Preparation and storage of liposomal RNA preparations suitable for treatment.

Abstract

Provided is a formulation for delivering biologically active RNA to a target tissue, in which the delivered RNA is efficiently translated into its encoded peptide or protein. [Solution] A composition is provided which comprises RNA lipoplex particles comprising RNA encoding a peptide or protein containing at least one epitope, at least one cationic lipid, and at least one additional lipid, wherein the ratio of positive charges to negative charges within the RNA lipoplex particles is about 1:2 to about 1.9:2, or about 1.3:2.0; sodium chloride at a concentration of 0 mM to about 40 mM; and a stabilizer.

Description

[Technical Field]

[0001] This disclosure relates to methods for preparing RNA lipoplex particles for delivery of 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 that enable the preparation of RNA lipoplex particles in accordance with industrial GMP. Furthermore, this disclosure relates to methods and compositions for storing RNA lipoplex particles without substantial loss of product quality, particularly without substantial loss of RNA activity. The RNA lipoplex particle formulations described herein can be frozen or dehydrated by lyophilization, spray drying, or related methods, which enable an extended shelf life of the product compared to liquid storage. In one embodiment, the RNA lipoplex particles contain 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 is translated into an encoded peptide or protein that can exhibit its physiological activity. The target peptide or protein may be a peptide or protein comprising one or more epitopes for inducing or enhancing an immune response directed to one or more epitopes. The methods and compositions described herein are suitable for use in accordance with pharmaceutical requirements, more specifically, GMP manufacturing requirements and pharmaceutical quality requirements for parenteral administration. [Background technology]

[0002] The use of RNA for delivering 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 or be integrated into the host genome for expression, thus eliminating the risk of carcinogenicity.

[0003] RNA can be delivered by so-called lipoplex formulations, which involve binding RNA to liposomes, a mixture of cationic lipids and helper lipids, to form injectable nanoparticle formulations. However, the need to develop formulations that deliver biologically active RNA to target tissues even after the formulation has been stored remains unmet. In addition, the need to develop methods for manufacturing long-shelf-life, GMP-compliant injectable RNA lipoplex particle formulations also remains unmet.

[0004] Therefore, there is a need to provide a formulation for delivering biologically active RNA to target tissues, wherein the delivered RNA is efficiently translated into the peptide or protein it encodes. Furthermore, there is a need to provide a formulation that can be stored at room temperature without substantial loss of product quality, and in particular without substantial loss of the biological activity of the RNA.

[0005] To our surprise, the inventors have found that the RNA lipoplex particle formulation described herein satisfies the requirements mentioned above. [Overview of the Initiative]

[0006] I. Method for preparing RNA lipoplex particles, RNA lipoplex particles, and compositions containing RNA lipoplex particles In a first embodiment, 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 the delivery of RNA to target tissues after parenteral administration, particularly after intravenous administration. RNA lipoplex particles are prepared using liposomes obtained by injecting a solution with a high concentration of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the RNA lipoplex product is approximately 1 nm in size. -1A single Bragg peak was observed at this point, with a peak width of 0.2 nm. -1 Characterized by smaller, specific patterns in X-ray scattering.

[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, but when combined with DOTMA, it has a solubility of 100 mM or more. A lipid solution for forming liposomes from which a highly active lipoplex can have a total lipid concentration of 270 mM or more (e.g., 90 mM or more of DOPE). By increasing the temperature, a solution with an even higher concentration in ethanol can be obtained. Liposomes obtained from lipid solutions where the DOPE concentration exceeds the equilibrium solubility are significantly larger than liposomes derived from lipid solutions where the DOPE concentration is below the equilibrium solubility. Liposome size increases monotonically with increasing concentration in ethanol.

[0008] Liposomes prepared in accordance with 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 a specific ionic strength required to increase the activity of the lipoplex. The lipoplexes formed from these large liposomes exhibit markedly high bioactivity, as confirmed by in vitro and in vivo experiments. These highly active lipoplexes can obviously be distinguished from less active lipoplexes by certain physicochemical parameters, such as (i) a low peak width of the Bragg peak and (ii) different separation profiles in dispersive analytical methods for sizing, such as field flow fractionation. Less active lipoplexes are, on average, smaller. In addition, less active lipoplexes also have different elution profiles, possibly related to parameters such as molecular conformation, shape, and interaction with the bulk phase.

[0009] Accordingly, in this embodiment, the present disclosure relates to a method for producing a liposome colloid, comprising injecting a lipid solution in ethanol into an aqueous phase to produce a liposome colloid, wherein the concentration of at least one lipid 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 approximately 180 mM to 600 mM, approximately 300 mM to 600 mM, or approximately 330 mM.

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

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

[0016] 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).

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

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

[0019] In one embodiment, the molar ratio of at least one cationic lipid to at least one further 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.

[0020] In one embodiment, the lipid solution 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.

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

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

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

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

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

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

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

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

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

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

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

[0032] In one embodiment, the liposome has a polydispersity index 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 further 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 further 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 further 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 further 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] This disclosure further relates to a method for preparing RNA lipoplex particles, the method comprising adding the liposome colloid described above to a solution containing RNA.

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

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

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

[0044] In one embodiment, the RNA lipoplex particle comprises at least one cationic lipid and at least one further lipid.

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

[0046] This disclosure is, RNA encoding a peptide or protein, containing at least one epitope, At least one cationic lipid and at least one further lipid Includes, The ratio of positive charges to negative charges within RNA lipoplex particles is approximately 1:2 to 1.9:2, or approximately 1.3:2.0. Approximately 1nm -1Characterized by a single Bragg peak, where the peak width is 0.2 nm. -1 Smaller RNA lipoplex particles The present invention relates to compositions containing the following:

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

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

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

[0050] In one embodiment, the RNA lipoplex particles described under I. of this embodiment 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 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 further 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 further 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 further 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 concentration of EDTA 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] One embodiment of this disclosure uses a fluidic system for GMP-compliant manufacturing of pharmaceutical RNA lipoplex particle products, enabling precise control of the RNA-to-liposome mixing ratio, which is critical to product quality. In one embodiment, the fluidic system includes mixing a liposome solution and an RNA solution in a 1:1 (volume / volume) ratio, where the component concentrations are selected to precisely maintain the desired charge ratio. In one embodiment, the RNA is incubated with NaCl before mixing to adjust a specific ionic strength required for lipoplex activity. In one embodiment, a Y-shaped mixing setup is achieved entirely based on a single material used. Fluid dynamics are optimized to maintain particle properties and avoid clogging. In contrast, when using commercially available microfluidic devices, clogging occurs after a certain period of time, making GMP compliance impossible.

[0065] In one embodiment, the lipoplex is produced by incubating RNA with cationic liposomes, in which case the mixing ratio and mixing 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. Since the pistons of both pumps are moved forward by the same drive, the relative volumes mixed are precisely controlled. In the selected processing conditions, since identical syringes are used for both solutions, precise 1:1 (v / v) mixing conditions are possible. Then, 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 and 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 RNA-containing solution and the cationic liposome-containing solution being mixed.

[0073] In one embodiment, the mixing ratio of RNA to cationic liposomes is controlled by using the same mixed 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 the RNA lipoplex particles while avoiding clogging.

[0075] In one embodiment, the method includes using a Y-shaped mixed element or a T-shaped 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 includes using a mixing element in which fluids from two tubes or hoses converge and remix, such as a Y-shaped or T-shaped mixing element, a channel with alternating herringbone-shaped channels, a zigzag-shaped channel, or a twisted-shaped channel, or a three-dimensional meandering path, where there is no internal static mixing element. The mixing element may have a diameter between 1.2 and 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 holder or two holders, and the device's piston 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 velocity sensor with a feedback loop for online control and real-time adjustment of the flow velocity.

[0080] In one embodiment, 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.

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

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

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

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

[0085] The present disclosure further relates to a composition comprising RNA lipoplex particles obtained 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 within the RNA lipoplex particles is about 1:2 to about 1.9:2, or about 1.3:2.0.

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

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

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

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

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

[0093] In one embodiment, the RNA lipoplex particles have a polydispersity index 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 further 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 further 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 further 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 concentration of EDTA 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 storing RNA lipoplex particles In a third aspect, the disclosure relates to methods and compositions for storing RNA lipoplex particles without substantial loss of product quality, in particular without substantial loss of RNA activity. In particular, the disclosure relates to formulations that enable freezing, lyophilization, or spray drying of RNA lipoplex particles without substantial loss of product quality, in particular without substantial loss of RNA activity.

[0106] The RNA lipoplex particle formulations described herein may be frozen or dehydrated by lyophilization, spray drying, or related methods, which allows for an extended shelf life of the product compared to liquid storage.

[0107] To enable freezing, a stabilizer (cryoprotectant) is added. In one embodiment, after preparation, the lipoplex is diluted with a stabilizer (cryoprotectant) to adjust the ionic strength, preferably by reducing it, and to adjust the appropriate concentration of the stabilizer. To freeze the product, the stabilizer concentration may be higher than the value required to obtain the physiological osmolality. In this case, for administration, the product is diluted with a suitable aqueous phase (e.g., water for injection, physiological saline) to adjust the desired osmolality and ionic strength. Sugars such as glucose, sucrose, or trehalose may be used as stabilizers, but other compounds such as dextran may also be used.

[0108] Surprisingly, it has been found that, in accordance with this disclosure, RNA lipoplex formulations containing stabilizers, as described herein, can also be lyophilized. For lyophilization, the required concentration of stabilizer (lyophilization protectant) may be lower than that for freezing, and the acceptable NaCl concentration (ionic strength) may also be higher than that for freezing. Where cost-effective dehydration is required on a large scale, the product can also be spray-dried.

[0109] The pH of some RNA lipoplex formulations is adjusted to a value lower than the usual physiological range and the optimal pH for RNA storage in the bulk phase. The optimal pH is approximately 6.2, and the appropriate range is between approximately 5.7 and 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, the RNA lipoplex composition is frozen for storage. The composition can then be thawed, and optionally, an aqueous solution can be added to adjust the osmolality, ionic strength, and / or pH of the composition. The resulting composition can then be administered to a subject.

[0111] In embodiments of this disclosure, an RNA lipoplex composition is lyophilized or freeze-dried for storage. The composition can be reconstituted by adding an aqueous liquid, and optionally, the osmolality, ionic strength, and / or pH of the composition can be adjusted by adding an aqueous liquid. The resulting composition can 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) preparing 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 polyalcohols.

[0116] In one embodiment, preparing an aqueous composition containing RNA lipoplex particles and a stabilizer involves preparing an aqueous composition containing RNA lipoplex particles and adding the stabilizer to the aqueous composition containing RNA lipoplex particles. Therefore, a method for preparing a composition for freezing involves preparing an aqueous composition containing RNA lipoplex particles and adding the 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 an aqueous composition containing RNA lipoplex particles and the stabilizer is higher than the value required for the physiological osmolality.

[0119] In one embodiment, 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 storage of the composition at a temperature of about -15°C to about -40°C for at least 1 month, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0120] In one embodiment, the concentration of the stabilizer in the aqueous composition containing RNA lipoplex and the stabilizer is approximately 5% to approximately 35.0% (w / v), approximately 10% to approximately 30.0% (w / v), approximately 12.5% ​​to approximately 25.0% (w / v), or approximately 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 optimal pH value for storing RNA.

[0122] In one embodiment, the aqueous composition comprising RNA lipoplex and a stabilizer contains sodium chloride at a concentration of about 10 mM to about 50 mM, or contains 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 are obtained by the methods described under I. and II. above.

[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] This disclosure further relates to compositions comprising RNA lipoplex particles obtained by the method for preparing the above-described frozen compositions. This disclosure also relates to compositions comprising RNA lipoplex particles obtained by the above-described method for preparing a composition for freezing.

[0127] In one embodiment, the RNA lipoplex particle comprises at least one cationic lipid and at least one further lipid.

[0128] In one embodiment, the RNA encodes a peptide or protein containing at least one epitope, and the ratio of positive charge to negative charge within the RNA lipoplex particle is approximately 1:2 to approximately 1.9:2, or approximately 1.3:2.0.

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

[0130] This disclosure is, RNA encoding a peptide or protein, containing at least one epitope, At least one cationic lipid and at least one further lipid, Includes, 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, Stabilizer and The present invention relates to compositions containing the following:

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

[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 concentration of sodium chloride is approximately 20 mM to approximately 30 mM.

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

[0135] In one embodiment, the sodium chloride concentration 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 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 polyalcohols.

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

[0140] In one embodiment, the sucrose concentration is 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 optimal pH value for storing RNA.

[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 is, RNA encoding a peptide or protein, containing at least one epitope, at a concentration of approximately 0.05 mg / mL, DOTMA and DOPE in a molar ratio of approximately 2:1 Includes, 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 with 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 and The present invention relates to compositions containing the following:

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

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

[0153] In one embodiment, the frozen composition is stable for at least two months at a temperature of approximately -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 approximately -20°C.

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

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

[0158] This disclosure further relates to aqueous compositions containing RNA lipoplex particles obtained by thawing the above-mentioned frozen composition and optionally adding an aqueous liquid to adjust the osmolality and ionic strength.

[0159] In one embodiment, the osmolality of the composition is approximately 200 milliosmoles to approximately 450 milliosmoles.

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

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

[0162] The present disclosure relates to a method for preparing a composition comprising RNA lipoplex particles that has been dehydrated, for example, by freeze-drying or spray-drying, the method comprising (i) preparing an aqueous composition comprising RNA lipoplex particles and a stabilizer, and (ii) dehydrating the composition, for example by freeze-drying 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 polyalcohols.

[0164] In one embodiment, preparing an aqueous composition containing RNA lipoplex particles and a stabilizer includes preparing an aqueous composition containing RNA lipoplex particles and adding the stabilizer to the aqueous composition containing RNA lipoplex particles. Therefore, a method for preparing a composition for dehydration, such as freeze-drying or spray-drying, includes preparing an aqueous composition containing RNA lipoplex particles and adding the stabilizer to the aqueous composition containing RNA lipoplex particles.

[0165] 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.

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

[0167] In one embodiment, 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 storage of the composition 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 approximately 5% to approximately 35.0% (w / v), approximately 10% to approximately 30.0% (w / v), approximately 12.5% ​​to approximately 25.0% (w / v), or approximately 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 optimal pH value for storing RNA.

[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 contains 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 are obtained by the methods described under I. and II. above.

