclosure provides compositions comprising buffering agents. While we do not wish to be bound by theory, the use of buffering agents maintains the pH of the composition during its preparation, storage, and use. In certain embodiments of this disclosure, the buffering agents include 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), and 3-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl] The buffer may be amino-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholine-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), or phosphate-buffered saline (PBS). Other suitable buffers may be acetic acid in the salt, citric acid in the salt, boric acid in the salt, and phosphoric acid in the salt.

[0495] In some embodiments, the buffer has a pH of 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 preferred embodiments, HEPES has a pH of 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 exemplary embodiments, HEPES has a pH of about 6.2.

[0496] In yet another embodiment, the buffer has a concentration of about 2.5 mM to about 10 mM. In a particular embodiment 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. The concentrations are approximately 6.0 mM, 6.25 mM, 6.5 mM, 6.75 mM, 7.0 mM, 7.25 mM, 7.5 mM, 7.75 mM, 8.0 mM, 8.25 mM, 8.5 mM, 8.75 mM, 9.0 mM, 9.25 mM, 9.5 mM, 9.75 mM, or 10.0 mM. In a preferred embodiment, HEPES has a concentration of approximately 7.5 mM.

[0497] D. Chelating agents Certain embodiments of this disclosure intend to utilize chelating agents. A chelating agent is a chemical compound that can form at least two coordination-covalent bonds with a metal ion, thereby producing a stable, water-soluble complex. While we do not wish to be bound by theory, chelating agents in this disclosure reduce the concentration of free divalent ions that could normally induce accelerated RNA degradation. Examples of preferred chelating agents, but not limited to, include ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, sodium dithiocarb, penicillamine, calcium pentetate, sodium salts of pentetate, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic 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 exemplary embodiments, the chelating agent is EDTA disodium dihydrate.

[0498] In some embodiments, the EDTA concentration is approximately 0.25 mM to approximately 5 mM. In certain embodiments, the EDTA concentration is approximately 0.25 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, and 2.5 mM. The concentrations are approximately 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3.0 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4.0 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, or 5.0 mM. In a preferred embodiment, the EDTA concentration is approximately 2.5 mM.

[0499] E. Exemplary compositions of the present disclosure In one exemplary embodiment, the RNA lipoplex particle composition 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, where the charge ratio of positive to negative charges in the RNA lipoplex particle is about 1.3:2.0 at physiological pH, 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, and EDTA at a concentration of about 2.5 mM, with a pH of about 6.2. In a further specific embodiment, the RNA encodes five or ten epitopes.

[0500] In another exemplary embodiment, the RNA lipoplex particle composition 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, where the charge ratio of positive to negative charges in the RNA lipoplex particle is about 1.3:2.0 at physiological pH, 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, and EDTA at a concentration of about 2.5 mM, with a pH of about 6.2. In a further specific embodiment, the RNA encodes five or ten epitopes.

[0501] F. Stability of the compositions of this disclosure As used herein, “stable” refers to a composition in which measurements of various physicochemical parameters are within a defined range. In one embodiment, a composition is analyzed to evaluate its stability according to various parameters. According to this disclosure, stability parameters include, but are not limited to, the average diameter of RNA lipoplex particles, polydispersity index, RNA integrity, RNA content, pH, osmolality by weight, and the number of particles not visible to the naked eye. Those skilled in the art will be able to measure such parameters using commonly used experimental techniques and measuring means. For example, stability parameters may be evaluated using dynamic light scattering (DLS), photocanceling, spectroscopy, agarose gel electrophoresis, a bioanalyzer, or any other suitable technique. In one embodiment, the bioanalyzer is an Agilent 2100 Bioanalyzer (Agilent Technologies) capable of measuring both RNA integrity and RNA content. In one embodiment, the bioanalyzer is a Fragment Analyzer manufactured by Advanced Analytical.

[0502] While we do not wish to be bound by theory, DLS measurements are useful for analyzing parameters related to RNA lipoplex particles in this disclosure. In one embodiment, DLS may be used to determine the average diameter of RNA lipoplex particles, expressed with respect to Z-avg (a measure of average particle size). In another embodiment, DLS may be used to determine the polydispersity index of RNA lipoplex particles, indicating the size and weight distribution of RNA lipoplex particles.

[0503] In certain embodiments, the composition is stable if the measured values ​​of the stability parameters are within a specified range. In one embodiment of a stable composition, the RNA lipoplex particles, after storage, for example at a temperature of about -15°C to about -40°C, have an average diameter that differs from the original average diameter (i.e., the average diameter before freezing, lyophilization, or spray drying and thawing or reconstitution) by only ±20%, ±10%, ±5%, or ±3% or less. In one embodiment of a stable composition, the RNA lipoplex particles, after storage, for example at a temperature of about -15°C to about -40°C, have an average diameter that differs from the original average diameter (i.e., the average diameter before freezing, lyophilization, or spray drying and thawing or reconstitution) by 20%, 10%, 5%, or 3% or less. In another embodiment of the stable composition, the RNA lipoplex particles, after storage, for example at a temperature of approximately -15°C to approximately -40°C, have a polydispersity index that differs from the original polydispersity index (i.e., the polydispersity index before freezing, lyophilization, or spray drying and thawing or reconstitution) by only ±20%, ±10%, ±5%, or ±3% or less. In one embodiment, the stable composition, after storage, for example at a temperature of approximately -15°C to approximately -40°C, has 6,000 or fewer invisible particles with a diameter of 10 μm or more. In one embodiment, the stable composition, after storage, for example at a temperature of approximately -15°C to approximately -40°C, has 600 or fewer invisible particles with a diameter of 25 μm or more.

[0504] 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.

[0505] In one embodiment, the composition is stable at a storage temperature of about -15°C to about -40°C. In a particular embodiment, the composition is stable at temperatures 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 a preferred embodiment, the composition is stable at temperatures of about -15°C, about -20°C, about -30°C, or about -40°C.

[0506] 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 a preferred embodiment, the composition is stable at a temperature of about -15°C to about -25°C when the composition is protected from light.

[0507] In one embodiment, the composition is stable at a temperature of about -15°C to about -40°C for at least one month to about 24 months. In a particular embodiment, the composition is stable at a temperature of approximately -15°C to approximately -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.

[0508] In a preferred embodiment, the composition is stable at a temperature of about -15°C for at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months.

[0509] In another preferred embodiment, the composition is stable at a temperature of about -20°C for at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months.

[0510] In yet another preferred embodiment, the composition is stable at a temperature of about -30°C for at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months.

[0511] In one embodiment, the composition is stable after being frozen at a temperature of approximately -15°C to approximately -40°C and thawed at a temperature of approximately 4°C to approximately 25°C (ambient temperature). In another embodiment, the composition is stable after being frozen at a temperature of approximately -15°C to approximately -40°C through multiple freeze-thaw cycles and thawed at a temperature of approximately 4°C to approximately 25°C (ambient temperature).

[0512] G. Physical state of the compositions disclosed herein In embodiments, the compositions of the present disclosure are liquid or solid. Non-limiting examples of solids include frozen or freeze-dried forms. In preferred embodiments, the composition is liquid.

[0513] Pharmaceutical compositions of the present disclosure The compositions comprising RNA lipoplex particles described herein are useful as pharmaceutical compositions or agents for therapeutic or prophylactic treatments, or for preparing them.

[0514] The particles of this disclosure may be administered in the form of any suitable pharmaceutical composition.

[0515] The term “pharmaceutical composition” refers to a formulation comprising a therapeutically effective active substance, preferably 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 administration of the pharmaceutical composition to a target. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. In the context of this disclosure, a pharmaceutical composition includes RNA lipoplex particles as described herein.

[0516] The pharmaceutical compositions of this disclosure may preferably contain one or more adjuvants or be administered together with one or more adjuvants. The term “adjuvant” refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), inorganic compounds (e.g., alum), bacterial products (e.g., 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, and chemokines. Chemokines may 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, and LT-a. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides such as Pam3Cys.

[0517] The pharmaceutical compositions described herein are generally applied in "pharmaceutically effective amounts" and "pharmaceutically acceptable preparations."

[0518] The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the active ingredient of a pharmaceutical composition.

[0519] The term “pharmaceutically effective amount” refers to the amount, alone or in combination with additional doses, that achieves the desired response or effect. In the case of treating a particular disease, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. The desired response in the treatment of a disease may also be delaying or preventing the onset of the disease or condition described herein. 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 treatment, the type of accompanying treatment (if any), the specific route of administration, and similar factors. Therefore, the dosage of the particles or compositions described herein may depend on such various parameters. If the patient’s response is insufficient with the initial dose, a higher dose (or substantially higher dose achieved by a different, more localized route of administration) may be used.

[0520] The pharmaceutical compositions of this disclosure may contain salts, buffers, preservatives and optionally other therapeutic agents. In one embodiment, the pharmaceutical composition of this disclosure comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0521] Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0522] As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions of this 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 colorants.

[0523] The term “diluent” refers to a substance that dilutes and / or reduces a substance. Furthermore, the term “diluent” includes one or more fluids, liquid or solid suspensions, and / or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.

[0524] The term “carrier” refers to a component that may be natural, synthetic, organic, or inorganic, to which the active ingredient is combined to facilitate, enhance, or enable the administration of the pharmaceutical composition. As used herein, carriers may be one or more suitable solid or liquid fillers, diluents, or encapsulants suitable for administration to a target. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, naphthalene hydrogenation, and in particular biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of this disclosure contains an isotonic saline.

[0525] Pharmacopoeia-acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0526] The drug carrier, excipient, or diluent can be selected in relation to the intended route of administration and standard pharmacopoeia.

[0527] Route of administration of the pharmaceutical composition disclosed herein In one embodiment, the pharmaceutical composition described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may 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 intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration.

[0528] product In one embodiment, the RNA lipoplex particles described herein are present in a pharmaceutical composition. In another embodiment, the composition described herein is a pharmaceutical composition.

[0529] In one embodiment, this disclosure relates to a vial containing the pharmaceutical composition described herein. In another embodiment, this disclosure relates to a syringe containing the pharmaceutical composition described herein.

[0530] Use of the pharmaceutical composition disclosed herein The RNA lipoplex particles described herein may be used in the therapeutic or prophylactic treatment of various diseases, particularly those in which providing peptides or proteins to a target results in a therapeutic or prophylactic effect. For example, providing antigens or epitopes derived from a virus may be useful in the treatment of viral diseases caused by such viruses. Providing tumor antigens or epitopes may be useful in the treatment of cancerous diseases in which cancer cells express such tumor antigens.

[0531] The term “disease” refers to an abnormal condition affecting an individual’s body. Often, a disease is interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by external factors, such as infections, or by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death in the affected individual, or similar problems in those in contact with the individual. In this broader sense, disease sometimes includes injury, helplessness, disability, syndrome, infection, solitary symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Many diseases affect individuals not only physically but also emotionally, as suffering from and living with them can alter one’s outlook on life and personality.

[0532] In this context, the terms “treatment,” “to treat,” or “therapeutic intervention” relate to the management and care of an individual aimed at combating a condition such as a disease or disorder. The term is intended to encompass all range of treatments for a given condition in which an individual is afflicted, including the administration of therapeutically effective compounds to alleviate 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, as well as to prevent the condition, where prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder, and including the administration of active compounds to prevent the onset of symptoms or complications.

[0533] The term "therapeutic treatment" refers to any treatment that improves the health of an individual and / or extends (increases) its lifespan. Such treatments may eliminate disease in an individual, stop or delay the onset of disease in an individual, inhibit or delay the onset of disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in individuals that currently have or have previously had the disease.

[0534] The terms “preventive measures” or “preventive measures” refer to any treatment aimed at preventing disease from developing in an individual. The terms “preventive measures” and “preventive measures” are used interchangeably herein.

[0535] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) that is susceptible to, or prone to, a disease or disorder (e.g., cancer), and may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children and newborns. In embodiments of this disclosure, “individual” or “subject” is “patient.”

[0536] The term "patient" means an individual or subject for treatment, in particular an individual or subject that is afflicted.

[0537] In one embodiment of the present disclosure, the objective is to provide an immune response against disease cells expressing antigens, such as cancer cells expressing tumor antigens, and to treat diseases such as cancers involving cells expressing antigens such as tumor antigens.

[0538] A pharmaceutical composition comprising RNA lipoplex particles described herein, which comprises RNA encoding a peptide or protein containing one or more antigens or one or more epitopes, may be administered to a subject to induce an immune response to one or more antigens or one or more epitopes that may be therapeutic or partially or completely protective. Those 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 is produced by immunizing a subject with an antigen or epitope that is immunologically relevant with respect to the disease being treated. Thus, the pharmaceutical compositions described herein are applicable to induce or enhance an immune response. In this way, the pharmaceutical compositions described herein are useful for prophylactic and / or therapeutic treatment of diseases involving antigens or epitopes.

[0539] As used herein, “immune response” refers to an integrated bodily response to an antigen or a cell expressing an antigen, and includes cellular and / or humoral immune responses. Cellular immune responses include, but are not limited to, cellular responses directed to cells expressing an antigen and characterized by the presentation of the antigen by a class I or class II MHC molecule. Cellular responses involve T lymphocytes, which can be classified as helper T cells (also called CD4+ T cells) that play a central role in regulating the immune response, or killer cells (also called cytotoxic T cells, CD8+ T cells, or CTLs) that induce apoptosis in infected or cancer cells. In one embodiment, administration of a pharmaceutical composition of this disclosure involves stimulating an antitumor CD8+ T cell response to cancer cells expressing one or more tumor antigens. In certain embodiments, the tumor antigen is presented by a class I MHC molecule.

[0540] This disclosure envisions an immune response that may be protective, defensive, prophylactic, and / or therapeutic. As used herein, “inducing (or inducing) an immune response” may indicate that an immune response to a particular antigen was not present before induction, or that a baseline level of immune response to a particular antigen existed before induction and was enhanced after induction. Thus, “inducing (or inducing) an immune response” includes “enhancing (or enhancing) an immune response.”

[0541] The term "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0542] The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual with the aim of inducing an immune response, for example, for therapeutic or preventive reasons.

[0543] In one embodiment, this disclosure envisions an embodiment in which RNA lipoplex particles described herein are administered targeting spleen tissue. The RNA encodes a peptide or protein containing an antigen or epitope, for example, as described herein. The RNA is taken up by antigen-presenting cells in the spleen, such as dendritic cells, to express the peptide or protein. Following any processing and presentation by the antigen-presenting cells, an immune response to the antigen or epitope occurs, which may result in prophylactic and / or therapeutic treatment for a disease in which the antigen or epitope is involved. In one embodiment, the immune response induced by the RNA lipoplex particles described herein includes the presentation of its fragments, such as the antigen or epitope, by antigen-presenting cells such as dendritic cells and / or macrophages, and the activation of cytotoxic T cells by this presentation. For example, a peptide or protein encoded by RNA or its processing product may be presented by a major histocompatibility complex (MHC) protein expressed on an antigen-presenting cell. The MHC peptide complex can then be recognized by immune cells such as T cells or B cells, resulting in their activation.

[0544] Therefore, 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 splenic antigen-presenting cells. Therefore, in one embodiment, after administration of the RNA lipoplex particles, RNA delivery and / or RNA expression within the antigen-presenting cells occur. The antigen-presenting cells may be professional antigen-presenting cells or non-professional antigen-presenting cells. Professional antigen-presenting cells may be dendritic cells and / or macrophages, more preferably splenic dendritic cells and / or splenic macrophages.

[0545] Accordingly, this disclosure relates to RNA lipoplex particles as described herein, or pharmaceutical compositions comprising RNA lipoplex particles, for inducing or enhancing an immune response, preferably an immune response against cancer.

[0546] In further embodiments, the present disclosure relates to RNA lipoplex particles as described herein, or pharmaceutical compositions comprising RNA lipoplex particles, for use in prophylactic and / or therapeutic treatment of antigen-related diseases, preferably cancerous diseases.

[0547] In further embodiments, the present disclosure relates to a method for delivering an antigen or an epitope of an antigen to antigen-presenting cells, such as professional antigen-presenting cells in the spleen, or a method for expressing an antigen or an epitope of an antigen within antigen-presenting cells, such as professional antigen-presenting cells in the spleen, the method comprising administering RNA lipoplex particles or a pharmaceutical composition comprising RNA lipoplex particles as described herein to a target. In one embodiment, the antigen is a tumor antigen. In this embodiment, the antigen or epitope of an antigen is preferably encoded by RNA in the RNA lipoplex particles.

[0548] In one embodiment, systemic administration of the RNA lipoplex particles described herein, or a pharmaceutical composition containing RNA lipoplex particles, results in targeting and / or accumulation of the RNA lipoplex particles or RNA in the spleen, but no targeting and / or accumulation occurs in the lungs and / or liver. In one embodiment, the RNA lipoplex particles release RNA within the spleen and / or enter cells within the spleen. In one embodiment, systemic administration of the RNA lipoplex particles described herein, or a pharmaceutical composition containing RNA lipoplex particles, results in delivery of RNA to antigen-presenting cells in the spleen. In certain embodiments, the antigen-presenting cells in the spleen are dendritic cells or macrophages.

[0549] In further embodiments, the present disclosure relates to a method for inducing or enhancing an immune response to a target, comprising administering RNA lipoplex particles described herein, or a pharmaceutical composition comprising RNA lipoplex particles, to a target. In exemplary embodiments, the immune response is against cancer.

[0550] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf foreign pathogens within the macrophage and kill them through hydrolytic and oxidative attacks that lead to the degradation of the pathogens. Peptides derived from the degraded proteins are displayed on the surface of macrophage cells, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to the activation of T 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.

[0551] The term “dendritic cell” (DC) refers to another subtype of phagocytic cell belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells originate from hematopoietic myeloprogenitor cells. These progenitor cells first differentiate 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 for pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated and become mature dendritic cells, and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, breaking down their proteins into small fragments, and upon maturation, they use MHC molecules to present these fragments on their cell surface. 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 lymph nodes. Here, they function as antigen-presenting cells, activating helper T cells and killer T cells, as well as B cells by presenting antigens along with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T-cell or B-cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0552] The term "antigen-presenting cell" (APC) refers to one of the various cells capable of displaying, acquiring, and / or presenting at least one antigen or antigenic fragment on (or on) its cell surface. Antigen-presenting cells can be distinguished into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0553] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When T cells interact with the MHC class II molecular complex on the membrane of antigen-presenting cells, the antigen-presenting cells produce costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0554] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but express them in response to stimulation by certain cytokines, such as interferon-gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0555] "Antigen processing" refers to the breakdown of an antigen into processing products, which are fragments of the antigen (for example, breaking down a protein into a peptide), and the association (for example, 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.

[0556] The terms “antigen-related disease” or “epitope-related disease” refer to any disease in which an antigen or epitope is involved, for example, a disease characterized by the presence of an antigen or epitope. Antigen-related diseases may be infectious diseases, or cancerous diseases or simply cancer. As mentioned above, antigens can be disease-related antigens such as tumor-associated antigens, viral antigens, or bacterial antigens, and epitopes may originate from such antigens.

[0557] 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 factor (e.g., the common cold). Infectious diseases are well 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 may include, for example, hepatitis, sexually transmitted infections (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.

[0558] The term “cancer” or “cancer” refers to or describes a physiological condition in an individual typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, hematological cancers, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, genital and reproductive cancers, Hodgkin’s disease, esophageal cancer, small intestine cancer, endocrine cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), neuroectodermal carcinoma, spinal axial tumors, gliomas, meningiomas, and pituitary adenomas. The term “cancer” as used in this disclosure also includes cancer metastases.

[0559] Combination strategies in cancer treatment can be desirable due to the resulting synergistic effects, which can be considerably more potent than the effects of monotherapy approaches. In one embodiment, a pharmaceutical composition is administered together with an immunotherapy agent. As used herein, “immunotherapy agent” refers to any active substance that may be involved in the activation of a particular immune response and / or immune effector function(s). This disclosure intends to use antibodies as immunotherapy agents. While we do not wish to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or, by binding to complement proteins, may result in direct cytotoxicity known as complement-dependent cell-mediated cytotoxicity (CDC). Non-limiting examples of anticancer antibodies and potential antibody targets (in parentheses) that may be used in combination with this disclosure include: avagovomab (CA-125), absiximab (CD41), adecatumumab (EpCAM), aftuzumab (CD20), aracizumab pegol (VEGFR2), artumomab penteate (CEA), amatsuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), alsitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), vectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), and vibatuzumab meltansine (CD44). v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab meltansine (mucin CanAg), cantuzumab lavtansine (MUC1), capromab pendetide (prostate cancer cells), carrumab (CNT0888), catumakisomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab vogatox (EpCAM), thixumumab (IGF-1 receptor), clodiximab (claudin), cribatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), darotuzumab (insulin) (Lymphoma-like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), droditumab (DR5), eclomeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enabatuzumab (PDL192), encituximab (NPC-1C), epratuzumab (CD22), ertzumaxomab (HER2 / neu, CD3), etalacizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficratuzumab (SCH900105), Figitumumab (IGF-1 receptor), Frambotumab (glycoprotein 75), Fresolimmubab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab Ozogamicin (CD33), Gevokizumab (IL-Iβ), Gilentuximab (carbonic anhydrase 9 (CA-IX)), Grembatumumab Vedotin (GPNMB), ibritumomab tiuxetan (CD20), iculcumab (VEGFR-1), igoboma (CA-125), indatuximab tansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), rabetsumab (CEA), lexatumumab Br (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), rorbotuzumab meltansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matsuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitumomab (GD3 ganglion). Oside), mogamulizumab (CCR4), moxetumomab pasdotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R)a) Onartuzumab (human scattering factor receptor kinase), oportuzumab monatox (EpCAM), olegobomab (CA-125), oxerumab (OX-40), panitumumab (EGFR), patritumumab (HER3), pemtumoma (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), lacosumomab (N-glycolylneuraminic acid), radretumumab (fibronectin extradomain B), rafibirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor ), samalizumab (CD200), sibrotuzumab (FAP), siltuximab (IL-6), tabalumab (BAFF), takatuzumab tetraxetan (α-fetoprotein), tapritumomab paptox (CD19), tenatumomab (tenascin C), teprotumumab (CD221), tisilimmumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucozotsumab cermoloykin (EpCAM), ubrituximab (MS4A1), urerumab (4-1 Examples include BB), boroximab (integrin α5β1), botumumab (tumor antigen CTAA 16.88), saltumumab (EGFR), and zanolimmubab (CD4).

[0560] In one embodiment, the immunotherapy 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 for "PD-1" include CD279 and SLEB2. Aliases for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Aliases for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, a 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, a PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In other embodiments, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In certain embodiments, the PD-L2 binding partner is PD-1. The PD-1 binding antagonist may be an antibody, its antigen-binding fragment, 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.