[0173] In one embodiment, a method for preparing a dehydrated composition, such as a freeze-dried or spray-dried composition, further includes storing the composition containing freeze-dried or spray-dried 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 certain embodiments, 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.

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

[0175] In one embodiment, the RNA lipoplex particle comprises at least one cationic lipid and at least one further lipid.

[0176] In one embodiment, the RNA encodes a peptide or protein containing at least one epitope, and the ratio of positive charge to negative charge within the RNA lipoplex particle is approximately 1:2 to approximately 1.9:2, or approximately 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 is, RNA encoding a peptide or protein, containing at least one epitope, At least one cationic lipid and at least one further lipid Includes, 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, Stabilizer and The present invention relates to compositions containing the following:

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

[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 concentration of sodium chloride is approximately 20 mM to approximately 30 mM.

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

[0183] In one embodiment, the sodium chloride concentration 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 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 polyalcohols.

[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 is present at a concentration of 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 optimal pH value for storing RNA.

[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 is, RNA encoding a peptide or protein, containing at least one epitope, at a concentration of approximately 0.05 mg / mL, DOTMA and DOPE in a molar ratio of approximately 2:1 Includes, 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, EDTA at a concentration of approximately 2.5 mM and The present invention relates to compositions containing the following:

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

[0199] In one embodiment, the dehydrated composition, for example, a lyophilized or freeze-dried composition, is stable for at least 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, at about 25°C or about 4°C, or, for example, at room temperature.

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

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

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

[0203] In one embodiment, the osmolality of the composition is approximately 150 milliosmoles to approximately 450 milliosmoles.

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

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

[0206] In one embodiment, the RNA lipoplex particles described under Section III of this embodiment are approximately 1 nm in size. -1 Characterized by a single Bragg peak, where the peak width is 0.2 nm. -1 Smaller.

[0207] In one embodiment, the RNA lipoplex particles described under Section III of this embodiment 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 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 further 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 further 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 further 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 concentration of EDTA 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 present disclosure further relates to a method for preparing an aqueous composition containing RNA lipoplex particles, comprising thawing the frozen composition described above, or reconstituting the freeze-dried or spray-dried composition described above, and optionally adjusting the osmolality and ionic strength by adding an aqueous liquid.

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

[0222] In one embodiment, 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]

[0223] [Figure 1] This figure shows the correlation between lipid concentration in a lipid stock solution and liposome size. Liposomes were prepared by ethanol injection into water (filtration was not performed after ethanol injection). Liposome size increases with the lipid concentration in ethanol. Example: DOTMA / DOPE is a lipid mixture with a molar ratio of 66:33 (by percentage). [Figure 2] This figure shows the amount of particles present in DOTMA / DOPE liposome formulations prepared with different concentrations of different lipid solutions. [Figure 3] This figure shows the effect of liposome precursor (the unfiltered liposome colloid used) size on RNA lipoplex size. When small liposomes were used for their formation, small RNA lipoplexes were obtained. No clear correlation was found between liposome size and RNA lipoplex size within RNA lipoplexes prepared using all large liposomes. [Figure 4] This figure shows the amount of particles present in RNA lipoplex formulations prepared with different liposome precursors (unfiltered liposomes). The amount of 0.5 μm particles increases in RNA lipoplex formulations prepared with larger liposomes. The liposomes were prepared by ethanol injection using different lipid stocks at different lipid concentrations. [Figure 5] The figures show diffraction curves obtained from SAXS measurements of RNA lipoplexes formed from liposomes prepared with a charge ratio of 4 / 1 (top) and a charge ratio of 1.3 / 2, where the liposomes for lipoplex formation were obtained using lipid stock solutions of 400 mM, 300 mM, and 100 mM in ethanol. [Figure 6]This figure shows the correlation length and transfection efficiency in vitro (in human dendritic cells) of different RNA lipoplexes prepared with different liposome precursors. Biological activity (RNA transfection in vitro) increases monotonically with the correlation length of the RNA lipoplex. Liposomes were prepared by ethanol injection and by different lipid stocks prepared at different lipid concentrations. [Figure 7] This figure shows the AF4 measurement of lipoplexes derived from two different types of liposomes, prepared from either a 150 mM stock solution in ethanol or a 400 mM stock solution in ethanol. [Figure 8] This figure shows the in vitro transfection efficiency of different RNA lipoplexes within dendritic cells. The luciferase signal increases monotonically with the liposome size used for RNA lipoplex formation. [Figure 9] This figure shows the in vitro transfection efficiency of different RNA lipoplexes within dendritic cells. Luciferase signaling increases monotonically with liposome size. [Figure 10] This figure shows the in vivo transfection efficiency of RNA lipoplex formulations 6 hours after application. RNA lipoplexes were prepared using small and large liposomes (non-filterable liposomes). RNA lipoplexes prepared with large liposomes resulted in high luciferase expression. [Figure 11]This figure shows in vivo imaging of RNA lipoplexes 6 hours after application. RNA lipoplexes were prepared using small and large liposomes. A) This figure shows RNA lipoplexes prepared using small liposomes derived from biocolloids. B) This figure shows RNA lipoplexes prepared using large liposomes derived from biocolloids. C) This figure shows filtered RNA lipoplexes prepared using small liposomes. D) This figure shows filtered RNA lipoplexes prepared using large liposomes. This is a highly bioluminescent signal obtained from RNA lipoplexes prepared using large liposomes. [Figure 12] This diagram shows a general procedure for automated batch production of RNA lipoplex. [Figure 13] This figure shows the Z-mean and polydispersity of RNA lipoplexes prepared using a Y-shaped mixed element with an inner diameter of 3.2 mm at different flow rates. [Figure 14] This figure shows the Z-mean and polydispersity of RNA lipoplexes prepared using a Y-shaped mixed element with an inner diameter of 2.4 mm at different flow rates. [Figure 15] This figure shows the correlation between particle size and RNA concentration during lipoplex formation. PCS measurements were performed before freezing. [Figure 16] This figure shows the correlation between particle characteristics and RNA concentration during lipoplex formation after three freeze-thaw cycles. [Figure 17] This figure shows the correlation between particle properties and charge ratios (positive:negative) in the range of 1.0:2.0 to 2.1:2.0. [Figure 18] This figure shows the correlation between particle properties and charge ratios (positive:negative) in the range of 2.0:1.0 to 5.0:1.0. [Figure 19] This figure shows the correlation between particle characteristics and NaCl concentration during lipoplex formation. [Figure 20] This figure shows the correlation between biological activity and NaCl concentration during lipoplex formation. [Figure 21] This figure shows the measurement of RNA integrity in RNA (LIP) compositions without cryoprotection, using different buffer substances (HEPES; sodium acetate; sodium phosphate; sodium carbonate) at several pH values ​​after accelerated storage at +40°C. The different bars indicate increasing storage time from left (3 days) to right (21 days). [Figure 22] This figure shows the measurement of RNA integrity in RNA lipoplex formulations with HEPES as a buffer and sucrose as a cryoprotectant, at several pH values, after accelerated storage at +40°C. Different bars indicate increasing storage time from left (1 day) to right (21 days). [Figure 23] This figure shows the RNA integrity in lipoplexes with different EDTA content, stored at 40°C. [Figure 24] This schematic diagram shows that the concentrations of NaCl and the cryogenic protective agent were optimized in each step of the process. [Figure 25] This figure shows the Z-mean and polydispersity of RNA lipoplexes frozen in the presence of an alternative cryoprotectant that increases their volume. [Figure 26] This figure shows the number of invisible particles ≥10 μm in an RNA lipoplex formulation frozen in the presence of an alternative cryoprotectant that increases the volume. [Figure 27] This figure shows the measurement of 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] This figure shows the measurement of 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] This figure shows the measurement of particle size in RNA lipoplex formulations containing 50 mM NaCl and varying amounts (%w:v) of trehalose dihydrate after multiple freeze-thaw cycles. [Figure 30] This figure shows the measurement of particle size in RNA lipoplex formulations containing 70 mM NaCl and varying amounts (%w / v) of trehalose dihydrate after multiple freeze-thaw cycles. [Figure 31] This figure shows the measurement of particle size in RNA lipoplex formulations containing 90 mM NaCl and varying amounts (%w / v) of trehalose dihydrate after multiple freeze-thaw cycles. [Figure 32] This figure shows the measurement of particle size in RNA lipoplex formulations containing 5-20% w / v sucrose and various low NaCl concentrations after 8 months of storage at -15°C. Different bars indicate increasing storage period from left (0 months) to right (8 months). For sucrose / NaCl combinations with fewer than 8 bars, the particle size exceeded the specification at two subsequent time points, and the analysis was stopped. [Figure 33] This figure shows the measurement of particle size in RNA lipoplex formulations containing 5-20% w / v sucrose and various low NaCl concentrations after storage at -30°C for 8 months. Different bars indicate increasing storage period from left (0 months) to right (8 months). For sucrose / NaCl combinations with fewer than 8 bars, the particle size exceeded the specification at two subsequent time points, and the analysis was stopped. [Figure 34] This figure shows the measurement of particle size in RNA lipoplex formulations containing 5-20% w / v trehalose dihydrate and various low NaCl concentrations after 8 months of storage at -15°C. Different bars indicate increasing storage period from left (0 months) to right (8 months). For trehalose / NaCl combinations with fewer than 8 bars, the particle size exceeded the specifications at two subsequent time points, and the analysis was stopped. [Figure 35]This figure shows the measurement of particle size in RNA lipoplex formulations containing 5-20% w / v trehalose dihydrate and various low NaCl concentrations after storage at -30°C for 8 months. Different bars indicate increasing storage period from left (0 months) to right (8 months). For trehalose / NaCl combinations with fewer than 8 bars, the particle size exceeded the specifications at two subsequent time points, and the analysis was stopped. [Figure 36] This figure shows the measurement of particle size in RNA lipoplex formulations containing 22% w / v sucrose and various low NaCl concentrations after storage at -20°C. [Figure 37] This figure shows the measurement of particle size in RNA lipoplex formulations containing 22% w / v trehalose dihydrate and various low NaCl concentrations after storage at -20°C. [Figure 38] This figure shows the measurement of particle size in RNA lipoplex formulations containing 22% w / v glucose and various low NaCl concentrations after storage at -20°C. [Figure 39] This figure shows the measurement of particle size in an RNA lipoplex formulation containing 22% w / v glucose and 20 mM NaCl, which is frozen and stored at -15 to -40°C. [Figure 40] This figure shows the particle size measurements of RNA lipoplexes frozen in compositions containing the combinations of cryoprotective agents listed in Table 10, after storage at -20°C. [Figure 41] This figure shows the effect of trehalose concentration in RNA lipoplex formulations [RNA(lip)] formulations after freeze-thaw cycles, or after lyophilization and reconstitution. [Figure 42] This figure shows the change in particle size of a lyophilized RNA (lip) preparation prepared in 22% trehalose after reconstitution with a 0.9% NaCl solution or WFI (water for injection). [Figure 43]This figure shows in vitro luc-RNA transfection of lyophilized RNA lipoplex preparations [RNA(lip)] prepared in 22% trehalose at different NaCl concentrations. The lyophilized samples were reconstituted with a 0.9% NaCl solution (striped column: liquid control). [Figure 44] This figure shows the mean Z diameter of lyophilized RNA lipoplex formulations [RNA(lip)], formulated with different trehalose / NaCl ratios and stored at 2–8°C or 25°C, after reconstitution with a 0.9% NaCl solution. The formulations were reconstituted to their original volume after lyophilization. [Figure 45] This figure shows the RNA integrity (full-length RNA%) after reconstitution with a 0.9% NaCl solution of lyophilized RNA (lip) formulated with different trehalose / NaCl ratios and stored at 2–8°C or 25°C. The formulations were reconstituted to their original volume after lyophilization and diluted 1:1 with a 0.9% NaCl solution (0.01 mg / mL RNA) for cell culture experiments. [Modes for carrying out the invention]

[0224] While this disclosure is described in detail below, please understand that it is not limited to the specific methods, protocols, and reagents described herein, as they are subject to change. Also, please understand that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit the scope of this disclosure, which is limited only to the claims provided herein. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0225] Preferably, the terms used herein are defined as those set forth in "A multilingual glossary of biotechnological terms (IUPAC Recommendations)," edited by H.G. Wleuenberger, B. Nagel, and H. Kolbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

[0226] In implementing this disclosure, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA methods will be relied upon, as described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

[0227] The elements of this disclosure are described below. These elements are listed along with specific embodiments, but it should be understood that they can be combined in any form and in any number to create further embodiments. The various examples and embodiments described are not to be construed as limiting this disclosure to only the embodiments expressly described. This description is to be understood as disclosing and encompassing embodiments that combine the expressly described embodiments with any number of disclosed elements. Furthermore, any permutations and combinations of all described elements are to be considered disclosed by this description unless otherwise indicated by the context.

[0228] The term “approximately” means “about” or “nearly,” and in one embodiment, in the context of a number or range expressed herein, means ±20%, ±10%, ±5%, or ±3% of the number or range enumerated or claimed.

[0229] The terms “a,” “an,” and “that,” and similar references used in the context describing this disclosure (particularly in the context of the claims), shall be construed to refer to both singular and plural unless otherwise indicated herein or explicitly refuted by the context. The enumeration of value ranges herein is intended solely as a contraction of referring to each individual value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated herein as it would be if it were individually listed herein. Unless otherwise indicated herein or explicitly refuted by the context, all methods described herein may be performed in any suitable order. Any and all examples or illustrative expressions (e.g., “etc.”) presented herein are intended solely to better illustrate this disclosure and do not limit the claims. No expression herein shall be construed as referring to any unclaimed element essential to the practice of this disclosure.

[0230] Unless otherwise explicitly indicated, the term “including” in the context of this document is used to indicate that, in addition to the members of the list introduced by “including,” further members may optionally exist. However, in specific embodiments of this disclosure, it is also conceivable that the term “including” may also encompass the possibility that no further members exist; that is, for the purposes of this embodiment, “including” is understood to mean “consisting of.”

[0231] Several documents are referenced throughout this specification. Each of the documents referenced herein (including all patents, patent applications, academic publications, manufacturer specifications, instructions, etc.) is incorporated in whole by reference, whether above or below. Nothing in this specification shall be construed as an acceptance that this disclosure does not grant any prior rights to such disclosure.