[0561] In one embodiment, the PD-1 binding antagonist is an immunoadhesin containing an extracellular portion of PD-L1 or PD-L2 fused to a constant region or a PD-1 binding moiety. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg, which is PD-L2-Fc), a fusion soluble receptor described in International Publication Nos. 2010 / 027827 and 2011 / 066342.

[0562] In one embodiment, the PD-1 conjugated antagonist is an anti-PD-L1 antibody, but is not limited to, YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab).

[0563] In one embodiment, the immunotherapy agent is a PD-1 conjugated antagonist. In another embodiment, the PD-1 conjugated antagonist is an anti-PD-L1 antibody. In an exemplary embodiment, the anti-PD-L1 antibody is atezolizumab.

[0564] The references to documents and studies made herein are not intended as an endorsement that any of the above constitutes appropriate prior art. All statements relating to the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the contents of these documents.

[0565] The following description is provided to enable those skilled in the art to create and use various embodiments. Descriptions of specific apparatus, techniques, and applications are provided as examples 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 given a scope consistent with the claims. [Examples]

[0566] (Example 1) material The key materials used in the experiments described below were as follows: TIFF2026143672000003.tif105153

[0567] (Example 2) Preparation of lipid mixtures DOTMA / DOPE lipid mixtures were prepared with different lipid concentrations in ethanol, each with a lipid molar ratio of 2:1. The solutions were prepared as follows: Weigh the DOPE lipids. Calculate the amount of DOTMA and maintain a 2:1 DOTMA / DOPE percentage molar ratio. Weigh the DOTMA lipids. • Calculate the amount of anhydrous ethanol needed to dissolve the lipids. Weigh out the anhydrous ethanol. • The lipids are dissolved in ethanol using a 37°C aqueous bed.

[0568] The solubility of DOPE and DOPE / DOTMA in ethanol was investigated by preparing ethanol solutions of 300 mM DOPE or 330 mM DOPE / DOTMA (66:33). Lipids were 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.

[0569] Further lipid mixtures can be similarly prepared by following the basic steps described in this example, provided that other lipids are used as starting materials and / or that other molar ratios are calculated.

[0570] (Example 3) Liposome preparation Liposomes were prepared by ethanol injection as follows: Using a 1 mL syringe equipped with a 0.9 × 40 mm needle, 0.2 mL of DOTM / DOPE or (other lipid solution) ethanol solution was injected into 9.8 mL of water while stirring at 120 rpm. The liposome colloid was stirred for 30 minutes. The liposomes were filtered through a 0.45, 1.2, or 5 μm CA syringe filter, or not filtered. The liposome colloid was 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, in a 66:33% molar ratio.

[0571] (Example 4) Preparation of RNA lipoplex 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. Subsequently, 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. Various RNA lipoplex formulations were prepared, for example, 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 (of different sizes) were used in the preparation of the RNA lipoplexes.

[0572] (Example 5) Dynamic light scattering Liposome and RNA lipoplex sizes were measured using a Nicomp instrument (PSS.Santa Barbara.USA) by a known method of dynamic light scattering (DLS). Liposome samples were diluted with water to a total lipid concentration of 1 mM. RNA lipoplex samples were diluted 1:5 with 0.9% NaCl solution. Samples were measured in 5 × 50 mm culture tubes (Kimble.USA).

[0573] (Example 6) Optical occultation - dynamic light scattering Using an Accusizer A7000 instrument (PSS.Santa Barbara.USA), particle counting / measurement was performed from various liposomes and RNA lipoplex formulations with size ranges from 0.5 to 5 μm. Three measurements were performed with a volume of 5 mL (2.5 μL sample / 20 mL free particle water). The results obtained represent the average particle amount from the three measurements.

[0574] (Example 7) Small angle X-ray scattering The internal structure parameters of various RNA lipoplex formulations prepared using various liposome precursors were measured using small-angle X-ray scattering (SAXS). SAXS is a technique that can quantify nanoscale density differences in a sample by analyzing the elastic scattering behavior of X-rays as they pass through a material and recording these scatterings at small angles. Correlation length and d-interval parameters were calculated for all RNA formulations tested. RNA lipoplex formulations were prepared using an N / P ratio of 0.65, 0.1 mg / mL of RNA, and 112 mM NaCl.

[0575] (Example 8) Agarose gel electrophoresis The amount of free RNA in various RNA lipoplex samples was measured by gel electrophoresis. Measurements were performed using a 1% agarose gel containing sodium hypochlorite. RNA lipoplex samples were diluted 1:6 with DNA loading dye. 12 μL of diluted RNA lipoplex sample was carefully loaded onto the agarose gel. Electrophoresis was performed at 80 V for a run time of 40 minutes.

[0576] (Example 9) HPLC Lipid concentrations in various liposome formulations were measured by HPLC (Agilent Technologies, Santa Clara, USA) using a Sunfire C 18 2.5 μm 4.6 × 75 mm column (Waters, Massachusetts, USA) and a wavelength of 205 nm. 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. Liposome samples were diluted with water or left undiluted to a total lipid concentration of 3 mM.

[0577] (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 to 0.01 mg / mL of RNA using 0.9% NaCl solution. The RNA transfection efficiency of various RNA lipoplex formulations was investigated in human dendritic cells seeded in culture medium or whole blood.

[0578] (Example 11) Animal models: Spleen 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 lipoplex was injected retroorbitally, and luciferase expression in dendritic cells (spleen target) was measured 6 hours later. RNA lipoplexes were prepared using Luc-RNA, various liposome precursors, small or large liposomes obtained from biocolloids, and small and large liposomes after 0.45 μm filtration, with N / P ratios of 0.65 and 112 mM NaCl.

[0579] (Example 12) Solubility test High-concentration DOPE-containing solutions were prepared (in a supersaturated state) and tested for their suitability for ethanol injection for liposome production (see next section).

[0580] The results obtained from this series of solubility tests are shown in Tables 1 and 2:

[0581] [Table 1]

[0582] Solubility of DOPE in ethanol containing 220 mM DOTMA

[0583] [Table 2]

[0584] These results indicate that 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 significantly increases the equilibrium solubility, for example, to approximately 90–100 mM.

[0585] (Example 13) Manufacturing of liposomes of various sizes Liposomes were prepared by ethanol injection using the protocol described in Example 3. No filtration process was performed after ethanol injection. The lipid concentration in the ethanol was varied, while all other parameters remained fixed. Liposome size was measured by dynamic light scattering (DLS) as described in Examples 5 and 6.

[0586] 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) and Figures 1 and 2.

[0587] [Table 3]

[0588] As described above, the obtained liposome size (Z-mean) increases with the lipid concentration in the ethanol solution used for ethanol injection. Therefore, the lipid concentration in ethanol can be efficiently used to control liposome size. Interestingly, the size change was most pronounced when the DOPE concentration was below and above the equilibrium solubility of DOPE in ethanol alone. When the DOPE concentration exceeded 50 mM (150 mM total lipid concentration), the obtained liposome size increased dramatically from less than 50 nm to more than 500 nm (Figure 1). However, beyond the solubility limit, the liposome size increased monotonically with increasing lipid concentration.

[0589] All liposome formulations contained a 0.5 μm liposome fraction, which was high in liposomes prepared with a 300 mM lipid solution and decreased in liposomes prepared with lower or higher lipid concentrations. Furthermore, the 0.6 μm and 0.7 μm liposome fractions were measured in liposome formulations prepared with high lipid concentrations (Figure 2). Large liposomes were formed after ethanol injection of the high-concentration lipid solution. The total amount of formed liposomes was less compared to liposome formulations prepared with low lipid concentrations in the lipid solution. The results obtained represent the average particle size from three measurements.

[0590] (Example 14) Production of RNA lipoplexes from liposomes of various sizes RNA lipoplexes were prepared using various liposome precursors 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. The liposome size (z-mean) and polydispersity index (PDI) were determined during the dynamic light scattering (DLS) experiments described in Examples 5 and 6.

[0591] The results regarding the effect of lipid concentration for liposome preparation on RNA lipoplex size (Z-mean) (and consequently on liposome precursor size) are shown in Table 4 (below) and the corresponding Figures 3 and 4.

[0592] [Table 4]

[0593] 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 prepared using solutions of 300 mM or higher were approximately twice the size of those obtained from liposomes prepared using a 150 mM stock solution. In all cases of RNA lipoplexes prepared using large liposomes, no correlation was observed between liposome size and RNA lipoplex size (Figure 3).

[0594] The amount of RNA-lipoplexes obtained increased with the size of the liposomes used in their formation. Formulations prepared using large liposomes showed a large amount of large RNA-lipoplex particles, supported by data obtained from dynamic light scattering measurements (Figure 4). The results represent the average particle amount from three measurements.

[0595] (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 of various concentrations derived from stock solutions. 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 prepared by ethanol injection from lipid concentrations in 100 mM, 300 mM, and 400 mM ethanol, as described.

[0596] Figure 5 shows diffraction curves obtained from small-angle X-ray scattering measurements of RNA lipoplexes formed using liposomes prepared with a charge ratio of 4 / 1 (top) and a charge ratio of 1.3 / 2, where the diffraction curves obtained using mM lipid stock solutions in 400 mM, 300 mM, and 100 mM ethanol for lipoplex formation.

[0597] The scattering pattern is approximately 1 nm. -1 This includes a single Bragg peak. This is a typical diffraction pattern for spleen-targeted lipoplexes that use excess (negatively charged) RNA for lipoplex formation. If the lipoplex is not negatively charged, the scattering profile is completely different. As an example, when an RNA lipoplex with a charge ratio of + / - 4 / 1 was measured, a much less prominent peak was observed. Similarly, secondary peaks (though less intense) are also discernible. In fact, a general feature of the X-ray scattering profile of lipoplexes is that, depending on the phase state, several peaks that could be equidistant may have other spacings. 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. Higher concentrations in ethanol resulted in lower peak widths. The Bragg peak indicates that the lipoplex is regularly arranged, where the repetition distance (d interval) is given from the peak position as follows:

number

[0598] Here, q is momentum transfer,

number

[0599] The peak width Δq decreases as the number of iteration units in the stack increases. In the case of a liquid crystal array, the correlation length can be given as follows:

number

[0600] A clear correlation can be derived between the scattering pattern of the lipoplex product, the aforementioned lipid concentration in ethanol for liposome production, and its biological activity. While the peak position remains constant, the peak width changes 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 peak width, and therefore an increase in correlation length. Simultaneously, biological activity increases with increasing correlation length. Thus, the above-mentioned lipoplex with improved activity, produced from liposomes using a high-concentration lipid stock solution in ethanol, can be identified by distinct structural characteristics. Furthermore, the above-mentioned lipoplex with improved activity can also be distinguished from the less active lipoplex by other methods such as asymmetric field flow fractionation (AF4).

[0601] (Example 16) Transfection efficiency of RNA lipoplexes in human dendritic cells Luc-RNA lipoplexes were prepared as described using various liposome precursors, in which case the size of the liposomes for their formation was varied (by changing the starting lipid concentration for their formation), while all other parameters were kept fixed. Subsequently, the RNA transfection efficiency of the various RNA lipoplex formulations was investigated in human dendritic cells seeded in culture medium or whole blood.

[0602] Figure 6 shows the results of in vitro transfection efficiency (in human dendritic cells) determined by measuring the luciferase expression and corresponding biological activity of various RNA lipoplexes prepared using various liposome precursors.

[0603] 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.

[0604] (Example 17) Asymmetric flow field flow fractionation of various lipoplexes Today, we used asymmetric flow field-flow fractionation (AF4), a common and state-of-the-art method for fractionating and separating particles in suspensions, to determine yet another difference in RNA lipoplexes. According to AF4 theory, particles with similar properties and equal size should elute simultaneously.

[0605] Figure 7 shows some results regarding AF4 measurements of lipoplexes obtained from two different types of liposomes prepared from either a 150 mM stock solution in ethanol or a 400 mM stock solution in ethanol.

[0606] In summary, field flow fractionation measurements demonstrate that lipoplexes derived from liposomes at a 150 mM lipid concentration in ethanol differ qualitatively and quantitatively from those derived from lipoplexes at a 400 mM lipid concentration. The 150 mM lipoplexes are smaller, but intuitively, the slower elution suggests there must be qualitative differences between the two (morphology, medium interactions, charge). The RNA lipoplexes obtained from the 150 mM ethanol solution are smaller in size but elute more slowly. This indicates that RNA lipoplexes produced from liposomes using a 150 mM lipid solution in ethanol are, on average, smaller than those obtained from 400 mM lipid in ethanol. Even at the same size, the 150 mM lipoplexes have different physicochemical properties than the 400 mM lipoplexes. These differences in size and physicochemical properties correlate with the relatively higher biological activity of the 400 mM RNA lipoplexes.

[0607] (Example 18) Biological activity of RNA lipoplexes in vitro As determined by cell culture transfection experiments (dendritic cells) using various sizes of RNA lipoplexes encoding luciferase, prepared from liposomes, various lipid stocks, and / or various lipid concentrations, the luciferase signal, and thus its biological activity, increases monotonically with the lipid starting concentration and, consequently, the liposome size used for RNA lipoplex formation. The corresponding results are shown in Figures 6, 8, and 9.

[0608] In summary, RNA lipoplexes generated from high lipid concentrations for liposome production exhibit significantly higher activity in vitro.

[0609] (Example 19) In vivo biological activity of RNA lipoplexes Cell culture transfection experiments (in dendritic cells) using RNA lipoplexes encoding luciferase, prepared from liposomes of various sizes, have determined that RNA lipoplexes prepared using larger liposomes result in significantly higher luciferase expression and corresponding biological activity.

[0610] Non-limiting examples of this series of tests are shown in Figures 10 and 11. RNA lipoplexes prepared using large liposomes obtained from 360 mM biocolloids yielded significantly better in vivo expression signals than small RNA lipoplexes obtained from 200 mM biocolloids.

[0611] (Example 20) Automated production of RNA lipoplex A generally applicable procedure for automated batch production of RNA lipoplexes was developed and is shown in Figure 12. All steps are performed using pre-sterilized, disposable fluid pathways that allow for safe, aseptic handling of materials. First, the RNA concentration is adjusted to match the liposome concentration, and NaCl is added to concentrate the RNA. This adjusts the RNA solution to an RNA concentration that allows for mixing of equal volumes of RNA and liposomes. Both RNA and liposome solutions are transferred to large-capacity syringes, and both syringes are attached to a single syringe pump, driving both pistons of the syringes simultaneously. After RNA lipoplex formation, a cryoprotectant solution is added to adjust the final concentration of the formulation. After filling the formulation into glass vials, the formulation is frozen as a concentrate for long-term storage.

[0612] A key quality attribute of RNA lipoplexes is the charge ratio, which is adjusted by the mixing ratio of RNA to liposomes. We have developed an automated and scalable industrial manufacturing process for RNA lipoplexes that enables efficient control of the mixing ratio. In small-scale (≤10 liters) manufacturing processes, control of the mixing of two identical volumes of aqueous solutions containing liposomes and RNA is achieved by using a single perfusion pump that simultaneously drives two large-volume syringes filled with RNA or liposomes. To similarly pump larger volumes (≥10 liters) as pressurized vessels, membrane pumps, gear pumps, magnetic levitation pumps, or peristaltic pumps are used in combination with flow sensors that have a feedback loop for online control and real-time adjustment of flow rate.

[0613] Automated RNA lipoplex production requires a static mixing element to ensure efficient mixing of aqueous solutions containing RNA and liposomes. Commercial microfluidic mixing elements containing meandering channels and embedding structures to facilitate mixing, as well as prototype mixing elements with equivalent structures, were found to clog during production. Therefore, these mixing elements are not suitable for automated RNA lipoplex production. Y-type and T-type mixing elements with diameters ranging from 1.2 mm to 50.0 mm were found to be suitable for automated RNA lipoplex production.

[0614] Methods: RNA lipoplexes were prepared using various mixed elements (Table 1). During lipoplex production, the mixed elements were observed by the operator, and material deposition or clogging was recorded.

[0615] Results: With commercially available microfluidic chips (NanoAssemblr®, Precision Nanosystems, Vancouver, Canada), clogging was observed after the preparation of 3 mL of RNA lipoplex. Similar observations were made during testing of additional prototype microfluidic mixing elements (Table 5). Deposition, particularly element clogging, was observed with all (micro)fluidic mixing elements having a structure equivalent to the commercially available elements, whereas this was not observed when using Y-type or T-type mixing elements. In addition to the Y-type mixing element with a diameter of 2.4 mm described above, mixing elements with larger diameters were also tested. Since no limitations were found on the diameter of the Y-type mixing element, the above method is considered suitable for the preparation of RNA lipoplex with Y-type mixing elements with a diameter of up to 50 mm.

[0616] [Table 5]

[0617] When using Y-type or T-type mixed elements, a minimum flow rate is required to achieve sufficient mixing. RNA lipoplex preparation can be carried out by mixing two aqueous solutions containing RNA and liposomes using Y-type or T-type mixed elements with an inner diameter of 1.6 to 50 mm. To ensure efficient mixing, the Reynolds number resulting from the flow rate for mixing should not be less than approximately 300. To ensure efficient mixing, the ratio of flow rate to the diameter of the mixed element should not be less than approximately 150. Experimental testing of Reynolds numbers up to approximately 2100 found it suitable for automated production. The data supports the possibility of even higher flow rates. This stems from the fact that at a Reynolds number of 2100, the conditions are already in turbulent mode, and similar mixing conditions can be expected even at higher flow rates.

[0618] Methods: RNA lipoplexes were prepared by pumping RNA solution and liposome solution using a single perfusion pump. RNA lipoplex formation was performed using representative Y-type mixed elements with inner diameters of 2.4 and 3.2 mm. Photon correlation spectroscopy (PCS) measurements were used to analyze the particle size and polydispersity of the RNA lipoplexes. The Reynolds number resulting from the investigated combinations of flow rate and mixed element diameter was theoretically calculated using the formula (Figure 13). The ratio of flow rate to mixed element diameter is given by the flow rate (cm 3 The calculation was performed by dividing the coefficient ( / min) by the diameter (cm) of the mixed element. The coefficient is given as a dimensionless number.

[0619] Results: When RNA lipoplexes were produced using flow rate and mixed element combinations that resulted in a theoretically calculated Reynolds number of less than approximately 300, RNA lipoplexes with increased particle size and polydispersity were formed (Figure 13 and Table 6). To ensure the reproducible formation of RNA lipoplexes with the desired particle characteristics, the theoretical Reynolds number should be at least approximately 300 (Figures 13 and 14 and Table 6), and the flow rate-to-mixed element diameter ratio should be at least approximately 150. Mixed elements of 2.4 mm were found to enable efficient and reproducible mixing of RNA and liposomes over a wide range of flow rates (60–240 mL / min). No upper limit was found during these tests, so no upper limit is predicted for the Reynolds number or the flow rate-to-mixed element diameter ratio.

[0620] [Table 6]

[0621] (Example 21) Effect of RNA concentration RNA lipoplexes were prepared using y-type mixed elements with typical dimensions (2.4 mm). To determine the concentration range in which RNA lipoplexes can be prepared using 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 formulations were adjusted to 0.05 mg / mL RNA, 22% sucrose, and 20 mM NaCl, and the formulations were frozen three times.

[0622] RNA lipoplex formation at RNA concentrations of 0.1–0.5 mg / mL yields comparable particle properties (Figure 15). These particle properties are also preserved after three freezes, demonstrating a highly robust manufacturing process with respect to variations in RNA concentration (Figure 16). Since there are no indications of limitations on RNA concentration during RNA lipoplex preparation, the described settings are considered suitable for RNA lipoplex preparation at RNA concentrations up to 5 mg / mL.

[0623] The described process for the automated production of RNA lipoplexes enables the reproducible preparation of stable RNA lipoplexes with various charge ratios.

[0624] Methods: To demonstrate the robustness of semi-automated preparation of RNA lipoplexes with respect to 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).

[0625] Results: At charge ratios of 1.0:2.0 to 2.1:2.0, no effect of charge ratio variation on RNA lipoplex size and polydispersity was observed (Figure 17). Therefore, the range of 1.0:2.0 to 2.1:2.0 is considered to yield RNA lipoplex preparations of comparable quality. Furthermore, at charge ratios of 3.0:1.0 to 5.0:1.0, stable particles with specified size and polydispersity were formed (Figure 18).

[0626] (Example 22) Salt concentration during RNA lipoplex formation Automated production of RNA lipoplexes with high biological activity requires controlled ionic conditions during RNA lipoplex formation. To ensure biological activity, RNA lipoplex formation must occur in the presence of 45–300 mM NaCl. Other ionic compounds, such as EDTA and HEPES, can contribute to ionic strength and potentially lower the minimum required concentration of NaCl.

[0627] Methods: RNA lipoplexes were automatically prepared using various concentrations of NaCl during RNA lipoplex formation. Particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS). Furthermore, the biological activity of the lipoplexes was investigated by measuring the luciferase signal in vitro.

[0628] Results: Particle properties could be controlled by adjusting the ionic strength. Increasing the salt concentration during manufacturing induced a slight increase in particle size (Figure 19). Salt concentration was found to affect biological activity. Increasing the salt concentration during manufacturing induced an increase in biological activity (Figure 20). Therefore, the NaCl concentration during RNA lipoplex formation should not be less than 45 mM NaCl.

[0629] (Example 23) Stabilization of RNA lipoplex To stabilize RNA in RNA lipoplexes both in and out of the presence of cryoprotective agents, buffer systems such as HEPES, acetate / sodium acetate, and sodium phosphate can be used in the pH range of 5.5–6.7. Sodium carbonate systems were found not to provide a comparable stabilization effect.

[0630] Methods: To investigate the optimal pH range and test the suitability of various buffers encompassing different pH ranges (HEPES pH 6.8–8.2, acetate / sodium acetate pH 3.7–5.6, sodium phosphate pH 5.8–8.0, and sodium carbonate pH 6.2–8.6), RNA lipoplexes were initially incubated under stress conditions (40°C) in the absence of cryoprotectants. RNA integrity was analyzed by capillary electrophoresis over 21 days. To investigate the optimal pH range in the presence of exemplary cryoprotectants, RNA lipoplexes were incubated at 40°C in the presence of HEPES and sucrose, and RNA integrity was analyzed for 21 days.

[0631] Results: Similar results were obtained with the buffer systems HEPES, acetate / sodium acetate, and sodium phosphate, but the carbonate systems did not provide comparable RNA stabilization (Figure 21). RNA integrity depends on the pH value of the formulation. The optimal pH range was identified as pH 5.5–7.4. In the presence of the exemplary cryoprotectant sucrose, a pH range of pH 5.5–8.0 was identified as providing the best RNA stabilization (Figure 22).

[0632] Divalent metal ions may arise from the RNA synthesis process, formulation excipients, or glass containers, potentially affecting 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. The described process allows for RNA lipoplex formation in the presence of EDTA (0-20 mM).