[0232] definition The following definitions apply to all aspects of this disclosure. Unless otherwise indicated, the following terms have the following meanings. Any unspecified terms have the meanings recognized in the art.

[0233] As used herein, terms such as “reduce” or “inhibit” mean the ability to cause an overall reduction of, for example, about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more. The term “inhibit” or similar phrases include complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0234] 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%.

[0235] As used herein, "physiological pH" refers to a pH of approximately 7.5.

[0236] As used in this disclosure, "%w / v" refers to weight / volume percentage, 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).

[0237] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic molecular species, in particular, the mathematical relationship between a solution and the respective charges of those molecules. Therefore, ionic strength I is given by the formula It can be mathematically expressed as TIFF0007860909000001.tif18170 [wherein c is the molar concentration of a particular ionic molecular species and z is the absolute value of its charge. The sum Σ is taken for all different kinds of ions (i) in the solution].

[0238] In one embodiment, the term “ionic strength” as used in this disclosure refers to the presence of monovalent ions. In one embodiment, with respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) is sufficiently low to prevent RNA degradation due to the presence of chelating agents. In one embodiment, the concentration or effective concentration of divalent ions is below 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.

[0239] "Osmolality" refers to the concentration of a particular solute, expressed as the number of osmoles of solute per kilogram of solvent.

[0240] The Reynolds number is in the following form: It is a dimensionless number that can be calculated using the formula TIFF0007860909000002.tif15170 [wherein ρ is the fluid density, ν is the fluid volume, l is the characteristic length (in this case, the inner diameter of the mixing element), and η is the viscosity].

[0241] The term "to freeze" usually refers to the solidification of a liquid by the removal of heat.

[0242] The term "freeze-drying" refers to the process of freeze-drying a substance by freezing it and then reducing the ambient pressure, which allows the medium within the frozen substance to sublimate directly from the solid phase to the gaseous phase.

[0243] The term "spray drying" refers to a substance that has been spray-dried by mixing a (heated) gas with a fluid that has been atomized (sprayed) in a container (spray dryer), where the solvent evaporates from the formed droplets, resulting in a dry powder.

[0244] The term "cryoprotectant" relates to substances added to a formulation to protect the active ingredient during the freezing stage.

[0245] The term "lyoprotectant" relates to substances added to a formulation to protect the active ingredient during the drying stage.

[0246] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0247] In the context of the present disclosure, the term "recombinant" means "made through genetic manipulation". In one embodiment, a "recombinant object" in the context of the present disclosure does not exist in nature.

[0248] 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 an organism (including viruses), can be isolated from natural sources, and are not artificially and intentionally modified in the laboratory are naturally occurring. The term "found in nature" means "naturally occurring" and includes objects that, in addition to known objects, have not been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0249] The term "equilibrium solubility" refers to the concentration of a solute at which the rate of dissolution of the solute is the same as the rate of deposition of the solute from the solution. In one embodiment, the term relates to each concentration at room temperature.

[0250] As used herein, the term "room temperature" refers to a temperature above 4°C, preferably a temperature of 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 would include 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0251] In the context of this disclosure, the term “particle” refers to a structured entity formed by molecules or molecular complexes. In one embodiment, the term “particle” refers to a micron-sized or nano-sized structure, such as a micron-sized or nano-sized dense structure.

[0252] 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 complexation and the spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and further lipids such as DOPE. In one embodiment, the RNA lipoplex particles are nanoparticles.

[0253] 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.

[0254] The term "average diameter" is a so-called Z, which involves the dimension of length. average This refers to the mean hydrodynamic diameter of a particle, measured by dynamic light scattering (DLS), accompanied by data analysis using a so-called cumulant algorithm that results in a dimensionless multidispersion index (PI) (Koppel, D., J. Chem. Phys., 57, 1972, pp. 4814-4820, ISO 13321). Here, the "mean diameter," "diameter," or "size" of the particle is Z average This value is used synonymously with this value.

[0255] In this specification, the term "polydispersion index" is used as a measure of the size distribution of a collection of particles, such as nanoparticles. The polydispersion index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0256] 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 using light occlusion, which indicates the degree of aggregation of RNA lipoplex particles in this disclosure. 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.

[0257] The term "ethanol injection method" refers to a method of rapidly injecting an ethanol solution containing lipids into an aqueous solution via a hypodermic needle. This method disperses the lipids throughout the solution and promotes lipid structure formation, such as lipid vesicle formation, including liposome formation. Generally, the RNA lipoplex particles described herein are obtained by adding RNA to a colloidal liposome dispersion. In one embodiment, such a colloidal liposome dispersion is formed using the ethanol injection method by injecting an ethanol solution containing lipids, such as cationic lipids like DOTMA, and further lipids into an aqueous solution under agitation, as follows. In one embodiment, the RNA lipoplex particles described herein are obtained without an extrusion step.

[0258] The terms "extrude" or "extrude" refer to the creation of particles with a specific cross-sectional profile. In particular, the terms "extrude" or "extrude" refer to the miniaturization of particles, forcing them to pass through a filter with defined pores.

[0259] Diameter of RNA lipoplex particles The RNA lipoplex particles described herein, in one embodiment, have an average diameter in the range of approximately 200 nm to approximately 1000 nm, approximately 200 nm to approximately 800 nm, approximately 250 nm to approximately 700 nm, approximately 400 nm to approximately 600 nm, approximately 300 nm to approximately 500 nm, or approximately 350 nm to approximately 400 nm. In specific embodiments, RNA lipoplex particles have an average diameter of approximately 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, 900 nm, 925 nm, 950 nm, 975 nm, or 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 the exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0260] RNA lipoplex particles described herein, for example, RNA lipoplex particles produced by the methods described herein, exhibit polydispersity indices less than approximately 0.5, less than approximately 0.4, or less than approximately 0.3. For example, RNA lipoplex particles may exhibit polydispersity indices in the range of approximately 0.1 to approximately 0.3.

[0261] Lipids In one embodiment, the lipid solution, 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 to negatively charged RNA through electrostatic interactions with the lipid matrix. Generally, cationic lipids have lipophilic moieties such as sterols, acyl or diacyl chains, and lipid head groups, which typically possess a positive charge. Examples of cationic lipids 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-di(tetradecoxy)prop This includes, but is not limited to, 2-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1(l),2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1(l)-propanamium trifluoroacetic acid (DOSPA). DOTMA, DOTAP, DODAC, and DOSPA are preferred. In specific embodiments, at least one cationic lipid is DOTMA and / or DOTAP.

[0262] Further lipids can be incorporated to adjust the overall positive charge-to-negative charge ratio and the physical stability of the RNA lipoplex particles. In certain embodiments, the further lipids are neutral lipids. As used herein, “neutral lipids” refers to lipids with 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 specific embodiments, the second lipid is DOPE, cholesterol, and / or DOPC.

[0263] In certain embodiments, RNA lipoplex particles contain both cationic lipids and further lipids. In exemplary embodiments, the cationic lipid is DOTMA and the further 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 further lipid can 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 further 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 specific 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 further lipid is approximately 2:1.

[0264] RNA In this disclosure, the term “RNA” refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA, to the least of our knowledge, includes partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides, including double-stranded RNA, single-stranded RNA, and isolated RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of RNA. It is also assumed herein that the nucleotides within RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0265] In certain embodiments of this disclosure, RNA is messenger RNA (mRNA) relating to an RNA transcript encoding a peptide or protein. As is established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is prepared by in vitro transcription or chemical synthesis. In one embodiment, mRNA is prepared by in vitro transcription using a DNA template in which DNA refers to a nucleic acid containing deoxyribonucleotides.

[0266] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a vector appropriate for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0267] In certain embodiments of the present disclosure, the RNA in the RNA lipoplex composition described herein is at a concentration of about 0.01 mg / mL to about 1 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL. In specific embodiments, the RNA is at a concentration of about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL, or about 0.50 mg / mL. In an exemplary embodiment, the RNA is at a concentration of 0.05 mg / mL.

[0268] In one embodiment, RNA may have modified ribonucleotides. Examples of modified ribonucleotides, to the extent not limited to them, include 5-methylcytidine and pseudouridine.

[0269] 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 can be modified with a 5' cap analog. The term "5' cap" refers to a structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide attached to the RNA via a 5'-to-5' triphosphate linkage. In one embodiment, this guanosine is methylated at position 7. Imposing a 5' cap or a 5' cap analog on the RNA can be achieved by in vitro transcription, in which case the 5' cap can be expressed on the RNA chain by co-transcription, or it can be attached to the RNA post-transcriptionally using a capping enzyme.

[0270] In some embodiments, 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 to a corresponding region within the RNA molecule, such as an amino acid sequence or an mRNA molecule. Untranslated regions (UTRs) may exist on the 5' side (upstream) of an open reading frame (5'-UTR) and / or on the 3' side (downstream) of an open reading frame (3'-UTR). 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 end codon of the protein-coding region, although the term “3'-UTR” preferably does not include a poly(A) tail. Therefore, the 5'-UTR is upstream of the poly(A) sequence (if any) and, for example, directly adjacent to the poly(A) sequence.

[0271] In some embodiments, the RNA according to this disclosure includes a 3'-poly(A) sequence. The term "poly(A) sequence" typically refers to a sequence of adenyl (A) residues located at the 3' end of an RNA molecule. In one embodiment according to this disclosure, the poly(A) sequence includes at least about 20, at least about 40, at least about 80, or at least about 100 A nucleotides, and up to about 500, up to about 400, up to about 300, up to about 200, or up to about 150 A nucleotides, and in particular, about 120 A nucleotides.

[0272] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code within a DNA sequence is transcribed into RNA. RNA can then be translated into peptides or proteins.

[0273] In relation to RNA, the terms "expression" or "translation" refer to the process within a cell's ribosome in which a strand of mRNA directs the assembly of amino acid sequences to produce a peptide or protein.

[0274] 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 yield a peptide or protein. In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells, such as professional antigen-presenting cells, within the spleen. In one embodiment, the target cells are dendritic cells within the spleen. Therefore, 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 in a subject, including the administration of the RNA lipoplex particles described herein to a subject. 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 yield a peptide or protein.

[0275] In one embodiment, RNA encodes a pharmaceutically active peptide or protein.

[0276] As used in this disclosure, the term “RNA encodes a ~” means that, when RNA is present in a suitable environment, such as within the cells of a target tissue, it can, in the process of translation, direct the assembly of amino acids to produce the peptide or protein it encodes. In one embodiment, RNA can interact with an intracellular translation mechanism that enables the translation of the peptide or protein. The cell may produce the encoded peptide or protein within the cell (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or produce it on its surface.

[0277] As per 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 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked to one another via peptide bonds. The term “protein” refers to larger peptides, in particular peptides having at least about 151 amino acids, but as used herein, the terms “peptide” and “protein” are typically used as synonyms.

[0278] A "pharmaceutically active peptide or protein" is one that, when administered to a subject in a therapeutically effective dose, exerts an appropriate or beneficial effect on the subject's condition or disease. In one embodiment, a pharmaceutically active peptide or protein has therapeutic or mitigating properties and is administered to improve, reduce, alleviate, suppress, 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 can be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes whole proteins or whole polypeptides, and also refers to pharmaceutically active fragments thereof. The term "pharmaceutically active peptide or protein" may also include pharmaceutically active analogs of peptides or proteins.

[0279] Examples of pharmaceutically active proteins include 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, adrenaline, seretins, homing receptors, T cell receptors, immunoglobulins, soluble major tissue complex antigens, bacterial antigens, parasitic antigens, or viral antigens, allergens, autoantigens, autoantibodies, etc., immunologically active). Antigens), hormones (insulin, thyroid hormones, catecholamines, gonadotrophins, trophotrophins, prolactin, oxytocin, dopamine, bovine somatotrophin, 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 biosynthesis enzymes or cholesterol-degrading enzymes, steroid-producing enzymes, kinases, etc.) Phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylyl cyclase or guanylate cyclase, neuraminidases, 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., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin) This includes, but is not limited to, cytokines and immune system proteins such as fibrinogen, elastin, tubulin, actin, and myosin, blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willembrandt factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF), or modified factor VIII, and anticoagulants.

[0280] The term "immunologically active compound" refers to any compound that modifies the immune response, for example, by inducing and / or suppressing the maturation of immune cells, by inducing and / or suppressing cytokine biosynthesis, and / or by stimulating antibody production by B cells, thereby altering humoral immunity. Immunologically active compounds include, but are not limited to, antiviral and antitumor activity, and possess potent immunostimulatory activity that can also downmodulate other aspects of the immune response, for example, by shifting the immune response away from the TH2 immune response useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants.

[0281] In one embodiment, a pharmaceutically active peptide or protein contains 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 to one or more antigens or one or more epitopes that is therapeutic or partially or completely defensive.

[0282] 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, the antigen is presented by cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. In one embodiment, this processing product, such as an antigen or T cell epitope, is bound to an immunoglobulin molecule, such as a T cell receptor or B cell receptor, or an antibody. Thus, the antigen or this 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.

[0283] 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 an antigen-specific, cellular immune response and / or humoral antibody response to a disease. Therefore, disease-associated antigens or this epitope can be used for therapeutic purposes. Disease-associated antigens may be associated with microorganisms, typically infections caused by microbial antigens, and may also be associated with cancer, typically tumors.

[0284] The term "tumor antigen" refers to components of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus. In particular, "tumor antigen" refers to antigens produced within cells or as surface antigens on tumor cells.

[0285] The term "viral antigen" refers to any viral component that possesses antigenic properties, that is, is capable of eliciting an immune response in an individual. Viral antigens can be viral ribonucleoproteins or viral envelope proteins.

[0286] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties, that is, is capable of eliciting an immune response in an individual. Bacterial antigens can originate from the bacterial cell wall or cell membrane.

[0287] 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 can contain a continuous or discontinuous portion of the antigen and can be between approximately 5 and 100 amino acids in length. In one embodiment, an epitope is between approximately 10 and 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0288] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that includes MHC class I and MHC class II molecules and are present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or infected cells in immune responses, in which case 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 surface of cells and present both self-antigens (peptide fragments derived 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 binding peptide is typically about 8 to 10 amino acids long, but longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the binding peptide is typically about 10 to 25 amino acids long, and especially about 13 to 18 amino acids long, but longer and shorter peptides can also be effective.