[0633] Methods: RNA lipoplexes were formed in the presence of gradually increasing concentrations of EDTA (up to 18 mM), diluted, and incubated at 40°C with decreasing EDTA content (0.1 mM to 5.4 mM). RNA integrity was analyzed as an important physicochemical parameter over 21 days.

[0634] Results: Forming RNA lipoplexes in the presence of high concentrations of EDTA (up to 18 mM) resulted in particle properties equivalent to those obtained in lower concentrations. No significant differences were observed between different groups containing 0.01% (w / v) (0.26 mM) to 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 can reduce RNA integrity, the presence of EDTA concentrations up to 20 mM during RNA lipoplex formation is considered advantageous.

[0635] (Example 24) Optimization of NaCl and cryoprotectant content The controlled ionic conditions need to be adjusted during the production, long-term storage, and application of RNA lipoplexes to patients (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 a frozen state, and 80-150 mM after thawing and dilution with physiological saline.

[0636] For each NaCl concentration of ≤70 mM, the respective content of cryoprotectants that should not be reduced to ensure stabilization of particle properties during multiple freezes was identified. Monosaccharides and disaccharides can be used as cryoprotectants, including triols as glucose, sucrose, mannitol, trehalose, sorbitol, and glycerin, and mixtures thereof at concentrations of 12.5–35.0% (w / v). The stabilizing effect of sorbitol is lower compared to the latter compounds, and arginine does not efficiently stabilize RNA lipoplexes during freezing.

[0637] [Table 7]

[0638] [Table 8]

[0639] Methods: To identify suitable cryoprotectants, various representative compounds were investigated for cryoprotectant systems containing monosaccharides (glucose and sorbitol), disaccharides (sucrose and trehalose), amino acids (arginine and proline), triols (glycerin), and mixtures of different sugars (mannitol and sucrose) (Figures 25 and 26). RNA lipoplexes were then frozen in the presence of these compounds at progressively increasing concentrations. To determine the minimum content of cryoprotectant at specific concentrations of NaCl, RNA lipoplexes were frozen in progressively increasing amounts of sucrose or trehalose as representative cryoprotectants (Table 7). Samples were initially frozen once to determine the concentration range of cryoprotectants to be investigated in detail. In a third experiment, the effect of particle size stabilization was examined by freezing RNA lipoplexes up to 10 times in the presence of the cryoprotectant trehalose (Table 8).

[0640] Results: Arginine clearly destabilizes RNA lipoplexes, but other cryoprotective agents are generally applicable to stabilizing RNA lipoplexes during freezing (Figures 25 and 26). Glycerin, mannitol, and sucrose (1:1, w:w), proline, and sorbitol can be used as cryoprotective agents for RNA lipoplexes. In addition to arginine, all of the additional stabilizers tested appeared suitable, indicating that a wide range of amino acids, sugars, and mixtures of such compounds are suitable for stabilizing RNA lipoplexes during freezing. The stabilizing effect of sorbitol was lower compared to the latter compounds.

[0641] A detailed investigation of the required amount of cryoprotectant at various NaCl concentrations (Table 8) revealed a direct correlation between the NaCl concentration and the required concentration of cryoprotectant after a single freezing step (Figures 27 and 28). With 20% (w / v) sucrose or trehalose dihydrate, acceptable particle size preservation was observed after a single freezing step with 0–60 mM NaCl. However, at even lower concentrations of cryoprotectant (e.g., ≤15% for 60 mM NaCl; ≤10% for 40 mM NaCl; ≤5% for 20 mM NaCl), insufficient stabilization was observed. Within the investigated range, no significant difference was found between sucrose and trehalose.

[0642] The particle size of RNA lipoplexes after 1, 2, 3, 5, and 10 freezes in the presence of the NaCl and trehalose combinations shown in Table 8 was analyzed, yielding the following results: At a concentration of 50 mM NaCl, ≥12.5% ​​trehalose was sufficient to stabilize the RNA lipoplex particle characteristics even after 10 freezes (Figure 29). However, at 70 mM NaCl, ≥12.5% ​​trehalose was required for minimum stabilization, and ≥22.5% was required for accurate particle size preservation (Figure 30). At 90 mM NaCl, ≥15.0% trehalose was required for minimum stabilization, and accurate particle size preservation could not be achieved even at the highest investigated concentration of 27.5% (Figure 31).

[0643] (Example 25) A combination of salt and freeze protectants for long-term preservation. For long-term storage at a given temperature, the combinations of NaCl and cryoprotectants listed in Table 9 should be used.

[0644] method: To investigate the minimum amount of cryoprotectant required for long-term storage at -15°C to -30°C at specific concentrations of NaCl, RNA lipoplexes were frozen in the presence of either NaCl and either sucrose or trehalose. Samples were frozen at -30°C and then transferred to their respective storage temperatures (-15°C or -30°C). After the specified storage time, samples were analyzed, and the preservation of colloidal stability was assessed by measuring particle size using PCS. These experiments were performed at a total RNA concentration of 0.05 mg / mL.

[0645] Results: While the particle properties of RNA lipoplexes could be preserved during freezing in the presence of up to 70 mM NaCl, these experiments revealed additional effects contributing to the destabilization of colloidal stability. For example, a combination of 60 mM NaCl and 20% sucrose also resulted in acceptable stabilization of particle properties after freezing, and at a storage temperature of -15°C, the particle size of these formulations significantly increased over time (Figures 32 and 33). This effect was delayed at storage at -30°C (Figures 34 and 35).

[0646] The stability of RNA lipoplexes at a given NaCl content depends on the amount of cryoprotectant. The amount of cryoprotectant required for stabilization increases with increasing salt content in the storage solution. This effect is independent of the type of sugar used as the cryoprotectant. RNA lipoplex compositions for long-term storage at -15°C or -30°C should contain the cryoprotectant content listed in Table 9.

[0647] [Table 9]

[0648] (Example 26) Long-term preservation using various cryoprotectants Using 10–40 mM NaCl, stabilization for up to 9 months is possible in the presence of 22% (w / v) monosaccharide or disaccharide. These formulations can be frozen at -15–-40°C and maintained at their respective temperatures for long-term storage. 10–30 mM NaCl is achievable in the presence of 12.6–16.8% (w / v) dextran. These experiments were performed at a total RNA concentration of 0.05 mg / mL.

[0649] Methods: RNA lipoplexes were frozen with fixed cryoprotectant content in combination with various NaCl concentrations to investigate the minimum content of representative cryoprotectants, such as mixtures containing sucrose, trehalose, glucose, and dextran, required for long-term storage at -20°C. For monomeric or dimeric cryoprotectants as glucose, sucrose, and trehalose, concentrations of 22% (w / v) were adjusted. Long-term stability was investigated by freezing these formulations, storing them at -15 to -40°C, and measuring particle size using PCS after a specified storage time.

[0650] For formulations containing polymer dextran mixtures, the compositions listed in Table 10 were prepared. The samples were frozen and stored at -20°C. After the specified storage time, the samples were analyzed, and the preservation of colloidal stability was assessed by measuring the particle size.

[0651] Results: For all monomeric or dimeric cryoprotective agents investigated, a maximum NaCl concentration that should not be exceeded to ensure long-term stability of RNA lipoplexes in a frozen state was identified. 60 and 80 mM NaCl resulted in rapid destabilization of the lipoplexes, but colloidal properties could be preserved for at least 9 months when the NaCl concentration was ≤40 mM at -20°C (Table 10 and Figures 36-38). No difference in stabilization effect was observed for sucrose, trehalose, and glucose. Formulations containing 20 mM NaCl showed no difference in long-term stability when samples were frozen and stored at -15 to -40°C (Figure 39).

[0652] Further investigation of formulations containing dextran with even lower cryoprotectant content (12.6-16.8% (w / v)) revealed that the stabilizing effect was equivalent to or even better than that of monosaccharides or disaccharides (Figure 40).

[0653] [Table 10]

[0654] (Example 27) Freeze drying a) Freezing and thawing To determine the most effective cryoprotective / lyophilization protective agent concentration, freeze-thaw tests were performed. RNA lipoplex formulations were frozen and thawed in 5 mM HEPES, 80 mM NaCl, and 2.6 mM EDTA with 10%, 15%, 20%, 25%, and 30% trehalose added. Particle size was determined before and after storage at -20°C. Particle aggregation was observed in formulations frozen in the absence of trehalose, and their particle size could not be determined. As shown in Figure 41, formulations frozen with cryoprotective / lyophilization protective agents showed concentration-dependent cryoprotection, and particle size increased as the lyophilization protective agent / cryoprotective agent concentration decreased. At 22% w / v trehalose, only a minimal increase in particle size was observed, and the 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 formulation after lyophilization and reconstitution.

[0655] b) Reconfiguration and particle stability RNA lipoplex formulations prepared with 5 mM HEPES, 2.6 mM EDTA, NaCl concentrations from 0 mM to 80 mM, and 10% or 22% trehalose concentrations were lyophilized. All lyophilized samples showed a good cake appearance. The samples were reconstituted to their original volume using a 0.9% NaCl solution. All lyophilized RNA lipoplex formulations dissolved immediately upon reconstitution with a 0.9% NaCl solution or water-based filtration (WFI). The particle size change of lyophilized RNA lipoplex formulations prepared in 22% trehalose after reconstitution with a 0.9% NaCl solution or water was determined.

[0656] As shown in Figure 42, the particle size of lyophilized RNA lipoplex formulations containing trehalose remained nearly stable after lyophilization and reconstitution. When lyophilized samples were reconstituted using 0.9% NaCl, only a slight decrease in RNA lipoplex size was observed compared to lyophilized samples reconstituted with water. A correlation was observed between the ratio of NaCl to trehalose and particle stability. Formulations prepared with low trehalose concentrations and high NaCl concentrations resulted in increased RNA lipoplex particle size.

[0657] c) Cell culture experiments using freeze-dried RNA lipoplex preparations In vitro transfection experiments were performed on lyophilized RNA lipoplex preparations encoding luciferase, prepared in 22% trehalose at various NaCl concentrations. The lyophilized samples were reconstituted using a 0.9% NaCl solution.

[0658] As shown in Figure 43, lyophilized RNA lipoplex preparations prepared with 22% trehalose and various NaCl concentrations showed similar levels of Luc-RNA transfection in dendritic cells. No correlation was observed between the NaCl concentration present in the RNA lipoplex preparations and in vitro RNA transfection. Lyophilized samples showed similar or even better Luc-RNA transfection compared to fresh RNA lipoplex controls.

[0659] d) Stability test For stability testing at 4°C, 25°C, and 40°C (1 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 a 0.9% NaCl solution, the samples were characterized for particle size and RNA integrity (total RNA%).

[0660] As shown in Figures 44 and 45, the sizes of various RNA lipoplexes did not change significantly over time, regardless of the formulation or storage temperature. Interestingly, a small amount of cryoprotectant (e.g., 10%) was sufficient to maintain particle stability in lyophilized formulations, while a larger amount (e.g., 22%) was required for frozen formulations. RNA integrity (full-length RNA%) in lyophilized samples after 6 months of storage at 4°C varied between 94% and 100%, and for RNA(lip) formulations stored at 25°C, it varied between 85% and 94%. However, no correlation was identified with the ratio of trehalose to NaCl concentration or with storage time.

[0661] (Example 28) Preparation and testing of RNA lipoplex particles Preparation of RNA lipoplex For automated batch production of RNA lipoplexes, each step is performed using pre-sterilized, disposable fluid pathways that enable safe, aseptic handling of materials. First, the RNA concentration is adjusted to match the liposome concentration, and the RNA is concentrated by adding NaCl. This adjusts the RNA solution to an RNA concentration that allows for mixing of equal volumes of RNA and liposomes. Both the RNA and liposome solutions are transferred to large-capacity syringes, and both syringes are attached to a single syringe pump, driving the two pistons of the syringes 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.

[0662] A key quality attribute of RNA lipoplexes is the charge ratio, which is adjusted by the mixing ratio of RNA to liposomes. We have developed an automated and scalable industrial manufacturing process for RNA lipoplexes that enables efficient control of the mixing ratio. In small-scale (≤10 liters) manufacturing processes, control of the mixing of two identical volumes of aqueous solutions containing liposomes and RNA is achieved by using a single perfusion pump that simultaneously drives two large-volume syringes filled with RNA or liposomes. To similarly pump larger volumes (≥10 liters) as pressurized vessels, membrane pumps, gear pumps, magnetic levitation pumps, or peristaltic pumps are used in combination with flow sensors that have a feedback loop for online control and real-time adjustment of flow rate.

[0663] Automated RNA lipoplex production requires a mixing element to ensure efficient mixing of the aqueous solution containing RNA and liposomes. Commercial microfluidic mixing elements containing meandering channels and embedding structures to facilitate mixing, as well as prototype mixing elements with equivalent structures, were found to clog during production. Therefore, these mixing elements are not suitable for automated RNA lipoplex production. Y-type and T-type mixing elements with diameters ranging from 1.2 mm to 50.0 mm were found to be suitable for automated RNA lipoplex production.

[0664] 1. Example composition 1.1 Composition of the preparation Formulation 1 includes the following: • Approximately 10% (10% or less) of trehalose / sucrose • ≤ 10mM NaCl • ≤7.5 mM HEPES (or histidine as a second choice) pH 6.5 or pH 6.7 • ≤3.5mM EDTA.

[0665] Formulation 1 allows for the freezing of RNA lipoplex under buffering conditions, enabling direct parenteral administration to patients without further dilution or other procedures. Low salt content does not reduce activity. The weight osmolality of the formulation is suitable for direct intravenous injection.

[0666] The formulation is obtained by mixing equal volumes of RNA solution and liposome solution in a fluid pathway setting with optimized concentration and buffering conditions. The concentrations of cationic lipids in the liposomes and RNA are in a precisely defined molar ratio (charge ratio) of 1.3:2. The liposome and RNA solutions are provided under the following conditions.

[0667] 1.2 RNA composition • RNA concentration of approximately 0.3 mg / mL (however, in the exact molar ratio to cationic lipids in liposomes) • Approximately 18mM of HEPES • Approximately 18 mM EDTA*2Na*2H2O pH 6.2~7.0

[0668] To compensate for the pH shift caused by acetic acid present in the liposomes, the pH of the RNA drug substance buffer is adjusted to pH 7.0.

[0669] The RNA concentration is adjusted to match the DOTMA concentration in the liposomes by dilution with (normal) physiological saline.

[0670] 1.3 Composition of liposomes ·About 0.5mM~0.7mM DOTMA • Approximately 0.2-0.4 mM DOPE • ≤ 5 mM acetic acid (preferably 2 mM)

[0671] Adding acetic acid extends the shelf life of liposomes.

[0672] 2. Examples of more suitable buffers and optimal pH ranges To stabilize RNA in RNA lipoplexes both in and out of the presence of cryoprotective agents, buffer systems such as HEPES, histidine, and acetate / sodium acetate can be used to stabilize the RNA in the lipoplex within a pH range of 5.5 to 7.0.

[0673] Methods: To investigate the optimal pH range and test the suitability of various buffers, buffers encompassing a wide range of pH levels (HEPES pH 6.0–7.2, histidine pH 5.8–7.0, acetate pH 5.5–5.8, MES pH 6.0–7.0) were tested. RNA lipoplexes were incubated under accelerated conditions (25°C) in the presence of exemplary cryoprotectants. RNA integrity was analyzed by capillary electrophoresis over 60 days. Furthermore, RNA lipoplexes were incubated for up to 2 years under predicted storage conditions (-15°C) in the presence of exemplary cryoprotectants. RNA integrity was analyzed by capillary electrophoresis.

[0674] Results: Comparable results were obtained in terms of preservation of particle size and polydispersity of RNA lipoplexes combined with reduced amounts of sucrose and NaCl in both liquid (Figures 46, 47, 54, and 55) and frozen (Figures 50, 51, and 58-60) buffer systems HEPES, histidine, acetate, and MES. RNA integrity depended on the pH value of the formulation. The optimal pH range was identified as pH 6.5–7.0 (Figures 48, 49, 53, 57, and 62). All buffer types tested were found to efficiently preserve the adjusted pH (Figures 52, 56, and 61).

[0675] Example 2.1: HEPES, histidine, and acetate are suitable for stabilizing RNA lipoplexes. The following tests investigated the ability of three buffer systems (HEPES, histidine, and acetate) to stabilize RNA lipoplexes formed using liposomes containing 5 mM acetate. Each RNA lipoplex was tested under accelerated conditions (25°C) and frozen conditions (-15°C). The pH range of 5.5–7.0 was tested, encompassing different pH ranges for each buffer system (HEPES pH 6.2–7.0, histidine pH 5.8–7.0, and acetate pH 5.5–5.8). *Accelerated testing only

[0676] [Table 11]

[0677] Liquid storage at 25°C (accelerated) Figures 46 and 47 demonstrate that particle size and polydispersity are sufficiently maintained in all systems during storage under accelerated conditions, even in the presence of any buffer system.

[0678] Figure 48 demonstrates that the adjusted pH is adequately maintained in all systems during storage under accelerated conditions, even in the presence of any buffer system.

[0679] Figure 49 shows that under accelerated conditions (25°), the best stability was found in samples containing HEPES buffer, while the stability of RNA in RNA lipoplexes was clearly reduced with histidine and acetate buffers.

[0680] In the HEPES buffer system, pH values ​​above 6.2 show significantly improved stability compared to lower values. The optimal stability was identified at a pH of around 6.5–7.0.

[0681] Similar pH dependence is observed for histidine. However, overall stability is lower compared to formulations buffered with HEPES. Acetate shows lower integrity than both other systems, with pH 5.8 showing better stability than pH 5.5.

[0682] Cryopreservation (-15℃) Figures 50 and 51 show the colloidal stability of the buffered samples in the same system previously investigated in liquid state at 25°C. Figures 50 and 51 demonstrate that HEPES, histidine, and acetate are equally suitable for preserving the particle size and polydispersity of RNA lipoplexes in a frozen state (-15°C). Furthermore, in the presence of low concentrations of NaCl, low concentrations of sucrose are found to be sufficient for efficient preservation of the polydispersity of RNA lipoplexes in a frozen state.

[0683] Figure 52 shows the pH values ​​of the samples buffered in the same system as previously investigated in the liquid state at 25°C. This demonstrates that all buffer systems (HEPES, histidine, and acetate) can maintain their pH within the adjustable range even in the frozen state (-15°C).

[0684] Figure 53 shows the stability of samples buffered in the same system previously investigated in liquid state at 25°C. Overall, there is no clear trend regarding the preservation of RNA integrity in the frozen state (-15°C). Certain 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, as no degradation over time is observed in this system.

[0685] Example 2.2: MES is also suitable for stabilizing RNA lipoplexes. The following tests compare the two most suitable buffer systems identified in the previous tests (HEPES and histidine) with an additional buffer system (MES). RNA lipoplexes were formed using liposomes containing 2 mM acetate. Each RNA lipoplex was investigated under accelerated conditions (25°C) and frozen conditions (-15°C). Here, we focused on the pH range of 6.0–7.0, which was found to be the optimal range in the previous tests.

[0686] [Table 12]

[0687] Liquid storage at 25°C (accelerated) As shown in Figure 55, no differences in polydispersity and particle size were observed between buffer systems during storage under accelerated conditions in this new test. MES exhibited similar behavior to histidine and HEPES.

[0688] As shown in Figure 56, all buffer systems can maintain the pH within the adjustable range during storage under accelerated conditions. Therefore, MES, like HEPES and histidine, is suitable for fixing the pH of each RNA lipoplex formulation.

[0689] Figure 57 shows stability data obtained from various buffer systems during storage under accelerated conditions (25°C). HEPES and MES showed comparable stabilization of RNA integrity, while histidine showed slightly reduced stability. The optimal pH for HEPES, MES, or histidine buffers was found to be approximately 6.5–7.0.

[0690] Cryopreservation (-15℃) Figure 58 demonstrates that particle size and polydispersity are well maintained during freezing of the formulation in the presence of any buffer system, and that in the presence of low concentrations of NaCl, low concentrations of sucrose are sufficient to efficiently stabilize the particle size of the RNA lipoplex during freezing.

[0691] Figures 59 and 60 demonstrate that particle size and polydispersity are well maintained during storage in a frozen state in the presence of any buffer system. Furthermore, it is found that in the presence of low concentrations of NaCl, low concentrations of sucrose are sufficient to efficiently stabilize the colloidal properties of RNA lipoplexes in a frozen state.

[0692] Figure 61 demonstrates that all of the buffer systems investigated (HEPES, histidine, and MES) can maintain the pH within the adjustment range even when frozen.

[0693] Figure 62 shows stability data obtained from various buffer systems under frozen conditions. All of the buffer systems investigated (HEPES, MES, or histidine) showed comparable stabilization of RNA integrity, and there was no clear trend indicating an optimal pH within the previously optimized pH range investigated.

[0694] 3. Examples of more suitable buffering agent concentrations For long-term stabilization of RNA lipoplexes in both liquid and frozen states, buffer concentrations were optimized to ensure both pH stabilization and RNA integrity preservation. To ensure pH stabilization and RNA integrity, RNA lipoplexes were formulated using 10% (w / v) sucrose and 6.5 mM NaCl. Samples were stored under accelerated conditions in the presence of an exemplary buffer system HEPES with the following concentrations: 2.5mM HEPES 5.0mM HEPES 7.5mM HEPES 10.0mM HEPES The stabilization effect was investigated using the following exemplary pH values: • pH 6.2 pH 6.7 pH 7.2

[0695] Methods: Each RNA lipoplex was automatically prepared in a single batch, and different concentrations of HEPES were prepared by adding cryoprotection solutions containing different amounts of HEPES. Particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS). Furthermore, RNA integrity was analyzed by capillary electrophoresis, and pH was measured.

[0696] Results: All tested buffer concentrations were found to efficiently preserve the particle size and polydispersity of the RNA lipoplex in liquid state (Figures 63 and 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). In conclusion, buffer concentrations of 2.5 mM to 10.0 mM were found to equally and efficiently stabilize RNA integrity in lipoplex formulations. The optimal pH range was found to be pH 6.7 to 7.2.

[0697] The following tests investigated the concentration of HEPES required to stabilize the pH of RNA lipoplexes. RNA lipoplexes were formed using liposomes containing 5 mM acetic acid, and various concentrations of HEPES were prepared using different cryoprotection solutions. Each RNA lipoplex was then tested under accelerated conditions (25°C). The pH range of 6.2 to 7.2 was tested.

[0698] [Table 13]

[0699] Liquid storage at 25°C (accelerated) Figures 63 and 64 show that the particle size and polydispersity of RNA lipoplexes are preserved at their respective pH values ​​under accelerated storage conditions (25°C) for all buffer concentrations investigated.

[0700] Figure 65 shows the measured pH values ​​of RNA lipoplexes. Samples were stored in the presence of HEPES ranging from 2.5 mM to 10.0 mM. It was found that a low HEPES concentration of 2.5 mM was sufficient to stabilize the adjusted pH volume.