[0289] 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, which is generally known to be expressed in a variety of cancers. The tumor antigen may also be a “neoantigen,” which is specific to the tumor of an individual and has not yet been 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, resulting in an amino acid change. Examples of tumor antigens, to name few, include p53, ART-4, BAGE, beta-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN (CLAUD Cell surface proteins of the claudin family, such as GAN-6, CLAUDIN-18.2, and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MA GE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A1 2, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 Includes 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.

[0290] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) represent attractive targets in the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes capable of inducing a robust immune response within the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) in the spleen represent target antigen-presenting cells in particular for expressing RNA of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutanomers for personalized vaccines may present multiple epitopes, optionally separated by linkers, which may be encoded by RNA described herein, for example, as a single polypeptide. 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 an RNA encoding at least five epitopes (referred to as a "pentatope") and an RNA encoding at least ten epitopes (referred to as a "decatope").

[0291] 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 (moles)) × (total number of positive charges in the cationic lipid)] / [(concentration of RNA (moles)) × (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 conventional methods.

[0292] In the first embodiment, at physiological pH, the charge ratio of positive charges to negative charges within RNA lipoplex particles is approximately 1.9:2 to approximately 1:2. In specific embodiments, at physiological pH, the charge ratio of positive charges to negative charges within RNA lipoplex particles is approximately 1.9:2.0, approximately 1.8:2.0, approximately 1.7:2.0, approximately 1.6:2.0, approximately 1.5:2.0, approximately 1.4:2.0, approximately 1.3:2.0, approximately 1.2:2.0, approximately 1.1:2.0, or approximately 1:2.0. In one embodiment, at physiological pH, the charge ratio of positive charges to negative charges within RNA lipoplex particles is 1.3:2.0. In another embodiment, the RNA lipoplex particles described herein may have an equal number of positive and negative charges at physiological pH, thus resulting in RNA lipoplex particles with a net neutral charge ratio.

[0293] In the second embodiment, at physiological pH, the charge ratio of positive charges to negative charges within RNA lipoplex particles is approximately 6:1 to approximately 1.5:1. In specific embodiments, the charge ratio of positive charges to negative charges within 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. The ratios are approximately 0:1.0, 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.

[0294] For RNA-based immunotherapy, precise organ targeting, such as the spleen, is necessary to avoid autoimmune responses and potential toxicity in other organs. According to this disclosure, RNA can be targeted to different cells, tissues, or organs.

[0295] It has been found that RNA lipoplex particles having a charge ratio according to the first embodiment can preferentially target antigen-presenting cells, particularly spleen tissue or spleen cells, such as dendritic cells. Thus, in one embodiment, RNA accumulation and / or RNA expression occurs in the spleen after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression does not occur in the lungs and / or liver after administration of RNA lipoplex particles. In one embodiment, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells, in the spleen after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0296] It has been found that RNA lipoplex particles having a charge ratio according to the second embodiment can preferentially target lung tissue or lung cells. Thus, in one embodiment, RNA accumulation and / or RNA expression occurs in the lungs after administration of RNA lipoplex particles. Thus, the RNA lipoplex particles of this disclosure can be used to express RNA in the lungs. Accordingly, in the embodiments described herein, which relate to a charge ratio according to the first embodiment, e.g., a charge ratio of about 1:2 to about 1.9:2, if RNA expression in tissues other than the spleen is desired, a charge ratio according to the second embodiment, e.g., 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. In these embodiments and other embodiments, RNA other than peptide or protein-encoding RNA containing at least one epitope is described herein, e.g., pharmaceutically active peptide or protein-encoding RNA as described herein can be used. In one embodiment, the pharmaceutically active peptide or protein is a cytokine and / or is intended for the treatment of lung cancer.

[0297] Composition containing RNA lipoplex particles A. Salt strength and ionic strength In accordance with this disclosure, the compositions described herein may include salts such as sodium chloride. While we do not wish to be bound by theory, sodium chloride functions as an ionic osmolality agent for pretreatment of RNA before mixing with at least one cationic lipid. Certain embodiments assume alternative organic or inorganic salts for sodium chloride in this disclosure. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0298] 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 contains ionic strengths corresponding to such sodium chloride concentrations.

[0299] Generally, compositions for forming RNA lipoplex particles from RNA and liposomes, and compositions obtained therefrom, such as those described herein, contain high sodium chloride concentrations 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 contains ionic strength corresponding to such sodium chloride concentrations.

[0300] In general, compositions for storing RNA lipoplex particles, such as compositions for freezing RNA lipoplex particles, and compositions such as those described herein, contain a low sodium chloride concentration or a low ionic strength. 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 specific 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, 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 contains ionic strengths corresponding to such sodium chloride concentrations.

[0301] Generally, compositions obtained by thawing a frozen RNA lipoplex particle composition and optionally adding an aqueous liquid to adjust the osmolality and ionic strength contain high sodium chloride concentrations 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 contains ionic strengths corresponding to such sodium chloride concentrations.

[0302] 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 of frozen compositions, lyophilized compositions, or spray-drying compositions, and during storage. Such compositions are also referred to herein as stabilizers. Typically, the stabilizers are present before the freezing, lyophilization, or spray-drying process and persist in the resulting frozen, lyophilized, or freeze-dried preparation. The compositions described herein can be used to protect RNA lipoplex particles during freezing, lyophilization, or spray-drying of frozen, lyophilized, or freeze-dried preparations, and during storage, thereby reducing or preventing damage such as aggregation, particle disintegration, RNA degradation, and / or other types of damage.

[0303] In one embodiment, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0304] In some embodiments, the stabilizer is a monosaccharide. As used herein, the term “monosaccharide” refers to a single carbohydrate unit (e.g., a monosaccharide) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide stabilizers include glucose, fructose, galactose, xylose, and ribose.

[0305] In some embodiments, the stabilizer is a disaccharide. As used herein, the term “disaccharide” refers to a compound or chemical part formed by two monosaccharide units linked together via glycosidic linkages, for example, via 1-4 or 1-6 linkages. Disaccharides can be hydrolyzed to two monosaccharides. Exemplary disaccharide stabilizers include sucrose, trehalose, lactose, and maltose.

[0306] The term "trisaccharide" refers to three sugars linked together to form a single molecule. Examples of trisaccharides include raffinose and melegitose.

[0307] In some embodiments, the stabilizer is an oligosaccharide. As used herein, the term “oligosaccharide” refers to a compound or chemical portion formed by 3 to about 15, preferably 3 to about 10, monosaccharide units linked together via glycosidic linkages, for example, via 1-4 or 1-6 linkages, to form a linear, branched, or cyclic structure. Exemplary oligosaccharide stabilizers include cyclodextrin, raffinose, melegitose, maltotriose, stachyose, acarbose, and the like. Oligosaccharides can be oxidized or reduced.

[0308] In some embodiments, the stabilizer is a cyclic oligosaccharide. As used herein, the term “cyclic oligosaccharide” refers to a compound or chemical portion formed by three to about 15, preferably six, seven, eight, nine, or ten monosaccharide units linked together to form a cyclic structure, for example, via glycosidic linkages, such as 1-4 or 1-6 linkages. Exemplary cyclic oligosaccharide stabilizers include cyclic oligosaccharides, which are individual compounds such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

[0309] Other exemplary cyclic oligosaccharide stabilizers include compounds containing a cyclodextrin moiety within a larger molecular structure, such as a polymer containing a cyclic oligosaccharide moiety. Cyclic oligosaccharides can be oxidized and reduced, for example, to a dicarbonyl form. As used herein, the term “cyclodextrin moiety” refers to a cyclodextrin group (e.g., α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin) that is incorporated into or is part of a larger molecular structure, such as a polymer. The cyclodextrin moiety can be directly bonded to one or more other moieties, or bonded via an optional linker. The cyclodextrin moiety can be oxidized and reduced, for example, to a dicarbonyl form.

[0310] 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).

[0311] Exemplary stabilizers are polysaccharides. As used herein, the term “polysaccharide” refers to a compound or chemical moiety formed by at least 16 monosaccharide units linked together via glycosidic linkages, for example, via 1-4 or 1-6 linkages, to form a linear, branched, or cyclic structure, and includes polymers containing polysaccharides as part of their skeletal structure. Within the skeleton, the polysaccharide may be linear or cyclic. Exemplary polysaccharide stabilizers include glycogen, amylase, cellulose, dextran, and maltodextrin.

[0312] In some embodiments, the stabilizer is a sugar alcohol. As used herein, the term “sugar alcohol” refers to the reduction product of “sugars,” and a “polyol” is a sugar alcohol in which all oxygen atoms within a monosaccharide alcohol molecule are present in the form of hydroxyl groups. This term refers to compounds containing three or more hydroxyl groups and is synonymous with another general term, polyhydric alcohol. Examples of sugar alcohols include, but are not limited to, sorbitol, mannitol, maltitol, lactitol, erythritol, glycerin, xylitol, or inositol.

[0313] In accordance with this disclosure, pharmaceutical compositions comprising sucrose as a stabilizer are presented. While we do not wish to be bound by theory, sucrose functions to promote cold protection of the composition, thereby preventing aggregation of RNA lipoplex particles and maintaining the chemical and physical stability of the composition. Certain embodiments assume alternative stabilizers to sucrose in this disclosure. Alternative stabilizers include, but are not limited to, trehalose, glucose, fructose, arginine, glycerin, mannitol, proline, sorbitol, glycine betaine, and dextran. In specific embodiments, the alternative stabilizer to sucrose is trehalose.

[0314] 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 one embodiment 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.

[0315] In accordance with this disclosure, the RNA lipoplex particle compositions described herein have stabilizer concentrations suitable for the stability of the composition, particularly stabilizer concentrations suitable for the stability of the RNA lipoplex particles and for the stability of the RNA.

[0316] C. pH and buffer In accordance with this disclosure, the RNA lipoplex particle compositions described herein have a pH suitable for the stability of RNA lipoplex particles, and in particular for the stability of RNA. In one embodiment, the RNA lipoplex particle compositions described herein have a pH of about 5.7 to about 6.7. In specific embodiments, the compositions have 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.

[0317] In accordance with this disclosure, compositions comprising buffers are presented. While we do not wish to be bound by theory, the use of buffers maintains the pH of the composition during its preparation, storage, and use. In certain embodiments of this disclosure, the buffer is 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 (tri The buffers may be 3-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]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 salt, citrate in salt, boric acid in salt, and phosphoric acid in salt.

[0318] In some embodiments, the buffer has a pH of about 5.7 to about 6.7. In specific embodiments, the buffer has a pH of about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7. In one embodiment, the buffer is HEPES. In a preferred embodiment, HEPES has a pH of about 5.7 to about 6.7. In specific embodiments, HEPES has a pH of about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7. In an exemplary embodiment, HEPES has a pH of about 6.2.

[0319] In yet another embodiment, the buffer has a concentration of about 2.5 mM to about 10 mM. In a specific embodiment where HEPES is the buffer, the concentrations of HEPES are 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, and about 5.75 mM. The concentrations are approximately mM, about 6.0 mM, about 6.25 mM, about 6.5 mM, about 6.75 mM, about 7.0 mM, about 7.25 mM, about 7.5 mM, about 7.75 mM, about 8.0 mM, about 8.25 mM, about 8.5 mM, about 8.75 mM, about 9.0 mM, about 9.25 mM, about 9.5 mM, about 9.75 mM, or about 10.0 mM. In a preferred embodiment, HEPES is at a concentration of about 7.5 mM.

[0320] D. Chelating agents Certain embodiments of this disclosure assume the use of a chelating agent. A chelating agent is a compound capable of forming at least two coordination-covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. While we do not wish to be bound by theory, chelating agents reduce the concentration of free divalent ions, which otherwise could induce accelerated RNA degradation in this disclosure. Examples of suitable chelating agents, to the extent that they are not limited, include ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, sodium dithiocarb, penicillamine, calcium pentoteate, sodium salt 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.

[0321] In some embodiments, the EDTA concentration is approximately 0.25 mM to approximately 5 mM. In specific embodiments, the EDTA concentrations are 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 is at a concentration of approximately 2.5 mM.

[0322] 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, and RNA at a concentration of about 0.05 mg / mL encoding at least one epitope, in which case the charge ratio of positive charges 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 with a pH of about 6.2; and EDTA at a concentration of about 2.5 mM. In a further specific embodiment, the RNA encodes five or ten epitopes.

[0323] 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, and RNA at a concentration of about 0.05 mg / mL encoding at least one epitope, in which case the charge ratio of positive charges 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 with a pH of about 6.2; and EDTA at a concentration of about 2.5 mM. In a further specific embodiment, the RNA encodes five or ten epitopes.

[0324] F. Stability of the compositions of this disclosure As used herein, “stable” refers to a composition in which measurements of various physiological and chemical 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, to the extent not limited, the mean diameter of RNA lipoplex particles, polydispersity index, RNA integrity, RNA content, pH, osmolality, and the number of undetectable particles. Those skilled in the art will be able to measure such parameters using conventional laboratory methods and instruments. For example, stability parameters can be evaluated using dynamic light scattering (DLS), light occlusion, spectrophotometric methods, 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.

[0325] While we do not wish to be bound by theory, DLS measurements are useful for analyzing the RNA lipoplex particles and associated parameters of this disclosure. In one embodiment, DLS can be used to determine the average diameter of RNA lipoplex particles, expressed in relation to the Z-mean (a measure of average particle size). In another embodiment, DLS can be used to determine the polydispersity index of RNA lipoplex particles, indicating the size and weight distribution of the RNA lipoplex particles.

[0326] In certain embodiments, a composition is stable if a measure of stability parameters falls within a specified range. In one embodiment of a stable composition, RNA lipoplex particles, after storage, for example, after storage 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, lyophilizing, or spray-drying and thawing or reconstitution) by no more than ±20%, ±10%, ±5%, or ±3%. In one embodiment of a stable composition, RNA lipoplex particles, after storage, for example, after storage at a temperature of about -15°C to about -40°C, have an average diameter that does not exceed 20%, 10%, 5%, or 3% compared to the original average diameter (i.e., the average diameter before freezing, lyophilizing, or spray-drying and thawing or reconstitution). In another embodiment for a stable composition, the RNA lipoplex particles, after storage, for example, after storage at a temperature of about -15°C to about -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 before thawing or reconstitution) by ±20%, ±10%, ±5%, or ±3% or less. In one embodiment, the stable composition, after storage, for example, after storage at a temperature of about -15°C to about -40°C, has 6,000 or fewer invisible particles with a diameter of 10 μm or more. In another embodiment, the stable composition, after storage, for example, after storage at a temperature of about -15°C to about -40°C, has 600 or fewer invisible particles with a diameter of 25 μm or more.