[0701] Figure 66 shows stability data obtained from various buffer concentrations. Under accelerated conditions, all buffer concentrations investigated were found to equally stabilize RNA integrity. The best stabilization of RNA integrity was observed at a pH of approximately 6.7–7.2. RNA lipoplexes stored at pH 6.2 showed slightly reduced RNA integrity.

[0702] 4. Examples of more suitable concentrations of NaCl and cryoprotectant The following examples illustrate situations where the adjusted ionic conditions for RNA lipoplexes may be identical during long-term storage and patient application. Dilution or other modification of the product after thawing is not required to obtain an injectable product. In addition to the previously investigated NaCl concentration range 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 without requiring additional dilution steps.

[0703] For NaCl concentrations ≤ 10 mM, the following w / v content levels of cryoprotective agents were found that should not be reduced in order to ensure the stabilization of particle properties during multiple freezing cycles.

[0704] Methods: Sucrose was investigated as a representative cryoprotectant at a concentration of 10% (w / v). A detailed description of the experiment is given in Section 2.

[0705] Results: Analysis of RNA lipoplex particle size during storage in a frozen state revealed that exemplary concentrations of 10% (w / v) sucrose combined with a NaCl concentration of ≤10.0 mM were sufficient for efficient long-term stabilization of the RNA lipoplex. Accelerated (Figures 49, 57, 66, and 70) and frozen (Figures 53 and 62) stability tests confirmed colloidal stability in a frozen state (Figures 50, 51, 58-60, and 71) and the RNA degradation rate in RNA lipoplexes stabilized by the reported formulation. Reducing the sugar content and using histidine as a buffer in combination with a reduced NaCl content yielded results comparable to the present formulation (22% sucrose and 20 mM NaCl).

[0706] Example 4.1: Stabilization of RNA lipoplex with reduced sucrose and NaCl concentrations In the following tests, the long-term stability of RNA lipoplexes in a 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.

[0707] [Table 14]

[0708] Liquid storage at 25°C (accelerated) Figures 67 and 68 demonstrate that in systems with reduced NaCl and sucrose content, particle size and polydispersity are well maintained during storage under accelerated conditions. The particle size and polydispersity are equivalent to those of the formulation containing 22% sucrose and 20 mM NaCl.

[0709] Figure 69 demonstrates that the summarized system can maintain pH within the adjustable range during storage under accelerated conditions. A malfunction of the pH meter used was identified as the explanation for the slight pH increase observed.

[0710] Figure 70 shows stability data obtained from formulations containing low concentrations of sucrose and NaCl. Compared to the formulation containing 20 mM NaCl and 22% sucrose, the new formulation exhibits improved RNA integrity. Consistent with previous studies, this improved stabilization may be due to the improved pH value of 6.5. Consistent with previous studies, RNA integrity is even better preserved when using HEPES compared to histidine.

[0711] Cryopreservation (-15℃) Figure 71 demonstrates that the described system can stabilize the colloidal properties of the formulation as particle size. All formulations can be frozen with minimal change in particle size.

[0712] (Example 29) Preparation and testing of RNA lipoplex particles 1. Materials and Methods The key materials used in the experiments described below were as follows:

[0713] [Table 15]

[0714] Preparation of lipid mixtures DOTMA / DOPE lipid mixtures were prepared with different lipid concentrations in ethanol, each with a lipid molar ratio of 2:1. The solutions were prepared as follows: Weigh the DOPE lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Calculate the amount of DOTMA and maintain a 2:1 DOTMA / DOPE percentage molar ratio. Weigh the DOTMA lipid (1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt)). • Calculate the amount of anhydrous ethanol needed to dissolve the lipids. Weigh out the anhydrous ethanol. • Dissolve the lipids in ethanol using a 37°C water bath.

[0715] The solubility of DOTMA / DOPE in ethanol was investigated by preparing a 330 mM DOTMA / DOPE (66:33) ethanol solution. Lipids were dissolved by incubating the lipid solution at 37°C for 360 minutes. The DOPE concentration was measured by HPLC. The DOTMA / DOPE solution was filtered through a 0.22 μm PES filter, and the lipid concentration in the filtrate was measured by HPLC.

[0716] Liposome preparation Liposomes were prepared by ethanol injection as follows: Using a 60 mL syringe equipped with a 0.9 × 152 mm needle, 60 mL of DOTMA / DOPE ethanol solution was injected into 3000 mL of water while stirring at 150 rpm. The liposome colloid was stirred for 60 minutes. The liposomes were filtered through a 0.45 μm filter. The liposome colloid was stored at 4–8°C. A DOTMA / DOPE lipid solution was prepared in a molar ratio of 66:33%. The liposomes were diluted with water to a total concentration of 0.9 mM. The DOTMA / DOPE concentration was measured by HPLC.

[0717] Dynamic light scattering RNA lipoplex size was measured using a Nicomp instrument (PSS.Santa Barbara.USA) by a known method of dynamic light scattering (DLS).

[0718] The RNA lipoplex sample was diluted before measurement. 1. Dilute with water from 1 to 20 (BM1; INEST 2.1) 2. Dilute with water from 1 to 8 (all other samples; INEST 2.X)

[0719] Particle size and polydispersity were measured using the Nicomp 380 ZLS dynamic light scattering particle sizing system. CDZW 388 version 2.14 software was used to separate closely spaced bimodal and native particle populations from aggregate tails, and the data were plotted as Gaussian or multimodal distributions. Measurements were performed at room temperature, wavelength 660 nm, in 5 × 50 mm disposable borosilicate glass culture tubes (Kimble, USA). The liquid refractive index was pre-set to 1.333 and the liquid viscosity to 0.933 cp. Analysis was performed as an intensity-weighted Gaussian distribution analysis of solid particles with an external fiber angle of 90°. Before measuring the samples, the functionality of the Nicomp particle sizing system was confirmed by measuring a 150 nm nanosphere standard diluted 1:500 with water. Functionality was guaranteed when the measured average particle size of the standard was 150 nm ± 7.5 nm.

[0720] HPLC Lipid concentrations in various liposome formulations were measured by HPLC (Agilent Technologies, Santa Clara, USA) using a Sunfire C 18 2.5 μm 4.6 × 75 mm column (Waters, Massachusetts, USA) and a wavelength of 205 nm. 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. Liposome samples were diluted with water to a total lipid concentration of 3 mM.

[0721] Cell culture: RNA transfection in dendritic cells in vitro For cell culture experiments, RNA lipoplex was diluted to 0.01 mg / mL of RNA using a 0.9% NaCl solution. The RNA transfection efficiency of various RNA lipoplex formulations was investigated in human dendritic cells seeded in culture medium.

[0722] Animal models: Spleen targeting and RNA transfection in dendritic cells The transfection efficiency of various RNA lipoplex formulations was investigated in BALB / c mice. 2 μg of formulated RNA lipoplex was injected retroorbitally, and luciferase expression in dendritic cells (spleen target) was measured 6 hours later.

[0723] Formulation INEST 2.1 (Figure 81B) was diluted with 0.9% NaCl to an RNA concentration of 0.01 mg / mL. A 200 μL injection volume was applied to mice. For formulation INEST 2.X, 100 μL was injected (Figure 81B).

[0724] Asymmetric flow field flow fractionation The AF4 method is a fractionation technique for characterizing and separating nanoparticles, polymers, and proteins based on their diffusion coefficients. Separation is achieved using hydrodynamic forces applied to a flat separation channel. Within the channel, a parabolic flow profile is generated by the laminar flow of the mobile phase under a vertical force field. Separation was performed using an elution injection program with an injection flow rate of 0.2 mL / min, a detector flow rate of 0.5 mL / min, and a cross-flow of 1.5 mL / min. Eluted particles were detected by a multi-angle light scattering (MALS) detector (HELLEOS II, Wyatt Technology Corp., Santa Barbara, CA, USA). LS traces were recorded using ASTRA Software version 7.1.3.25 (Wyatt Technology Europe, Dernbach, Germany).

[0725] Automated production of RNA lipoplex A generally applicable procedure was developed for the automated batch production of RNA lipoplexes. Each step is performed using pre-sterilized, disposable fluid pathways that allow for safe, aseptic handling of materials. First, the RNA concentration is adjusted to match the liposome concentration, and the RNA is concentrated by adding NaCl. This adjusts the RNA solution to an RNA concentration that allows for mixing of equal volumes of RNA and liposomes. Both the RNA and liposome solutions are transferred to large-capacity syringes, and both syringes are mounted on a single syringe pump, driving both pistons simultaneously. Afterward, the liposome and RNA-NaCl solutions are mixed in a Y-type mixer (for 2.4 mm inner diameter tubing) to form the RNA lipoplex. The RNA lipoplex formulations were incubated at room temperature for 10 minutes and then processed directly. Various RNA lipoplex formulations were prepared with an N / P ratio of 0.65 between positively charged DOTMA groups and negatively charged phosphate groups in the RNA backbone. The RNA concentration in these various RNA lipoplex formulations was 0.15 mg / mL, and the NaCl concentration was approximately 50 mM.

[0726] Current formulation (INEST 2.1 / BM): After RNA lipoplex formation, cryoprotectant solution is added in a 1:1.5 ratio, the RNA concentration is measured, and then the final concentration of the formulation is adjusted with a formulation buffer.

[0727] Proposed formulations (INEST 2.X / all others): After RNA lipoplex formation, add cryoprotectant solution until the desired RNA concentration is reached.

[0728] Finally, the RNA lipoplex was filtered through a 5 μm filter.

[0729] A key quality attribute of RNA lipoplexes is the charge ratio, which is adjusted by the mixing ratio of RNA to liposomes. We have developed an automateable and scalable industrial manufacturing process for RNA lipoplexes that enables efficient control of the mixing ratio. In small-scale (≤10 liters) manufacturing processes, control of the mixing of two identical volumes of aqueous solutions containing liposomes and RNA is achieved by using a single perfusion pump that simultaneously drives two large-volume syringes filled with RNA or liposomes. To similarly pump larger volumes (≥10 liters) as pressurized vessels, membrane pumps, gear pumps, magnetic levitation pumps, or peristaltic pumps are used in combination with flow sensors that have or do not have feedback loops for online flow control and, optionally, real-time adjustment.

[0730] Automated RNA lipoplex production requires a mixing element to ensure efficient mixing of aqueous solutions containing RNA and liposomes. Commercially available microfluidic mixing elements, including serpentine channels and embedded structures to facilitate mixing, as well as prototype mixing elements with equivalent structures, were found to cause material deposition and potential clogging during production. Therefore, these mixing elements are unsuitable for automated RNA lipoplex production. Y-type and T-type mixing elements with diameters ranging from 1.2 mm to 50.0 mm were found to be suitable for automated RNA lipoplex production.

[0731] RNA integrity measurement To analyze the integrity of the RNA, it was necessary to isolate the RNA from the RNA lipoplex. The cryoprotected RNA lipoplex was mixed with RNeasy kit (Qiagen) RLT buffer in a 1:3.5 ratio. Then, ethanol was added in a ratio of 1 part ethanol to 1.8 parts LPX-RLT.

[0732] Further RNA isolation was performed using the RNeasy Mini Kit protocol and the Qiagen RNeasy Mini Kit.

[0733] RNA integrity was analyzed using an Advanced Analytical 48-capillary fragment analyzer automated system (AATI) according to the manufacturer's instructions. The results were analyzed using PROSize 2.0 version 2.0.051 data analysis software.

[0734] (Example 29.1) Summary of changes to pharmaceutical formulations Figure 72A summarizes the differences between the current formulation and the proposed formulation.