[0327] 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.

[0328] In one embodiment, the composition is stable at a storage temperature of about -15°C to about -40°C. In a specific 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.

[0329] In one embodiment, when the pharmaceutical composition is protected from light, the composition is stable at a temperature of about -15°C to about -40°C. In a preferred embodiment, when the composition is protected from light, the composition is stable at a temperature of about -15°C to about -25°C.

[0330] In one embodiment, the composition is stable for at least one month, up to about 24 months, at a temperature of about -15°C to about -40°C. In specific embodiments, the composition is stable at a temperature of about -15°C to about -40°C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, at least 25 months, at least 26 months, at least 27 months, at least 28 months, at least 29 months, at least 30 months, at least 31 months, at least 32 months, at least 33 months, at least 34 months, at least 35 months, or at least 36 months.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] In one embodiment, the composition is stable after being frozen at a temperature of approximately -15°C to approximately -40°C and then thawed at a temperature of approximately 4°C to approximately 25°C (room temperature). In another embodiment, the composition is stable after being frozen at a temperature of approximately -15°C to approximately -40°C and then thawed at a temperature of approximately 4°C to approximately 25°C (room temperature) through multiple freeze-thaw cycles.

[0335] G. Physical state of the compositions disclosed herein In some embodiments, the compositions of the present disclosure are liquid or solid. Examples of solids, not limited to those described above, include frozen or freeze-dried forms. In preferred embodiments, the composition is liquid.

[0336] Pharmaceutical composition of the present disclosure Compositions comprising RNA lipoplex particles as described herein are useful as pharmaceutical compositions or pharmaceutically acceptable for therapeutic or prophylactic treatments, or for preparing such compositions.

[0337] The particles of this disclosure may be administered in the form of any suitable pharmaceutical composition.

[0338] The term “pharmaceutical composition” relates to a formulation comprising a therapeutically effective agent, preferably in combination with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful, upon administration to a subject, for treating, preventing, or reducing the severity of a disease or disorder. In the art, pharmaceutical compositions are also known as pharmaceutical formulations. In the context of this disclosure, a pharmaceutical composition includes RNA lipoplex particles as described herein.

[0339] The pharmaceutical compositions of this disclosure preferably comprise one or more adjuvants, or are administered together with one or more adjuvants. The term “adjuvant” relates to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a diverse group of compounds, such as oily emulsions (e.g., Freund’s adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants, to the extent not limited to, include 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, or LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA51. Other adjuvants suitable for use in this disclosure include lipopeptides such as Pam3Cys.

[0340] Pharmaceutical compositions relating to this disclosure are generally applied in a "pharmaceutically acceptable amount" in a "pharmaceutically acceptable preparation."

[0341] The term "pharmaceutically acceptable" refers to a non-toxic material that does not interact with the active ingredients of a pharmaceutical composition.

[0342] The term "pharmaceutically effective dose" refers to the amount, either alone or in combination with further doses, that achieves the desired response or effect. In the case of treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, in particular interrupting or suppressing its progression. The desired response in the treatment of a disease may also be the delay of the onset of the disease or condition, or the prevention of its onset. The effective dose of the particles or compositions described herein will depend on the individual patient's parameters, including the condition being treated, the severity of the disease, age, physiological state, height and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration, and similar factors. Therefore, the dose administered of the particles or compositions described herein may depend on these parameters. If the initial dose does not produce an adequate response in the patient, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) may be used.

[0343] The pharmaceutical compositions of this disclosure may contain a salt, a buffer, a preservative, 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.

[0344] Preservatives suitable for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0345] 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.

[0346] The term “diluent” refers to a diluting and / or thinning agent. Furthermore, the term “diluent” includes any one or more fluids, liquids, or solid suspensions and / or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.

[0347] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, that mixes the active ingredient therein to facilitate, enhance, or enable the administration of a pharmaceutical composition. As used herein, carriers may be one or more suitable, solid or liquid fillers, diluents, or encapsulants suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of this disclosure contains isotonic saline.

[0348] In the field of pharmaceutical technology, pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known and are described, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Co. (edited by AR Gennaro, 1985).

[0349] Pharmaceutical carriers, excipients, or diluents can be selected in light of the intended route of administration and standard pharmaceutical practice.

[0350] Route of administration of the pharmaceutical compositions of this disclosure In one embodiment, the pharmaceutical compositions described herein are administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration may include enteral administration with absorption through the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” refers to any form of administration other than administration 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.

[0351] 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.

[0352] 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.

[0353] Use of the pharmaceutical composition of this disclosure The RNA lipoplex particles described herein can be used in therapeutic or prophylactic treatments for a variety of diseases, particularly those in which the provision of peptides or proteins to a target results in a therapeutic or prophylactic effect. For example, the provision of a virus-derived antigen or epitope may be useful in the treatment of viral diseases caused by such virus. The provision of a tumor antigen or epitope may be useful in the treatment of cancerous diseases in which cancer cells express such tumor antigen.

[0354] The term "disease" refers to an abnormal condition that affects an individual's body. Often, a disease is interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external pathogens, such as infectious diseases, or by internal dysfunction, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual, or similar problems in individuals who come into contact with the affected individual. In this broader sense, "disease" may include, in some cases, injury, physical impairment, disability, syndrome, infection, sporadic symptoms, deviant behavior, and atypical changes in structure and function, but in other contexts and for other purposes, "disease" is considered a distinguishable category. Many diseases, and living with them, can alter one's outlook on life and alter one's personality; therefore, diseases typically have not only physical but also emotional effects on an individual.

[0355] In this context, the terms “treatment,” “treating,” or “therapeutic intervention” relate to the management and care of an individual aimed at counteracting a condition such as a disease or disorder. The terms refer to a full spectrum of treatments for a given condition that an individual suffers from, including the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of the disease, disorder, or condition, alleviate or reduce symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, or prevent the condition, and prevention is understood to be the management and care of the individual aimed at counteracting the disease, condition, or disorder, and are intended to include treatments that involve the administration of active compounds to prevent the onset of symptoms or complications.

[0356] The term “therapeutic treatment” refers to any treatment that improves the health of an individual and / or extends (increases) the lifespan of an individual. Such treatment may eliminate a disease in an individual, stop or slow the onset of a disease in an individual, inhibit or slow the onset of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual that currently has or has previously had the disease.

[0357] The terms “preventive measures” or “preventive measures” refer to any measures intended to prevent disease from occurring in an individual. In this specification, the terms “preventive measures” and “preventive measures” are used interchangeably.

[0358] In this specification, the terms “individual” and “subject” are used interchangeably. The terms “individual” and “subject” refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), and is susceptible to such diseases or disorders. In many embodiments, “individual” is 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 some embodiments of this disclosure, “individual” or “subject” is “patient.”

[0359] The term "patient" means an individual or subject for treatment, in particular an affected individual or subject.

[0360] In one embodiment of the present disclosure, the objective is to induce an immune response against diseased cells that express antigens, such as cancer cells that express tumor antigens, and to treat diseases such as cancer that involve cells that express antigens such as tumor antigens.

[0361] A pharmaceutical composition comprising RNA lipoplex particles described herein, comprising an RNA-encoding peptide or protein containing one or more antigens or one or more epitopes, is administered to a subject to induce an immune response in the subject to one or more antigens or one or more epitopes, which is therapeutic or partially or completely protective. Those skilled in the art will know that the principles of immunotherapy and vaccination are based on the fact that an immune defense response against a disease is brought about by immunizing a subject with an antigen or epitope that is immunologically relevant with respect to the disease being treated. Therefore, the pharmaceutical compositions described herein are applicable to the induction or enhancement of an immune response. Accordingly, the pharmaceutical compositions described herein are useful in prophylactic and / or therapeutic treatment of diseases involving antigens or epitopes.

[0362] 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, to the extent not limited to, cellular responses directed to a cell expressing an antigen and characterized by the presentation of the antigen accompanied by a class I MHC molecule or a class II MHC molecule. Cellular responses relate to T lymphocytes, which may be classified as helper T cells (also referred to as CD4+ T cells) that play a central role in modulating the immune response, or killer cells (also referred to as cytotoxic T cells, CD8+ T cells, or CTLs) that induce apoptosis in infected or cancer cells. In one embodiment, administration of the pharmaceutical composition of this disclosure is accompanied by stimulation of an antitumor CD8+ T cell response to cancer cells expressing one or more tumor antigens. In a specific embodiment, the tumor antigen is presented by a class I MHC molecule.

[0363] This disclosure assumes an immune response that may be defensive, preventive, prophylactic, and / or therapeutic. As used herein, “inducing an immune response” may mean that no immune response to a particular antigen was present before induction, or that a basal level immune response to a particular antigen was present before induction but was enhanced after induction. Thus, “inducing an immune response” includes “enhancing an immune response.”

[0364] The term "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen-based immunization or antigen-based vaccination.

[0365] The terms "immunization" or "vaccination" describe methods of administering antigens to an individual for the purpose of inducing an immune response, for example, for therapeutic or preventive reasons.

[0366] In one embodiment, this disclosure envisions an embodiment in which RNA lipoplex particles, as described herein, are administered to target spleen tissue. The RNA encodes a peptide or protein, for example, an antigen or epitope, 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 optional processing and presentation by antigen-presenting cells, an immune response to the antigen or epitope occurs, which may result in prophylactic and / or therapeutic treatment for diseases associated with the antigen or epitope. In one embodiment, the immune response induced by the RNA lipoplex particles described herein includes the presentation of this fragment, such as the antigen or epitope, by antigen-presenting cells such as dendritic cells and / or macrophages, and the resulting activation of cytotoxic T cells. For example, the peptide or protein encoded by the RNA or this procession product may be presented by major histocompatibility complex (MHC) proteins expressed on antigen-presenting cells. Subsequently, the MHC peptide complex can be recognized by immune cells such as T cells or B cells, potentially leading to their activation.

[0367] Therefore, in one embodiment, the RNA in the RNA lipoplex particles described herein is delivered to and / or expressed in the spleen after administration. In one embodiment, the RNA lipoplex particles are delivered to the spleen to activate antigen-presenting cells in the spleen. Therefore, in one embodiment, after administration of the RNA lipoplex particles, delivery of RNA and / or expression of RNA occurs within the antigen-presenting cells. 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, and more preferably splenic dendritic cells and / or splenic macrophages.

[0368] Accordingly, this disclosure relates to RNA lipoplex particles as described herein, or to pharmaceutical compositions comprising RNA lipoplex particles, for inducing or enhancing an immune response, preferably an immune response against cancer.

[0369] In further embodiments, the disclosure relates to RNA lipoplex particles as described herein, or pharmaceutically active compositions comprising RNA lipoplex particles, for use in prophylactic and / or therapeutic treatment of antigens, preferably diseases associated with cancer.

[0370] In further embodiments, the 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 for expressing an antigen or an epitope of an antigen within antigen-presenting cells, such as professional antigen-presenting cells, in the spleen, to a subject. The Disclosure relates to administering RNA lipoplex particles, or a pharmaceutical composition comprising RNA lipoplex particles, as described herein, to a subject. 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.

[0371] In one embodiment, systemic administration of RNA lipoplex particles, or a pharmaceutical composition containing RNA lipoplex particles, as described herein, results in the targeting and / or accumulation of RNA lipoplex particles or RNA in the spleen, and not in the lungs and / or liver. In one embodiment, the RNA lipoplex particles release RNA into the spleen and / or enter cells within the spleen. In one embodiment, systemic administration of RNA lipoplex particles, or a pharmaceutical composition containing RNA lipoplex particles, as described herein, delivers RNA to antigen-presenting cells in the spleen. In a specific embodiment, the antigen-presenting cells in the spleen are dendritic cells or macrophages.

[0372] In further embodiments, the disclosure relates to a method for inducing or enhancing an immune response in a subject, comprising administering to the subject RNA lipoplex particles described herein, or a pharmaceutical composition comprising RNA lipoplex particles. In exemplary embodiments, the immune response is against cancer.

[0373] The term "macrophage" refers to a subgroup of phagocytic cells resulting from the differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage through hydrolytic and oxidative attacks, resulting in the degradation of 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, resulting in 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.

[0374] The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells originate from bone marrow hematopoietic progenitor cells. These progenitor cells first transform into immature dendritic cells. These immature cells are characterized by their high phagocytic activity and potential for low T cell activation. Immature dendritic cells constitutively sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with antigens they can present, immature dendritic cells become activated into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, breaking down their proteins into fragments, and upon maturation, they use MHC molecules to present these fragments on their cell surface. Simultaneously, DCs upmodulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, significantly enhancing their ability to activate T cells. DCs also upregulate CCR7, a chemotactic receptor that induces dendritic cells to migrate to the spleen via the bloodstream or to lymph nodes via the lymphatic system. DCs act as antigen-presenting cells, activating B cells as well as helper T cells and killer T cells by presenting antigens to them along with non-antigen-specific co-stimulatory signals. Thus, dendritic cells can actively induce T-cell-associated or B-cell-associated immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0375] The term "antigen-presenting cell" (APC) refers to one of several types of cells capable of displaying, taking up, and / or presenting at least one antigen or antigenic fragment on its cell surface (or on its cell surface). Antigen-presenting cells can be classified into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0376] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules required 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.

[0377] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but express them when stimulated 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.

[0378] "Antigen processing" refers to the breakdown of an antigen into procession products, which are fragments of the antigen (for example, the breakdown of a protein into a peptide), and the association (for example, via binding) of one or more of these fragments with MHC molecules by a cell such as an antigen-presenting cell for presentation to a specific T cell.

[0379] The terms “disease-associated antigen” or “disease-associated epitope” refer to any disease associated with an antigen or epitope, such as a disease characterized by the presence of an antigen or epitope. Diseases associated with an antigen or epitope may be infectious diseases, cancerous diseases, or simply cancer. As mentioned above, antigens can be disease-associated antigens, such as tumor-associated antigens, viral antigens, or bacterial antigens, and epitopes may originate from such antigens.

[0380] The term “infectious disease” refers to any disease caused by a microbial agent (e.g., the common cold) that may be transmitted from individual to individual or from organism to organism. In the art, infectious diseases are known and include, for example, viral diseases, bacterial diseases, or parasitic diseases 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.

[0381] The terms “cancer disease” or “cancer” typically refer to or describe a physiological condition in an individual characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, such examples of cancer include bone cancer, blood cancer, 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, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the genitals, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, tumors of the central nervous system (CNS), neuroectodermal carcinoma, spinal tumors, glioma, meningioma, and pituitary adenoma. The term “cancer” as used in this disclosure also includes cancer metastases.

[0382] Combination strategies in cancer treatment may be desirable due to the resulting synergistic effects, which can be significantly more potent than those of monotherapy. In one embodiment, a pharmaceutical composition is administered together with an immunotherapy agent. As used herein, “immunotherapy agent” refers to any agent that may be involved in the activation of a specific immune response and / or immune effector function. This disclosure assumes the use of 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 may induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or bind to complement proteins, resulting 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), pentetate altumomab (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), alsitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), vectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF- A) Vibatuzumab meltansine (CD44v6), blinatumomab (CD19), brentuximab vedotin (TNFRSF8, which is CD30), cantuzumab meltansine (mucin CanAg), cantuzuma bravatansine (MUC1), capromab pendetide (prostate cancer cells), carrumab (CNT0888), catumakisomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab vogatox (EpCAM), cyclostomumab (IGF-1 receptor), claudiximab (Claudin), cribatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40),Darotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B-cell 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), phaletuzumab (folate receptor 1), FBTA05 (CD20), ficratuzumab (SCH90) 0105), Figitumumab (IGF-1 receptor), Frambotumab (glycoprotein 75), Fresolimmab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab Ozogamisin (CD33), Gevokizumab (IL-Iβ), Gilentuximab (carbonic anhydrase 9 (CA-IX)), Grembatumumab Vedotin (GPNMB), Ibritumomab Tiuxetan (CD20), Icurcumab (VEGFR-1), Igobomab (CA-125), Indatuximab Tansine (SDC1), Intetumumab ( CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD152), Iratumumab (CD30), Labetuzumab (CEA), Lexatumumab (TRAIL-R2), Ribivirumab (Hepatitis B surface antigen), Lintuzumab (CD33), Rorbotuzumab meltansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR), Mepolizumab (IL-5), Miratuzumab (CD74), Mitsumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moki Setumomab pasdotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumumab (PDGF-Ra), onarutuzumab (human scattering factor receptor kinase), oportuzumab monatox (EpCAM), olegobomab (CA-125), oxerumab (OX-40), panitumumab (EGFR), patritumumab (HER3), pemtumomab (MUC1),Pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), lacosumomab (N-glycolylneuraminic acid), radretumumab (fibronectin extracellular domain B), rafibirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samarizumab (CD200), sibrotuzumab (FAP), silituximab (IL-6), tabalumab (BAFF), tacutuzumab tetraxetan (alpha-fetoprotein), tapritumomab paptox This includes (CD19), tenatumomab (tenascin C), teprotumumab (CD221), tisilimmab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucozouzumab cermoloykin (EpCAM), ubirituximab (MS4A1), urerumab (4-1BB), borosiximab (integrin α5β1), botumumab (tumor antigen CTAA16.88), saltumumab (EGFR), and zanolimmumab (CD4).

[0383] 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. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In specific embodiments, the binding partners of the PD-1 ligand are PD-L1 (PD-L1) and / or PD-L2. In another embodiment, a PD-L1 (PD-Ll) binding antagonist is a molecule that inhibits the binding of PD-L1 (PD-Ll) to its binding partner. In a specific embodiment, the binding partner of PD-L1 (PD-Ll) is PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific embodiment, the binding partner of PD-L2 is PD-1. The PD-1 binding antagonist can be an antibody, an immunoadhesin which is the antigen-binding fragment of this antibody, 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, to name few, include MDX-1106 (nivolumab, OPDIVO), Merck3475 (MK-3475, pembrolizumab, KEYTRUDA), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.

[0384] In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising an extracellular portion or a PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DC Ig and PD-L2-Fc), a fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0385] In one embodiment, the PD-1 binding antagonist is an anti-PD-L1 (PD-L1) antibody, including, but not limited to, YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab).

[0386] In one embodiment, the immunotherapy agent is a PD-1 binding antagonist. In another embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody. In an exemplary embodiment, the anti-PD-L1 antibody is atezolizumab.

[0387] The references to the literature and studies referred to herein are not intended to constitute an acceptance that any of the foregoing constitutes relevant prior art. All statements regarding the contents of these documents are based on information available to the applicants and do not constitute any acceptance of the accuracy of the contents of these documents.

[0388] The following description is provided to enable those skilled in the art to perform and use a variety of embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Those skilled in the art will readily see a variety of modifications to the examples described herein, and the general principles set forth herein may be applied to other examples and applications, provided that they do not deviate 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 are given a scope corresponding to the claims. [Examples]

[0389] Example 1 material The essential materials used for the experiments described later in this specification are: The filename was TIFF0007860909000003.tif115170.

[0390] Example 2 Preparation of lipid mixtures Prepare DOTMA / DOPE lipid mixtures with different lipid concentrations in ethanol, each with a lipid molar ratio of 2:1. Prepare the solutions as follows: Weigh the DOPE lipids. Calculate the amount of DOTMA needed to maintain a molar ratio of 2:1 between DOTMA and DOPE. Weigh the DOTMA lipids. • 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 (bad).

[0391] The solubility of DOPE and DOPE / DOTMA in ethanol was investigated by preparing a 300 mM DOPE solution or a 330 mM DOPE / DOTMA (66:33) solution in ethanol. 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 was measured by HPLC in the supernatant. The DOTMA / DOPE solution was filtered through a 0.22 μm PES syringe filter, and the lipid concentration was measured by HPLC in the filtrate.

[0392] Further lipid mixtures can also be prepared by following the basic steps described in this embodiment, provided that other lipids are used as starting materials and / or that the molar ratios of other lipids are calculated.

[0393] 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) was injected into 9.8 mL of water in ethanol under 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 without such filters. The liposome colloid was stored at 4–8°C. The DOTMA / DOPE lipid solution was prepared in a molar ratio of 66:33% at different total lipid concentrations ranging from 100–400 mM, with intermediate steps of 50 mM each.

[0394] Example 4 Preparation of RNA lipoplex RNA lipoplex formulations were prepared as follows: First, an RNA solution (e.g., luc-RNA solution) was mixed with an NaCl solution to pre-concentrate the luc-RNA. Then, liposome colloids were mixed with the luc-RNA-NaCl solution to form RNA lipoplexes. The RNA lipoplex formulations were incubated at room temperature for 10 minutes and stored at 4-8°C. Different RNA lipoplex formulations were prepared, for example, with an N / P ratio of 0.65. The concentration of RNA in these different RNA lipoplex formulations was 0.1 mg / mL, and the NaCl concentration was 50 mM. Different liposome precursors (of different sizes) were used to prepare the RNA lipoplexes.

[0395] Example 5 Dynamic light scattering Liposome and RNA lipoplex sizes were measured using a known dynamic light scattering (DLS) method with a Nicomp analyzer (PSS, Santa Barbara, USA). 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).

[0396] Example 6 Optical occultation: dynamic light scattering Particle counting / measurement for different liposome and RNA lipoplex formulations within a size range of 0.5–5 μm was performed using an Accusizer A7000 analyzer (PSS, Santa Barbara, USA). Three measurements were performed for a 5 mL volume (2.5 μL sample / 20 mL free particle water). The results obtained represent the average particle quantity from the three measurements.

[0397] Example 7 Small angle X-ray scattering The internal structure parameters of different RNA lipoplex formulations prepared with different liposome precursors were measured by small-angle X-ray scattering (SAXS). SAXS is a technique that analyzes the elastic scattering behavior of X-rays as they pass through a material and quantifies nanoscale density differences in a sample by recording these small-angle scatterings. For all tested RNA formulations, the correlation between the length parameter and the d-interval parameter was calculated. The RNA lipoplex formulations were prepared with an N / P ratio of 0.65, 0.1 mg / mL of RNA, and 112 mM NaCl.

[0398] Example 8 Agarose gel electrophoresis The amount of free RNA in different 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 trial time of 40 minutes.

[0399] Example 9 HPLC Lipid concentrations in different liposome formulations were measured by HPLC (Agilent Technologies, Santa Clara, USA) using a Sunfire C18 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 either diluted with water to a total lipid concentration of 3 mM or left undiluted.

[0400] Example 10 Cell cultures: RNA transfection in dendritic cells in vitro RNA lipoplex formulations were prepared using different liposome precursors and luc-RNA. For cell culture experiments, the RNA lipoplexes were diluted to 0.01 mg / mL of RNA in a 0.9% NaCl solution. The RNA transfection efficiency of the different RNA lipoplex formulations was explored in human dendritic cells seeded in culture medium or whole blood.

[0401] Example 11 Animal models: Spleen targeting and RNA transfection in dendritic cells The transfection efficiencies of different RNA lipoplex formulations were explored in BALB / c mice. 20 μg of formulated RNA lipoplex was injected retroorbitally, and luciferase expression in dendritic cells (spleen target) was measured after 6 hours. RNA lipoplexes were prepared using luc-RNA and different liposome precursors, small or large liposomes obtained from biocolloids, as well as small and large liposomes after 0.45 μm filtration, an N / P ratio of 0.65, and 112 mM NaCl.

[0402] Example 12 Solubility test We prepared a high-concentration (supersaturated) DOPE-containing solution and investigated whether it could be used for ethanol injection (see next section) to produce liposomes. The results obtained in this solubility test series are shown in Tables 1 and 2. TIFF0007860909000004.tif75170TIFF0007860909000005.tif78170

[0403] Based on these results, DOPE purchased from different suppliers has an equilibrium solubility in ethanol of approximately 50–60 mM (room temperature). However, when preparing a co-solution with the cationic lipid DOTMA, the equilibrium solubility increases significantly, for example, to approximately 90–100 mM when using a DOTMA / DOPE co-solution with a molar ratio of 66:33.

[0404] Example 13 Manufacturing of liposomes of different sizes Liposomes were prepared by ethanol injection using the protocol described in Example 3. Filtration was not performed after ethanol injection. The lipid concentration in the ethanol was varied while keeping all other parameters fixed. Liposome size was measured by dynamic light scattering (DLS) as described in Examples 5 and 6.

[0405] As an example, the results regarding 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. TIFF0007860909000006.tif109170

[0406] As can be seen, the size of the resulting liposomes (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 change in size 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 resulting liposome size increased significantly from <50 nm to over 500 nm (Figure 1). However, even above the solubility limit, the liposome size continued to increase monotonically with increasing lipid concentration.

[0407] All liposome formulations contained a 0.5 μm liposome fraction, which increased in liposomes prepared with a 300 mM lipid solution and decreased in liposomes prepared with low and high lipid concentrations. Furthermore, 0.6 μm and 0.7 μm liposome fractions were also measured in liposome formulations prepared with high lipid concentrations (Figure 2). Larger liposomes were formed after ethanol injection into high-concentration lipid solutions. The total amount of formed liposomes was lower compared to liposome formulations prepared with low lipid concentrations in the lipid solution. The results represent the average particle size from three measurements.

[0408] Example 14 Production of RNA lipoplexes from liposomes of different sizes RNA lipoplexes were prepared as described in Example 4, using different liposome precursors to vary the size of the liposomes for their formation while keeping all other parameters fixed. Liposome size (z-mean) and polydispersity index (PDI) were determined over the course of dynamic light scattering (DLS) experiments as described in Examples 5 and 6.

[0409] The results regarding the effect of lipid concentration on the RNA lipoplex size (Z-mean) (and therefore on the liposome precursor size) of liposome preparations are shown in Table 4 (below) and the corresponding Figures 3 and 4. TIFF0007860909000007.tif138170

[0410] According to this series of tests, RNA lipoplexes obtained from small liposomes were smaller than RNA lipoplexes derived from large liposomes. RNA lipoplexes obtained from liposomes prepared with 300 mM and higher solutions were approximately twice as large as those obtained from liposomes prepared with a 150 mM stock solution. No correlation was found between liposome size and RNA lipoplex size in all RNA lipoplexes prepared with large liposomes (Figure 3).

[0411] The amount of RNA lipoplexes obtained increased with the size of the liposomes used for their formation. The data obtained from dynamic light scattering measurements was confirmed by the measurement of high amounts of large RNA lipoplex particles in formulations prepared with large liposomes (Figure 4). The results represent the average amount of particles from three measurements.

[0412] Example 15 Small-angle X-ray scattering (SAXS) by different lipoplexes Small-angle X-ray scattering experiments were performed as described using RNA lipoplexes obtained from liposomes derived from stock solutions of different concentrations. For example, RNA lipoplexes were formed from liposomes with a DOTMA / DOPE ratio of 2 / 1 (mol / mol) and RNA with a charge ratio of 1.3:2. The liposomes were prepared by ethanol injection as described, with lipid concentrations in ethanol of 100 mM, 300 mM, and 400 mM.

[0413] Figure 5 shows diffraction curves obtained from small-angle X-ray scattering measurements of RNA lipoplexes formed from liposomes prepared with a charge ratio of 4 / 1 (top) and a charge ratio of 1.3 / 2, where the liposomes for lipoplex formation were obtained from lipid stock solutions of 400 mM, 300 mM, and 100 mM in ethanol.

[0414] The scattering pattern is approximately 1 nm. -1 It includes a single Bragg peak. This is a typical diffraction pattern for a spleen-targeting lipoplex, which uses an excess amount of (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 more ambiguous peak was found. Similarly, a secondary peak is also discernible (albeit at low intensity). In fact, a general feature of the X-ray scattering profile of a lipoplex is the presence of several peaks, which may be equidistant or have other spacings depending on the phase state. Here, in contrast, only a single peak is determined. Furthermore, the peak width also changes depending on the concentration of the stock solution originally used to produce the liposomes. At higher concentrations in ethanol, the peak width was smaller. The Bragg peak indicates that the lipoplex is regularly arranged, where the repeat distance (d interval) is TIFF0007860909000008.tif15170[In the formula, q is of degree n, q maxLet the position where each Bragg peak is maximum be defined as the wavelength, λ as the wavelength, and θ as the angle. This is momentum transfer via TIFF0007860909000009.tif14170. This indicates that the peak position is given by the Bragg peak. The d-interval that can originate from the Bragg peak is on the order of 6.5 nm.