[0735] Figure 72B shows the range investigated for the development of the proposed formulation. The table includes the investigated concentration ranges and pH ranges for RNA, DOTMA, DOPE, HEPES, EDTA disodium salt, sucrose, NaCl, and acetic acid.

[0736] Results: In order to simplify the manufacturing process and reduce production costs, the following aspects were investigated, and the following changes should be implemented: Acidification with 1.1 mM acetic acid improved liposome stability. • Modification of the RNA drug buffer to simplify the process (addition of 100 mM NaCl to compensate for the pH shift induced by acetate, and a change in pH to 7.0, as well as improvement of RNA stability). Reducing the RNA concentration in the formulation from 0.05 mg / mL to 0.025 mg / mL makes it possible to create a ready-to-use product that can be administered directly without dilution. This eliminates the need for bedside dilution and simplifies the handling of low doses. • A reduction in ionic strength from 7.5 mM HEPES to 5.0 mM HEPES without affecting pH stability. The EDTA disodium dihydrate content remained unchanged, and it was found to contribute as a buffer in addition to its RNA stabilization function (chelation of potentially present divalent metal ions). EDTA disodium stabilizes the pH during the manufacturing of the formulation. • The NaCl content for RNA conditioning remains unchanged. The decrease in NaCl in the formulation allows for a decrease in sucrose content due to a reduction in the ionic strength of the solution. Reducing the concentration to 13% (w / v) sucrose leads to cost savings without affecting stability and is essential for achieving nearly physiological osmolality. Bedside dilution is not required. • Increase the pH value to pH 6.7 to improve RNA stabilization.

[0737] (Example 29.2) Investigation of the stability of DOPE in liposomes at different acetic acid concentrations as a function of time, measured at three different temperatures. Figure 73A: Liposomes were stored at 4°C with 0 mM to 3.0 mM acetic acid.

[0738] Figure 73B: Liposomes were stored at 25°C with 0 mM to 1.6 mM acetic acid.

[0739] Figure 73C: Liposomes were stored at 40°C with 0 mM to 3.0 mM acetic acid.

[0740] The average value calculated from the initial DOPE integrity value of samples immediately after manufacturing (0 months) was set as the initial integrity value of 1 for each test group. The following values ​​were normalized to this starting value.

[0741] Methods: Liposomes were prepared as described above. To acidify the liposomes, various volumes of 1 M stock acetic acid were added to the liposomes at a total lipid concentration of 0.9 mM. Subsequently, the liposomes were stored at various acetic acid concentrations ranging from 0 mM to 3 mM at various temperatures (4°C, 25°C, and 40°C) for up to 6 months.

[0742] Results: Acidification was found to lead to a decrease in the hydrolysis rate of DOPE ester in liposomes. Stability increased with increasing acetic acid concentration at all measurement temperatures. An acetic acid concentration of 1.1 mM is preferred as it can be achieved by slightly modifying existing liposome manufacturing processes. The stabilizing effect can already be observed at low concentrations of acetic acid. There is a linear correlation of the stabilizing effect with the addition of acetic acid, which plateaus above an acetic acid concentration of approximately 1 mM. The stabilizing effect can be correlated with the pH shift induced by acetic acid. Therefore, other acids, such as hydrochloric acid, are also suitable for stabilizing DOPE in liposomes.

[0743] (Example 29.3) RNA drug buffer and RNA concentration: Adjustments for process simplification Figure 74 summarizes the differences between current RNA drug substance formulations and proposed formulations.

[0744] Results: The proposed RNA API formulation allows for the processing of the RNA API into an RNA lipoplex without the need for concentration and tonic adjustments. Fixed and slightly increased buffer concentrations, EDTA disodium salt concentration, and pH adjustments enhance process reproducibility and allow for compensation of pH shifts caused by acetate in liposomes (predictable pH shifts during RNA lipoplex formation). Process parameters such as RNA concentration and ionic conditions remain similar during RNA lipoplex formation, with a low risk of change. The stability of the RNA API is improved at pH 7.0.

[0745] (Example 29.4) RNA concentration adjustment to 0.025 mg / mL Figure 75 shows the typical dose volume of the active pharmaceutical ingredient at an RNA concentration of 0.025 mg / mL.

[0746] Results: An RNA concentration of 25 μg / mL in the formulation results in dose volumes that correspond to the large markings on a 1 mL syringe for all scenarios under consideration. These dose volumes are easier for HCPs to accurately measure and administer.

[0747] (Example 29.5) HEPES content: Reduced to 5.0 mM. Figure 76A shows the RNA integrity of the formulation over time at various pH levels and under various buffer systems (HEPES, histidine, and sodium acetate) after incubation at 25°C for 61 days. Each dot represents the mean RNA integrity value of the formulation in two vials, with the associated standard deviation. The mean value calculated from the initial RNA integrity value of the sample immediately after production (time point T0) was set as 100% as the initial integrity value for each test group. The following values ​​were normalized to this starting value. The dotted line indicates the specification limit of ≥80% complete full-length RNA for the current frozen formulation (BM1).

[0748] Methods: BM1 RNA lipoplex was prepared as described above. All other RNA lipoplexes were prepared as described above using liposomes containing 5 mM acetate. RNA was supplied in the following manner to generate various test groups: a) For BM1, use 18 mM HEPES and 18 mM EDTA disodium salt at pH 6.2. b) For the HEPES group, use 18 mM HEPES and 18 mM EDTA disodium salt at pH 7.0. c) For the histidine group, 20 mM histidine, 18 mM EDTA disodium salt, pH 7.0 d) For the sodium acetate group, 20 mM sodium acetate, 18 mM EDTA disodium salt, pH 6.6

[0749] The BM1 RNA lipoplex was cryoprotected and the RNA concentration was adjusted as described above. All other RNA lipoplexes were cryoprotected by adding cryoprotective ...

Claims

1. RNA, and At least one cationic lipid and at least one additional lipid, RNA lipoplex particles containing, Sodium chloride at a concentration of approximately 10 mM or less, A stabilizer with a concentration exceeding approximately 10% by weight / volume (%w / v) and less than approximately 15% by weight / volume (%w / v), A composition containing a buffering agent.

2. The composition according to claim 1, wherein the sodium chloride is concentrated to a concentration of about 5 mM to about 10 mM.

3. The composition according to claim 1 or 2, wherein the sodium chloride is present at a concentration of approximately 8.5 mM or less.

4. The composition according to any one of claims 1 to 3, wherein the sodium chloride is concentrated at a concentration of approximately 8.2 mM.

5. The composition according to any one of claims 1 to 4, wherein the concentration of the salt and / or stabilizer in the composition is approximately the value required for the physiological osmolality by weight.

6. The composition according to any one of claims 1 to 5, wherein the concentration of the stabilizer in the composition is about 12 to about 14% (w / v), preferably about 13% (w / v).

7. The composition according to any one of claims 1 to 6, wherein the stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols.

8. The composition according to any one of claims 1 to 7, wherein the stabilizer is sucrose or trehalose.

9. The composition according to any one of claims 1 to 8, wherein the stabilizer is sucrose in a concentration of about 12 to about 14% (w / v).

10. The composition according to claim 8 or 9, wherein the sucrose is present at a concentration of about 13% (w / v).

11. The composition according to any one of claims 1 to 8, wherein the stabilizer is trehalose at a concentration of about 12 to about 14% (w / v).

12. The composition according to claim 8 or 11, wherein the trehalose is present at a concentration of about 13% (w / v).

13. The composition according to any one of claims 1 to 12, wherein the buffering agent is selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), histidine, acetate / sodium acetate, and MES (2-(N-morpholino)ethanesulfonic acid).

14. The composition according to any one of claims 1 to 13, wherein the buffering agent is HEPES, histidine, or MES.

15. The composition according to any one of claims 1 to 14, wherein the buffering agent is HEPES or MES.

16. The composition according to any one of claims 1 to 15, wherein the buffering agent is HEPES.

17. The composition according to any one of claims 1 to 16, having a pH of 6.0 to 7.5, 6.5 to 7.5, 6.5 to 7.3, 6.5 to 7.2, 6.7 to 7.2, or 6.5 to 7.

0.

18. A composition according to any one of claims 1 to 17, having a pH of approximately 6.

7.

19. The composition according to any one of claims 1 to 18, wherein the buffering agent is present at a concentration of 2.5 mM to 10 mM.

20. The composition according to any one of claims 1 to 19, wherein the buffering agent is present at a concentration of about 5 mM.

21. The composition according to any one of claims 1 to 20, wherein the buffer is HEPES with a pH of about 6.7 and a concentration of about 5 mM or less.

22. The composition according to any one of claims 1 to 21, wherein the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

23. The composition according to any one of claims 1 to 22, wherein the 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).

24. The composition according to any one of claims 1 to 23, wherein the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

25. The composition according to any one of claims 1 to 24, wherein the molar ratio of the at least one cationic lipid to the 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.

26. The composition according to any one of claims 1 to 25, wherein the RNA lipoplex particles contain 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.

27. The composition according to any one of claims 1 to 26, further comprising a chelating agent.

28. The composition according to claim 27, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

29. The composition according to claim 28, wherein the EDTA is at a concentration of about 3.5 mM or less, or about 0.25 mM to about 3.5 mM, or about 0.25 mM to about 2.5 mM.

30. The composition according to any one of claims 1 to 29, wherein 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 about 1:2 to about 1.9:2, or about 1.3:2.

0.

31. RNA encoding a peptide or protein containing at least one epitope, DOTMA and DOPE in a molar ratio of approximately 2:

1. RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge in the composition is approximately 1.3:2.0, Sodium chloride at a concentration of approximately 8.2 mM, Sucrose at a concentration of approximately 13% (w / v), HEPES at a concentration of approximately 5 mM with a pH of approximately 6.7, A composition containing EDTA at a concentration of approximately 2.5 mM.

32. The composition according to any one of claims 1 to 31, 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.

33. The composition according to any one of claims 1 to 32, wherein 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 0.025 mg / mL.

34. The composition according to any one of claims 1 to 33, further comprising an amount of acid that stabilizes liposomes.

35. The composition according to any one of claims 1 to 34, which is in a liquid state, a frozen state, or a dehydrated state.

36. The freezing composition according to claim 35, which is stable for at least one month at a temperature of approximately -15°C.

37. The freezing composition according to claim 35, which is stable for at least two months at a temperature of approximately -15°C.

38. The freezing composition according to claim 35, which is stable for at least four months at a temperature of approximately -15°C.

39. The freezing composition according to claim 35, which is stable at a temperature of approximately -15°C for at least six months.

40. A liquid composition comprising RNA lipoplex particles, which can be obtained by thawing the frozen composition according to any one of claims 35 to 39.

41. A liquid composition comprising RNA lipoplex particles, which can be obtained by dissolving the dehydrated composition described in claim 35.

42. The liquid composition according to claim 35, 40, or 41, which is an aqueous composition.

43. The composition according to claim 42, which can be administered directly to the target.

44. A pharmaceutical composition, as described in any one of claims 1 to 43.

45. A composition according to any one of claims 1 to 44, formulated for systemic administration.

46. The composition according to claim 45, wherein the systemic administration is by intravenous administration.

47. A composition according to any one of claims 1 to 46 for therapeutic use.

48. A method for preparing a liquid composition for direct administration to a subject, comprising thawing a frozen composition according to any one of claims 35 to 39.

49. A method for preparing a liquid composition for direct administration to a target, comprising dissolving the dehydrated composition according to claim 35.

50. The method according to claim 48 or 49, wherein the liquid composition is an aqueous composition.

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