[0415] The peak width, Δq, decreases with increasing number of iteration units in the stack. For liquid crystal arrays, the correlation length is: This may be given as TIFF0007860909000010.tif12170. In the lipoplex products described herein, a clear correlation can be derived between the scattering pattern, the aforementioned lipid concentration in ethanol for the production of liposomes, and the biological activity. The peak position remains constant, but the peak width changes monotonically with the lipid concentration in the applied 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 to a larger correlation length. Simultaneously, the biological activity also increases with increasing correlation length. Thus, the described lipoplex with improved activity, produced using a high concentration of lipid stock solution in ethanol for its production and from the liposomes used, can be distinguished by clear structural characteristics. In addition, the described lipoplex with improved activity can also be distinguished from the low-activity lipoplex by other methods, such as asymmetric field flow fractionation (AF4).

[0416] Example 16 Transfection efficiency of RNA lipoplexes in human dendritic cells The luc-RNA lipoplexes were prepared as described using different liposome precursors, varying the size of the liposomes for their formation (by varying the starting lipid concentration for their formation), while keeping all other parameters fixed. Subsequently, the RNA transfection efficiency of the different RNA lipoplex formulations was explored in human dendritic cells seeded in culture medium or whole blood.

[0417] Figure 6 shows illustrative results illustrating transfection efficiency in vitro (in human dendritic cells), determined by measuring luciferase expression and the corresponding biological activity of different RNA lipoplexes prepared with different liposome precursors.

[0418] Biological activity (RNA transfection in vitro) increases monotonically with the correlation length of the RNA lipoplex. A large correlation length indicates a more homogeneous population of lipid bilayers within the RNA lipoplex.

[0419] Example 17 Asymmetric flow field flow fractionation of different lipoplexes Further differences in RNA lipoplexes were determined using asymmetric fluid-field fractionation (AF4), a common and state-of-the-art method for fractionating and separating particles in suspension. According to AF4 theory, particles with similar properties and comparable size should elute simultaneously.

[0420] Figure 7 shows some of the results regarding AF4 measurement for lipoplexes derived from two different types of liposomes, prepared from either a 150 mM stock solution in ethanol or a 400 mM stock solution in ethanol.

[0421] In summary, field-fluid fractionation measurements confirm that lipoplexes derived from liposomes with a lipid concentration of 150 mM in ethanol are qualitatively and quantitatively different from those derived from lipoplexes with a lipid concentration of 400 mM. Although the lipoplexes derived from 150 mM ethanol are smaller, counterintuitively they elute later, suggesting that qualitative differences (shape, interaction with culture medium, charge) should be observed between the two parts. RNA lipoplexes from 150 mM ethanol elute later, but are smaller in size. This indicates that RNA lipoplexes derived from liposomes using a 150 mM lipid solution in ethanol are, on average, smaller than RNA lipoplexes derived from 400 mM lipid in ethanol. Even at the same size, lipoplexes derived from 150 mM ethanol possess different physicochemical properties than those derived from 400 mM ethanol. These differences in size and physicochemical properties correlate with the high biological activity of RNA lipoplexes derived from 400 mM.

[0422] Example 18 Bioactivity of RNA lipoplexes in vitro As determined by transfection experiments (dendritic cells) into cell cultures of different sizes of luciferase-encoding RNA lipoplexes prepared from liposomes, different lipid stocks, and / or different lipid concentrations, the luciferase signal, and therefore the biological activity, increases monotonically with the starting lipid concentration and, consequently, with the liposome size used for RNA lipoplex formation. The corresponding results are shown in Figures 6, 8, and 9.

[0423] In summary, RNA lipoplexes produced from high-lipid concentrations for liposome manufacturing exhibit remarkably high activity in vitro.

[0424] Example 19 Bioactivity of RNA lipoplexes in vivo As determined by transfection experiments (in dendritic cells) of RNA lipoplexes encoding luciferase, prepared from liposomes of different sizes, RNA lipoplexes prepared from larger liposomes result in markedly high luciferase expression and corresponding biological activity.

[0425] Non-limiting examples of this experimental series are shown in Figures 10 and 11. RNA lipoplexes prepared with large liposomes derived from 360 mM biocolloids yielded a more pronounced in vivo expression signal than small RNA lipoplexes derived from 200 mM biocolloids.

[0426] Example 20 Automated production of RNA lipoplex For the automated batch production of RNA lipoplexes, a generally applicable procedure, as shown in Figure 12, was developed. All steps are performed using pre-sterilized, single-use fluid channels that allow for safe, aseptic handling of materials. First, the RNA concentration is adjusted to the liposome concentration, and NaCl is added to concentrate the RNA. This allows for the mixing of equal volumes of RNA and liposomes by adjusting the RNA solution to the RNA concentration. 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, a cryoprotectant solution is added to adjust the final concentration of the drug product. After filling the drug product into glass bottles, the drug product is frozen as a concentrate for long-term storage.

[0427] A critical 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, scalable, industrial manufacturing method for RNA lipoplexes that enables efficient control of the mixing ratio. For small-scale (≤10 liters) production, the method uses a single perfusion pump to simultaneously drive two high-volume syringes filled with RNA or liposomes, thereby achieving control of the mixing of two identical volumes of aqueous solutions containing liposomes and RNA. For high-volume (≥10 liters) isotope ejection, a pumping system such as a pressurized vessel, membrane pump, gear pump, magnetic levitation pump, or peristaltic pump is used in combination with a flow rate sensor with a feedback loop for online control and real-time adjustment of the flow rate.

[0428] Automated RNA lipoplex production requires static mixing elements to ensure efficient mixing of aqueous solutions containing RNA and liposomes. Commercially available microfluidic mixing elements containing serpentine pathways and embedded structures to enhance mixing, as well as prototype mixing elements with equivalent architectures, were found to become clogged during production. Therefore, these mixing elements are unsuitable for automated RNA lipoplex production. Y-shaped and T-shaped mixing elements with diameters between 1.2 and 50.0 mm were found to be suitable for automated RNA lipoplex production.

[0429] Methods: RNA lipoplexes were prepared using different mixed elements (Table 1). During lipoplex preparation, the operator observed the mixed elements and recorded any deposition or clogging of the material.

[0430] Results: With commercially available microfluidic chips (NanoAssemblr®, Precision Nanosystem, Vancouver, Canada), clogging was observed after preparing 3 mL of RNA lipoplex. Similar observations were made during further testing of prototype microfluidic mixing elements (Table 5). Deposition, particularly element blockage, was observed with all (micro)fluidic mixing elements, including those with architectures equivalent to commercially available elements, whereas this was not observed when using Y-shaped or T-shaped mixing elements. In addition to the described Y-shaped mixing element with a diameter of 2.4 mm, larger diameter mixing elements were also investigated. No limitations were found on the diameter of the Y-shaped mixing element, so the described method is considered suitable for RNA lipoplex preparation with Y-shaped mixing elements up to a maximum diameter of 50 mm. TIFF0007860909000011.tif119170

[0431] When using Y-shaped or T-shaped mixing 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-shaped or T-shaped mixing elements with an inner diameter of 1.6–50 mm. To ensure efficient mixing, the Reynolds number derived from the mixing flow rate should not be lower than approximately 300. To ensure efficient mixing, the ratio of flow rate to the diameter of the mixing element should not be lower than approximately 150. Experiments with Reynolds numbers up to approximately 2100 were found to be suitable for automated production. The data also support the possibility of higher flow rates. This stems from the fact that at a Reynolds number of 2100, the conditions are already in turbulent mode, and therefore, similar mixing conditions are expected even at higher flow rates.

[0432] Methods: RNA lipoplexes were prepared by ejecting RNA and liposome solutions using a single perfusion pump. RNA lipoplex formation was carried out using representative Y-shaped mixed elements with inner diameters of 2.4 and 3.2 mm. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS) measurements. The Reynolds number obtained from exploring combinations of flow velocity and mixed element diameter was theoretically calculated using the formula (Figure 13). The ratio of flow velocity to mixed element diameter is given by the flow velocity (cm²). 3 The calculation was performed by dividing the fraction ( / min) by the diameter (cm) of the mixed element. The factors are given as dimensionless numbers.

[0433] Results: Combinations of flow rate and mixing elements that result in theoretically calculated Reynolds numbers below approximately 300 lead to the formation of RNA lipoplexes with increased particle size and polydispersity (Figure 13 and Table 6). Theoretically, to ensure the reproducible formation of RNA lipoplexes with desired particle characteristics, the Reynolds number should be a minimum of approximately 300 (Figures 13 and 14 and Table 6), and the ratio of flow rate to mixing element diameter should be a minimum of approximately 150. A 2.4 mm mixing element was found to enable efficient and reproducible mixing of RNA and liposomes over a wide range of flow rates (60–240 mL / min). Since no upper limit was found in these studies, no upper limit is predicted for the Reynolds number or the ratio of flow rate to mixing element diameter. TIFF0007860909000012.tif106170

[0434] Example 21 Effect of RNA concentration RNA lipoplexes were prepared using Y-shaped mixed elements with typical dimensions (2.4 mm). To identify the concentration range in which RNA lipoplexes can be prepared under current settings, the RNA concentration was systematically varied from 0.05 mg / mL to 0.5 mg / mL. To explore the stability of the formed lipoplexes, the final formulations were adjusted to 0.05 mg / mL of RNA, 22% sucrose, and 20 mM NaCl, and the formulations were frozen three times.

[0435] RNA lipoplex formation using RNA concentrations between 0.1 and 0.5 mg / mL results in comparable particle characteristics (Figure 15). These particle characteristics were also preserved during 3 hours of freezing, indicating an extremely robust manufacturing method with respect to RNA concentration fluctuations (Figure 16). Since no limit on RNA concentration during RNA lipoplex preparation is indicated, the described settings are considered suitable for preparing RNA lipoplexes with a maximum RNA concentration of 5 mg / mL.

[0436] The described method for the automated production of RNA lipoplexes enables the reproducible preparation of stable RNA lipoplexes with different charge ratios.

[0437] Methods: To demonstrate the robustness of the charge ratio (ration) of the semi-automated preparation of RNA lipoplexes, this parameter was systematically varied between 1.0:2.0–2.1:2.0 and 2.0:1.0–5.0:1:0. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS).

[0438] Results: No effect of charge ratio variation on RNA lipoplex size and polydispersity was observed between charge ratios of 1.0:2.0 and 2.1:2.0 (Figure 17). Therefore, the range between 1.0:2.0 and 2.1:2.0 is considered to result in RNA lipoplex preparations of comparable quality. In addition, stable particles with defined size and polydispersity were also formed at charge ratios between 3.0:1.0 and 5.0:1.0 (Figure 18).

[0439] Example 22 Salt concentration during RNA lipoplex formation Automated production of RNA lipoplexes containing high biological activity requires control of ionic conditions during RNA lipoplex formation. To ensure biological activity, RNA lipoplex formation must be carried out in the presence of 45-300 mM NaCl. Other ionic compounds, such as EDTA and HEPES, can contribute to ionic strength and reduce the required minimum concentration of NaCl.

[0440] Methods: RNA lipoplexes were automatically prepared at different NaCl concentrations during RNA lipoplex formation. The particle size and polydispersity of the RNA lipoplexes were analyzed by photon correlation spectroscopy (PCS). In addition, the biological activity of the lipoplexes was investigated in vitro by measuring the luciferase signal.

[0441] Results: Particle characteristics could be controlled by adjusting the ionic strength. Increasing the salt concentration during production induced a slight increase in particle size (Figure 19). Salt concentration was found to affect biological activity. Increasing the salt concentration during production induced an increase in biological activity (Figure 20). Therefore, the NaCl concentration during RNA lipoplex formation should not be lower than 45 mM NaCl.

[0442] Example 23 Stabilization of RNA lipoplex To stabilize RNA within an RNA lipoplex, both in the presence and absence of a cryoprotectant, buffer systems such as HEPES, acetate / sodium acetate, and sodium phosphate can be used to stabilize the RNA within the lipoplex within a pH range of 5.5–6.7. Sodium carbonate systems were found not to yield comparable stabilization effectiveness.

[0443] Methods: To explore the optimal pH range and investigate different buffering agents targeting 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 first incubated under stress conditions (40°C) in the absence of cryoprotectants. RNA integrity was analyzed by capillary electrophoresis for 21 days. To explore 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.

[0444] Results: Comparable results were obtained for buffering systems, HEPES, acetate / sodium acetate, and sodium phosphate, whereas carbonate systems did not result in comparable RNA stabilization (Figure 21). RNA integrity depends on the pH value of the formulation. The optimal pH range was identified as being between pH 5.5 and 7.4. In the presence of sucrose, an exemplary cryoprotectant, a pH range of 5.5 to 8.0 was identified as resulting in the best RNA stabilization (Figure 22).

[0445] RNA synthesis methods may generate divalent metal ions, and excipients or glass containers and cans in the formulation can affect RNA stability. Disodium EDTA forms a stable, water-soluble complex with alkaline earth metals and heavy metal ions. Disodium EDTA contributes to the concentration of ions present during RNA lipoplex formation, thereby reducing the concentration of NaCl required to prepare a biologically active RNA lipoplex. RNA lipoplex formation in the presence of EDTA (0-20 mM) is possible by the described method.

[0446] 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 reduced EDTA content (0.1 mM to 5.4 mM). RNA integrity was analyzed for 21 days using key physicochemical parameters.

[0447] Results: It was found that RNA lipoplex formation in the presence of high concentrations of EDTA (up to 18 mM) resulted in particle properties equivalent to those of preparations in the presence of low concentrations of EDTA. No significant differences were found among different groups containing EDTA between 0.01% (w:v) (0.1 mM) and 0.2% (w:v) (5.4 mM) during storage (Figure 23). Since disodium EDTA can contribute to the ion concentration present during RNA lipoplex formation and act as a scavenger for divalent metal ions that may reduce RNA integrity, the presence of EDTA concentrations up to 20 mM during RNA lipoplex formation is considered advantageous.

[0448] Example 24 Optimization of NaCl and cryogenic protective agent content The ionic conditions to be adjusted must be controlled during the production of the RNA lipoplex, during long-term storage, and during application to patients (Figure 24). The concentration of NaCl can be 45–300 mM during the formation of the RNA lipoplex; 10–50 mM during long-term storage of the RNA lipoplex in a frozen state; and 80–150 mM after thawing and dilution with physiological saline.

[0449] To ensure the stabilization of particle properties during multiple freezes, we identified cryoprotective agents with concentrations of ≤70 mM for each NaCl concentration, which should not be lower than this. As cryoprotective agents, monomolecular or dimolecular sugars such as glucose, sucrose, mannitol, trehalose, sorbitol, triols as glycerin, and mixtures thereof can be used in concentrations between 12.5% ​​and 35.0% (w:v). The stabilizing effectiveness of sorbitol is lower compared to the previously mentioned compound, and arginine does not efficiently stabilize RNA lipoplexes during freezing. TIFF0007860909000013.tif68170TIFF0007860909000014.tif68170

[0450] Methods: To identify suitable cryoprotective agents, cryoprotective systems containing different compounds, representative of monosaccharides (glucose and sorbitol), disaccharides (sucrose and trehalose), amino acids (arginine and proline), and triols (glycerin), as well as mixtures of different sugars (mannitol and sucrose), were explored (Figures 25 and 26). Accordingly, RNA lipoplexes were frozen in the presence of these compounds at increasing concentrations. To identify the minimum content of cryoprotective agents at a specific concentration of NaCl, RNA lipoplexes were frozen in the presence of sucrose or trehalose, representative cryoprotective agents, at increasing concentrations (Table 7). To determine the concentration range of the cryoprotective agents being explored in detail, samples were first frozen once. In a third experiment, the effectiveness of particle size stabilization was examined by freezing RNA lipoplexes up to 10 times in the presence of trehalose, a cryoprotective agent (Table 8).

[0451] Results: Arginine clearly destabilizes the RNA lipoplex, while other cryoprotectants are generally applicable to stabilizing the RNA lipoplex during freezing (Figures 25 and 26). Glycerin, mannitol, and sucrose (1:1, w:w), proline, and sorbitol can be used as cryoprotectants for RNA lipoplexes. Since all further test stabilizers, in addition to arginine, are considered suitable, it is indicated that a wide range of amino acids, sugars, and mixtures of such compounds are suitable for stabilizing RNA lipoplexes during freezing. The stabilizing effectiveness of sorbitol is lower compared to the previously mentioned compound.

[0452] Detailed investigations into the required amount of cryogenic protectant for each concentration of NaCl (Table 8) revealed a direct correlation between NaCl concentration and the required concentration of cryogenic protectant after a single freezing step (Figures 27 and 28). Acceptable preservation of particle size was observed after a single freezing step for 0–60 mM NaCl and 20% (w:v) sucrose or trehalose dihydrate, whereas insufficient stabilization was found with low percentages of cryogenic protectant (e.g., ≤15% for 60 mM NaCl; ≤10% for 40 mM NaCl; ≤5% for 20 mM NaCl). No significant differences were found between sucrose and trehalose within the explored range.

[0453] The particle size of RNA lipoplexes was analyzed in the presence of NaCl and trehalose combinations, as shown in Table 9.3.2, after 1, 2, 3, 5, and 10 freezes. The following results were obtained: At a concentration of 50 mM NaCl, ≥12.5% ​​trehalose was sufficient to stabilize the properties of RNA lipoplex particles even after 10 hours of freezing (Figure 29). At 70 mM NaCl, ≥12.5% ​​was required for minimal stabilization, and ≥22.5% was required for accurate preservation of particle size (Figure 30). At 90 mM NaCl, ≥15.0% trehalose was required for minimal stabilization, and accurate preservation of particle size could not be achieved even at the highest exploratory concentration of ≥27.5% (Figure 31).

[0454] Example 25 A combination of salt and chilling agent for long-term storage. For long-term storage at a given temperature, the combinations of NaCl and cryogenic protective agents listed in Table 9 should be used.

[0455] Methods: RNA lipoplexes were frozen in the presence of NaCl and a combination of sucrose or trehalose at -15 to -30°C to explore the minimum content of cryogenic protective agents for long-term storage at specific concentrations of NaCl. Samples were frozen at -30°C and then transitioned to their respective storage temperatures (-15 or -30°C). After the specified storage period, the samples were analyzed, and the preservation of colloidal stability was assessed by measuring particle size using PCS.

[0456] Results: These experiments showed that freezing in the presence of up to 70 mM NaCl could preserve the particle properties of RNA lipoplexes, but also revealed further effects that contributed to the destabilization of colloidal stability. Furthermore, while acceptable stabilization of particle properties after freezing was confirmed with combinations such as 60 mM NaCl and 20% sucrose, the particle size of these formulations significantly increased over time at a storage temperature of -15°C (Figures 32 and 33). This effect was reduced at storage at -30°C (Figures 34 and 35).

[0457] Given a NaCl content, the stability of the RNA lipoplex depends on the amount of cryoprotectant. The required amount of cryoprotectant increases with increasing salt content in the storage solution. This effect is independent of the type of sugar used as the cryoprotectant. For long-term storage in a frozen state at -15 or -30°C, the RNA lipoplex composition should contain the cryoprotectant in the amounts listed in Table 9. TIFF0007860909000015.tif76170

[0458] Example 26 Long-term storage using different cryogenic protective agents Stabilization for 9 months in the presence of 22% (w:v) monomolecular or bimolecular sugars is possible with 10–40 mM NaCl. These formulations can be frozen at -15 to -40°C and maintained at their respective temperatures for long-term storage. In the presence of 12.6–16.8% (w:v) dextran, 10–30 mM NaCl can be used.

[0459] Methods: To explore the minimum content of representative cryoprotectants required for long-term storage at -20°C, RNA lipoplexes were frozen with varying NaCl concentrations in combination with a constant cryoprotectant content. The concentrations of monomer or dimer cryoprotectants, such as glucose, sucrose, and trehalose, were adjusted to 22% (w:v). These formulations were frozen and stored at -15 to -40°C. After a specified storage time, long-term stability was explored by measuring particle size using PCS.

[0460] Formulations containing mixtures of dextran compositions, which are polymers, as listed in Table 9.5.1 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 determined by measuring the particle size.

[0461] Results: To ensure long-term stability of RNA lipoplexes in a frozen state, a maximum NaCl concentration that should not be exceeded was identified for all explored monomer or dimer molecule cryoprotectants. While 60 and 80 mM NaCl resulted in rapid destabilization of the lipoplex, NaCl concentrations of ≤40 mM allowed for preservation of colloidal properties for a minimum of 9 months when stored at -20°C (Table 10 and Figures 36-38). No difference in stabilization effectiveness was found for sucrose, trehalose, and glucose. For formulations containing 20 mM NaCl, no difference in long-term stability was found when samples were frozen and stored at -15 to -40°C (Figure 39).

[0462] When we explored formulations containing dextran with low levels of cryogenic protective agents (12.6-16.8% (w:v)), we found that their stabilizing effectiveness was comparable to or better than that of monomolecular or bimolecular sugars (Figure 40). TIFF0007860909000016.tif84170

[0463] Example 27 Freeze drying a) Freeze-thaw To determine the most effective cryogenic / lyophilization protective agent concentration, freeze-thaw studies were conducted. RNA lipoplex formulations were freeze-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 therefore their particle size was not determined. As shown in Figure 41, formulations frozen with cryogenic / lyophilization protective agents showed concentration-dependent cryogenic protection, with particle size increasing at lower lyophilization / lyophilization protective agent concentrations. At 22% w / v trehalose, only a minimal increase in particle size was observed, with an Sf / Si ratio of 1.04, which is still considered acceptable as it is less than 1.3 (Sf = final size, Si = initial size). At low trehalose concentrations, the Sf / Si ratio increased. The Sf / Si ratio obtained from freeze-thaw studies correlated with the Sf / Si ratio obtained from the same formulation after lyophilization and reconstitution.

[0464] b) Reconfiguration and particle stability RNA lipoplex formulations prepared in 5 mM HEPES, 2.6 mM EDTA, 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, and 80 mM NaCl, as well as 5%, 10%, 15%, and 22% trehalose, were lyophilized. All lyophilized samples exhibited a good cake-like appearance. Samples were reconstituted to their original volume with a 0.9% NaCl solution. All lyophilized RNA lipoplex formulations were rapidly dissolved during reconstitution with a 0.9% NaCl solution or water-based filtration (WFI). Changes in particle size in the lyophilized RNA lipoplex formulation prepared in 22% trehalose were determined after reconstitution with a 0.9% NaCl solution or water.

[0465] As shown in Figure 42, particle size remained stable in all lyophilized RNA lipoplex formulations containing trehalose after lyophilization and reconstitution. When lyophilized samples were reconstituted with 0.9% NaCl, 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. RNA lipoplex particle size increased in formulations prepared with low trehalose and high NaCl concentrations.

[0466] c) Cell culture experiments using freeze-dried RNA lipoplex preparations In vitro transfection experiments were performed on lyophilized luciferase-encoding RNA lipoplex preparations prepared in 22% trehalose at different NaCl concentrations. The lyophilized samples were reconstituted with a 0.9% NaCl solution.

[0467] As shown in Figure 43, lyophilized RNA lipoplex formulations prepared with 22% trehalose and different NaCl concentrations showed similar levels of luc-RNA transfection in dendritic cells. No correlation was found between the NaCl concentration present in the RNA lipoplex formulations and RNA transfection in vitro. Lyophilized samples showed similar, or even better, luc-RNA transfection compared to untransfected RNA lipoplex controls.

[0468] d) Stability studies Lyophilized RNA lipoplex formulations containing 10% trehalose, 22% trehalose, and 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM NaCl were explored for stability studies at 4°C, 25°C, and 40°C (for 1 month and 6 months). After reconstitution to the original volume with a 0.9% NaCl solution, the samples were characterized for particle size and RNA integrity (full-length RNA%).

[0469] As shown in Figures 44 and 45, the size of different RNA lipoplexes did not change significantly over time, independently of formulation or storage temperature. Interestingly, low amounts of cryoprotectant (e.g., 10%) were sufficient to maintain particle stability in lyophilized formulations, while high amounts (e.g., 22%) were 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 between 85% and 94% for RNA(lip) formulations stored at 25°C. However, no correlation was identified between trehalose, its ratio to NaCl concentration, or storage time.

Claims

1. RNA encoding a peptide or protein, containing at least one epitope, At least one cationic lipid and at least one further lipid, RNA lipoplex particles containing, The at least one cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and the at least one further lipid is DOPE or DOTMA. RNA lipoplex particles in which the ratio of positive charge to negative charge is 1:2 to 1.9:2, or 1.3:2.0, Sodium chloride at concentrations of 20 mM to 40 mM, A buffer and / or chelating agent wherein the buffer is 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) and the chelating agent is ethylenediaminetetraacetic acid (EDTA), Stabilizer and A composition containing the following:

2. The amount of RNA in the composition is 0.01 mg / mL to 1 mg / mL, 0.05 mg / mL to 0.5 mg / mL, or 0.05 mg / mL, and / or Sodium chloride is at a concentration of 20 mM to 30 mM, a concentration of 20 mM, or a concentration of 30 mM, and / or The concentration of the stabilizer in the composition is higher than the value required for the physiological osmolality, and / or The composition according to claim 1, wherein the concentration of the stabilizer in the composition is 5 to 35 wt / volume percent (% w / v) or 12.5 to 25 wt / volume percent (% w / v).

3. The stabilizer is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyalcohols, or The composition according to claim 1 or 2, wherein the stabilizer is sucrose at a concentration of 5 to 25% by weight / volume (% w / v), or sucrose at a concentration of 15% (w / v) to 25% (w / v), or sucrose at a concentration of 20% (w / v) to 25% (w / v), or sucrose at a concentration of 22% (w / v) or 20% (w / v).

4. The composition according to any one of claims 1 to 3, having a pH of 5.7 to 6.7, 5.7 to 6.2, or 6.

2.

5. The composition according to any one of claims 1 to 4, further comprising a chelating agent, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

6. RNA encoding a peptide or protein, containing at least one epitope, at a concentration of 0.05 mg / mL, 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio of 2:1 RNA lipoplex particles containing, RNA lipoplex particles in which the ratio of positive charge to negative charge is 1.3:2.0, Sodium chloride at a concentration of 20 mM, Sucrose at a concentration of 22% (w / v), HEPES at a pH of 6.2 and a concentration of 7.5 mM, EDTA at a concentration of 2.5 mM and A composition containing the following:

7. The composition according to any one of claims 1 to 6, which, when frozen, is stable at a temperature of -15°C to -40°C for at least one month, or stable at a temperature of -15°C for at least one month, or at a temperature of -15°C for at least two months, or at a temperature of -20°C for at least one month, or at a temperature of -20°C for at least two months, or at a temperature of -30°C for at least one month, or at a temperature of -30°C for at least two months.

8. RNA lipoplex particles are 1 nm －１ Characterized by a single Bragg peak, where the peak width is 0.2 nm. －１ Smaller, and / or The composition according to any one of claims 1 to 7, wherein the RNA lipoplex particles have an average diameter in the range of 200-800 nm, 250-700 nm, 400-600 nm, 300-500 nm, or 350-400 nm.

9. At least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and at least one further lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), and / or The molar ratio of at least one cationic lipid to at least one further lipid is 10:0 to 1:9, 4:1 to 1:2, 3:1 to 1:1, or 2:1, and / or The composition according to any one of claims 1 to 5, 7, and 8, wherein the RNA lipoplex particles contain DOTMA and DOPE in a molar ratio of 10:0 to 1:9, 4:1 to 1:2, 3:1 to 1:1, or 2:1, and the charge ratio of the positive charge in DOTMA to the negative charge in RNA is 1:2 to 1.9:

2.

10. The composition according to any one of claims 1 to 9, further comprising an adjuvant.

11. A composition according to any one of claims 1 to 10, formulated for systemic administration.

12. The composition according to claim 11, wherein systemic administration is by intravenous administration.

13. A composition according to any one of claims 1 to 12 for therapeutic use.

14. A method for preparing an aqueous composition containing RNA lipoplex particles, comprising: thawing the composition according to any one of claims 7 to 13, which is in a frozen state; and adjusting the osmolality and ionic strength by adding an aqueous liquid.

15. The method according to claim 14, wherein an aqueous liquid is added to obtain a composition with an osmolality of 200 mmol to 450 mmol, and / or sodium chloride with a concentration of 80 mM to 150 mM.

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